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Handbook of Biochemistry and Molecular Biology, Fourth Edition
F O U R T H E D I T I O N HANDBOOK OF BIOCHEMISTRY AND MOLECULAR BIOLOGY 9168_Book.indb 1 4/30/10 10:58 AM F O U
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F O U R T H
E D I T I O N
HANDBOOK OF
BIOCHEMISTRY AND MOLECULAR BIOLOGY
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F O U R T H
E D I T I O N
HANDBOOK OF
BIOCHEMISTRY AND MOLECULAR BIOLOGY EDITED BY
ROGER L. LUNDBLAD FIONA M. MACDONALD
Boca Raton London New York
CRC Press is an imprint of the Taylor & Francis Group, an informa business
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CRC Press Taylor & Francis Group 6000 Broken Sound Parkway NW, Suite 300 Boca Raton, FL 33487-2742 © 2010 by Taylor and Francis Group, LLC CRC Press is an imprint of Taylor & Francis Group, an Informa business No claim to original U.S. Government works Printed in the United States of America on acid-free paper 10 9 8 7 6 5 4 3 2 1 International Standard Book Number: 978-0-8493-9168-2 (Hardback) This book contains information obtained from authentic and highly regarded sources. Reasonable efforts have been made to publish reliable data and information, but the author and publisher cannot assume responsibility for the validity of all materials or the consequences of their use. The authors and publishers have attempted to trace the copyright holders of all material reproduced in this publication and apologize to copyright holders if permission to publish in this form has not been obtained. If any copyright material has not been acknowledged please write and let us know so we may rectify in any future reprint. Except as permitted under U.S. Copyright Law, no part of this book may be reprinted, reproduced, transmitted, or utilized in any form by any electronic, mechanical, or other means, now known or hereafter invented, including photocopying, microfilming, and recording, or in any information storage or retrieval system, without written permission from the publishers. For permission to photocopy or use material electronically from this work, please access www.copyright.com (http://www.copyright.com/) or contact the Copyright Clearance Center, Inc. (CCC), 222 Rosewood Drive, Danvers, MA 01923, 978-750-8400. CCC is a not-for-profit organization that provides licenses and registration for a variety of users. For organizations that have been granted a photocopy license by the CCC, a separate system of payment has been arranged. Trademark Notice: Product or corporate names may be trademarks or registered trademarks, and are used only for identification and explanation without intent to infringe. Library of Congress Cataloging‑in‑Publication Data Handbook of biochemistry and molecular biology / edited by Roger L. Lundblad and Fiona M. Macdonald. p. ; cm. Includes bibliographical references and index. ISBN-13: 978-0-8493-9168-2 (alk. paper) ISBN-10: 0-8493-9168-7 (alk. paper) 1. Biochemistry--Handbooks, manuals, etc. 2. Molecular biology--Handbooks, manuals, etc. I. Lundblad, Roger L. II. Macdonald, Fiona. [DNLM: 1. Biochemical Phenomena--Tables. QU 16 H2363 2010] QH345.H347 2010 572--dc22
2009042039
Visit the Taylor & Francis Web site at http://www.taylorandfrancis.com and the CRC Press Web site at http://www.crcpress.com
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This work is dedicated to the many scientists of the “The Greatest Generation” who contributed to the base of our knowledge of biochemistry and molecular biology. Roger L. Lundblad, Ph.D. To my parents, Pat and Paul Macdonald, whose unwavering love and support has been my guiding light. Fiona M. Macdonald, Ph.D., F.R.S.C.
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Table of contents Foreword...............................................................................................................................................................................................................................xi Preface................................................................................................................................................................................................................................ xiii Acknowledgments.............................................................................................................................................................................................................xv Editors............................................................................................................................................................................................................................... xvii
Section 1: Amino Acids, Peptides, and Proteins
Properties of Amino Acids.................................................................................................................................................................................... 3 Data on the Naturally Occurring Amino Acids............................................................................................................................................... 7 Structures and Symbols for Synthetic Amino Acids Incorporated into Synthetic Polypeptides........................................................43 Unnatural Amino Acids for Incorporation into Proteins.............................................................................................................................53 Properties of the α-Keto Acid Analogs of Amino Acids...............................................................................................................................55 α,β-Unsaturated Amino Acids............................................................................................................................................................................57 Amino Acid Antagonists......................................................................................................................................................................................59 Coefficients of Solubility Equations of Certain Amino Acids in Water....................................................................................................65 Heat Capacities, Absolute Entropies, and Entropies of Formation of Amino Acids and Related Compounds................................67 Heat of Combustion, Enthalpy and Free Energy of Formation of Amino Acids and Related Compounds.......................................69 Solubilities of Amino Acids in Water at Various Temperatures.................................................................................................................71 Heats of Solution of Amino Acids in Aqueous Solution at 25°C................................................................................................................75 Free Energies of Solution and Standard Free Energy of Formation of Amino Acids in Aqueous Solution at 25°C........................77 Far Ultraviolet Absorption Spectra of Amino Acids.....................................................................................................................................79 UV Absorption Characteristics of N-Acetyl Methyl Esters of the Aromatic Amino Acids, Cystine and of N-Acetylcysteine.......................................................................................................................................................................81 Numerical Values of the Absorbances of the Aromatic Amino Acids in Acid, Neutral and Alkaline Solutions............................83 Luminescence of the Aromatic Amino Acids.................................................................................................................................................89 Luminescence of Derivatives of the Aromatic Amino Acids.......................................................................................................................91 Luminescence of Proteins Lacking Tryptophan.............................................................................................................................................93 Luminescence of Proteins Containing Tryptophan......................................................................................................................................95 Hydrophobicities of Amino Acids and Proteins.............................................................................................................................................99 Chemical Specificity of Reagents for Protein Modification ......................................................................................................................115 Reagents for the Chemical Modification of Proteins . ................................................................................................................................117 Protein pK Values.................................................................................................................................................................................................137 Protease Inhibitors and Protease Inhibitor Cocktails.................................................................................................................................141 Assay of Solution Protein Concentration.......................................................................................................................................................155 Spectrophotometric Determination of Protein Concentration in the Short-Wavelength Ultraviolet.............................................161
Section 2: Lipids
A Comprehensive Classification System for Lipids......................................................................................................................................165 Properties of Fatty Acids and Their Methyl Esters......................................................................................................................................189 Densities, Specific Volumes, and Temperature Coefficients of Fatty Acids from C8 to C12. ..............................................................191 Composition and Properties of Common Oils and Fats.............................................................................................................................193 Androgens.............................................................................................................................................................................................................199 Bile Acids...............................................................................................................................................................................................................201 Corticoids............................................................................................................................................................................................................. 203 Estrogens............................................................................................................................................................................................................... 205 Progestogens........................................................................................................................................................................................................ 207 Sterols .................................................................................................................................................................................................................. 209 Prostaglandins and Related Fatty-Acid Derived Materials........................................................................................................................221
Section 3: Vitamins and Coenzymes
Properties of Vitamins........................................................................................................................................................................................225 Biological Characteristics of Vitamins...........................................................................................................................................................237 Properties for Ascorbic Acid and Ascorbate-2-Sulfate...............................................................................................................................241 Vitamers.................................................................................................................................................................................................................243 Vitamin Names Discarded................................................................................................................................................................................251
Section 4: Nucleic Acids
UV Spectral Characteristics and Acidic Dissociation Constants of 280 Alkyl Bases, Nucleosides, and Nucleotides....................................................................................................................................................................................................255 Ultraviolet Absorbance of Oligonucleotides Containing 2¢-O-Methylpentose Residues...................................................................263 v ii
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Table of Contents
viii
Spectrophotometric Constants of Ribonucleotides.....................................................................................................................................265 Purines, Pyrimidines, Nucleosides, and Nucleotides: Physical Constants and Spectral Properties................................................269 Chemical Modification of Nucleic Acids........................................................................................................................................................359 Transfection Technologies................................................................................................................................................................................373
Section 5: Carbohydrates
Introduction to Carbohydrates.........................................................................................................................................................................383 Natural Alditols, Inositols, Inososes, and Amino Alditols and Inosamines......................................................................................... 407 Natural Acids of Carbohydrate Derivation....................................................................................................................................................417 Natural Aldoses....................................................................................................................................................................................................431 Natural Ketoses....................................................................................................................................................................................................457 Carbohydrate Phosphate Esters........................................................................................................................................................................473 The Naturally Occurring Amino Sugars........................................................................................................................................................491 Oligosaccharides (Including Disaccharides).................................................................................................................................................499 Mucopolysaccharides (Glycosaminoglycans)................................................................................................................................................533
Section 6: Physical and Chemical Data
Recommendations for Nomenclature and Tables in Biochemical Thermodynamics..........................................................................537 Standard Transformed Gibbs Energies of Formation for Biochemical Reactants................................................................................551 Enthalpy, Entropy, and Free Energy Values for Biochemical Redox Reactions......................................................................................555 Oxidation-Reduction Potentials, Absorbance Bands and Molar Absorbance of Compounds Used in Biochemical Studies.......................................................................................................................................................................................557 Calorimetric ΔH Values Accompanying Conformational Changes of Macromolecules in Solution..............................................565 Free Energies of Hydrolysis and Decarboxylation........................................................................................................................................579 Deci-Normal Solutions of Oxidation and Reduction Reagents.................................................................................................................585 Guidelines for Potentiometric Measurements in Suspensions Part A. The Suspension Effect..........................................................587 Ionization Constants of Acids and Bases.......................................................................................................................................................595 Guidelines for NMR Measurements for Determination of High and Low pKa Values........................................................................637 Measurement and Interpretation of Electrokinetic Phenomena............................................................................................................. 645 Measurement of pH Definition, Standards, and Procedures.....................................................................................................................675 General Comments on Buffers..........................................................................................................................................................................693 List of Buffers........................................................................................................................................................................................................695 Brønsted Acidities...............................................................................................................................................................................................707 Measurement of pH.............................................................................................................................................................................................709 Buffer Solutions....................................................................................................................................................................................................715 Amine Buffers Useful for Biological Research..............................................................................................................................................719 Preparation of Buffers for Use in Enzyme Studies.......................................................................................................................................721 Buffer for Acrylamide Gels (Single-Gel Systems).........................................................................................................................................725 Buffer for Acrylamide Gels with More Than One Layer.............................................................................................................................726 Starch Gels............................................................................................................................................................................................................729 Indicators for Volumetric Work and pH Determinations..........................................................................................................................731 Acid and Base Indicators....................................................................................................................................................................................735 Specific Gravity of Liquids.................................................................................................................................................................................739 Viscosity and Density Tables.............................................................................................................................................................................743 A Listing of Log P Values, Water Solubility, and Molecular Weight for Some Selected Chemicals.................................................747 Chemicals Commonly Used in Biochemistry and Molecular Biology and Their Properties.............................................................751 Common Detergents Used in Biochemical Research..................................................................................................................................811 Some Properties of Detergents and Surfactants Used in Biochemistry and Molecular Biology.......................................................813 Some Biological Stains and Dyes......................................................................................................................................................................819 Mordant Dyes...................................................................................................................................................................................................... 843 Metal Chelating Agents . ...................................................................................................................................................................................847 Water ...................................................................................................................................................................................................................855 Stability of Solutions for GLP and cGMP Use...............................................................................................................................................857 General Information on Spectroscopy............................................................................................................................................................859 Microplates............................................................................................................................................................................................................861 Plastics ...................................................................................................................................................................................................................863 Chemical and Physical Properties of Various Commercial Plastics........................................................................................................869 Generic Source-Based Nomenclature for Polymers.....................................................................................................................................871 Definitions of Terms Relating to Reactions of Polymers and to Functional Polymeric Materials....................................................877 Definitions of Terms Related to Polymer Blends, Composites, and Multiphase Polymeric Materials.............................................887 Organic Name Reactions Useful in Biochemistry and Molecular Biology.............................................................................................899 Enzymes in Synthetic Organic Chemistry ....................................................................................................................................................927
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Table of Contents
i x
Therapeutic Enzymes..........................................................................................................................................................................................939 Weights of Cells and Cell Constituents......................................................................................................................................................... 943 Particle Diameter................................................................................................................................................................................................ 945 Appendix A: Abbreviations and Acronyms......................................................................................................................................... 947 Appendix B: Glossary of Terms Useful in Biochemistry.................................................................................................................... 959 Index....................................................................................................................................................................................................... 1075
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Foreword For almost a century, CRC Press has been a leader in providing concise compilations of scientific data for researchers, teachers, and students. The CRC Handbook of Chemistry and Physics, which first appeared in 1913 and is now in its 90th edition, is the basic source of physical science data that most chemists, physicists, and engineers turn to. Other widely used handbooks from CRC Press cover materials science, engineering, and mathematics. Many specialized handbooks have also appeared under the CRC imprint, ranging from semiconductors to lipids. The 1968 publication of the CRC Handbook of Biochemistry, edited by Herbert A. Sober, marked a milestone in bioscience data. Appearing just 15 years after Watson and Crick elucidated the structure of DNA, the subtitle of this work, Selected Data for Molecular Biology, was a recognition that molecular biology
was the new frontier of the biosciences. This was followed by the multivolume Handbook of Biochemistry and Molecular Biology, edited by Gerald D. Fasman, and its single volume abridged version Practical Handbook of Biochemistry and Molecular Biology, which appeared in 1989. The intervening 20 years has seen an explosion of data in this field and an exponential growth in the translation of recent discoveries into new technology. This new Handbook of Biochemistry, edited by Roger Lundblad and Fiona M. Macdonald, is thus a welcome addition to the list of handbooks available through CRC Press. I am sure it will find heavy use in both basic research and biotechnology. David R. Lide Editor-in-Chief, CRC Handbook of Chemistry and Physics
x i
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Preface This is the fourth edition of the Handbook of Biochemistry and Molecular Biology. The first edition was published as a single volume in 1968 under the guidance of Herbert Sober. The second edition appeared in 1970 and the third, with Gerald Fasman as editor, appeared in eight volumes published in 1975–6. This increase in size reflected the rapid advances in knowledge in the then relatively new field of molecular biology. It is intended that current Handbook of Biochemistry and Molecular Biology be a companion volume to the CRC Handbook of Chemistry and Physics—a single volume ready-reference work that will find a home on the bookshelves of biochemists and molecular biologists everywhere. This fourth edition contains materials from the previous editions as well as extensive new material. Staying within the confines of a single volume has meant difficult decisions on which tables to include, and the editors welcome feedback from readers. The advent of electronic media allows for more frequent updating and it is hoped that any infelicities in our selection may be
readily rectified. Additionally, suggestions on new topics for this Handbook and notification of errors are always appreciated. Address all comments to Editor, Handbook of Biochemistry and Molecular Biology, Taylor & Francis Group, 6000 Broken Sound Parkway NW, Suite 300, Boca Raton, FL 33487. Much of the current content is derived from the research of the giants of biochemistry and molecular biology in the three decades following World War II. While it seems the vogue to develop new names and descriptions for old, established concepts, biochemistry and molecular biology continue to be the mainstays of current biomedical research. Roger L. Lundblad [email protected] Fiona M. Macdonald [email protected] March 2010
x iii
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ACknowledgments This work would not have been possible without the outstanding support of Jill Jurgensen and Glen Butler of Taylor & Francis. The support of Professor Edward A. Dennis is acknowledged. The help of various research librarians at the University of North Carolina at Chapel Hill is also
acknowledged. Professors Charles Craik of the University of California at San Francisco and Bryce Plapp of the University of Iowa provided advice on selection of materials to be included. However, the editors take all responsibility for the selection of content.
x v
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Editors Roger L. Lundblad, PhD
Fiona M. Macdonald, PhD, F.R.S.C.
Roger L. Lundblad is a native of San Francisco, California. He received his undergraduate education at Pacific Lutheran University and his PhD degree in biochemistry at the University of Washington. After postdoctoral work in the laboratories of Stanford Moore and William Stein at the Rockefeller University, he joined the faculty of the University of North Carolina at Chapel Hill. He joined the Hyland Division of Baxter Healthcare in 1990. Currently Dr. Lundblad is an independent consultant and writer in biotechnology in Chapel Hill, North Carolina. He is an adjunct professor of Pathology at the University of North Carolina at Chapel Hill and editor-in-chief of the Internet Journal of Genomics and Proteomics.
Fiona M. Macdonald received her BSc in chemistry from Durham University, UK. She obtained her PhD in inorganic biochemistry at Birkbeck College, University of London, studying under Peter Sadler. Having spent most of her career in scientific publishing, she is now at Taylor & Francis and is involved in developing chemical information products.
x vii
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Section I Amino Acids, Peptides, and Proteins
•
Properties of Amino Acids. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Data on the Naturally Occurring Amino Acids. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 Structures and Symbols for Synthetic Amino Acids Incorporated into Synthetic Polypeptides . . . 43 Unnatural Amino Acids for Incorporation into Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53 Properties of the α-Keto Acid Analogs of Amino Acids. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55 α,β-Unsaturated Amino Acids. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57 Amino Acid Antagonists. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59 Coefficients of Solubility Equations of Certain Amino Acids in Water. . . . . . . . . . . . . . . . . . . . . . . 65 Heat Capacities, Absolute Entropies, and Entropies of Formation of Amino Acids and Related Compounds. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67 Heat of Combustion, Enthalpy and Free Energy of Formation of Amino Acids and Related Compounds. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69 Solubilities of Amino Acids in Water at Various Temperatures. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71 Heats of Solution of Amino Acids in Aqueous Solution at 25°C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75 Free Energies of Solution and Standard Free Energy of Formation of Amino Acids in Aqueous Solution at 25°C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77 Far Ultraviolet Absorption Spectra of Amino Acids. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79 UV Absorption Characteristics of N-Acetyl Methyl Esters of the Aromatic Amino Acids, Cystine and of N-Acetylcysteine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81 Numerical Values of the Absorbances of the Aromatic Amino Acids in Acid, Neutral and Alkaline Solutions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83 Luminescence of the Aromatic Amino Acids. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89 Luminescence of Derivatives of the Aromatic Amino Acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91 Luminescence of Proteins Lacking Tryptophan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93 Luminescence of Proteins Containing Tryptophan. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95 Hydrophobicities of Amino Acids and Proteins. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99 Chemical Specificity of Reagents for Protein Modification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115 Reagents for the Chemical Modification of Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117 Protein pK Values. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137 Protease Inhibitors and Protease Inhibitor Cocktails. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141 Assay of Solution Protein Concentration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155 Spectrophotometric Determination of Protein Concentration in the Short-Wavelength Ultraviolet. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161
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Properties of Amino Acids This table gives selected properties of some important amino acids and closely related compounds. The first part of the table lists the 20 “standard” amino acids that are the basic constituents of proteins. The second part includes other amino acids and related compounds of biochemical importance. Within each part of the table the compounds are listed by name in alphabetical order. Structures are given in the following table. Symbol : Three-letter symbol for the standard amino acids Mr : Molecular weight tm: Melting point pKa, pKb, pKc, pKd : Negative of the logarithm of the acid dissociation constants for the COOH and NH2 groups (and, in some cases, other groups) in the molecule (at 25°C) pI: pH at the isoelectric point S: Solubility in water in units of grams of compound per kilogram of water; a temperature of 25°C is understood unless otherwise stated in a superscript. When quantitative data are not available, the notations sl.s. (for slightly soluble), s. (for soluble), and v.s. (for very soluble) are used. V2 0: Partial molar volume in aqueous solution at infinite dilution (at 25°C) Data on the enthalpy of formation of many of these compounds are included in the table “Heat of Combustion, Enthalpy and Free Symbol
Name
Ala
l-Alanine
Arg Asn Asp Cys Gln Glu Gly His Ile Leu Lys Met Phe Pro Ser Thr Trp Tyr Val
l-Arginine l-Asparagine l-Aspartic acid l-Cysteine l-Glutamine l-Glutamic acid Glycine l-Histidine l-Isoleucine l-Leucine l-Lysine l-Methionine l-Phenylalanine l-Proline l-Serine l-Threonine l-Tryptophan l-Tyrosine l-Valine N-Acetylglutamic acid N6-Acetyl-l-lysine b-Alanine
Mol. Form C3H7NO2
Mr
tm/°C
Energy of Formation of Amino Acids and Related Compounds” on p. 69 of this Handbook. Absorption spectra and optical rotation data can be found in Reference 3. Partial molar volume is taken from Reference 5; other thermodynamic properties, including solubility as a function of temperature, are given in References 3 and 5. Most of the pK values come from References 1, 6, and 7.
References 1. Dawson, R. M. C., Elliott, D. C., Elliott, W. H., and Jones, K. M., Data for Biochemical Research, Third Edition, Clarendon Press, Oxford, 1986. 2. O’Neil, Maryadele J., Ed., The Merck Index, Fourteenth Edition, Merck & Co., Rahway, NJ, 2006. 3. Sober, H. A., Ed., CRC Handbook of Biochemistry. Selected Data for Molecular Biology, CRC Press, Boca Raton, FL, 1968. 4. Voet, D., and Voet, J. G., Biochemistry, Second Edition, John Wiley & Sons, New York, 1995. 5. Hinz, H. J., Ed., Thermodynamic Data for Biochemistry and Biotechnology, Springer–Verlag, Heidelberg, 1986. 6. Fasman, G. D., Ed. Practical Handbook of Biochemistry and Molecular Biology, CRC Press, Boca Raton, FL, 1989. 7. Smith, R. M., and Martell, A. E., NIST Standard Reference Database 46: Critically Selected Stability Constants of Metal Complexes Database, Version 3.0, National Institute of Standards and Technology, Gaithersburg, MD, 1997. 8. Ramasami, P., J. Chem. Eng. Data, 47, 1164, 2002. pKa
pKb
89.09
297
2.33
9.71
C6H14N4O2 C4H8N2O3 C4H7NO4 C3H7NO2S C5H10N2O3 C5H9NO4 C2H5NO2 C6H9N3O2 C6H13NO2 C6H13NO2 C6H14N2O2 C5H11NO2S C9H11NO2 C5H9NO2 C3H7NO3 C4H9NO3 C11H12N2O2 C9H11NO3 C5H11NO2 C7H11NO5
174.20 132.12 133.10 121.16 146.14 147.13 75.07 155.15 131.17 131.17 146.19 149.21 165.19 115.13 105.09 119.12 204.23 181.19 117.15 189.17
244 235 270 240 185 160 290 287 284 293 224 281 283 221 228 256 289 343 315 199
2.03 2.16 1.95 1.91 2.18 2.16 2.34 1.70 2.26 2.32 2.15 2.16 2.18 1.95 2.13 2.20 2.38 2.24 2.27
9.00 8.73 9.66 10.28 9.00 9.58 9.58 9.09 9.60 9.58 9.16 9.08 9.09 10.47 9.05 8.96 9.34 9.04 9.52
C8H16N2O3 C3H7NO2
188.22 89.09
265 200
2.12 3.51
9.51 10.08
pKc 12.10 3.71 8.14 4.15 6.04
10.67
10.10
pKd
pI
S/g kg-1
V2 0/cm3 mol-1
6.00
166.9
60.54
10.76 5.41 2.77 5.07 5.65 3.22 5.97 7.59 6.02 5.98 9.74 5.74 5.48 6.30 5.68 5.60 5.89 5.66 5.96
182.6 25.1 5.04 v.s. 42.5 8.6 250.2 43.5 34.2 22.0 5.8 56 27.9 1622 250 98.1 13.2 0.46 88 s.
127.42 78.0 74.8 73.45
723.6
58.28
89.85 43.26 98.3 105.80 107.77 108.5 105.57 121.5 82.76 60.62 76.90 143.8 90.75
3
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Handbook of Biochemistry and Molecular Biology
4
Properties of Amino Acids (Continued) Name
Mol. Form
Mr
2-Aminoadipic acid dl-2-Aminobutanoic acid dl-3-Aminobutanoic acid 4-Aminobutanoic acid 10-Aminodecanoic acid 7-Aminoheptanoic acid 6-Aminohexanoic acid l-3-Amino-2methylpropanoic acid 2-Amino-2-methylpropanoic acid 9-Aminononanoic acid 8-Aminooctanoic acid 5-Amino-4-oxopentanoic acid 5-Aminopentanoic acid o-Anthranilic acid Azaserine Canavanine l-g-Carboxyglutamic acid Carnosine Citrulline Creatine l-Cysteic acid l-Cystine 2,4-Diaminobutanoic acid 3,5-Dibromo-l-tyrosine 3,5-Dichloro-l-tyrosine 3,5-Diiodo-l-tyrosine Dopamine l-Ethionine N-Glycylglycine Guanidinoacetic acid Histamine l-Homocysteine Homocystine l-Homoserine 3-Hydroxy-dl-glutamic acid 5-Hydroxylysine trans-4-Hydroxy-l-proline l-3-Iodotyrosine l-Kynurenine l-Lanthionine Levodopa l-1-Methylhistidine l-Norleucine l-Norvaline l-Ornithine O-Phosphoserine l-Pyroglutamic acid Sarcosine Taurine l-Thyroxine
C6H11NO4 C4H9NO2 C4H9NO2 C4H9NO2 C10H21NO2 C7H15NO2 C6H13NO2 C4H9NO2
161.16 103.12 103.12 103.12 187.28 145.20 131.17 103.12
C4H9NO2
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tm/°C
pKa
pKb
pKc
207 304 194.3 203 188.5 195 205 185
2.14 2.30 3.43 4.02
4.21 9.63 10.05 10.35
9.77
103.12
335
2.36
C9H19NO2 C8H17NO2 C5H9NO3
173.26 159.23 131.13
191 192 118
C5H11NO2 C7H7NO2 C5H7N3O4 C5H12N4O3 C6H9NO6 C9H14N4O3 C6H13N3O3 C4H9N3O2 C3H7NO5S C6H12N2O4S2 C4H10N2O2 C9H9Br2NO3 C9H9Cl2NO3 C9H9I2NO3 C8H11NO2 C6H13NO2S C4H8N2O3 C3H7N3O2 C5H9N3 C4H9NO2S C8H16N2O4S2 C4H9NO3 C5H9NO5 C6H14N2O3 C5H9NO3 C9H10INO3 C10H12N2O3 C6H12N2O4S C9H11NO4 C7H11N3O2 C6H13NO2 C5H11NO2 C5H12N2O2 C3H8NO6P C5H7NO3 C3H7NO2 C2H7NO3S C15H11I4NO4
117.15 137.14 173.13 176.17 191.14 226.23 175.19 131.13 169.16 240.30 118.13 338.98 250.08 432.98 153.18 163.24 132.12 117.11 111.15 135.19 268.35 119.12 163.13 162.19 131.13 307.08 208.21 208.24 197.19 169.18 131.17 117.15 132.16 185.07 129.12 89.09 125.15 776.87
157 dec 146 150 172 167 260 222 303 260 260 118.1 245 247 213 273 263 282 83 232 264 203 209 274 205 194 294 277 249 301 307 140 166 162 212 328 235
10.21
4.05
8.90
2.05
4.95 8.55 6.60 9.90 9.35 9.30 14.30 8.70 8.80 8.24
2.50 1.70 2.51 2.32 2.63 1.89 1.50 1.85
2.12
9.25 4.75 6.76
3.20
1.30 2.05 10.44
8.03
6.16 8.88 13.10
2.15 1.59 2.27
9.83 8.57 9.44 9.28
6.11 10.38 2.54
2.13 1.82 2.20
8.85 9.47 9.10
9.83
2.32 1.69 2.31 2.31 1.94 2.14 3.32 2.18 -0.3 2.20
8.72 8.85 9.68 9.65 8.78 9.80 9.97 9.06 10.01
pI
S/g kg-1
3.18 6.06 7.30
2.240 210 1250 971
7.29
v.s. 863 s.
5.72
137
V20/cm3 mol-1 75.6 76.3 73.2 167.3 120.0 104.2 77.55 151.3 136.1
9.10 10.36 9.05 8.10
2.18 3.13 2.82
pKd
5.92
8.52
8.70
9.96 6.48
10.52 5.70
6.45
7.93
9.27
5.55 6.17 3.28 9.15 5.74
11.79 6.09 9.73
s. 3.514 v.s. v.s.
87.6
322 s. 16 v.s. 0.11 s. 2.72 1.97 0.62 s. 225 5 v.s. s. 0.2 1100
361 sl.s. sl.s. 1.5 520 200 15 107 v.s.
84.49
107.7 91.8
428 105 sl.s.
4/16/10 1:10 PM
Properties of Amino Acids
5 Structures of Common Amino Acids
O OH
H2N
N H
NH2 -Alanine (Ala)
OH
OH
-Aspartic acid (Asp)
OH O
O
H2N
NH2
NH2 -Glutamine (Gln)
NH2 -Cysteine (Cys)
NH2 OH
O
O
O
HO
NH2
-Asparagine (Asn)
OH
O
OH
OH O
NH2
NH2
O
H2N
-Arginine (Arg)
O HS
O
O
NH
OH Glycine (Gly)
-Glutamic acid (Glu)
O HO NH2
-Histidine (His)
NH2
-Isoleucine (Ile)
-Leucine (Leu)
O
OH
OH
OH
-Phenylalanine (Phe) O
O
HO
N O H -Proline (Pro)
NH2
-Methionine (Met)
-Serine (Ser)
O
NH2 N H -Tryptophan (Trp)
OH -Threonine (Thr)
O
NH OH
OH
NH2
HO
NH2
-Tyrosine (Tyr)
-Valine (Val) OH
N H
NH2
O O N-Acetylglutamic acid
O OH
O
HO
OH NH2
OH
NH2
O HO
-Lysine (Lys)
O
NH2
OH
OH NH2
NH2
O S
H2N
OH
OH
N H
O
O
O
N
O
OH H2N
OH β-Alanine
N 6-Acetyl--lysine
O
O NH2
OH
2-Aminoadipic acid
O NH2 -2-Aminobutanoic acid
OH -3-Aminobutanoic acid NH2
O H 2N
OH
-3-Amino-2-methylpropanoic acid
OH
OH
NH2 O
OH
H2N
O 4-Aminobutanoic acid
H2N
O
6-Aminohexanoic acid
O
OH
O 2-Amino-2-methylpropanoic acid
H2N
OH
O 5-Amino-4-oxopentanoic acid
OH
H2N
O 5-Aminopentanoic acid OH
O
O O
HO NH2
Azaserine
9168_Book.indb 5
N
O
N
OH
NH O
HO NH2
N H
Canavanine
NH2
OH O
O H2N
O OH -γ-Carboxyglutamic acid
N
O H 2N
NH O
N H
Carnosine
4/16/10 1:10 PM
Handbook of Biochemistry and Molecular Biology
6
Structures of Common Amino Acids (Continued) O
O
NH2
N H
HO NH2
NH2
HN
N O Creatine
Citrulline
I
O
OH
HO
OH
NH2
O
NH2 2,4-Diaminobutanoic acid
OH
H2N
NH2
OH
OH
S
NH2
OH Dopamine H2N
HN
N H
Histamine
HO
O OH
S
HO
OH
Homocystine
OH
N
NH2
O
N N H
O Guanidinoacetic acid
NH2
-Homocysteine
HO
OH
NH2
HO
NH2
NH2
O
O
HS
-Cystine
3,5-Diiodo--tyrosine
N-Glycylglycine
O
OH NH2
I
O
-Ethionine
S
O
NH2
Br
H N
O S
OH
HO
3,5-Dibromo--tyrosine
O S
HO
O
Br H2N
NH2
O O S OH HO NH2 O -Cysteic acid
OH
O H trans-4-Hydroxy--proline
-Homoserine
O O
OH
O
OH
NH2
HO
O
NH2 HO
O
NH2
I -3-Iodotyrosine
O S NH2
-Kynurenine
OH H
NH2
NH2
HO
OH Levodopa
-Lanthionine
O HO
HO O HO
-Norleucine
-Norvaline
H2N
OH NH2 -Ornithine
O OH
O
NH2
O
N H
OH
O -Pyroglutamic acid
O-Phosphoserine
H N
H2N
O
Sarcosine
OH
O S OH O Taurine
I I
HO NH2
O I -Thyroxine
9168_Book.indb 6
NH2
NH2
-1-Methylhistidine
O OH
OH
N
O P
O
O
N NH2
OH I
4/16/10 1:11 PM
Data On The Naturally Occurring Amino Acids Elizabeth Dodd Mooz The amino acids included in these tables are those for which reliable evidence exists for their occurrence in nature. These tables are intended as a guide to the primary literature in which the isolation and characterization of the amino acids are reported. Originally, it was planned to include more factual data on the chemical and physical properties of these compounds; however, the many different conditions employed by various authors in measuring these properties (i.e., chromatography and spectral data) made them difficult to arrange into useful tables. The rotation values are as given in the references cited; unfortunately, in some cases there is no information given on temperature, solvent, or concentration. The investigator employing the data in these tables is urged to refer to the original articles in order to evaluate for himself the reliability of the information reported. These references are intended to be informative to the reader rather than to give credit to individual scientists who published the original reports. Thus not all published material is cited.
The compounds listed in Sections A to N are known to be of the l configuration. Section O contains some of the d amino acids which occur naturally. This last section is not intended to be complete since most properties of the d amino acids correspond to those of their l enantiomorphs. Therefore, emphasis was placed on including those d amino acids whose l isomers have not been found in nature. The reader will find additional information on the d amino acids in the review by Corrigan 263 and in the book by Meister.1 Compilation of data for these tables was completed in December 1974. Appreciation is expressed to Doctors L. Fowden, John F. Thompson, Peter Müller, and M. Bodanszky who were helpful in supplying recent references and to Dr. David Pruess who made review material available to me prior to its publication. A special word of thanks to Dr. Alton Meister who made available reprints of journal articles which I was not able to obtain.
7
9168_Book.indb 7
4/16/10 1:11 PM
9168_Book.indb 8
No.
Amino Acid (Synonym)
Source
Formula (Mol Wt)
Melting Point °Ca
[α]Db
pKa
Isolation and Purification
8
DATA ON THE NATURALLY OCCURRING AMINO ACIDS (Continued) Chromatography
References Chemistry
Spectral Data
A. l-MONOAMINO, MONOCARBOXYLIC ACIDS 2
3
4
4
3.55 10.24
5
5
5
–
2.29 9.83
6
7
6
–
9, 10
11
–
11
11
12
12
–
13
13
13
13
–
–
14
14
–
–
200° (dec) (14a)
–
–
14a
–
–
14a
C6H11NO2 (129.17)
–
–
–
15
15
–
–
Euphoria longan
C7H11NO3 (157.19)
–
−2720 (c 2, H2O) −820 (c 1, 5 N HCl) (15a)
–
15a
15a
–
15a
2-Amino-6-hydroxy-4methyl-4-hexenoic acid
Aesculus California seeds
C7H13NO3 (159.21)
–
−3120 (c 2.2, H2O) +220 (c 1.1, 5 N HCl) (23b)
–
23b
23b
–
15b
8c
2-Amino-4-hydroxy-5methyl hexenoic acid
Euphoria longan
C7H11NO3 (157.19)
–
−2720 (c 2, H2O) −1320 (c 1, 5 N HCl) (15a)
–
15a
–
15a
15a
8d
2-Amino-3-hydroxymethyl-3-pentenoic acid
Bankera fuligineoalba
C6H11NO3 (145.18)
160–161° (dec) (13)
+18225 (c 0.8, H2O) +20125 (c 0 8, 0 3 N HCl) (13)
–
13
13
13
13
1
C3H7NO2 (89.09)
+1.825 (c 2, H2O) (1) +14.625 (c 2, 5 N HCl) (1)
2.34
Alanine (α-aminopropionic acid)
Silk fibroin
2
β-Alanine (β-aminopropionic acid)
Iris tingitana
C3H7NO2 (89.09)
196° (dec)
–
3
α-Aminobutyric acid
Yeast protein
C4H9NO2 (103.12)
292° (dec)
+20.525 (c 1–2, 5 N HCl) (290) +9.325 (c 1–2, H2O) (290) +4225 (c 1–2, gl acetic) (290)
4
γ-Aminobutyric acid (piperidinic acid)
Bacteria
C4H9NO2 (103.12)
203° (dec)
–
4.03 10.56 (290)
5
1-Aminocyclopropane-1carboxylic acid
Pears and apples
C4H7NO2 (101.11)
–
–
–
6
2-Amino-3-formyl-3pentenoic acid
Bankera fulgineoalba (a mushroom)
C6H9NO3 (143.15)
–
–
7
α-Aminoheptanoic acid
Claviceps purpures
C7H15NO2 (145.21)
–
7a
2-Amino-4,5hexadienoic acid
Amanita solitaria
C6H9NO2 (127.16)
8
2-Amino-4-hexenoic acid
Ilamycin
8a
2-Amino-4-hydroxyhept-6-ynoic acid
8b
297°
9.69
8–10
Handbook of Biochemistry and Molecular Biology
4/16/10 1:11 PM
Melting Point °Ca
Isolation and Purification
Chromatography
Spectral Data
16
–
–
17
17
17
17
–
17a
17b
–
17a, 17b
–
–
18
18
18
–
–
−220 (c 1, H2O) (19) +2420 (c 0.87, 5 N HCl) (19)
–
19
19
19
19
–
−6120 (c 2.4, H2O) (19) −36 (c 1.2, 6 N HCl) (19)
–
19
19
19
19
260° (dec) (19a)
−9.6° (c 1.78, H2O) +5.7° (c 0.7, 1 N HCl) (99a)
–
19a
19a
–
19a
C7H13NO2 (143.19)
260–270° (dec) (22a)
−45.923 (c 0.47, H2O) −723 (c 0.4, 1 N HCl) (22a)
–
22, 22a
22a
–
22a
Euphoria longan
C7H11NO2 (141.19)
–
−3320 (c 2, H2O) −2720 (c 1, 5 N HCl) (15a)
–
15a
–
15a
15a
α-Amino-octanoic acid
Aspergillus atypigue
C8H17NO2 (159.23)
–
–
–
23
23
23
23
15a
2-Amino-4-pentynoic acid
Streptomyces sp. #8–4
C5H7NO2 (113.13)
241–242° (dec) (23a)
−31.125 (c 1, H2O) −5.525 (c 1, 5 N HCl) (23a)
–
23a
23a
23a
23a
15a′
cis-α-(Carboxycyclopropyl)glycine
Aesculus parviflora
C6H9NO4 (159.16)
–
+2520 (c 1, H2O) +58 (c 0.5, 5 N HCl) (23a′)
–
23a′
23a′
–
23a′
15b
trans-α-(Carboxycyclopropyl)glycine
Blighia sapida
C6H9NO4 (159.16)
–
+10720 (c 2, H2O) +14620 (c 1, 5 N HCl) (23a′)
–
23a′
23a′
–
23a′
Amino Acid (Synonym)
Source
Formula (Mol Wt)
9
α-Aminoisobutyric acid
Iris tingitana, muscle protein
C4H9NO2 (103.12)
200° (dec)
10
β-Aminoisobutyric acid
Iris tingitana
C4H9NO2 (103.12)
179° (17)
10a
2-Amino-4-methoxytrans-3-butenoic acid
Pseudomonas aeruginosa
C6H9NO3 (131.15)
11
γ-Amino-α-methylene butyric acid
Arachis hypogaea (groundnut plants)
C4H9NO2 (115.13)
12
2-Amino-4-methylhexanoic acid (homoisoleucine)
Aesculus californica seeds
C7H15NO2 (145.21)
13
2-Amino-4-methyl-4hexenoic acid
Aesculus californica seeds
C7H13NO2 (143.19)
13a
2-Amino-4-methyl-5hexenoic acid
Streptomyces species
C7H13NO2 (143.21)
14
2-Amino-5-methyl-4hexenoic acid
Leucocortinarius bulbiger
14a
2-Amino-4-methyl-5hexenoic acid
15
– 152° (18)
pKa
–
2.36 10.21 (290)
16
–
–
−2126 (c 0.43, H2O) (17)
9
Chemistry
No.
[α]Db
References
Data on the Naturally Occurring Amino Acids
9168_Book.indb 9
DATA ON THE NATURALLY OCCURRING AMINO ACIDS (Continued)
4/16/10 1:11 PM
9168_Book.indb 10
No.
Amino Acid (Synonym)
Source
Formula (Mol Wt)
Melting Point °Ca
15b′
trans-α-(2-Carboxymethylcyclopropyl) glycine
Blighia unijugata
C7H11NO4 (173.19)
15c
γ-Glutamyl-2-amino-4methylhex-4-enoic acid
Aesculus californica seeds
C12H20N2O5 (272.34)
Isolation and Purification
[α]Db
pKa
–
+12 (c 1, H2O) +4520 (c 0.5, 5 N HCl) (99a)
–
99a
–
+1720 (c 3, H2O) (23b)
–
23b
20
10
DATA ON THE NATURALLY OCCURRING AMINO ACIDS (Continued) Chromatography
References Chemistry
Spectral Data
99a
–
99a
23b
–
–
B. l-MONOAMINO, DICARBOXYLIC ACIDS Glycine (α-aminoacetic acid)
Gelatin hydrolyzate
C2H6NO2 (75.07)
290° (dec) (1)
–
2.35 9.78 (290)
24
3
25
25
17
Hypoglycin A [α-aminoβ-(2-methylene cyclopropyl)propionic acid]
Blighia sapida
C7H11NO2 (141.18)
280–284° (26)
+9.2 (c 1, H2O) (26)
–
26
26
27
27
18
Isoleucine (α-amino-βmethylvaleric acid)
Sugar beet molasses
C6H13NO2 (131.17)
284° (1)
+39.525 (c 1, 5 N HCl) (290) +12.425 (c 1, H2O) (290)
2.36 9.68
3
9
29
1
19
Leucine (α-aminoisocaproic acid)
Muscle fiber, wool
C3H13NO2 (131.17)
337° (1)
−1125 (c 2, H2O) +1625c 2, 5 N HCl) (1)
2.36 960 (1)
30
3
31
31
19a
N-Methyl-γ-methylalloisoleucine
Etamycin
C8H17NO2 (159.26)
–
–
–
31a
31a
–
–
19b
β-Methyl-β-(methylenecyclopropyl)alanine
Aesculus californica seeds
C8H13NO2 (155.22)
–
1.520 (c 2, H2O) +4520 (c 1, 5 N HCl) (23b)
– 960 (1)
23b
23b
–
15b
20
α-(Methylene cyclopropyl)glycine
Litchi chinensis
C6H9NO2 (127.15)
–
+4322.5 (c 0.5, 5 N HCl) (32)
–
32
32
32
32
21
β-(Methylenecyclopropyl)-βmethylalanine
Aesculus californica
C8H13NO2 (155.19)
–
+1.520 (c 2, H2O) +4520 (c 1, 5 N HCl)
–
19
19
19
19, 21
21a
β-Methylenenorleucine
Amanita vaginata
C7H13NO2 (143.21)
+15820 (c 0.51, 1 N HCl) +14920 (c 0.56, H2O) (35a)
–
–
35a
35a
–
171° (35a)
Handbook of Biochemistry and Molecular Biology
16
4/16/10 1:11 PM
Melting Point °Ca
[α]Db
pKa
Isolation and Purification
Chromatography
References Chemistry
Spectral Data
3
35, 36
36
37
37
37
–
–
38
38
38
38
–
39
39
39
–
2.02 8.80 (1)
40
3
41
42
1.88 3.65 9.60 (1)
43
3
41
41
–
45
45
45
45
+2120 (c 2.8, H2O) (47) +38.320 (c 1.4, 6 N HCl) (47)
–
46
46
–
46
229° (48)
–
–
48
–
48
–
C5H9NO4 (147.13)
249° (1)
+1225 (c 2, H2O) +31.828 (c 2, 5 N HCl) (1)
2.19 4.25 9.67 (1)
49
3
50
50
C5H10N2O3 (146.15)
185° (1)
+6.325 (c 2, H2O) +31.825 (c 2, 1 N HCl) (1)
2.17 9.13 (1)
51
3
50
50
No.
Amino Acid (Synonym)
Source
Formula (Mol Wt)
22
Valine (α-amino isovaleric acid
Casein
C5H11NO2 (117.15)
292–295° (1)
+28.3 (c 1, 2, 5 N HCl) +5.6325 (c 1–2, H2O) (290)
2.32 9.62 (1)
35
23
α-Aminoadipic acid
Pisum sativum
C6H11NO4 (161.18)
195° (37)
+3.225 (c 2, H2O (290) +2322 (c 2, 6 N (HCl) (37)
2.14 4.21 9.77 (290)
24
3-Aminoglutaric acid
Chondria armata
C5H9NO4 (162.13)
280–282° (38)
±0c (c 2, 5 N HCl) (38)
25
α-Aminopimelic acid
Asplenium septentrionale
C7H13NO4 (175.19)
204° (39)
26
Asparagine (α-aminosuccinamic acid)
Asparagus
C4H8N2O3 (132.12)
236° (1)
+5.0625 (c 2, H2O) (290) +33.2 (3 N HCl) (1)
27
Aspartic acid (α-aminosuccinic acid)
Conglutin, legumin
C4H7NO4 (133.10)
270° (1)
+5.0525 (c 2, H2O) +25.425 (c 2, 5 N HCl) (1)
28
Ethylasparagine
Ecballium elaterium, Bryonia dioica
C6H12N2O3 (160.19)
–
–
29
γ-Ethylideneglutamic acid
Mimosa
C7H11NO4 (173.18)
–
30
N-Fumarylalanine
Penicillium recticulosum
C7H9NO5 (187.16)
31
Glutamic acid (α-aminoglutaric acid)
Gluten-fibrin hydrolyzates
32
Glutamine (α-aminoglutaramic acid)
Beet juice
25
–
Data on the Naturally Occurring Amino Acids
9168_Book.indb 11
DATA ON THE NATURALLY OCCURRING AMINO ACIDS (Continued)
11
4/16/10 1:11 PM
9168_Book.indb 12
Melting Point °Ca
+7.1 (c 1.7, H2O) (53)
–
53
−4.2 (c 5.5, H2O) (54)
–
53
–
–
54
–
–
3.5 9.9 (290)
55
55
55
–
–
–
56
56
–
–
196° (57)
–
–
57
57
57
–
173–182° (57)
–
–
57
57
57
–
–
–
–
59
59
59
59
–
−8720 (c 8, H2O) −3520 (c 4, 6 N HCl) (59a)
–
59a
59a
59a
59a
60
60
60
–
Formula (Mol Wt)
33
N -Isopropylglutamine
Lunaria annua
C8H16N2O3 (188.23)
34
N4-Methylasparagine
–
35
β-Methylaspartic acid
Clostridium tetanomorphum
C5H9NO4 (147.13)
–
36
γ-Methylglutamic acid
Phyllitis scolopendrium
C6H11NO4 (141.17)
–
37
γ-Methyleneglutamic acid
Arachis hypogaea
C6H9NO4 (159.15)
38
γ-Methyleneglutamine
Arachis hypogaea
C6H10N2O3 (158.17)
39
Theanine (α-amino-γ-Nethylglutaramic acid)
Xerocomus hadius
C7H14N2O3 (174)
39a
β-N-Acetyl-α, βdiaminopropionic acid (β-acetamido-l-alanine)
Acacia armata seeds
C5H10N2O3 (146.17)
– 241–244° (54)
−10 (c 0.4, H2O) +12.4 (c 3, 1 N HCl) +13.3 (c 3, 5 N HCl) (55)
C. l-DIAMINO, MONOCARBOXYLIC ACIDS 40
N-Acetylornithine
Asplenium species
C7H14N2O3 (174.11)
200° (dec) (60)
–
41
α-Amino-γ-Nacetylaminobutyric acid
Latex of Euphorbia pulcherrima
C6H12N2O3 (160.18)
220–222° (dec) (61)
–
4.45 (33)
61
61
–
–
42
N-ε-(2-Amino-2carboxyethyl)lysine
Alkali-treated protein
C9H19N3O4 (233.28)
–
–
2.2 6.5 8.8 9.9 (62)
62
62
62
–
43
N-δ-(2-Amino-2carboxyethyl)ornithine
Alkali-treated wool
C8H17N3O4 (273.72)
–
–
–
63
63
63
–
44
2-Amino-3-dimethylaminopropionic acid
Streptomyces neocaliberis
C5H12N2O2 (117.15)
–
−17.825 (c 1, H2O) +18.1 (c 1, HCl pH 3) (64)
–
64
64
64
64
45
α-Amino-β-methylaminopropionic acid
Cycas circinalis
C4H11N2O2 (105.15)
–
–
65
65
65
65
165–167° (65)
–
Handbook of Biochemistry and Molecular Biology
53
Source
C5H10N2O3 (146.15)
pKa
22
References Spectral Data
Amino Acid (Synonym)
[α]Db
Chromatography
Chemistry
No.
5
Isolation and Purification
12
DATA ON THE NATURALLY OCCURRING AMINO ACIDS (Continued)
4/16/10 1:11 PM
pKa
Isolation and Purification
Chromatography
No.
Amino Acid (Synonym)
Source
Formula (Mol Wt)
Melting Point °Ca
45a
α-Amino-β-oxalylaminopropionic acid
Crotalaria
C5H8N2O5 (176.15)
–
–
–
–
46
Canaline
Canavalia ensiformis
C4N10N2O3 (134.14)
–
–
2.40 3.70 9.20 (20)
46a
threo-α,βDiaminobutyric acid
Amphomycin hydrolyzate
C4H10N2O2 (118.16)
213–214° (dec) (67a)
+27.125 (c 2, 5 N HCl) (67a)
47
α,γ-Diaminobutyric acid (γ-aminobutyrine)
Glumamycin
C4H10N2O2 (134.14)
–
48
3,5-Diaminohexanoic acid
Clostridium stricklandii
C6H14N2O2 (146.19)
48a
2,6-Diamino-7hydroxyazelaic acid
Bacillus brevis (edeine A and B)
C9H18N2O15 (234.29)
49
α,β-Diaminopropionic acid (β-aminoalanine)
Mimosa
C3H8N2O2 (104.11)
49a
Nε,Nε-Dimethyllysine
Human urine
49b
N5-Iminoethylornithine
50
Chemistry
Spectral Data
65a
–
–
66
–
67
–
–
67b
67b
67a
67b
+7.225 (c 2, H2O) +14.618 (c 3.67, H2O)g (290) +14.610 (c 3.67, H2O)g (290)
1.85 10.50 (20) 8.28
68
68
68
–
–
–
69
69
69
69
–
–
–
69a
69a
69a
–
–
–
1.23 6.73 9.56 (20)
70
70
70
–
C8H18N2O2 (174.28)
214–216° (dec) (70a)
–
–
70a
70a
–
70a
Streptomyces broth
C7H15N3O2 (173.25)
226–229° (70b)
+20.625 (c 1, 5 N HCl) (70b)
1.97 8.86 11.83 (70b)
70b
70b
–
70b
Lathyrus factor (β-N-(γ-glutamyl)aminopropionitrile)
Lathyrus pusillus
C8H13N3O3 (199.22)
193–194° (72)
+2818 (c 1, 6 N HCl) (72)
71
72
72
72
51
Lysine (α,εdiaminocaproic acid)
Casein
C6H14N2O2 (146.19)
224–225° (dec) (73)
+14.620 (H2O) (73)
2.16 9.18 10.79 (290)
74
3
75
–
52
β-Lysine (isolysine; β,ε-diaminocaproic acid)
Viomycin
C6H14N2O2 (146.19)
240–241° (76)
–
76
76
76
76
204–208° (69)
[α]Db
References
–
2.2 9.14
Data on the Naturally Occurring Amino Acids
9168_Book.indb 13
DATA ON THE NATURALLY OCCURRING AMINO ACIDS (Continued)
13
4/16/10 1:11 PM
9168_Book.indb 14
Melting Point °Ca
pKa
Isolation and Purification
Chromatography
–
78
–
References Chemistry
Spectral Data
78
77
77
79
79
–
79
1.71 8.69 10.76 (290)
60
60
81
81
–
–
81a
81a
–
81a
206° (dec) (82)
−36.927 (c 0.66, 4 N HCl) (82)
1.95 2.95 9.25 (82)
82
82
82
82
250–251° (dec) (82a)
–
3.2 9.4 11.2 (82a)
82a
82a
–
82a
–
83
83
83
83
1.8 2.2 9.9 8.8 (290)
84
84
85
–
–
86
86
86
86
No.
Amino Acid (Synonym)
Source
Formula (Mol Wt)
53
Lysopine N2-(d-1-carboxyethyl)lysine
Calf thymus histone
C9H18N2O4 (204.25)
54
ε-N-Methyllysine
Calf thymus histone
C7H16N2O2 (160.23)
–
55
Ornithine (α,δdiaminovaleric acid)
Asplenium nidus
C5H12H2O2 (132.16)
–
55a
4-Oxalysine
Streptomyces
C5N12N2O3 (148.19)
–
56
β-N-Oxalyl-α,βdiaminopropionic acid
Lathyrus sativus
C5H8N2O3 (176.13)
56a
β-Putreanine [N-(4aminobutyl)-3aminopropionic acid]
Bovine brain
C7H16N2O2 (160.25)
157–160° (77)
[α]Db
14
DATA ON THE NATURALLY OCCURRING AMINO ACIDS (Continued)
+18 (c 1.4, H2O) (77) – +12.1 (c 2, H2O) +28.4 (c 2, 5 N HCl) (1)
D. l-DIAMINO, DICARBOXYLIC ACIDS Acetylenic dicarboxylic acid diamide
Streptomyces chibaensis
C4H4N2O2 (112.09)
58
α,ε-Diaminopimelic acid
Pine pollen
C7H14N2O4 (190.20)
59
2,3-Diaminosuccinic acid
Streptomyces rimosus
C4H8N2O4 (148.10)
–
216–218° (dec) –
+8.125 (c 5, H2O) +4526 (c 1, 1 N HCl) +45.124 (c 2.6, 5 N HCl) (290) –
240–290° (dec)
E. l-KETO, HYDROXY, AND HYDROXY SUBSTITUTED AMINO ACIDS
4/16/10 1:11 PM
60
O-Acetylhomoserine
Pisum
C6H11NO4 (161.17)
–
60a
Threo-α-amino-β,γdihydroxybutyric acid
Streptomyces
C4H9NO4 (135.14)
–
87
87
87
–
–
87a
–
–
–
61
2-Amino-4,5-dihydroxy pentanoic acid
Lunaria annua
C5H11NO4 (149.15)
–
–
–
88
88
88
–
61a
2-Amino-3-formyl-3pentenoic acid
Bankera fuligineoalba
C6H9NO3 (143.16)
–
–
–
88a
88a
88a
88a
62
α-Amino-γ-hydroxyadipic acid
Vibrio comma
C6H11NO5 (177.17)
–
–
–
89
–
–
–
210°(dec)
– −13.3 (c 1, H2O) −1.125 (c 1, 2.2 N HCl) (87a) 25
Handbook of Biochemistry and Molecular Biology
57
[α]Db
pKa
Isolation and Purification
+6.3 (c 5, H2O) +23.910 (c 5.1, 1 N HCl) (90)
–
90
–
–
–
–
–
Melting Point °Ca
Chromatography
References Chemistry
Spectral Data
90
90
–
91
91
91
–
–
–
92
–
–
−3020(c 2.2, H2O) +220 (c 1.1, 5 N HCl (19)
–
19
19
19
19, 21
+18225 (c 0.8, H2O) +20125 (c 0.8, 0.3N HCl (13)
–
13
13
13
13
–
–
96
96
–
–
+625 (c 2.65, H2O) +2.425365 (c 2.65, H2O) (97)
–
97
97
97
97
–
–
98
–
–
–
–
–
99
99
99
99
–
–
99a
–
99a
–
–
100
100
100
100
–
–
7.2 8.6 (100b)
100a
–
–
100
–
–
–
101
101
101
–
Amino Acid (Synonym)
Source
Formula (Mol Wt)
63
2-Amino-6-hydroxyaminohexanoic acid
Mycobacterium phlei
C6H14N2O3 (162.19)
64
α-Amino-γ-hydroxybutyric acid
Escherichia coli mutants
C4H9NO3 (119.12)
65
γ-Amino-β-hydroxybutyric acid
Escherichia coli mutants
C4H9NO3 (119.12)
66
2-Amino-6-hydroxy-4methyl-4-hexenoic acid
Aesculus californica
C7H13NO3 (159.19)
67
2-Amino-3-hydroxymethyl-3-pentenoic acid
Bankera fuligineoalba
C6H11NO3 (145.17)
68
α-Amino-γ-hydroxypimelic acid
Asplenium seplentrionale
C7H13NO5 (191.19)
69
α-Amino-δ-hydroxyvaleric acid
Canavalia ensiformis
C5H11NO3 (133.15)
70
α-Amino-β-ketobutyric acid
Mikramycin A
C4H7NO3 (117.11)
71
α-Amino-β-methyl-γ, δ-dihydroxyisocaproic acid
Phalloidin
C7H15NO4 (177.21)
71a
2-Amino-5-methyl-6hydroxyhex-4-enoic acid
Blighia unijugata
C7H13NO3 (159.21)
72
O-Butylhomoserine
Soil bacterium
C8H17NO3 (175.23)
72a
Dihydrorhizobitoxine[O(2-amino-3hydroxypropyl)homoserine)
Rhizobium japonicum
C7H16N2O4 (192.25)
73
β,γ-Dihydroxyglutamic acid
Rheum rhaponticum
C5H9NO6 (179.13)
74
β,γ-Dihydroxyisoleucine
Thiostrepton
C6H15N04 (165.20)
–
–
–
102
–
–
–
75
γ,δ-Dihydroxyleucine
Phalloin
C6H13NO4 (163.18)
–
–
–
103
–
103
103
75a
δ,ε-Dihydroxynorleucine
Bovine tendon
C6H13NO4 (163.20)
–
–
–
103a
–
103a
–
76
O-Ethylhomoserine
Soil bacterium
C6H13NO3 (147.18)
–
100
100
100
100
20
199° (91)
160–161° (13)
– 216° (dec) (97)
– 208–210° (99) – 267° (100)
262° (100)
4/16/10 1:11 PM
−1430 (c 2.5, H2O) (100)
99a
15
No.
Data on the Naturally Occurring Amino Acids
9168_Book.indb 15
DATA ON THE NATURALLY OCCURRING AMINO ACIDS (Continued)
9168_Book.indb 16
[α]Db
pKa
Isolation and Purification
–
–
–
2.71 9.62 (290)
105
–
–
–
–
–
–
C6H13NO3 (147.18)
–
Human urine
C4H8O4N2 (148.12)
Azotobacter
C4H7NO5 (149.10)
Melting Point °Ca
–
–
104a
105
–
–
106
–
–
–
91
91
–
–
–
–
108
–
–
–
–
109
109
–
–
238–240° (dec) (110)
–
2.09 8.29 (20)
110
110
110
110
–
+41.4 (c 2.42, H2O) +53.0 (c 2.46, 1 N HCl) (290)d
1.91
111
111
–
–
–
111a
–
–
111a
−2.920 (c 5, H2O) (112)
–
112
112
112
112
+5.819 (c 1.8, H2O) (88)
–
88
88
88
–
+8.69 (H2O) +30.820 (c 2, 20% HCl) (290)d
–
114
114
–
–
Source
Formula (Mol Wt)
76a
β-Guanido-γhydroxyvaline
Viomycin
C6H14N4O3 (190.24)
77
Homoserine (α-amino-γhydroxybutyric acid)
Pisum sativum
C4H9NO3 (119.12)
–
78
α-Hydroxyalanine
Peptides of ergot
C3H7NO3 (105.10)
–
79
α-Hydroxy-γ-aminobutyric acid
E. coli mutants
C4H9NO3 (119.12)
–
80
β-Hydroxy-γ-aminobutyric acid
Mammalian brain
C4H9NO3 (119.12)
81
α-Hydroxy-ε-aminocaproic acid
Neurospora crassa
82
β-Hydroxyasparagine
83
β-Hydroxyaspartic acid
C5H11NO3 (133.17)
84
N4-(2-Hydroxyethyl)asparagine
Bryonia dioica
C6H12N2O4 (176.20)
85
N5-(2-Hydroxyethyl)glutamine
Lunaria annua
C7H14N2O4 (190.23)
86
β-Hydroxyglutamic acid
Mycobacterium tuberculosis
C5H9NO5 (163.13)
182°(dec) (104a)
– 199–200° (112) – 187° (dec) (290)
−8.825 (c 1–2, H2O) +18.326 (c 2, 2 N HCl) (290)
–
3.51 9.11 (20)
87
γ-Hydroxyglutamic acid
Linaria vulgaris
C5H9NO5 (163.13)
–
–
–
115
115
115
–
88
γ-Hydroxyglutamine
Phlox decussata
C5H10N2O4 (162.15)
163–164° (dec) (116)
–
–
116
116
116
–
89
ε-Hydroxylaminonorleucine (α-amino-εhydroxyaminohexanoic acid) 4-Hydroxyisoleucine
Mycobacterium phlei
C5H12N2O3 (148.17)
223–225° (dec) (117)
–
117
117
117
–
Trigonella foenumgraecum
C6H13NO3 (147.20)
+6.320 (c 5, H2O) +23.918 (c 5.1, N HCl) (117) +3120 (c 1, H2O) (117a)
–
117a
117a
–
117a
89a
–
Handbook of Biochemistry and Molecular Biology
Rumen protozoa
References Spectral Data
Amino Acid (Synonym)
N-(2-Hydroxyethyl)alanine
Chromatography
Chemistry
No.
83a
16
DATA ON THE NATURALLY OCCURRING AMINO ACIDS (Continued)
4/16/10 1:11 PM
No.
Amino Acid (Synonym)
Source
Formula (Mol Wt)
Melting Point °Ca
90
δ-Hydroxy-γketonorvaline
Streptomyces akiyoshiensis novo
C5H9NO4 (147.13)
–
91
δ-Hydroxyleucenine (δ-ketoleucine)
Phalloidin
C6H11NO3 (145.17)
–
–
–
92
β-Hydroxyleucine
Antibiotic from Paecilomyces strain
C6H13NO3 (147.17)
–
–
93
δ-Hydroxyleucine
Paecilomyces
C6H13NO3 (147.17)
–
–
94
threo-β-Hydroxyleucine
Deutzia gracilis
C6H13NO3 (147.17)
–
95
α-Hydroxylysine (α,ε-diamino-αhydroxycaproic acid)
Silvia officinalis
C6H15N2O3 (162.19)
–
96
δ-Hydroxylysine (α,ε-diamino-δhydroxycaproic acid)
Fish gelatin
C6H15N2O3 (162.19)
–
96a
β-Hydroxynorvaline
Streptomyces species
C5H11NO3 (133.17)
97
γ-Hydroxynorvaline
Lathyrus odoratus
C5H11NO3 (133.15)
98
γ-Hydroxyornithine
Vicia sativa
99
α-Hydroxyvaline
100
118
References Spectral Data
118
118
118
119
119
119
119
–
16
16
16
–
–
121
121
121
121
–
–
122
122
122
122
–
2.13 8.62 9.67 (20)
123
123
–
–
–
–
124
111
–
–
–
–
124a
124a
–
124a
–
+2220 (c 5, H2O) +32 (c 2.5, gl. acetic) (126)
–
126
126
126
–
C5H12N2O3 (148.16)
–
–
–
127
127
–
–
Ergot
C5H11NO3 (133.15)
–
–
2.55 9.77 (20)
106
–
–
–
γ-Hydroxyvaline
Kalanchoe daigremontiana
C5H11NO3 (133.15)
228° (dec) (129)
–
129
129
–
–
100a
Hypusine
Bovine brain
C10H23N3O3 (233.27)
234–238° (dec) (129a)
–
–
129a
–
129a
129a
100b
Isoserine
Bacillus brevis (edeine A and B)
C3H7NO3 (105.11)
–
–
129b
129b
129b
–
101
4-Ketonorleucine (2-amino-4-ketohexanoic acid)
Citrobacter freundii
C6H11NO3 (145.17)
–
–
131
131
130
130
102
γ-Methyl-γ-hydroxy glutamic acid
Phyllitis scolopendrium
C6H11NO5 (157.17)
–
–
56
56
–
–
– 142–143° (130) –
−8.2 (c 3.4, H2O) (118) 17
+1020 (H2O) (129)
pKa
Chromatography
Chemistry
244° (dec) (124a)
[α]Db
Isolation and Purification
2.0 9.1
Data on the Naturally Occurring Amino Acids
9168_Book.indb 17
DATA ON THE NATURALLY OCCURRING AMINO ACIDS (Continued)
17
4/16/10 1:11 PM
9168_Book.indb 18
pKa
Isolation and Purification
Chromatography
Amino Acid (Synonym)
Source
Formula (Mol Wt)
103
Pantonine (α-amino-β, β-dimethyl-γhydroxybutyric acid)
Escherichia coli
C6H13NO3 (147.18)
–
–
–
132
103a
threo-β-Phenylserine
Canthium eurysides
C9H11NO3 (181.21)
–
–
–
–
103b
Pinnatanine [N5-(2-hydroxymethylbutadienyl)-alloγ-hydroxyglutamine)
Staphylea pinnata
C10H16N2O5 (244.28)
165° (dec) (132a)
+3.227 (c 0.5, H2O) (132a)
104
O-Propylhomoserine
Soil bacterium
C7H15NO3 (161.21)
265° (100)
−1130 (c 2, H2O) (100)
104a
Rhizobitoxine [2-amino4-(2-amino-3hydroxypropoxy)but3-enoic acid]
Rhizobium japonicum
C7H15N2O4 (191.24)
105
Serine (α-amino-βhydroxypropionic acid)
Silk fibroin
C3H7NO3 (105.09)
106
O-Succinylhomoserine
Escherichia coli
C8H13NO6 (219.20)
107
Tabtoxinine (α,εdiamino-βhydroxypimelic acid)
Pseudomonas tabaci
C7H14N2O5 (186.20)
108
Threonine (α-amino-βhydroxybutyric acid)
Fibrin hydrolyzate
C4H9NO3 (119.12)
Chemistry
Spectral Data
132
132
–
–
–
252a
–
132a
132a
132a
–
100
100
100
–
–
132b
–
–
228° (dec) (1)
−7.525 (c2, H2O) +15.125 (c2. 5 N HCl) (290)
2.19 9.21 (290)
134
3
134
134
180–181° (135)
–
4.4 9.5 (135)
135
135
135
–
–
–
136
137
137
–
−2825 (c 1–2, H2O) −1525 (c 1–2, 5 N HCl) (290)
2.09 9.10 (290)
138
3
138, 139
139
140
–
–
253° (1)
9.1 (132a)
References
100 132c
F. l-AROMATIC AMINO ACIDS 109
α-Amino-β-phenylbutyric acid
Streptomyces bottropensis
C10H13NO2 (187)
109a
β-Amino-β-phenylpropionic acid
Roccella canariensis hydrolyzate
C9H11NO2 (165.21)
–
110
3-Carboxy-4-hydroxyphenylalanine (m-carboxytyrosine)
Reseda odorata
C10H11NO5 (165.15)
–
176–177° (140)
–
–
140
140
140
–
–
–
–
–
2 3.4 9.3 12–13 (297)
141
141
141
−7.725 (c 0.9, 1 N NaOH) −29.924 (c 0.6, 0.2 M PO4, pH 7) (297)
140a
141
Handbook of Biochemistry and Molecular Biology
No.
Melting Point °Ca
[α]Db
18
DATA ON THE NATURALLY OCCURRING AMINO ACIDS (Continued)
4/16/10 1:11 PM
Isolation and Purification
Chromatography
1.5 3.9 (142)
142
–
+19.718 (1 N HCl) (144) −14.3 (1 N HCl) (146)
Melting Point °Ca
[α]Db
pKa
–
– –
References Chemistry
Spectral Data
142
142
142
142a
142a
–
142a
–
144
144
144
144
2.32e 8.68e 9.88e
145
1
146
–
–
–
147
147
147
147
+42.525 (c 0.67, 0.1 N NaOH) (147a)
–
147a
–
–
147a
–
–
–
147b
147b
–
147b
C10H13NO4 (211.24)
–
−3620 (c 1, H2O) −420 (c 0.67,1 N NaOH) (147b)
–
147b
147b
–
147b
Human urine
C10H12N2O4 (224.21)
–
–
–
148
148
–
–
p-Hydroxymethylphenylalanine
Escherichia coli
C10H13NO3 (195.24)
231–233° (dec) (148a)
–
–
148b
148b
148a
116
m-Hydroxyphenylglycine
Euphorbia helioscopia
C8H9NO3 (177.16)
212–214° (147)
–
147
147
147
147
117
Kynurenine (β-anthraniloyl-αaminopropionic acid)
Rabbit urine
C10H12N2O3 (208.21)
191° (dec) (290)
−30.525 (c 1, H2O) (290)
–
148
–
148
148
118
O-Methyltyrosine (β-(p-methoxyphenyl)alanine)
Puromycin
C10H13NO3 (195.22)
191° (150)
−5–9546 (HCl) (150) −3.2546 (1 N NaOH) (150)
–
149
–
150
–
119
Phenylalanine
Lupinus luteus
C9H11NO2 (165.19)
284° (1)
−34.525 (c 1–2, H2O) (290) −4.525 (c 1–2, 5 N HCl) (290)
2.16 9.18 (290)
151
3
152
152
No.
Amino Acid (Synonym)
Source
Formula (Mol Wt)
111
m-Carboxyphenylalanine
Iris bulbs
C10H11NO4 (191.26)
111a
2,3-Dihydroxy-Nbenzoylserine
Escherichia coli
C10H11NO6 (241.22)
193–194° (142a)
112
2,4-Dihydroxy-6methylphenylalanine
Agrostemma githago
C10H13NO4 (211.23)
252° (144)
113
3,4-Dihydroxyphenylalanine (DOPA)
Vicia faba
C9H11NO4 (209.21)
114
3,5-Dihydroxyphenylglycine
Euphorbia helioscopia
C8H9NO4 (183.16)
230–232° (147)
114a
γ-Glutaminyl-4hydroxybenzene
Agaricus bisporus
C11H14N2O4 (238.27)
225–226° (147a)
114b
3-Hydroxymethylphenylalanine
Caesalpinia tinctoria
C10H13NO3 (195.24)
114c
4-Hydroxy-3hydroxymethylphenylalanine
Caesalpinia tinctoria
115
3-Hydroxykynurenine
115a
–
−32.520 (148a)
–
Data on the Naturally Occurring Amino Acids
9168_Book.indb 19
DATA ON THE NATURALLY OCCURRING AMINO ACIDS (Continued)
19
4/16/10 1:11 PM
9168_Book.indb 20
No.
Amino Acid (Synonym)
Source
Formula (Mol Wt)
120
Tyrosine (α-amino-βhydroxyphenyl propionic acid)
Casein, alkaline hydrolyzate
C9H11NO3 (181.19)
120a
β-Tyrosine
Bacillus brevis (edeine A and B)
C9H11NO3 (181.21)
121
m-Tyrosine
Euphorbia myrsinites L
C9H11NO3 (181.19)
Melting Point °Ca 344° (1)
– 272–274° (155)
[α]Db −10 (c 2, 5 N HCl)(1) 25
pKa 2.20 9.11 (1) 10.13 (290)
– −14.522 (70% EtOH) +8.9 (70% EtOH, 2 N HCl) (155)
Isolation and Purification
20
DATA ON THE NATURALLY OCCURRING AMINO ACIDS (Continued) Chromatography
153
3
References Chemistry
Spectral Data
154
154
–
129b
129b
129b
–
–
155
155
155
155
G. l-UREIDO AND GUANIDO AMINO ACIDS N-Acetylarginine
Cattle brain
C7H16N4O3 (204.27)
270° (155a)
–
–
155a
155a
155a
155a
122
Albizziine (2-amino-3ureidopropionic acid)
Mimosaceae
C4H9NO3 (119.12)
–
−6626 (c 2, H2O) (157)
–
156
157
157
157
123
Arginine (amino-δguanidinovaleric acid)
Lupinus luteus
C6H14N4O2
158
3
159
159
124
Canavanine (α-amino(O-guanidyl)-γhydroxybutyric acid)
Canavalia ensiformis
C5H12N4O3 (176.19)
161
160
160
162
125
Canavanosuccinic acid
Canavalia ensiformis
C9H16N4O7 (292.27)
163
163
–
–
126
Citrulline
Watermelon
C6H13N3O3
202° (164)
+425 (c 2, H2O) +24.225 (c 2, N HCl) +10.8 (c 1.1, 0.1 N NaOH) (290)
164
165
164
–
127
Desaminocanavanine
Canavanine
C5H9N3O3
256–257° (166)
+26.6121 (H2O) (166)
–
166
–
166
–
127a
NG, NG-Dimethylarginine
Bovine brain
C8H18N4O2 (202.30)
198–201° (70a)
–
–
166a
166a
–
166a
127b
NG, N’G-Dimethylarginine
Bovine brain
C8H18N4O2 (202.30)
237–239° (dec) (70a)
–
–
166a
166a
70a
166a
238° (1)
172° (160)
–
+12.525 (c 2, H2O) +27.625 (c 2, 5 N HCl) (1) +18.618.5 (c 7.8, H2O) (160) –
1.82 8.99 12.48 (290) 2.50 6.60 9.25 (33) – 2.43 9.41 (290)
Handbook of Biochemistry and Molecular Biology
121a
4/16/10 1:11 PM
No.
[α]Db
pKa
Isolation and Purification
References Chromatography Chemistry
Spectral Data
Source
Formula (Mol Wt)
Gymnogongrus flabelliformis Lathyrus species
C7H15N5O3 (217.25) C7H16N4O2 (188.25)
–
–
–
167
167
167
167
129
Gigartinine (α-amino-δ(guanylureido)valeric acid) Homoarginine
–
–
168
168
168
168
130
Homocitrulline
Human urine
–
–
169
169
–
–
131
γ-Hydroxyarginine
Vicia sativa
–
–
–
170
170
170
170
131a
N5-Hydroxyarginine
Bacillus species
C7H14N3O3 (189.22) C6H14N4O3 (190.20) C6H14N4O3 (190.24)
+42.4− (c 0.452, 1.02 N HCl)f (168) –
–
170a
170a
170a
170a
132
γ-Hydroxyhomoarginine (α-aminoε-guanidino-γ-hydroxyhexanoic acid) Indospicine (α-amino-ε-amidino caproic acid) ω-N-Methylarginine (guanidinomethylarginine)
Lathyrus tingitanus
C7H16N4O3 (204.23)
–
171
171
171
171
Endigofera spicata Bovine brain
C7H15N3O2 (173.23) C7H16N4O2 (188.27)
–
173
173
173
–
–
166a
166a
–
166a
(174)
174
–
174
174
8.55 (34)
175
–
175
175
1.7 7.4 (177)
176
176
177
177
–
177a
177a
177a
177a
–
178
178
178
178
–
179
–
179
179
–
180a
180b
180b
180b
−5725 (c 0.65, H2O) (180)
–
180
180
180
–
+826 (H2O) (184, 185)
–
184, 185
–
184, 185
184, 185
128
133 133a
Amino Acid (Synonym)
Melting Point °Ca
206–212° (dec) (170a) –
+2125 (c 1, 5 N HCl) (170a)
131–134° (173) –
+1822 (c 1.1, 5 N HCl) (173) –
–
Data on the Naturally Occurring Amino Acids
9168_Book.indb 21
DATA ON THE NATURALLY OCCURRING AMINO ACIDS (Continued)
H. l-AMINO ACIDS CONTAINING OTHER NITROGENOUS GROUPS Streptomyces alanosinicus
C3H7N3O4 (149.11)
135
Alanosine [2-amino-3(N-nitrosohydroxyamino) propionic acid] Azaserine (O-diazoacetylserine)
Streptomyces
C5H7N3O4 (173.14)
136
β-Cyanoalanine
Vicia sativa
C4H6N2O2 (114.11)
136a
γ-Cyano-α-aminobutyric acid
C5H8N2O2 (128.15)
221–223° (177a)
137
ε-Diazo-δ-ketonorleucine
Chromobacterium violaceum Streptomyces
C6H9N3O3 (171.17)
138
Hadacidin (N-formyl-Nhydroxyaminoacetic acid)
Penicillium frequentans
C3H5NO4 (119.08)
145–155° (178) 205–210° (179)
134
– 146–162° (175) 214.5° (176)
+8 (1 N HCl) −46 (0.1 N NaOH) (174) −0.527.5 (c 8.46, H2O) (175) −2.926 (c 1.4, 1 N acetic acid) (177) +32.121 (c 0.38, 1 N HOAc) (177a) 2126 (c 5.4, H2O) (178) –
I. l-HETEROCYCLIC AMINO ACIDS 2-Alanyl-3-isoxazolin-5-one
Pea seedlings
C6H8N2O4 (172.16)
203–205° (dec) (180b)
139
Allohydroxyproline
Santalum album
C5H9NO3 (131.13)
248° (180)
140
Allokainic acid (3-carboxymethyl-4isopropenyl proline)
Digenea simplex
C10H15NO4 (213.24)
–
–
21
4/16/10 1:11 PM
138a
9168_Book.indb 22
DATA ON THE NATURALLY OCCURRING AMINO ACIDS (Continued)
Amino Acid (Synonym)
Source
Formula (Mol Wt)
140a
1-Amino-2-nitrocyclopentane carboxylic acid
Aspergillus wentii
C6H10N2O4 (174.18)
141
4-Aminopipecolic acid
Strophanthus scandens
C6H11N2O2 (143.18)
141a
cis-3-Aminoproline
Morchella esculenta
C5H10N2O2 (130.17)
150° (dec) (185a) – 215° (dec) (186a)
References Chromatography Chemistry
22
No.
Melting Point °Ca
[α]Db
pKa
Isolation and Purification
–
–
185a
185a
–
–
186
186
–
–
186a
186a
–
186a
4.3 10.1 (186b)
186b
186b
–
186b
+5.820 (c 2, H2O)
185a
Spectral Data 185a –
+23.020 (c 2, 5 N HCl) (186a) 141b
Anticapsin
Streptomyces griseoplanus
C9H13NO4 (199.23)
240° (dec) (186b)
+12525 (c 1, H2O) (186b)
142
Ascorbigen
Cabbage
C15H15NO6 (305.30)
–
–
–
187
188
189
–
143
Azetidine-2-carboxylic acid
Convallaria majalis
C4H7NO2 (101.11)
–
–
–
190
190
190
190
143a
Azirinomycin (3-methyl-2hydroazirine carboxylic acid)
Streptomyces aureus
C4H5NO2 (99.10)
–
–
–
189a
189a
144
Baikiain (1,2,3,6-tetrahydropyridine-α-carboxylic acid)
Baikiaea plurijuga
C6H9NO2, (127.15)
273–274° (183)
–
–
181
182
183
144a
N-Carbamoyl-2(p-hydroxyphenyl)glycine
Vicia faba
C9H10N2O4 (210.21)
194–195° (dec) (190a)
–
–
190a
–
–
190a
144b
3-Carboxy-6,7-dihydroxy1,2,3,4-tetrahydroisoquinoline
Mucuna mutisiana
C10H11NO4 (209.22)
286–288° (190b)
−114.925 (c 1.65, 20% HCl) (190b)
–
190b
–
190b
190b
144c
Clavicipitic acid
Claviceps (ergot fungus)
C16H18N2O2 (270.36)
262° (dec) (190c)
–
–
190c
–
–
145
Cucurbitine (3-amino-3-carboxypyrrolidine
Cucurbita moschata
C5H10NO2 (116.14)
−19.7627 (c 9.3, H2O) (191)
–
191
191
191
191
145a
N-Dihydro jasmonoylisoleucine
Gibberella fujikuroi
C18H31NO4 (325.50)
140–141° (191a)
–
–
–
191a
191a
191a
145b
2,5-Dihydrophenylalanine (1,4-cyclohexadiene-1alanine)
Streptomycete X-13, 185
C9H13NO2 (167.23)
206–208° (191b)
−33.725 (c 1, 5 N HCl) (191b)
–
191b
145c
2-N,6-N-Di-(2,3-dihydroxybenzoyl)lysine
Azobacter vinelandii
C20H22N2O8 (418.44)
–
4.8 9 (191c)
191c
145d
cis-3,4-trans-3,4Dihydroxyproline
Diatom cell walls
C5H9NO4 (147.15)
262° (dec) (191d)
–
191d
145e
β-(2,6-Dihydroxypyrimidin1-yl) alanine
Pea seedlings
C7H9N3O4 (199.19)
230° (dec) (191f )
–
191f
–
−61.220 (c 0.5, H2O) (191d) –
–
189a 183
190c
–
191b
191c
–
191c
191d
–
191e
191f
191f
–
Handbook of Biochemistry and Molecular Biology
–
–
4/16/10 1:11 PM
No.
Amino Acid (Synonym)
Source
Formula (Mol Wt)
Melting Point °Ca
References Chromatography Chemistry
[α]Db
pKa
Isolation and Purification
–
–
191g
191g
191g
191g
–
192
192
–
192
2.20 3.72 4.93 9.82 (193)
193
193
193
193
–
194
–
194
194
–
194a
–
194a
194a′
194a′
Spectral Data
145f
4,6-Dihydroxyquinoline-2carboxylic acid
Tobacco leaves
C10H7NO4 (205.18)
287° (dec) (191g)
146
Dihydrozanthurenic acid (8-hydroxy-1,2,3,4tetrahydro-4-ketoquinaldic acid)
Lepidoptera
C10H9NO4 (207.19)
185–190° (192)
−4520 (c 0.9, MeOH) +1820 (c 0.9, MeOH-HCl) (192)
147
Domoic acid (2-carboxy-3-carboxymethyl4-1-methyl-2-carboxy-1,3hexadienyl-pyrrolidine)
Chondria armata
C15H21NO6 (311.35)
217° (193)
−109.612 (c 1.314, H2O) (193)
148
Echinine (2-tert-pentenyl-5,7diisopentenyltryptophan)
Aspergillus glaucus
C26H36N2O2 (408.50)
169–172° (194)
148a
Enduracididine [α-amino-β-(2iminoimidazolidinyl)propionic acid]
Enduracidin hydrolyzate
C6H12N4O2 (172.22)
148a′
Furanomycin (α-amino-(2,5dihydro-5-methyl)furan-2acetic acid]
Streptomyces L-803
C7H11NO3 (157.19)
148a″
Furosine [ε-N-(2-furoylmethyl)lysine]
Heated milk
C12H18N2O4 (254.32)
–
–
–
194a″
194b
–
194a″
148b
γ-Glutaminyl-3,4benzoquinone
Agaricus bisporus
C11H12N2O5 (252.25)
–
–
–
194b
194b
–
194b
149
Guvacine
Areca cathecu
C6H9NO2 (127.15)
–
150
Histidine
Protamine from sturgeon sperm
C6H9N3O2 (155.16)
150a
β-Hydroxyhistidine
Bleomycin A2 (antibiotic)
C6H9N3O3 (171.18)
–
220–223° (dec) (194a′)
–
+63.322 (1 M HCl) +57.622 (1 M NaOH) (194a)
2.5 8.3 12 (94a)
+136.127 (c 1, H2O) (194a’)
2.4 9.1 (194a′)
–
194a′
Data on the Naturally Occurring Amino Acids
9168_Book.indb 23
DATA ON THE NATURALLY OCCURRING AMINO ACIDS (Continued)
194a′
–
195, 196
–
–
–
277° (1)
−38.525 (H2O) +11.8 (5 N HCl(1)
1.82 6.00 9.17 (1)
197
3
198
198
205° (dec) (198a)
+4028 (c 1, H2O) (198a)
11
1 2 3 1
Carboxy, cys β 93 SH
References 1. Janssen, Willekens, De Bruin, and van Os, Eur. J. Biochem., 45, 53 (1974). 2. Snow, Biochem. J., 84, 360 (1962). 3. Guidotti, J. Biol. Chem., 242, 3673 (1967).
Reference 1 1
β0145 10.6 β3130 10.6 β335 >10.6
References 1. Hermans, Jr., Biochemistry, 1, 193 (1962). 2. Nagel, Ranney, and Kucinskis, Biochemistry, 5, 1934 (1966).
9168_Book.indb 140
1
Table 5: pK Values for Human Hb Cys β 93 SH
Tanford, Hauenstein, and Rands, J. Am. Chem. Soc., 77, 6409 (1955). Tanford and Epstein, J. Am. Chem. Soc., 76, 2163 (1954). Tanford and Roberts, Jr., J. Am. Chem. Soc., 74, 2509 (1952). Fromageot and Schnek, Biochem. Biophys. Acta,6, 113 (1950): Tanford and Wagner, J. Am. Chem. Soc., 76, 2331 (1954). 5. Karplus, Snyder, and Sykes, Biochemistry, 12, 1323 (1973).
No. of Residues
10.3 11.5 >12.8 10.3 11.5 >12.8
1. Hermans, Jr., Biochemistry, 1., 193 (1962).
1. 2. 3. 4.
Species
Reference
Reference
References
pK
2
2 2 2
Table 6: Carboxyl Side Chain pK Values Estimated in Lysozymes Residue
Range of pK Values1
Gin 35 Asp 101 Asp 66 Asp 52
6–6.5 4.2–4.7 1.5–2 3–4.6
Reference 1. Imoto, Johnson, North, Phillips, and Rupley, in The Enzymes, Vol. VII, 3rd ed. Bayer, Ed., Academic Press, New York, 1972, 665.
4/16/10 1:13 PM
Protease Inhibitors and Protease Inhibitor Cocktails associated with proteases such as the modification of tyrosine by DFP or PMSF.6 In addition, some of the protease inhibitors such as DFP and PMSF are subject to hydrolysis under conditions (pH ≥ 7.0) used for modification. For those unfamiliar with the history of DFP, DFP is a potent neurotoxin (inhibitor of acetyl cholinesterase) and should be treated with considerable care; a prudent investigator has a DFP repair kit in close proximity (weak base and pralidoxime-2-chloride [2-PAM]). Given these various issues, it is critical to validate that, in fact, the sample is being protected against proteolysis.
While protease inhibitor cocktails have been in use for some time,1 there are few rigorous studies examining their effect on proteolysis and very few concerned with proteolytic degradation during the processing of material for analysis or during purification.2 It is usually assumed that proteolysis can be a problem and protease inhibitors or protease inhibitor cocktails are usually included as part of a protocol without the provision of justification. There are several excellent review articles in this area. Salveson and Nagase3 discuss the inhibition of proteolytic enzymes in great detail including much practical information that should be considered in experimental design. The discussion of the relationship between inhibitor concentration, inhibitor/enzyme binding constants (association constants, binding constants, t1/2, inhibition constants, etc.), and enzyme inhibition is of particular importance. For example, with a reversible enzyme inhibitor (such as benzamidine), if the Ki value is 100 nM, a 100 µM concentration of inhibitor would be required to decrease protease activity by 99.9%. Salveson and Nagase3 also note the well-known differences in the reaction rates of inhibitors such as DFP and PMSF with the active site of serine proteases. DFP is much faster than PMSF with trypsin but equivalent rates are seen with chymotrypsin. PMSF is included in commercial protease inhibitor cocktails because of its lack of toxicity compared to DFP; 3,4-dichloroisocoumarin (3,4-DCI), as described by Powers and colleagues,4 is faster than either DFP or PMSF. Also enzyme inhibition occurs in the presence of substrate (proteins), which will influence the effectiveness of both irreversible and reversible enzyme inhibitors. In addition, some protease inhibitor cocktails include both PMSF and benzamidine. Benzamidine is a competitive inhibitor of trypticlike serine proteases and slows the rate of inactivation of such enzymes by reagents such as PMSF.5 The investigator is also advised to consider the modification of proteins and other biological compounds by protease inhibitors in reactions not
References 1. Takei, Y., Marzi, I., Kauffman, F.C. et al., Increase in survival time of liver transplants by protease inhibitors and a calcium channel blocker, nisoldipine. Transplantation 50, 14–20, 1990. 2. Pyle, L.E., Barton, P., Fujiwara, Y., Mitchell, A., and Fidge, N., Secretion of biologically active human proapolipoprotein A-1 in a baculovirus-insect cell system: protection from degradation by protease inhibitors, J. Lipid Res. 36, 2355–2361, 1995. 3. Salveson, G. and Nagase, H., Inhibition of proteolytic enzymes, in Proteolytic Enyzymes: Practical Approaches, 2nd ed., R. Benyon and J.S. Bond, Eds., Oxford University Press, Oxford, UK, pp. 105–130, 2001. 4. Harper, J.W., Hemmi, K., and Powers, J.C., Reaction of serine proteases with substituted isocoumarins: discovery of 3,4-dichloroisocoumarin, a new general mechanism-based serine protease inhibitor, Biochemistry 24, 1831–1841, 1985. 5. Lundblad, R.L., A rapid method for the purification of bovine thrombin and the inhibition of the purified enzyme with phenylmethylsulfonyl fluoride, Biochemistry 10, 2501–2506, 1971. 6. Lundblad, R.L., Chemical Reagents for Protein Modification, CRC Press, Boca Raton, FL, 2004.
Characteristics of Selected Protease Inhibitors, Which Can be Used in Protease Inhibitor Cocktailsa Common Name Amastatin CH3
NH2
CH H3C
O
CH C H2
CH
NH
CH H3C
M.W.
N-[(2S,3R)-3-amino-2hydroxy-5-methyl hexanoyl]-l-valyl-lvalyl-l-aspartic acid
529.0
Primary Design Inhibitor of some aminopeptidases.
O
CH OH
Other Nomenclature
C CH3
NH
O
CH CH H3C
C CH3
Amastatin
NH
O
CH C
H2C
OH
C HO
O
141
9168_Book.indb 141
4/16/10 1:14 PM
Handbook of Biochemistry and Molecular Biology
142
Characteristics of Selected Protease Inhibitors, Which Can be Used in Protease Inhibitor Cocktails (Continued) Common Name
Other Nomenclature
M.W.
Primary Design
Amastatin is a complex peptidelike inhibitor of aminopeptidases obtained from Actinoycetes culture. Amastatin is a competitive inhibitor of aminopeptidase A, aminopeptidase M, and other aminopeptidases. Amastatin has been used for the affinity purification of aminopeptidases. Amastatin has been shown to inhibit amino acid iosomerases. Amastatin is structurally related to bestatin and has been described as an immunomodulatory factor. See Aoyagi, T., Tobe, H., Kojima, F. et al., Amastatin, an inhibitor of aminopeptidase A, produced by actinomycetes, J. Antibiot. 31, 636–638, 1978; Tobe, H., Kojima, F., Aoyagi, T., and Umezawa, H., Purification by affinity chromatography using amastatin and properties of he aminopeptidase A from pig kidney, Biochim. Biophys. Acta 613, 459–468, 1980; Rich, D.H., Moon, B.J., and Harbeson, S., Inhibition of aminopeptidases by amastatin and bestatin derivatives. Effect of inhibitor structure on slow-binding processes, J. Med. Chem. 27, 417–422 , 1984; Meisenberg, G. and Simmons, W.H., Amastatin potentiates the behavioral effects of vasopressin and oxytocin in mice, Peptides 5, 535–539, 1984; Wilkes, S.H. and Prescott, J.M., The slow, tight binding of bestatin and amastatin to aminopeptidases, J. Biol. Chem. 260, 13154– 13162, 1985; Matsuda, N., Katsuragi, Y., Saiga, Y. et al., Effects of aminopeptidase inhibitors actinonin and amastatin on chemotactic and phagocytic responses of human neutrophils, Biochem. Int. 16, 383–390, 1988; Orawski, A.T. and Simmons, W.H., Dipeptidase activities in rat brain synaptosomes can be distinguished on the basis of inhibition by bestatin and amastatin: identification of a kyotrophin (Tyr-Arg)-degrading enzyme, Neurochem. Res. 17, 817–820, 1992; Kim, H. and Lipscomb, W.N., X-ray crystallographic determination of the structure of bovine lens leucine aminopeptidase complexed with amastatin: formation of a catalytic mechanism, featuring a gem-diolate transition state, Biochemistry 32, 8365–8378, 1993; Bernkop-Schnurch, A., The use of inhibitory agents to overcome the enzymatic barrier to perorally administered therapeutic peptides and proteins, J. Control. Release 52, 1–16, 1998; Fortin, J.P., Gera, L., Bouthillier, J. et al., Endogenous aminopeptidase N decreases the potency of peptide agonists and antagonists of the kinin B1 receptors in the rabbit aorta, J. Pharmacol. Exp. Ther. 312, 1169–1176, 2005; Olivo Rdo, A., Teixeira Cde, R., and Silveira, P.F., Representative aminopeptidases and prolyl endopeptidase from murin macrophages; comparative activity levels in resident and elicited cells, Biochem. Pharmacol. 69, 1441–1450, 2005; Gera. L., Fortin, J.P., Adam, A. et al., Discovery of a dual-function peptide that combines aminopeptidase N inhibition and kinin B1 receptor antagonism, J. Pharmacol. Exp. Ther. 317, 300–308, 2006; Krsyanovic, M., Brgles, M., Halassy, B. et al., Purification and characterization of the l,(l/d)-aminopeptidase from guinea pig serum, Prep. Biochem. Biotechnol. 36, 175–195, 2006; Torres, A.M., Tsampazi, M., Tsampazi, C. et al., Mammalian l to d-amino-acid-residue isomerase from platypus venom, FEBS Lett. 580, 1587–1591, 2006. Aprotinin 6512 Protein protease inhibitor. Basic pancreatic trypsin inhibitor; Kunitz pancreatic trypsin inhibitor; Trasylol®. This protein inhibits some but not all trypticlike serine proteinases and is included in some protease inhibitor cocktails. See Hulsemann, A.R., Jongejan, R.C., Rolien Raatgeep, H. et al., Epithelium removal and peptidase inhibition enhance relaxation of human airways to vasoactive intestinal peptide, Am. Rev. Respir. Dis. 147, 1483–1486, 1993; Cornelius, R.M. and Brash, J.L., Adsorption from plasma and buffer of single- and two-chain high molecular weight kininogen to glass and sulfonated polyurethane surfaces, Biomaterials 20, 341–350, 1999; Lafleur, M.A., Handsley, M.M., Knauper, V. et al., Endothelial tubulogenesis with fibrin gels specifically requires the activity of membrane-type-matrix metalloproteinases (MT-MMPs), J. Cell Sci. 115, 3427–3438, 2002; Shah, R.B., Palamakula, A., and Khan, M.A., Cytotoxicity evaluation of enzyme inhibitors and absorption enhancers in Caco-2 cells for oral delivery of salmon calcitonin, J. Pharm. Sci. 93, 1070–1982; Spens, E. and Häggerström, L., Protease activity in protein-free (NS) myeloma cell cultures, In Vitro Cell Dev. Biol. 41, 330–336, 2005. As it is a potent inhibitor of plasmin, aprotinin is frequently included in fibrin gel-based cultures to preserve the fibrin gel structure. See Ye, Q., Zund, G., Benedikt, P. et al., Fibrin gel as a three-dimensional matrix in cardiovascular tissue engineering, Eur. J. Cardiothorac. Surg. 17, 587–591, 2000; Krasna, M., Planinsek, F., Knezevic, M. et al., Evaluation of a fibrin-based skin substitute prepared in a defined keratinocyte medium, Int. J. Pharm. 291, 31–37, 2005; Sun, X.T., Ding, Y.T., Yan, X.G. et al., Antiangiogenic synergistic effect of basic fibroblast growth factor and vascular endothelial growth factor in an in vitro quantitative microcarrier-based three-dimensional fibrin angiogenesis system, World J. Gastroenterol. 10, 2524–2528, 2004; Gille, J., Meisner, U., Ehlers, E.M. et al., Migration pattern, morphology and viability of cells suspended in or sealed with fibrin glue: a histomorphology study, Tissue Cell 37, 339–348, 2005; Yao, L., Swartz, D.D., Gugino, S.F. et al., Fibrin-based tissue-engineered blood vessels: differential effects of biomaterial and culture parameters on mechanical strength and vascular reactivity, Tissue Eng. 11, 991–1003, 2005. Aprotinin is used therapeutically in the inhibition of plasmin activity both as a freestanding product and as a component of fibrin sealant products. Benzamidine HCI
H2N
NH
156.61
Inhibitor of trypticlike serine proteases.
Benzamidine An aromatic amidine derivative (Markwardt, F., Landmann, H., and Walsmann, P., Comparative studies on the inhibition of trypsin, plasmin, and thrombin by derivatives of benzylamine and benzamidine, Eur. J. Biochem. 6, 502–506, 1968; Guvench, O., Price, D.J., and Brooks, C.L., III, Receptor rigidity and ligand mobility in trypsin-ligand complexes, Proteins 58, 407–417, 2005), which is used as a competitive inhibitor of trypticlike serine proteases. It is not a particularly tight-binding inhibitor and is usually used at millimolar concentrations. Ensinck, J.W., Shepard, C., Dudl, R.J., and Williams, R.H., Use of benzamidine as a proteolytic inhibitor in the radioimmunoassay of glucagon in plasma, J. Clin. Endocrinol. Metab. 35, 463–467, 1972; Bode, W. and Schwager, P., The refined crystal structure of bovine beta-trypsin at 1.8 Å resolution. II. Crystallographic refinement, calciumbinding site, benzamidine-binding site, and active site at pH 7.0., J. Mol. Biol. 98, 693–717, 1975; Nastruzzi, C., Feriotto, G., Barbieri, R. et al., Differential effects of benzamidine derivatives on the expression of c-myc and HLA-DR alpha genes in a human B-lymphoid tumor cell line, Cancer Lett. 38, 297–305, 1988; Clement, B., Schmitt, S., and Zimmerman, M., Enzymatic reduction of benzamidoxime to benzamidine, Arch. Pharm. 321, 955–956, 1988; Clement, B., Immel, M., Schmitt, S., and Steinman, U., Biotransformation of benzamidine and benzamidoxime in vivo, Arch. Pharm. 326, 807–812, 1993; Renatus, M., Bode, W., Huber, R. et al., Structural and functional analysis of benzamidine-based inhibitors in complex with trypsin: implications for the inhibition of factor Xa, tPA, and urokinase, J. Med. Chem. 41, 5445–5456, 1998; Henriques, R.S., Fonseca, N., and
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Characteristics of Selected Protease Inhibitors, Which Can be Used in Protease Inhibitor Cocktails (Continued) Common Name
Other Nomenclature
M.W.
Primary Design
Ramos, M.J., On the modeling of snake venom serine proteinase interactions with benzamidine-based thrombin inhibitors, Protein Sci. 13, 2355–2369, 2004; Gustavsson, J., Farenmark, J., and Johansson, B.L., Quantitative determination of the ligand content in Benzamidine Sepharose® 4 Fast Flow media with ion-pair chromatography, J. Chromatog. A 1070, 103–109, 2005. Concentrated solutions of benzamidine will require pH adjustment prior to use. Bestatin
OH H2 C
O H N
CH
C
CH
C
CH
NH2
O
H2C
OH
N-[(2S,3R)-3-amino2-hydroxy-l-oxo-4phenylbutyl]-lleucine
344.8
Aminopeptidase inhibitor; also described as a metalloproteinase inhibitor.
CH3 CH
Bestatin
CH3
Bestatin is an inhibitor of some aminopeptidases and it was isolated from Actinomycetes culture. Bestatin was subsuently shown to have immunomodulatory activity and induces apoptosis in tumor cells. Bestatin is included in some proteaseinhibitor cocktails and has been demonstrated to inhibit intracellular protein degradation. See Umezawa, H., Aoyagi, T., Suda, H. et al., Bestatin, an inhibitor of aminopeptidase B, producted by actinomycetes, J. Antibiot. 29, 97–99, 1976; Suda, H., Takita, T., Aoyagi, T., and Umezawa, H., The structure of bestatin, J. Antibiot. 29, 100–101, 1976; Saito, M., Aoyagi, T., Umezawa, H., and Nagai, Y., Bestatin, a new specific inhibitor of aminopeptidases, enhances activation of small lymphocytes by concanavalin A, Biochem. Biophys. Res. Commun. 76, 526–533, 1976; Botbot, V. and Scornik, O.A., Degradation of abnormal proteins in intact mouse reticulocytes: accumulation of intermediates in the presence of bestatin, Proc. Natl. Acad. Sci. USA 76, 710–713, 1979; Botbol, V. and Scornik, O.A., Peptide intermediates in the degradation of cellular proteins. Bestatin permits their accumulation in mouse liver in vivo, J. Biol. Chem. 258, 1942–1949, 1983; Rich, D.H., Moon, B.J., and Harbeson, S., Inhibition of aminopeptidases by amastatin and bestatin derivatives. Effect of inhibitor structure on slow-binding processes, J. Med. Chem. 27, 417–422, 1984; Wilkes, S.H. and Prescott, J.M., The slow, tight binding of bestatin and amastatin to aminopeptidases, J. Biol. Chem. 260, 13154–13160, 1985; Patterson, E.K., Inhibition by bestatin of a mouse ascites tumor dipeptidase. Reversal by certain substrates, J. Biol. Chem. 264, 8004–8011, 1989; Botbol, V. and Scornik, O.A., Measurement of instant rates of protein degradation in the livers of intact mice by the accumulation of bestatin-induced peptides, J. Biol. Chem. 266, 2151–2157, 1991; Tieku, S. and Hooper, N.M., Inhibition of aminopeptidases N, A, and W. A re-evaluation of the actions of bestatin and inhibitors of angiotensin converting enzyme, Biochem. Pharmacol. 44, 1725–1730, 1992; Taylor, A., Peltier, C.Z., Torre, F.J., and Hakamian, N., Inhibition of bovine lens leucine aminopeptidase by bestatin: number of binding sites and slow binding of this inhibitor, Biochemistry 32, 784–790, 1993; Schaller, A., Bergey, D.R., and Ryan, C.A., Induction of wound response genes in tomato leaves by bestatin, an inhibitor of aminopeptidases, Plant Cell 7, 1893–1898, 1995; Nemoto, H., Ma, R., Suzuki, I.I., and Shibuya, M., A new one-pot method for the synthesis of alpha-siloxyamides from aldehydes or ketones and its application to the synthesis of (-)bestatin, Org. Lett. 2, 4245–4247, 2000; van Hensbergen, Y., Brfoxterman, H.J., Peters, E. et al., Aminopeptidase inhibitor bestatin stimulates microvascular endothelial cell invasion in a fibrin matrix, Thromb. Haemost. 90, 921–929, 2003; Stamper, C.C., Bienvenue, D.L., Bennett, B. et al., Spectroscopic and X-ray crystallographic characterization of bestatin bound to the aminopeptidase from Aeromonas(Vibrio)proteolytica, Biochemistry 43, 9620–9628, 2004; Zheng, W., Zhai, Q., Sun, J. et al., Bestatin, an inhibitor of aminopeptidases, provides a chemical genetics approach to dissect jasmonate signaling in Aribidopsis, Plant Physiol. 141, 1400–1413, 2006; Hui, M. and Hui, K.S., A novel aminopeptidase with highest preference for lysine, Neurochem. Res. 31, 95–102, 2006. Cystatins
Protein Inhibitors of Inhibitors of cysteine proteinases. Cysteine Proteases Cystatin refers to a diverse family of protein cysteine protease inhibitors. There are three general types of cystatins: Type 1 (stefens), which are primarily found in the cytoplasm but can appear in extracellular fluids; Type 2, which are secreted and found in most extracellular fluids; and Type 3, which are multidomain protease inhibitors containing carbohydrates and that include the kininogens. Cystatin 3 is used to measure renal function in clinical chemistry. See Barrett, A.J., The cystatins: a diverse superfamily of cysteine peptidase inhibitors, Biomed. Biochim. Acta 45, 1363–1374, 1986; Katunuma, N., Mechanisms and regulation of lysosomal proteolysis, Revis. Biol. Cellular 20, 35–61, 1989; Gauthier, F., Lalmanach, G., Moeau, T. et al., Cystatin mimicry by synthetic peptides, Biol. Chem. Hoppe Seyler 373, 465–470, 1992; Bobek, L.A. and Levine, M.J., Cystatins — inhibitors of cysteine proteineases, Crit. Rev. Oral Biol. Med. 3, 307–332, 1992; Calkins, C.C., and Sloane, B.F., Mammalian cysteine protease inhibitors: biochemical properties and possible roles in tumor progression, Biol. Chem. Hoppe Seyler 376, 71–80, 1995; Turk, B., Turk, V., and Turk, D., Structural and functional aspects of papainlike cysteine proteinases and their protein inhibitors, Biol. Chem. 378, 141–150, 1997; Kos, J., Stabuc, B., Cimerman, N., and Brunner, N., Serum cystatin C, a new marker of glomerular filtration rate, is increased during malignant progression, Clin. Chem. 44, 2556–2557, 1998; Vray, B., Hartman, S., and Hoebeke, J., Immunomodulatory properties of cystatins, Cell. Mol. Life Sci. 59, 1503–1512, 2002; Arai, S., Matsumoto, I., Emori, Y., and Abe, K., Plant seed cystatins and their target enzymes of endogenous and exogenous origin, J. Agric. Food Chem. 50, 6612–6617, 2002; Abrahamson, M., Alvarez-Fernandez, M., and Nathanson, C.M., Cystatins, Biochem. Soc. Symp. 70, 179–199, 2003; Dubin, G., Proteinaceous cysteine protease inhibitors, Cell. Mol. Life Sci. 62, 653–669, 2005; Righetti, P.G., Castagna, A., Antonucci, F. et al., Proteome analysis in the clinical chemistry laboratory: myth or reality? Clin. Chim. Acta 357, 123–139, 2005; Overall, C.M. and Dean, R.A., Degradomics: systems biology of the protease web. Pleiotropic roles of MMPs in cancer, Cancer Metastasis Rev. 25, 69–75, 2006; Kotsylfakis, M., Sá-Nunes, A., Francischetti, I.M.B. et al., Antiinflammatory and immunosuppressive activity of sialostatin L, a salivary cystatin from Tick Ixodes scapularis, J. Biol. Chem. 281, 26298–26307, 2006. DCI was developed by James C. Powers and coworkers at Georgia Institute of Technology (Harper, J.W., Hemmi, K., and Powers, J.C., Reaction of serine proteases with substituted isocoumarins: discovery of 3,4-dichloroisocoumarin, a new general mechanism-based serine protease inhibitor, Biochemistry 24, 1831–1841, 1985). This inhibitor is reasonably specific, although side reactions have been described. As with the sulfonyl fluorides and DFP, the modification is slowly reversible and enhanced by basic solvent conditions and/or nucleophiles. DCI has been used as a proteosome inhibitor. See Rusbridge, N.M. and Benyon, R.J., 3,4-dichloroisocoumarin, a serine protease inhibitor, inactivates glycogen phosphorylase b, FEBS Lett. 30, 133–136, 1990; Weaver,
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Characteristics of Selected Protease Inhibitors, Which Can be Used in Protease Inhibitor Cocktails (Continued) Common Name
Other Nomenclature
3,4-Dichloroisocoumarin
DCI
O
M.W. 215
Primary Design Mechanism-based inhibitor of serine proteases.
O
Cl Cl 3,4-dichloroisocoumarin V.M., Lach, B., Walker, P.R., and Sikorska, M., Role of proteolysis in apoptosis: involvement of serine proteases in internucleosomal DNA fragmentation in immature thymocytes, Biochem. Cell Biol. 71, 488–500, 1993; Garder, A.M., Aviel, S., and Argon, Y., Rapid degradation of an unassembled immunoglobulin light chain is mediated by a serine protease and occurs in a pre-Golgi compartment, J. Biol. Chem. 268, 25940–25947, 1993; Lu, Q. and Mellgren, R.L., Calpain inhibitors and serine protease inhibitors can produce apoptosis in HL-60 cells, Arch. Biochem. Biophys. 334, 175–181, 1996; Adams, J. and Stein, R., Novel inhibitors of the proteosome and their therapeutic use in inflammation, Annu. Rep. Med. Chem. 31, 279–288, 1996; Olson, S.T., Swanson, R., Patston, P.A., and Bjork, I., Apparent formation of sodium dodecyl sulfate-stable complexes between serpins and 3,4-dichloroisocoumarin-inactivated proteinases is due to regeneration of active proteinase from the inactivated enzyme, J. Biol. Chem. 272, 13338–13342, 1997; Mesner, P.W., Bible, K.C., Martins, L.M. et al., Characterization of caspase processing and activation in HL-60 cell cytosol under cell-free conditions — nucleotide requirement and inhibitor profile, J. Biol. Chem. 274, 22635–22645, 1999; Kam, C.M., Hudig, D., and Powers, J.C., Granzymes (lymphocyte serine proteases): characterization with natural and synthetic substrates and inhibitors, Biochim. Biophys. Acta 1477, 307–323, 2000; Rivett, A.J. and Gardner, R.C., Proteosome inhibitors: from in vitro uses to clinical trials, J. Pep. Sci. 6, 478–488, 2000; Bogyo, M. and Wang, E.W., Proteosome inhibitors: complex tools for a complex enzyme, Curr. Top. Microbiol. Immunol. 268, 185–208, 2002; Powers, J.C., Asgian, J.L., Ekici, O.D., and James, K.E., Irreversible inhibitors of serine, cysteine, and threonine proteases, Chem. Rev. 102, 4639– 4740, 2002; Pochet, L., Frederick, R., and Masereei, B., Coumarin and isocoumarin as serine protease inhibitors, Curr. Pharm. Des. 10, 3781–3796, 2004. Diisopropyl Phosphosphorofluoridate
H3C
CH3 H3C CH O
CH
H2 C
CH3
HO
Reaction at active site serine.
N H
CH
H2C
F Serine residue in protein
Diisopropylphosphorofluoridate
H3C
CH3 H3C CH O
CH3
CH O
O
CH3 H3C CH O
CH
O
CH
H2C
CH3
O
H2 C
P
H3C
184
NH
O P O
DFP; Diisopropyl Fluorophosphate
O
NH
N H
Disopropylphosphorylserine
O P O OH HO
H2C
9168_Book.indb 144
O
H2 C CH NH
N H
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Characteristics of Selected Protease Inhibitors, Which Can be Used in Protease Inhibitor Cocktails (Continued) Common Name
Other Nomenclature
M.W.
Primary Design
DFP was developed during World War II as a neurotoxin. DFP reacts with the active serine of serine proteases and was used to define the presence of this amino acid at the active sites of trypsin and chymotrypsin. DFP has been replaced by PMSF as a general reagent for inhibition of proteases although it is still used on occasion because of the ease of identification of the phosphoserine derivative. See Jansen, E.F., Jang, R., and Balls, A.K., The inhibition of purified, human plasma cholinesesterase with diisopropylfluorophosphate, J. Biol. Chem. 196, 247–253, 1952; Gladner, J.A. and Neurath, H.A., C-terminal groups in chymotrypsinogen and DFP-alpha-chymotrypsin in relation to the activation process, Biochim. Biophys. Acta 9, 335–336, 1952; Schaffer, N.K., May, S.C., Jr., and Summerson, W.H., Serine phosphoric acid from diisopropylphosphoryl chymotrypsin, J. Biol. Chem. 202, 67–76, 1953; Oosterbaan, R.A., Kunst, P., and Cohen, J.A., The nature of the reaction between diisopropylfluorophosphate and chymotrypsin, Biochim. Biophys. Acta. 16, 299–300, 1955; Wahlby, S., Studies on Streptomyces griseus protease. I. Separation of DFP-reacting enzymes and purification of one of the enzymes, Biochim. Biophys. Acta 151, 394–401, 1968; Hoskin, R.J. and Long, R.J., Purification of a DFP-hydrolyzing enzyme from squid head ganglion, Arch. Biochem. Biophys. 150, 548–555, 1972; Craik, C.S., Roczniak, S., Largman, C., and Rutter, W.J., The catalytic role of the active aspartic acid in serine proteases, Science 237, 909–913, 1987; D’Souza, C.A., Wood, D.D., She, Y.M., and Moscarello, M.A., Autocatalytic cleavage of myelin basic protein: an alternative to molecular mimicry, Biochemistry 44, 12905–12913, 2005. DFP is a potent neurotoxin and attention should be given to antidotes to organophosphates (Tuovinen, K., Kaliste-Korhonen, E., Raushel, F.M., and Hanninen, O., Phosphotriesterase, pralidoxime-2-chloride (2-PAM), and eptastigmine treatments and their combinations in DFP intoxication, Toxicol. Appl. Pharmacol. 141, 555–560, 1996; Auta, J., Costa, E., Davis, J., and Guidotti, A., Imidazenil: a potent and safe protective agent against diisopropyl fluorophosphate toxicity, Neuropharmacology 46, 397–403, 2004; Tuovinen, K., Organophosphate- induced convulsions and prevention of neuropathological damages, Toxicology 196, 31–39, 2004). E-64
NH H2N
O H N
N H
OH
N H
O
O E-64 from Aspergillus japonicus
l-transepoxysuccinylleucylamide-(4guanido)butane or N-[N-(l-transcarboxyoxiran-2carbonyl)-l-leucyl]agmatine
357.4
Inhibitor of sulfhydryl proteases.
E-64 is a reasonably specific inhibitor of sulfhydryl proteases and it functions by forming a thioether linkage with the active site cysteine. E-64 is frequently referred to as an inhibitor of lysosomal proteases and antigen processing. See Hashida, S., Towatari, T., Kominami, E., and Katunuma, N., Inhibition by E-64 derivatives of rat liver cathepsins B and cathepsin L in vitro and in vivo, J. Biochem. 88, 1805–1811, 1980; Grinde, B., Selective inhibition of lysosomal protein degradation by the thiol proteinease inhibitors E-64, Ep-459, and Ep-457 in isolated rat hepatocytes, Biochim. Biophys. Acta 701, 328–333, 1982; Barrett, A.J., Kembhavi, A.A., Brown, A.A. et al., L-trans-epoxysuccinyl-leucylamiodo (4-guanidino) butane (E-64) and its analogues as inhibitors of cysteine proteinases including cathepsins B, H, and L, Biochem. J. 201, 189–198, 1982; Ko, Y.M., Yamanaka, T., Umeda, M., and Suzuki, Y., Effects of thiol protease inhibitors on intracellular degradation of exogenous β-galactosidase in cultured human skin fibroblasts, Exp. Cell Res. 148, 525–529, 1983; Tamai, M., Matsumoto, K., Omura, S. et al., In vitro and in vivo inhibition of cysteine proteinases by EST, a new analog of E-64, J. Pharmacobiodyn. 9, 672–677, 1986; Shaw, E., Cysteinyl proteinases and their selective inactivation, Adv. Enzymol. Relat. Areas Mol. Biol. 63, 271–347, 1990; Mehdi, S., Cell-penetrating inhibitors of calpain, Trends Biochem. Sci. 16, 150–153, 1991; Min, K.S., Nakatsubo, T., Fujita, Y. et al., Degradation of cadmium metallothionein in vitro by lysosomal proteases, Toxicol. Appl. Pharmacol. 113 299–305, 1992; Schirmeister, T. and Klackow, A., Cysteine protease inhibitors containing small rings, Mini Rev. Med. Chem. 3, 585–596, 2003. EACA
NH2
NH2
CH2
CH2
131.2
Analogue of lysine; inhibitor of trypsinlike enzymes such as plasmin.
CH2
CH2 H2C
H2C
H2C
H2C CH2
NH2 CH
C O
ε-aminocaproic acid; 6-aminocaproic acid; 6-aminohexanoic acid; Amicar™
C OH
Epsilon-aminocaproic acid 6-aminohexanoic acid
OH
O Lysine
EACA is an inhibitor of trypticlike serine proteases. It has been used as a hemostatic agent that functions by inhibiting fibrinolysis. It is included in some protease inhibitor cocktails. See Soter, N.A., Austen, K.F., and Gigli, I., Inhibition by epsilon-aminocaproic acid of the activation of the first component of the complement system, J. Immunol. 114, 928–932, 1975; Burden, A.C., Stacey, R., Wood, R.F., and Bell, P.R., Why do protease inhibitors enhance leukocyte migration inhibition to the antigen PPD? Immunology 35, 959–962, 1978; Nakagawa, H., Watanabe, K., and Sato, K., Inhibitory action of synthetic proteinase inhibitors and substrates on the chemotaxis of rat polymorphonuclear leukocytes in vitro, J. Pharmacobiodyn. 11, 674–678, 1988; Hill, G.E., Taylor, J.A., and Robbins, R.A., Differing effects of aprotinin and ε-aminocaproic acid on cytokine-induced inducible nitric oxide synthase expression, Ann. Thorac. Surg. 63, 74–77, 1997; Stonelake, P.S., Jones, C.E., Neoptolemos, J.P., and Baker, P.R., Proteinase inhibitors reduce basement membrane degradation by human breast cancer cell lines, Br. J. Cancer 75, 951–959, 1997; Sun, Z., Chen, Y.H., Wang, P. et al., The blockage of the high-affinity lysine-binding sites of plasminogen by EACA significantly inhibits prourokinase-induced plasminogen activation, Biochim. Biophys. Acta 1596, 182–192, 2002.
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Other Nomenclature
M.W.
Ecotin
Primary Design Broad-spectrum protease inhibitor derived from Escherichia coli.
Ecotin is a broad-spectrum inhibitor of serine proteases that can be engineered to enhance inhibition of specific enzymes. See McGrath, M.E., Hines, W.M., Sakanari, J.A. et al., The sequence and reactive site of ecotin. A general inhibitor of pancreatic serine proteases from Escherichia coli, J. Biol. Chem. 266, 6620–6625, 1991; Erpel, T., Hwang, P., Craik, C.S. et al., Physical map location of the new Escherichia coli gene eco, encoding the serin protease inhibitor ecotin, J. Bacteriol. 174, 1704, 1992; Wang, C.I., Yang, Q., and Craik, C.S., Isolation of a high affinity inhibitor of urokinase-type plasminogen activator by phage display of ecotin, J. Biol. Chem. 270, 12250–12256, 1995; Yang, S.Q., Wang, C.T., Gilmor, S.A. et al., Ecotin: a serine protease inhibitor with two distinct and interacting binding sites, J. Mol. Biol. 279, 945–957, 1998; Gilmor, S.A., Takeuchi, T., Yang, S.Q. et al., Compromise and accommodation in ecotin, a dimeric macromolecular inhibitor of serine proteases, J. Mol. Biol. 299, 993–1003, 2000; Eggers, C.T., Wang, S.X., Fletterick, R.J., and Craik, C.S., The role of ecotin dimerization in protease inhibition, J. Mol. Biol. 308, 975–991, 2001; Wang, B., Brown, K.C., Lodder, M. et al., Chemical-mediated site-specific proteolysis. Alteration of protein–protein interaction, Biochemistry 41, 2805–2813, 2002; Stoop, A.A. and Craik, C.S., Engineering of a macromolecular scaffold to develop specific protease inhibitors, Nat. Biotechnol. 21, 1063–1068, 2003; Eggers, C.T., Murray, I.A., Delmar, V.A. et al., The periplasmic serine protease inhibitor ecotin protects bacteria against neutrophil elastase, Biochem. J. 379, 107–118, 2004. Ethylenediamine Tetraacetic Acid
HO
HO
EDTA
O
O H2 C
N
292.2
OH
N
Metal ion chelator; inhibitor of metalloenzymes.
OH
C H2 O O Edetic acid; EDTA; ethylenediaminetetraacetic acid; N, N’-1, 2-ethanediaminediylbis[N-(carboxymethylglycine)] (Ethylenedinitrilo)tetraacetic acid (ethylenediamine tetraacetic acid) chelates metal ions with a preference for divalent cations. EDTA functions as an inhibitor of metalloproteinases. See Manna, S.K., Bhattacharya, C., Gupta, S.K., and Samanta, A.K., Regulation of interleukin-8 receptor expression in human polymorphonuclear neutrophils, Mol. Immunol. 32, 883–893, 1995; Martin-Valmaseda, E.M., Sanchez-Yague, Y., Marcos, R., and Lianillo, M., Decrease in platelet, erythrocyte, and lymphocyte acetylcholinesterase activities due to the presence of protease inhibitors in the storage buffers, Biochem. Mol. Biol. Int. 41, 83–91, 1997; Oh-Ishi, M., Satoh, M., and Maeda, T., Preparative two-dimensional gel electrophoresis with agarose gels in the first dimension for high molecular mass proteins, Electrophoresis 21, 1653–1669, 2000; Shah, R.B., Palamakula, A., and Khan, M.A., Cytotoxicity evaluation of enzyme inhibitors and absorption enhancers in Caco-2 cells for oral delivery of salmon calcitonin, J. Pharm. Sci. 93, 1070–1082, 2004; Pagano, M.R., Paredi, M.E., and Crupkin, M., Cytoskeletal ultrastructure and lipid composition of I-Z-I fraction in muscle from pre- and postspawned female hake (Meriluccius hubbsi), Comp. Biochem. Physiol. B Biochem. Mol. Biol. 141, 13–21, 2005; Wei, G.X. and Bobek, L.A., Human salivary mucin MUC7 12-mer-L and 12-mer-D peptides: antifungal activity in saliva, enhancement of activity with protease inhibitor cocktail or EDTA, and cytotoxicity to human cells, Antimicrob. Agents Chemother. 49, 2336–2342, 2005. Iodoacetamide
I
H2C
185
I NH2
O Iodoacetamide
H2C
Primary reaction with sulfhydryl groups and slower reaction with other protein nucleophiles.
OH
O Iodoacetic acid
Iodoacetic acid and iodoacetamide can both be used to modify nucleophiles in proteins. The chloro- and bromo-derivatives can be used as well but the rate of modification is slower. The haloacetyl function can also be used as the reactive function for more complex derivatives. Iodoacetamide is neutral compared to iodoacetic acid and is less influenced by the local environment of the reactive nucleophile. See Janatova, J., Lorenz, P.E., and Schechter, A.N., Third component of human complement: appearance of a sulfhydryl group following chemical or enzymatic inactivation, Biochemistry 19, 4471–4478, 1980; Haas, A.L., Murphey, K.E., and Bright, P.M., The inactivation of ubiquitin accounts for the inability to demonstrate ATP, ubiquitin-dependent proteolysis in liver extracts, J. Biol. Chem. 260, 4694–4703, 1985; Molla, A., Yamamoto, T., and Maeda, H., Characterization of 73 kDa thiol protease from Serratia marcescens and its effect on plasma proteins, J. Biochem. 104, 616–621, 1988; Wingfield, P., Graber, P., Turcatti, G. et al., Purification and characterization of a methionine-specific aminopeptidase from Salmonella tyrphimurium, Eur. J. Biochem. 180, 23–32, 1989; Kembhavi, A.A., Buttle, D.J., Rauber, P., and Barrett, A.J., Clostripain: characterization of the active site, FEBS Lett. 283, 277–280, 1991; Jagels, M.A., Travis, J., Potempa, J. et al., Proteolytic inactivation of the leukocyte C5a receptor by proteinases derived from Porphyromas gingivalis, Infect. Immun. 64, 1984–1991, 1996; Tanksale, A.M., Vernekar, J.V., Ghatge, M.S., and Deshpande, V.V., Evidence for tryptophan in proximity to histidine and cysteine as essential to the active site of an alkaline protease, Biochem. Biophys. Res. Commun. 270, 910–917, 2000; Karki, P., Lee, J., Shin, S.Y. et al., Kinetic comparison of procapase-3 and caspases-3, Arch. Biochem. Biophys. 442, 125–132, 2005. The haloalkyl derivatives do react with thiourea and are perhaps less reliable than maleimides.
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Characteristics of Selected Protease Inhibitors, Which Can be Used in Protease Inhibitor Cocktails (Continued) Common Name
Other Nomenclature
M.W.
Primary Design
LBTI
Lima Bean Trypsin 6500 Protein protease inhibitor. Inhibitor Lima bean trypsin inhibitor is a protein/peptide with unusual stability. It is stable to heat (90°C for 15 minutes at pH 7 with no loss of activity) and acid (the original purification uses extraction with ethanol and dilute sulfuric acid). This is a reflection of the high content of cystine resulting in a “tight” structure. As a Bowman–Birk inhibitor, LBTI has seven disulfide bonds (Weder, J.K.P. and Hinkers, S.C., Complete amino acid sequence of the Lentil trypsin-chymotrypin inhibitor LCI-1.7 and a discussion of atypical binding sites of Bowman–Birk inhibitors, J. Agric. Food Chem. 52, 4219–4226, 2004). LBTI also inhibits both trypsin and chymotrypsin (Krahn, J. and Stevens, F.C., Lima bean trypsin inhibitor. Limited proteolysis by trypsin and chymotrypsin, Biochemistry 27, 1330–1335, 1970) as well as various other serine proteases. For additional information, see Fraenkel-Conrat, H., Bean, R.C., Ducay, E.D., and Olcott, H.S., Isolation and characterization of a trypsin inhibitor from lima beans, Arch. Biochem. Biophys. 37, 393–407, 1952; Stevens, F.C. and Doskoch, E., Lima bean protease inhibitor: reduction and reoxidation of the disulfide bonds and their reactivity in the trypsin-inhibitor complex, Can. J. Biochem. 51, 1021–1028, 1973; Nordlund, T.M., Liu, X.Y., and Sommer, J.H., Fluorescence polarization decay of tyrosine in lima bean trypsin inhibitor, Proc. Natl. Acad. Sci. USA 83, 8977–8981, 1986; Hanlon, M.H. and Liener, I.E., A kinetic analysis of the inhibition of rat and bovine trypsins by naturally occurring protease inhibitors, Comp. Biochem. Physiol. B 84, 53–57, 1986; Xiong, W., Chen, L.M., Woodley-Miller, C. et al., Identification, purification, and localization of tissue kallikrein in rat heart, Biochem. J. 267, 639–646, 1990; Briseid, K., Hoem, N.O., and Johannesen, S., Part of prekallikrein removed from human plasma together with IgG-immunoblot and functional tests, Scand. J. Clin. Lab. Invest. 59, 55–63, 1999; Yamasaki, Y., Satomi, S., Murai, N. et al., Inhibition of membrane-type serine protease 1/matriptase by natural and synthetic protease inhibitors, J. Nutr. Sci. Vitaminol. 49, 27–32, 2003. Leupeptin R
O
Transition-state inhibitor of proteinase.
H2 C
H
HO
N H
R1
(ac/pr-LeuLeuArginal)
O
N H
CH NH
O
H2C
Peptide aldehyde
Serine in peptide bond O
R
O
H2 C O
CH
N H
R1
N H
NH OH
H2C
Stabilized tetrahedral aldol CH3 H3C
CH3 H3C
CH CH2
HO
C O
CH
CH2 H N
C
C O
9168_Book.indb 147
N H
CH3
CH3 H3C
CH CH2
HO
CH
O Leupeptide A CH3
H3C
CH
CH
CH CH2
H N
C
O Leupeptin B
CH
N H
H2 C CH3
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Characteristics of Selected Protease Inhibitors, Which Can be Used in Protease Inhibitor Cocktails (Continued) Common Name
Other Nomenclature
M.W.
Primary Design
A tripeptide aldehyde (ac/pr-LeuLeuArginal) proteinase inhibitor isolated from Actinomycetes. It is a relatively common component of protease inhibitor cocktails used to preserve proteins during storage and purification. See Alpi, A. and Beevers, H., Proteinases and enzyme stability in crude extracts of castor bean endosperm, Plant Physiol. 67, 499–502, 1981; Ratajzak, T., Luc, T., Samec, A.M., and Hahnel, R., The influence of leupeptin, molybdate, and calcium ions on estrogen receptor stability, FEBS Lett. 136, 115–118, 1981; Takei, Y., Marzi, I., Kauffman, F.C. et al., Increase in survival time of liver transplants by protease inhibitors and a calcium channel blocker, nisoldipine, Transplantation 50, 14–20, 1990; Satoh, M., Hosoi, S., Miyaji, M. et al., Stable production of recombinant pro-urokinase by human lymphoblastoid Namalwa KJM-1 cells: host-cell dependency of the expressed-protein stability, Cytotechnology 13, 79–88, 1993; Hutchesson, A.C., Hughes, C.V., Bowden, S.J., and Ratcliffe, W.A., In vitro stability of endogenous parathyroid hormone-related protein in blood and plasma, Ann. Clin. Biochem. 31, 35–39, 1994; Agarwal, S. and Sohal, R.S., Aging and proteolysis of oxidized proteins, Arch. Biochem. Biophys. 309, 24–28, 1994; Yamada, T., Shinnoh, N., and Kobayashi, T., Proteinase inhibitors suppress the degradation of mutant adrenoleukodytrophy proteins but do not correct impairment of very long chain fatty acid metabolism in adrenoleukodystrophy fibroblasts, Neurochem. Res. 22, 233–237, 1997; Bi, M. and Singh, J., Effect of buffer pH, buffer concentration, and skin with or without enzyme inhibitors on the stability of [Arg(9)]-vasopressin, Int. J. Pharm. 197, 87–93, 2000; Bi, M. and Singh, J., Stability of luteinizing hormone-releasing hormone: effects of pH, temperature, pig skin, and enzyme inhibitors, Pharm. Dev. Technol. 5, 417–422, 2000; Ratnala, V.R., Swarts, H.G., VanOostrum, J. et al., Large-scale overproduction, functional purification, and ligand affinities of the His-tagged human histamine H1 receptor, Eur. J. Biochem. 271, 2636–2646, 2004. (p-Amidinophenyl) Methanesulfonyl Fluoride
aPMSF
NH2
163
Reaction at active site serine.
+ H2N
O S C H2
F O (p-amidinophenyl) methanesulfonyl fluoride
(p-Amidinophenyl) methanesulfonyl fluoride was developed by Bing and coworkers (Laura, R., Robison, D.J., and Bing, D.H., [p-Amidinophenyl] methanesulfonyl fluoride, an irreversible inhibitor of serine proteases, Biochemistry 19, 4859–4864, 1980) to improve the specificity of PMSF for trypticlike enzymes. aPMSF readily reacts with trypsin but is only poorly reactive with chymotrypsin. See Katz, I.R., Thorbecke, G.J., Bell, M.K. et al., Protease-induced immunoregulatory activity of platelet factor 4, Proc. Natl. Acad. Sci. USA 83, 3491–3495, 1986; Unson, C.G. and Merrifield, R.B., Identification of an essential serine residue in glucagon: implications for an active site triad, Proc. Natl. Acad. Sci. USA 91, 454–458, 1994; Nikai, T., Komori, Y., Kato, S., and Sugihara, H., Bioloical properties of kinin-releasing enzyme from Trimeresurus okinavensis(himehabu) venom, J. Nat. Toxins 7, 23–35, 1998; Ishidoh, K., Takeda-Ezaki, M., Watanabe, S. et al., Analysis of where and which types of proteinases participate in lysosomal proteinase processing using balifomycin A1 and Helicobacter pylori Vac A toxin, J. Biochem. 125, 770–779, 1999; Komori, Y., Tatematsu, R., Tanida, S., and Nikai, T., Thrombin-like enzyme, flavovilase, with kinin-releasing activity from Trimesurus flavoviridis(habu) venom, J. Nat. Toxins 10, 239–248, 2001; Luo, L.Y., Shan, S.J., Elliott, M.B. et al., Purification and characterization of human kallikrein 11, a candidate prostate and ovarian cancer biomarker, from seminal plasma, Clin. Cancer Res. 12, 742–750, 2006. Reaction at a residue other than a serine has not been demonstrated although it is not unlikely that, as with DFP and PMSF, reaction could occur at a serine residue. p-(Aminoethyl) Benzene Sulfonyl Fluoride
AEBSF; 4-(2-aminoethyl)benzenesulfonyl fluoride (Pefabloc™ SC)
H2 C
H2N C H2
O
165
Reaction at active site serine.
F S O 4-(2-aminoethyl)benzenesulfonyl fluoride
This reagent was developed to improve the reactivity of PMSF. It was originally considered to be somewhat more effective than PMSF; however, AEBSF has been shown to be somewhat promiscuous in its reaction pattern and care is suggested in its use during sample preparation. See Su, B., Bochan, M.R., Hanna, W.L. et al., Human granzyme B is essential for DNA fragmentation of susceptible target cells, Eur. J. Immunol. 24, 2073–2080, 1994; Helser, A., Ulrichs, K., and Muller-Ruchholtz, W., Isolation of porcine pancreatic islets: low trypsin activity during the isolation procedure guarantees reproducible high islet yields, J. Clin. Lab. Anal. 8, 407–411, 1994; Dentan, C., Tselepis, A.D., Chapman, M.J., and Ninio, E., Pefabloc, 4-[2-aminoethyl’benzenesulfonyl fluoride, is a new potent nontoxic and irreversible inhibitor of PAF-degrading acetylhydrolase, Biochim. Biophys. Acta 1299, 353–357, 1996; Sweeney, B., Proudfoot, K., Parton, A.H. et al., Purification of the T-cell receptor zeta-chain: covalent modification by 4-(2-aminoethyl)-benzenesulfonyl fluoride, Anal. Biochem. 245, 107–109, 1997; Diatchuk, V., Lotan, O., Koshkin, V. et al., Inhibition of NADPH oxidase activation by 4-(2-aminoethyl)benzenesulfonyl fluoride and related compounds, J. Biol. Chem. 272, 13292–13301, 1997; Chu, T.M. and Kawinski, E., Plasmin, subtilisin-like endoproteases, tissue plasminogen activator, and urokinase plasminogen activator are involved in activation of latent TGF-beta 1 in human seminal plasma, Biochem. Biophys. Res. Commun. 253, 128–134, 1998; Guo, Z.J., Lamb, C., and Dixon, R.A., A serine protease from suspension-cultured soybean cells, Phytochemistry 47, 547–553, 1998; Wechuck, J.B., Goins, W.F., Glorioso, J.C., and Ataai, M.M., Effect of protease inhibitors on yield of HSV-1-based viral vectors, Biotechnol. Prog. 16, 493–496, 2000; Baszk, S., Stewart, N.A., Chrétien, M., and Basak, A., Aminoethyl benzenesulfonyl fluoride and its hexapeptide (AC-VFRSLK) conjugate are both in vitro inhibitors of subtilisin kexin isozyme-1, FEBS Lett. 573, 186–194, 2004; King, M.A., Halicka, H.D., and Dzrzynkiewicz, Z., Pro- and anti-apoptotic effects of an inhibitor of chymotrypsin-like serine proteases, Cell Cycle 3, 1566–1571, 2004; Odintsova, E.S., Buneva, V.N, and Nevinsky, G.A., Casein-hydrolyzing activity of sIGA antibodies from human milk, J. Mol. Recog. 18, 413–421, 2005; Solovyan, V.T. and Keski-Oja, J., Proteolytic activation of latent TGF-beta precedes caspase-3 activation and enhances apoptotic death of lung epithelial cells, J. Cell Physiol. 207, 445–453, 2006.
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Characteristics of Selected Protease Inhibitors, Which Can be Used in Protease Inhibitor Cocktails (Continued) Common Name
Other Nomenclature
Pepstatin
M.W. 685.9
CH3
Primary Design Acid protease inhibitor.
H3C
O NH
O CH3
HN
CH3
O NH
H3C
O CH3 HN CH3 O NH
H3C
CH3 CH3 O HO Pepstatin
A group of pentapeptide acid protease inhibitors isolated from Streptomeyces (Umezawa, H., Aoyagi, T., Morishima, H. et al., Pepstatin, a new pepsin inhibitor produced by Actinomycetes, J. Antibiot. 23, 259–262, 1970; Aoyagi, T., Kunimoto, S., Morichima, H. et al., Effect of pepstatin on acid proteases, J. Antibiot. 24, 687–694, 1971). Pepstatins are frequently included in protease inhibitor cocktails and used for the stabilization of proteins during extraction, storage, and purification. See Takei, Y., Marzi, I., Kaufmann, F.C. et al., Increase in survival time of liver transplants by protease inhibitors and a calcium channel blocker, nisoldipine, Transplantation 50, 14–20, 1990; Liang, M.N., Witt, S.N., and McConnell, H.M., Inhibition of class II MHC-peptide complex formation by protease inhibitors, J. Immunol. Methods 173, 127–131, 1994; Deng, J., Rudick, V., and Dory, L., Lysosomal degradation and sorting of apolipoprotein E in macrophages, J. Lipid Res. 36, 2129–2140, 1995; Wang, Y.K., Lin, H.H., and Tang, M.J., Collagen gel overlay induces two phases of apoptosis in MDCK cells, Am. J. Physiol. Cell Physiol. 280, C1440–C1448, 2001; Lafleur, M.A., Handsley, M.M., Knaupper, V. et al., Endothelial tubulogenesis within fibrin gels specifically requires the activity of membrane-type-matrix-metalloproteinases (MT-MMPs), J. Cell Sci. 155, 3427–3438, 2002. Phenanthroline Monohydrate
1,10-phenanthroline N
198.2
Metal ion chelator; inhibitor of metalloenzymes; specificity for zinc-metalloenzymes.
N
o-Phenanthroline 1,10-Phenanthroline
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Characteristics of Selected Protease Inhibitors, Which Can be Used in Protease Inhibitor Cocktails (Continued) Common Name
Other Nomenclature
M.W.
Primary Design
1,10-phenanthroline, o-phenanthroline: an inhibitor of metalloproteinases and a reagent for the detection of ferrous ions. See Felber, J.P., Cooobes, T.L., and Vallee, B.L., The mechanism of inhibition of carboxypeptidase A by 1,10-phenanthroline, Biochemistry 1, 231–238, 1962; Hakala, M.T. and Suolinna, E.M., Specific protection of folate reductase against chemical and proteolytic inactivation, Mol. Pharmacol. 2, 465–480, 1966; Latt, S.A., Holmquist, B., and Vallee, B.L., Thermolysin: a zinc metalloenzyme, Biochem. Biophys. Res. Commun. 37, 333–339, 1969; Berman, M.B. and Manabe, R., Corneal collagenases: evidence for zinc metalloenzymes, Ann. Ophthalmol. 5, 1993–1995, 1973; Seltzer, J.L., Jeffrey, J.J., and Eisen, A.Z., Evidence for mammalian collagenases as zinc ion metalloenzymes, Biochim. Biophys. Acta 485, 179–187, 1977; Krogdahl, A. and Holm, H., Inhibition of human and rat pancreatic proteinases by crude and purified soybean trypsin inhibitor, J. Nutr. 109, 551–558, 1979; St. John, A.C., Schroer, D.W., and Cannavacciuolo, L., Relative stability of intracellular proteins in bacterial cells, Acta. Biol. Med. Ger. 40, 1375–1384, 1981; Kitjaroentham, A., Suthiphongchai, T., and Wilairat, P., Effect of metalloprotease inhibitors on invasion of red blood cells by Plasmodium falciparum, Acta Trop. 97, 5–9, 2006; Thwaite, J.E., Hibbs, S., Tritall, R.W., and Atkins, T.P., Proteolytic degradation of human antimicrobioal peptide LL-37 by Bacillus anthracis may contribute to virulence, Antimicrob. Agents Chemother. 50, 2316–2322, 2006. Phenylmethylsulfonyl Fluoride
PMSF
174
Reaction at active site serine.
O S C H2
F O
Phenylmethylsulfonyl fluoride (PMSF) Phenylmethylsulfonyl fluoride was developed by David Fahrney and Allen Gold and inhibits serine proteases such as trypsin and chymotrypsin in a manner similar to DFP. The rate of modification of trypsin and chymotrypsin with PMSF is similar to that observed with DFP; however, the reaction with acetylcholinesterase with PMSF is much less than that of DFP (>6.1 × 10−2 M−1min−1 vs. 1.3 × 104 M−1min−1)(Fahrney, D.E. and Gold, A.M., Sulfonyl fluorides as inhibitors of esterases. I. Rates of reaction with acetylcholinesterase, α-chymotrypsin, and trypsin, J. Amer. Chem. Soc. 85, 997–1000, 1963). For other applications see Lundblad, R.L., A rapid method for the purification of bovine thrombin and the inhibition of the purified enzyme with phenylmethylsulfonyl fluoride, Biochemistry 10, 2501–2506, 1971; Pringle, J.R., Methods for avoiding proteolytic artefacts in studies of enzymes and other proteins from yeasts, Methods Cell Biol. 12, 149–184, 1975; Bendtzen, K., Human leukocyte migration inhibitory factor (LIF). I. Effect of synthetic and naturally occurring esterase and protease inhibitors, Scand. J. Immunol. 6, 125–131, 1977; Carter, D.B., Efird, P.H., and Chae, C.B., Chromatin-bound proteases and their inhibitors, Methods Cell Biol. 19, 175–190, 1978; Hubbard, J.R. and Kalimi, M., Influence of proteinase inhibitors on glucocorticoid receptor properties: recent progress and future perspectives, Mol. Cell. Biochem. 66, 101–109, 1985; Kato, T., Sakamoto, E., Kutsana, H. et al., Proteolytic conversion of STAT3alpha to STAT3gamma in human neutrophils: role of granule-derived serine proteases, J. Biol. Chem. 279, 31076–31080, 2004; Cho, I.H., Choi, E.S., Lim, H.G., and Lee, H.H., Purification and characterization of six fibrinolytic serine proteases from earthworm Lumbricus rubellus, J. Biochem. Mol. Biol. 37, 199–205, 2004; Khosravi, J., Diamandi, A., Bodani, U. et al., Pitfalls of immunoassay and sample for IGF-1: comparison of different assay methodologies using fresh and stored serum samples, Clin. Biochem. 38, 659–666, 2005; Shao, B., Belaaouaj, A., Velinde, C.L. et al., Methionine sulfoxide and proteolytic cleavage contribute to the inactivation of cathepsin G by hypochlorous acid: an oxidative mechanism for regulation of serine proteinases by myeloperoxidase, J. Biol. Chem. 260, 29311–29321, 2005; Pagano, M.R., Paredi, M.E., and Crupkin, M., Cytoskeletal ultrastructural and lipid composition of 1-Z-1 fraction in muscle from pre- and post-spawned female hake (Merluccius hubbsi), Comp. Biochem. Physiol. B Biochem. Mol. Biol.141, 13–21, 2005. Although PMSF is reasonably specific for reaction with the serine residue at the active site of serine proteinases, as with DFP, reaction at tyrosine has been reported (De Vendittis, E., Ursby, T., Rullo, R. et al., Phenylmethanesulfonyl fluoride inactivates an archeael superoxide dismutase by chemical modification of a specific tyrosine residue. Cloning, sequencing, and expression of the gene coding for Sulfolobus solfataricus dismutase, Eur. J. Biochem. 268, 1794–1801, 2001). PMSF does have solubility issues and usually ethanol or another suitable water-miscible organic solvent is used to introduce this reagent. On occasion, the volume of ethanol required influences the reaction (see Bramley, T.A., Menzies, G.S., and McPhie, C.A., Effects of alcohol on the human placental GnRH receptor system, Mol. Hum. Reprod. 5, 777–783, 1999). SBTI
Soybean Trypsin 21,500 Protein protease inhibitor. Inhibitor Soybean trypsin inhibitor (SBTI, STI) usually refers to the inhibitor first isolated by Kunitz (Kunitz, M., Crystalline soybean trypsin inhibitor, J. Gen. Physiol. 29, 149–154, 1946; Kunitz, M., Crystalline soybean trypsin inhibitor. II. General properties, J. Gen. Physiol. 30, 291–310, 1947). This material is described as the Kunitz inhibitor and is reasonably specific for trypticlike enzymes. There are other protease inhibitors derived from soybeans; the Bowman–Birk inhibitor (Birk, Y., The Bowman–Birk inhibitor. Trypsin and chymotrypsin-inhibitor from soybeans, Int. J. Pept. Protein Res. 25, 113–131, 1985; Birk, Y., Protein proteinase inhibitors in legume seeds — overview, Arch. Latinoam. Nutr. 44 (4 Suppl. 1), 26S–30S, 1996) is the best known and, unlike the Kunitz inhibitor, inhibits both trypsin and chymotrypsin; the Bowman–Birk inhibitor is also a double-headed inhibitor having two reactive sites (see Frattali, V. and Steiner, R.F., Soybean inhibitors. I. Separation and some properties of three inhibitors from commercial crude soybean trypsin inhibitor, Biochemistry 7, 521–530, 1968; Frattali, V. and Steiner, R.F., Interaction of trypsin and chymotrypsin with a soybean proteinase inhibitor, Biochem. Biophys. Res. Commun. 34, 480–487, 1969; Krogdahl, A. and Holm, H., Inhibition of human and rat pancreatic proteinases by crude and purified soybean trypsin inhibitor, J. Nutr. 109, 551–558, 1979). Soybean trypsin inhibitor (Kunitz) is used as a model protein (Liu, C.L., Kamei, D.T., King, J.A. et al., Separation of proteins and viruses using two-phase aqueous micellar systems, J. Chromatog. B 711, 127–138, 1998; Higgs, R.E., Knierman, M.D., Gelfanova, Y. et al., Comprehensive label-free method for the relative quantification of proteins from biological samples, J. Proteome Res. 4, 1442–1450, 2005). The broad specificity of the Kunitz inhibitor for trypticlike serine proteases provides the basis for its use in the demonstration of protease processing steps (Hansen, K.K., Sherman, P.M., Cellars, L. et al., A major role for proteolytic and proteinase-activated receptor-3 in the pathogenesis of infectious colitis, Proc. Natl. Acad. Sci. USA 102, 8363–8368, 2005).
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Characteristics of Selected Protease Inhibitors, Which Can be Used in Protease Inhibitor Cocktails (Continued) Common Name
Other Nomenclature
M.W.
Tosyl-lysine Chloromethyl Ketone NH2
TLCK; 1-chloro-3tosylamido-7-amino2-heptanone
369.2 (HCl)
CH2
Primary Design Reaction at active site histidine residues of trypsinlike serine proteases.
CH2 CH2 CH2 H2C Cl
C
CH
O H N
S
O
CH3
O
Tosyl-lysine chloromethyl ketone Tosyl-lysine chloromethyl ketone (TLCK) was developed by Elliott Shaw and colleagues (Shaw, E., Mares-Guia, M., and Cohen, W., Evidence of an active center histidine in trypsin through use of a specific reagent, 1-chloro-3-tosylamido-7-amido-2-heptanone, the chloromethyl ketone derived from N-α tosyl-L-lysine, Biochemistry 4, 2219–2224, 1965). As with TPCK, reaction is not absolutely specific for trypticlike serine proteases (Earp, H.S., Austin, K.S., Gillespie, G.Y. et al., Characterization of distinct tyrosine-specific protein kinases in B and T lymphocytes, J. Biol. Chem. 260, 4351–4356, 1985; Needham, L. and Houslay, M.D., Tosyl-lysyl chloromethylketone detects conformational changes in the catalytic unit of adenylate cyclase induced by receptor and G-protein stimulation, Biochem. Biophys. Res. Commun. 156, 855–859, 1988). Reaction of this chloroalkyl compound with sulfydryl groups would be expected and it is possible that other protein nucleophilic centers would react, although this has not been unequivocally demonstrated. Attempts to synthesize the direct arginine analogue were unsuccessful; it was possible to make more complex arginine derivatives such as Ala-Phe-Arg-CMK, which was more effective with human plasma Kallikrein than the corresponding lysine derivatives (Ki = 0.078 µM vs. M vs. 4.9 µM) (Kettner, C. and Shaw, E., Synthesis of peptides of arginine chloromethyl ketone. Selective inactivation of human plasma kallikrein, Biochemistry 17, 4778–4784, 1978). Tosyl-phenylalanine Chloromethyl Ketone O H3C
S O
O NH
CH
C
H2 C
Cl
TPCK; l-1tosylamido-2phenylethyl chloromethyl ketone)
351.9
Reaction at active site histidine residues of chymotrypsinlike serine proteases.
CH2
Tosyl phenylalanine chloromethylketone Tosyl-phenylalanine chloromethyl ketone (TPCK) was developed by Guenther Schoellmann and Elliott Shaw (Schoellmann, G. and Shaw, E., Direct evidence for the presence of histidine in the active center of chymotrypsin, Biochemistry 2, 252–255, 1963). TPCK was developed as an affinity label (Plapp, B.V., Application of affinity labeling for studying structure and function of enzymes, Methods Enzymol. 87, 469–499, 1982) where binding to chymotrypsin is driven by the phenyl function with subsequent alkylation of the active site histidine. The chloroalkyl function was selected to reduce reactivity with other protein nucleophiles such as cysteine. TPCK does undergo a slow rate of hydrolysis to form the corresponding alcohol. TPCK inactivates proteases with chymotrypsinlike specificity. The rate of inactivation is relatively slow but is irreversible; reaction rates can be enhanced by a more elaborate peptide chloromethyl ketone structure. In the case of cucumisin, a plant serine proteinase, TPCK did not result in inactivation while inactivation was achieved with Z-Ala-Ala-Pro-Phe-chloromethyl ketone (Yonezawa, H., Uchikoba, T., and Kaneda, M., Identification of the reactive histidine of cucumisin, a plant serine protease: modification with peptidyl chloromethyl ketone derivative of peptide substrate, J. Biochem. 118, 917–920, 1995). There is, however, significant reaction of TPCK with other proteins at residues other than histidine (see Rychlik, I., Jonak, J., and Sdelacek, J., Inhibition of the EF-Tu factor by L-1-tosylamido-2-phenylethyl chloromethyl ketone, Acta Biol. Med. Ger. 33, 867–876, 1974); TPCK has been described as an inhibitor of cysteine proteinases (Bennett, M.J., Van Leeuwen, E.M., and Kearse, K.P., Calnexin association is not sufficient to protect T cell receptor proteins from rapid degradation in CD4+CD8+ thymocytes, J. Biol. Chem. 273, 23674–23680, 1998). TPCK has been suggested to react with a lysine residue in aminoacylase (Frey, J., Kordel, W., and Schneider, F., The reaction of aminoacylase with chloromethylketone analogs of amino acids, Z. Naturforsch. 32, 769–776, 1966). Other reactions continue to be described (McCray, J.W. and Weil, R., Inactivation of interferons: halomethyl ketone derivatives of phenylalanine as affinity labels, Proc. Natl. Acad. Sci. USA 79, 4829–4833, 1982; Conseiller, E.C. and Lederer, F., Inhibition of NADPH oxidase by aminoacyl chloromethane protease inhibitors in phorbol-ester-stimulated human neutrophils: A reinvestigation. Are proteases really involved? Eur. J. Biochem. 183, 107–114, 1989; Borukhov, S.I. and Strongin, A.Y.,
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152
Characteristics of Selected Protease Inhibitors, Which Can be Used in Protease Inhibitor Cocktails (Continued) Common Name
Other Nomenclature
M.W.
Primary Design
Chemical modification of the recombinant human α-interferons and β-interferons, Biochem. Biophys. Res. Commun. 167, 74–80, 1990; Gillibert, M., Dehry, Z., Terrier, M. et al., Another biological effect of tosylphenylalanylchloromethane (TPCK): it prevents p47(phox) phosphorylation and translocation upon neutrophil stimulation, Biochem. J. 386, 549–556, 2005). Peptide Halomethyl Ketones: While TPCK and TLCK represented a major advance in modifying active site residues in serine proteases, slow and relatively nonspecific reaction was a problem. The development of tripeptide halomethyl ketones provided a major advance in the value of such derivatives as presented in some specific examples below. However, even with these derivatives, reactions occur with “unexpected” enzymes. More general information can be obtained from the following references: Poulos, T.L., Alden, R.A., Freer, S.T. et al., Polypeptide halomethyl ketones bind to serine proteases as analogs of the tetrahedral intermediate. X-ray crystallographic comparison of lysine- and phenylalanine-polypeptide chloromethyl ketone-inhibited subtilisin, J. Biol. Chem. 251, 1097–1103, 1976; Powers, J.C., Reaction of serine proteases with halomethyl ketones, Methods Enzymol. 46, 197–208, 1977; Navarro, J., Abdel Ghany, M., and Racker, E., Inhibition of tyrosine protein kinases by halomethyl ketones, Biochemistry 21, 6138–6144, 1982; Conde, S., Perez, D.I., Martinez, A. et al., Thienyl and phenyl α-halomethyl ketones: new inhibitors of glycogen synthase kinase (GSK-3β) from a library of compound searching, J. Med. Chem. 46, 4631–4633, 2003. Peptide Fluoromethyl Ketones: Fluoroalkyl derivatives of the peptide chloromethyl ketones have been prepared in an attempt to improve specificity by reducing nonspecific alkylation at cysteine residues (Rasnick, D., Synthesis of peptide fluoromethyl ketones and the inhibition of human cathepsin B, Anal. Biochem. 149, 461–465, 1985). Nonspecific reaction with sulfydryl groups such as those in glutathione was reduced; there was still reaction with active site cysteine although at a slower rate than with the chloroalkyl derivative (16,200 M−1s−1 vs. 45,300 M−1s−1; t1/2 21.9 min. vs. 5.1 min.). Reaction also occurred with serine proteases (Shaw, E., Angliker, H., Rauber, P. et al., Peptidyl fluoromethyl ketones as thiol protease inhibitors, Biomed. Biochim. Acta 45, 1397–1403, 1986) where the modification occurred at a histidine residue (Imperiali, B. and Abeles, R.H., Inhibition of serine proteases by peptide fluoromethyl ketones, Biochemistry 25, 3760– 3767, 1986). The trifluoromethyl derivative was also an inhibitor but formed a hemiacetal derivative. The peptide fluoromethyl ketone, z-VAD-FMK, has proved to be a useful inhibitor of caspases D-Phe-Pro-Arg-chloromethyl Ketone
PPACK
Reaction at active site histidine residues of trypsinlike serine proteases.
D-Phe-Pro-Arg-chloromethyl ketone was one of the first complex peptide halomethyl ketones synthesized. These derivatives have the advantage of increased reaction rate and specificity (see Williams, E.B. and Mann, K.G., Peptide chloromethyl ketones as labeling reagents, Methods Enzymol. 222, 503–513, 1993; Odake, S., Kam, C.M., and Powers, J.C., Inhibition of thrombin by arginine-containing peptide chloromethyl ketones and bis chloromethyl ketone-albumin conjugates, J. Enzyme Inhib. 9, 17–27, 1995; Lundblad, R.L., Bergstrom, J., De Vreker, R. et al., Measurement of active coagulation factors in Autoplex®-T with colorimetric active site-specific assay technology, Thromb. Haemostas. 80, 811–815, 1998). With chymotrypsin, CHO-PheCH2Cl, kobsv./[I] = 0.55 M−1s−1 and Boc-Ala-Gly-Phe-CH2Cl, kobsv/[I] = 3.34 M−1s−1 (Kurachi, K., Powers, J.C., and Wilcox, P.E., Kinetics of the reaction of chymotrypsin A α with peptide chloromethyl ketones in relation to subsite specificity, Biochemistry 12, 771–777, 1973. See also Ketter, C. and Shaw, E., The selective affinity labeling of factor Xa by peptides of arginine chloromethyl ketone, Thromb. Res. 22, 645–652, 1981; Shaw, E., Synthetic inactivators of kallikrein, Adv. Exp. Med. Biol. 156, 339–345, 1983; McMurray, J.S. and Dyckes, D.F., Evidence for hemiketals as intermediates in the inactivation of serine proteinases with halomethyl ketones, Biochemistry 25, 2298–2301, 1986). There is a similar peptide chloromethyl ketone, PPACK II (D-Phe-Phe-Arg-CMK), which has been used to stabilize B-type natriuretic peptide (BNP) in plasma samples (Belenky, A., Smith, A., Zhang, B. et al., The effect of class-specific protease inhibitors on the stabilization of B-type natriuretic peptide in human plasma, Clin. Chim. Acta 340, 163–172, 2004). z-VAD-FMK CH3
O C H2
H N
O O
H3C
N H
CH CH
CH2
BenzyloxycarbonylVal-Ala-Asp(OMe) fluoromethyl ketone
O H N
CH O
C H2
Inhibitor of caspases.
F
O
H2C O
CH3
z-VADFMK
Benzyloxycarbonyl-Val-Ala-Asp(OMe) fluoromethyl ketone (z-VAD-FMK) is a peptide halomethyl ketone used for the inhibition of caspases and related enzymes. Because z-VAD-FMK is neutral, it passes the cell membrane and can inhibit intracellular proteolysis and is useful in understanding the role of caspases and related enzymes in cellular function. See Zhu, H., Fearnhead, H.O., and Cohen, G.M., An ICE-like protease is a common mediator of apoptosis induced by diverse stimuli in human monocytes THP.1 cells, FEBS Lett. 374, 303–308, 1995; Mirzoeva, O.K., Yaqoob, P., Knox, K.A., and Calder, P.C., Inhibition of ICE-family cysteine proteases rescues murine lymphocytes from lipoxygenase inhibitor-induced apoptosis, FEBS Lett. 396, 266–270, 1996; Slee, E.A., Zhu, H., Chow, S.C. et al., Benzyloxycarbonyl-Val-Ala-Asp(OMe) fluoromethylketone (z-VAD. FMK) inhibits apoptosis by blocking the processing of CPP32, Biochem. J. 315, 21–24, 1996; Gottron, F.J., Ying, H.S., and Choi, D.W., Caspase inhibition selectively reduces the apoptotic component of oxygen-glucose deprivation-induced cortical neuronal cell death, Mol. Cell. Neurosci. 9, 159–169, 1997; Longthorne, V.L. and Williams, G.T., Caspase activity is required for commitment to Fas-mediated apoptosis, EMBO J. 16, 3805–3812, 1997; Hallan, E., Blomhoff, H.K., Smeland, E.B., and Long, J., Involvement of ICE (Caspase) family in gamma-radiation-induced apoptosis of normal B lymphocytes, Scand. J. Immunol. 46, 601–608, 1997; Polverino, A.J. and Patterson, S.D., Selective activation of caspases during apoptotic induction in HL-60 cells. Effects of a tetrapeptide inhibitor, J. Biol. Chem. 272, 7013–7021, 1997; Cohen, G.M., Caspases: the executioners of apoptosis, Biochem. J. 328, 1–16, 1997; Sarin, A., Haddad, E.K., and Henkart, P.A., Caspase dependence of target cell damage induced by cytotoxic lymphocytes, J. Immunol. 161, 2810–2816, 1998; Nicotera, P., Leist, M., Single, B., and Volbracht, C., Execution of apoptosis: converging or diverging pathway? Biol. Chem. 380, 1035–1040, 1999; Grfaczyk, P.P., Caspase inhibitors as anti-inflammatory and antiapoptotic
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Protease Inhibitors and Protease Inhibitor Cocktails
153
Characteristics of Selected Protease Inhibitors, Which Can be Used in Protease Inhibitor Cocktails (Continued) Common Name
Other Nomenclature
M.W.
Primary Design
agents, Prog. Med. Chem. 39, 1–72, 2002; Blankenberg, F., Mari, C., and Strauss, H.W., Imaging cell death in vivo, Q. J. Nucl. Med. 47, 337–348, 2003; Srivastava, A., Henneke, P., Visintin, A. et al., The apoptotic response to pneumolysin in Toll-like receptor 4 dependent and protects against pneumococcal disease, Infect. Immun. 73, 6479–6489, 2005; Clements, K.M., Burton-Wurster, N., Nuttall, M.E., and Lust, G., Caspase-3/7 inhibition alters cell morphology in mitomycin-C treated chondrocytes, J. Cell Physiol. 205, 133–140, 2005; Coward, W.R., Marie, A., Yang, A. et al., Statininduced proinflammatory response in mitrogen-activated peripheral blood mononuclear cells through the activation of caspases-1 and IL-18 secretion in monocytes, J. Immunol. 176, 5284–5292, 2006. The protease inhibitor cocktails referred to herein are not to be confused with the protease inhibitor cocktails that are used for therapy for patients who have Acquired Immune Deficiency Syndrome (AIDS).
a
General references for inhibitors of proteolytic enzymes Albeck, A. and Kliper, S., Mechanism of cysteine protease inactivation by peptidyl epoxides, Biochem. J. 322, 879–884, 1997. Banner, C.D. and Nixon, R.A., Eds., Proteases and Protease Inhibitors in Alzheimer’s Disease Pathogenesis, New York Academy of Sciences, New York, 1992. Barrett, A.J. and Salvesen, G., Eds., Protease Inhibitors, Elsevier, Amsterdam, NL, 1986. Bernstein, N.K. and James, M.N., Novel ways to prevent proteolysis — prophytepsin and proplasmepsin II, Curr. Opin. Struct. Biol. 9, 684– 689, 1999. Birk, Y., Ed., Plant Protease Inhibitors: Significance in Nutrition, Plant Protection, Cancer Prevention, and Genetic Engineering, Springer, Berlin, 2003. Cheronis, J.C.D. and Repine, J.E., Proteases, Protease Inhibitors, and Protease-Derived Peptides: Importance in Human Pathophysiology and Therapeutics, Birkhäuser Verlag, Basel, Switzerland, 1993. Church, F.C., Ed., Chemistry and Biology of Serpins, Plenum Press, New York, 1997. Frlan, R. and Gobec, S., Inhibitors of cathepsin B, Curr. Med. Chem. 13, 2309–2327, 2006.
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Giglione, C., Boularot, A., and Meinnel, T., Protein N-terminal excision, Cell. Mol. Life Sci. 61, 1455–1474, 2004. Johnson, S.L. and Pellechhia, M., Structure- and fragment-based approaches to protease inhibition, Curr. Top. Med. Chem. 6, 317– 329, 2006. Kim, D.H., Chemistry-based design of inhibitors for carboxypeptidase A, Curr. Top. Med. Chem. 4, 1217–1226, 2004. Lowther, W.T., and Matthews, B.W., Structure and function of the methionine aminopeptidases, Biochim. Biophys. Acta 1477, 157–167, 2000. Magnusson, S., Ed., Regulatory Proteolytic Enzymes and Their Inhibitors, Pergamon Press, Oxford, UK, 1986. Powers, J.C. and Harper, J.W., Inhibition of serine proteinases, in Proteinase Inhibitors, Barrett, A.J. and Salvesen, G., Eds., Elsevier, Amsterdam, NL, chapter 3, pp. 55–152. Saklatvala, J., and Nagase, H., Eds., Proteases and the Regulation of Biological Processes, Portland Press, London, UK, 2003. Shaw, E., Cysteinyl proteinases and their selective inactivation, Adv. Enzymol. Relat. Areas Mol. Biol. 63, 271–347, 1990. Stennicke, H.R. and Salvesen, G.S, Chemical ligation — an unusual paradigm in protease inhibition, Mol. Cell. 21, 727–728, 2006. Tam, T.F., Leung-Toung, R., Li, W. et al., Medicinal chemistry and properties of 1,2,4-thiadiazoles, Mini Rev. Med. Chem. 5, 367–379, 2005. Vogel, R., Trautschold, I., and Werle, E., Natural Proteinase Inhibitors, Academic Press, New York, 1968.
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Assay of Solution Protein Concentration The determination of protein concentration is a somewhat overlooked procedure that is critical for the determination of the specific biological/therapeutic activity of most biopharmaceuticals, the “standardization” or normalization of samples for proteomic analysis and the comparison of cell homogenates. As such, it is unfortunate that most investigators do not recognize the limitations of the various procedures. The reader is recommended to some recent reviews of protein assay methods1-3. The purpose of this short section is to describe some commonly used techniques for the determination of protein concentration. Care must be taken with the use of these techniques several of the more frequently used techniques depend on protein quality as well as quantity. Thus the technique which is facile might not be accurate. It is noted that accuracy is an attribute in assay validation while facile is not. There are two issues which are common to any of the below assays. The first is the standard and the second is the solvent. The standard should be representative of the sample; albumin might not be the best choice. The concentration of the standard protein cannot be verified by preparation but must be verified by analysis. In other words, accurate dispensing of the standard protein and subsequent dissolution to a given volume does not ensure an accurate standard. The final concentration of a standard solution must be verified by analysis. For well-characterized proteins it is possible to employ ultraviolet spectroscopy using the known extinction coefficient for the standard protein. Thus the A280 of a 1 mg/mL of bovine serum albumin is 0.66 in a cuvette of 1 cm pathlength [126]. It is important to correct for any light scattering due to aggregated material, dust, etc., by recording the baseline over the range 400 to 310 nm where the protein does not absorb. While this procedure may seem somewhat tedious, it is necessary. It is possible to prepare a standard solution which can be used for a substantial period of time. The standard solution is best stored frozen in small aliquots, each of which is used once to calibrate the assay. The precise storage conditions used would require validation. Solvent can have an effect on the analytical response and should be selected for (1) lack of an effect on the signal and (2) the ability to be used for both standard and samples. The reader is directed to the List of Buffers (p. 695) for a discussion of the effect of various buffers on protein analysis.
Biuret assay The biuret assay measures the formation of a purple complex between copper salts and two or more peptide bonds under alkaline conditions. The assay was developed by Gornall and coworkers4 and modified to a microplate format by Jenzano and coworkers5. The biuret assay is not available in kit form and the preparation of the reagents requires some skill. The biuret assay also lacks the sensitivity of many of the other assays. The biuret assay is accurate as it is insensitive to protein quality2,3. Selected references on the use of this method with various proteins are provided in Table 1.
Bicinchoninic acid (BCA) protein assay This assay was developed by Smith and coworkers6. A modification for microplate use was developed by Jenzano and coworkers5, This procedure is a modification of the Lowry et al.7 reaction, but it is significantly easier and somewhat more sensitive6. The reaction is based on the formation of a complex of BCA with cuprous ion (Brenner, A.J. and Harris, E.D., A quantitative test for copper using
bicinchoninic acid, Anal.Biochem. 226, 80-84, 1995). The BCA reaction has the advantage of being able to measure protein bound to surfaces. This reaction is quite sensitive but it does reflect qualitative differences in proteins. As a reflection of the dependence on protein quality,2 it is critical to select a standard that is qualitatively similar, if not identical, with the samples. This is obviously difficult when the assay is used with heterogeneous mixtures such as saliva or serum. Selected references to the use of this method are given in Table 2. Information on the use of the Lowry assay is presented in Table 3.
Dye-binding assay for protein using Coomassie Brilliant Blue G-250 (Bradford assay) The dye-binding assay for proteins using Coomassie Brilliant Blue G-250 is likely the most sensitive and most extensively used protein assay at this time. It is also extremely easy to perform. The technique, as noted below, is extremely dependent on the quality of the protein. The procedure was developed by Bradford8. A modification for microplate technology is given by Jenzano and coworkers5. As noted above, this assay technique is likely the most sensitive and facile of the currently available procedures. Rigorous application of the dyebinding assay to the quantitative determination of a broad spectrum of proteins is difficult because of the marked influence of protein quality on the reaction. This is reflected by various studies attempting to modify the assay system to eliminate dependence on the quality of the protein.9-14. Examples of the application of the Coomassie Blue dye-binding assay are presented in Table 4.
Kjeldahl assay The Kjeldahl assay was developed in 188315 and is based on the determination of ammonia after hydrolysis of the sample in sulfuric acid. Most recent references to the use of this method relate to its use in food and environmental sciences16-20. It is our view that the Kjeldahl method remains a “gold” standard for protein assays21 but we also appreciate the issues of technical complexity and lack of sensitivity which make routine use difficult for biopharmaceuticals. In addition, problems can arise in the analysis of proteins which contain impurities which themselves contain significant quantities of nitrogen, or in the analysis of proteins of unusual amino acid composition where the usual conversion factors may not apply. There are numerous commercial sources for support of the Kjeldahl assay22-27. Zellmer et al.,28 have recently described an assay system which appears to have the accuracy of the Kjeldahl method with greatly improved sensitivity. Recent applications of the Kjeldahl assay are presented in Table 5.
Total amino acid analysis Current technology for total amino acid analysis certainly has the various analytical attributes (sensitivity, accuracy, ruggedness) and is sufficiently rapid for use in the analysis of protein concentration 29-34 and has been suggested as a reference procedure for the determination of total protein concentration 35,36 With a characterized biopharmaceutical such as a growth factor, the concentration of the protein can be 155
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156 determined by measuring the amounts of specific stable and abundant amino acids such as alanine and lysine with reference to an added internal standard such as norleucine 37. Application of amino acid analysis for total protein concentration are presented in Table 6.
Amido schwartz The above approaches are certainly worth considering. However, it is somewhat remarkable that more attention has not been given to the amido black (Amidoschwarz 10B) assay developed by Schaeffner and Weissman38. This assay is based on the quantitative precipitation of protein from solution with trichloroacetic acid, the capture of the precipitated material by filtration followed by quantitative measurement of captured protein with amido schwarz dye (amido black is the preferred term). This study has been cited 2356 times (ISI) since its publication in 1973. The original assay used the addition of an equal amount of 60% trichloroacetic acid to a final concentration of 10%. The precipitate is capture by filtration and stained with amido black dye in methanol/glacial acetic acid/H2O. The protein is visualized as a blue spot on an almost colorless background. The spot is excised from the filter, eluted with 25 mM NaOH-0.05 mM EDTA in 50% aqueous ethanol. The absorbance of the eluate at 630 nm is determined and concentration is determined by comparison with the results obtained with a standard protein. This assay has been used for the determination of protein concentration in grape juices and wines39, low concentration of protein in phospholipids40, and the Escherichia coli multidrug transporter EmrE41 in the presence of detergents. Of direct relevance to proteomic analyses are the studies of Eliane42 and coworkers on the determination of protein concentration of a Medicago truncatula root microsomal fraction with the amido black assay in a solution composed of 7.0 M urea-2.0 M thiourea-4% CHAPS (w/v)-0.1% (w/v) Triton X-100-2 mM tributylphosphine-2% ampholines. The reader is also commended to the study by Tate and coworkers41 who validated the amido black assay with quantitative amino acid analysis which has been suggested as a method of choice for accurate determination of protein concentration34,35. A list of some other applications of amido black for protein assay is given in Table 7. There has been limited application of fluorescent dyes for the determination of protein concentration (Table 8).
References 1. Dawnay, A. B. StJ., Hirst, A. D., Perry, D. E., and Chambers, R. E., A critical assessment of current analytical methods for the routine assay of serum total protein and recommendations for their improvement, Ann. Clin. Biochem., 28, 556, 1991. 2. Sapan, C. V., Price, N. C., and Lundblad, R. L., Colorimetric protein assay techniques, Biotechnol. Appl. Biochem., 29, 99-108, 1999. 3. Lundblad, R. L. and Price, N. C., Protein concentration determination. The Achilles’ heel of cGMP, Bioprocess International, January, 2004, 1-7. 4. Gornall, A. G., Bardawill, C. J., and David, M. M., Determination of serum proteins by means of the biuret reaction, J. Biol. Chem., 177, 751, 1949. 5. Jenzano, J. W., Hogan, S. L., Noyes, C. M., Featherstone, G. L., and Lundblad, R. L., Comparison of five techniques for the determination of protein content in mixed human saliva, Anal. Biochem., 159, 370, 1986. 6. Smith, P. K., Krohn, R. I., Hermanson, G. T., Mallia, A. K., Gartner, F. H., Provenzano, M. D., Fujimoto, E. K., Goeke, N. M., Olson, B. J., and Klenk, D. C., Measurement of protein using bicinchoninic acid, Anal. Biochem., 150, 76, 1985.
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7. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J., Protein measurement with the folin phenol reagent, J. Biol. Chem., 193, 265, 1951. 8. Bradford, M. M., A rapid and sensitive method for the determination of microgram quantities of protein utilizing the principle of proteindye binding, Anal. Biochem., 72, 248, 1976. 9. Wilmsatt, D. K. and Lott, J. A., Improved measurement of urinary total protein (including light-chain proteins) with a Coomassie Brilliant Blue G-250-sodium dodecyl sulfate reagent, Clin. Chem., 33, 2100, 1987. 10. Tal, M., Silberstein, A., and Nusser, E., Why does Coomassie Brilliant Blue R interact differently with different proteins, J. Biol. Chem., 260, 9976, 1985. 11. Pierce, J. and Suelter, C. H., An evaluation of the Coomassie Brilliant Blue G-250 dye-binding method for quantitative protein determination, Anal. Biochem., 81, 478, 1977. 12. Read, S. M. and Northcote, D. H., Minization of variation in the response to different proteins of the coomassie blue G dye-binding assay for protein, Anal. Biochem., 116, 53, 1981. 13. Sedmak, J. J. and Grossberg, S. F., A rapid, sensitive, and versatile assay for protein using coomassie brilliant blue G250, Anal. Biochem., 79, 544, 1977. 14. Stoscheck, C. M., Increased uniformity in the response of the Coomassie blue G protein assay to different proteins, Anal. Biochem., 184, 111, 1990. 15. Kjeldahl, J. Z., Zeitschrift für Analytische Chemie, 22, 366-382, 1883. 16. McPherson, T. N., Burian, S. J., Turin, H. J., Stenstrom, M. K. and Suffet, I. H., Water Sci. Technol. 45, 255-261, 2003. 17. Belloque, J. and Ramos, M., J. Dairy Res., 69, 411-418, 2002, 2002 18. Shan, S. B., Bhumbla, D. K., Basden, T. J. and Lawrence, L. D., J. Environ. Sci. Hlth., B 37, 493-505, 2002 19. Matttila, P., Salo-Vaananen, P., Konko, P., Aro, H. and Jalava, T. J., Agricul. Food Chem., 50, 6419-6422, 2002. 20. Thompson, M., Owen, L., Wilkinson, K., Wood, R. and Damant, A., Analyst 127, 1666-1668, 2002. 21. Johnson, A. M., Rohlfs, E. M. and Silverman, L. M., (1999), Proteins, in Teitz Textbook of Clinical Chemistry, ed. C. A. Burhs and E. R. Ashwood, W. B. Saunders Co., Philadelphia, PA., Chapter 20, pp. 524-525. 22. http://www.calixo.net/braun/biochimie/kjeldahl.htm. 23. http://www.labconco.com/pdf/kjeldahl/index.shtml. 24. http://www.buchi-analytical.com/haupt.asp?nv=3759. 25. http://www.storesonline.com/site/251298.page/73181. 26. http://www.slrsystems.com/products.htm. 27. http://www.voigtglobal.com/kjeldahl_flasks.htm. 28. Zellmer, S., Kaltenborn, G., Rothe, U., Lehnich, H., Lasch, J., and Pauer, H.-D., Anal. Biochem., 273, 163-167, 1999. 29. Anders, J. C., Parton, B. F., Petrie, G. E., Marlowe, R. L., and McEntire, J. E., Biopharm International, February, 30-37, 2003. 30. Weiss, M., Manneberg, M., Juranville, J.-F., Lahm, H.-W., and Fountaoulakis, M. Effect of the hydrolysis of method on the determination of the amino acid composition of proteins, J. Chromatog. A., 795, 263-275, 1998. 31. Fountoulakis, M. and Lahm, H.-W., Hydrolysis and amino acid composition of proteins, J. Chromatog. A., 826, 109-134, 1998. 32. Engelhart, W. G., Microwave hydrolysis of peptides and proteins for amino acid analysis, Am. Biotechnol. Lab., 8, 30-34, 1990. 33. Chiou, S. H. and Wang, K. T., Peptide and protein hydrolysis by microwave irradiation, J. Chromatog., 491, 424-431, 1989. 34. Bartolomeo, M. P. and Malsano, F., Validation of a reversed-phase method for quantitative amino acid analysis, J. Biomol. Tech., 17, 131-137, 2006. 35. Sittampalam, G. S., Ellis, R. M., Miner, D. J., et al., Evaluation of amino acid analysis as reference method to quantitate highly purified proteins, J. Assoc. Off. Anal. Chem., 71, 833-838, 1988. 36. Henderson, L. O., Powell, M. R., Smith, S. J., et al., Impact of protein measurements on standardization of assays of apolipoproteins A-I and B1, Clin.Chem., 36, 1911-1917, 1990. 37. Price, N. C. (1996) The determination of protein concentration, in Enzymology Labfax, ed. P. C. Engel, Bios Scientific Publishers, Oxford, UK, pp. 34-41.
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38. Schaeffner, W. and Weissman, C., A rapid, sensitive, and specific method for the determination of protein in dilute solutions, Anal. Biochem., 56, 502-514, 1973. 39. Weiss, K. C. and Bisson, L. F., Optimisation of the Amido Black assay for the determination of protein content of grape juices and wines, J. Science Food Agriculture, 81, 583-589, 2001. 40. Bergo, H. O. and Christiansen, C., Determination of low levels of protein impurities in phospholipids samples, Analyt. Biochem., 288, 225-227, 2001. 41. Butler, P. J. G., Ubarretxena-Belandia, I., Warne, T., and Tate, C. G., The Escherichia coli multidrug transporter EmrE is a dimer in the detergent solubilized state, J. Mol. Biol., 340, 797-808, 2004. 42. Valot, B., Gianinazzi, S., and Elaine, D.-G., Sub-cellular proteomic analysis of a Medicago truncatula root microsomal fraction, Phytochemistry, 65, 1721-1732, 2004.
Table 1: Biuret Assay Application
Reference
Dextran interferes with biuret assay of serum proteins Interference of amino acids with the biuret reaction for urinary peptides. These investigators showed that, contrary to “conventional wisdom”, the biuret assay showed cross-reaction with some amino acids and other compounds forming 5-membered and 6-membered complexes with copper. Measurement of protein content of apple homogenates in an allergenicity study Measurement of protein on surgical instruments Measurement of protein concentration in serum and synovial fluid from human patients with arthropathies
1 2
3 4 5
Table 4: Coomassie Blue Dye-Binding Assay (Bradford Assay) Application Measured soy protein extraction from various sources including soybean meal, soyprotein concentrate, and textured soy flake Measured protein in aqueous phase from oil-water distribution. The amount of protein measured with Coomassie blue dye correlated (r2 = 0.91) with protein concentration determined by tryptophan emission spectra (fourth derivative) Measured glomalin extraction from soil Automation of Coomassie dye-binding assay Resonance light scattering with Bordeaux red correlates with Coomassie method for the determination of protein concentration in human serum, saliva, and urine. Measured protein concentration in phenol extracts of plant tissues after precipitation with ammonium acetate in methanol Measured total protein concentration in rat tissue (pancreas, parotid gland, submandibular gland, lacrimal gland) extracts Measure protein release from alginate-dextran microspheres Protein release from Candida albicans secondary to microwave irradiation
Application Protein concentration in synovial fluid Measurement of protein concentration in liposomes Measurement of cells attached to hyaluronic surfaces Measurement of tear protein concentration Proteins released by venom digestion
Reference 6 7 8 9 10
16 17
18 19 20 21 22 23 24
Table 5: Kjeldahl Assay for Protein Concentration Application
Table 2: Bicinchoninic Acid Assay for Protein
Reference
Measurement of protein concentration in therapeutic protein concentrate. Kjeldahl used as the “gold standard.” The biuret assay gave comparable values while dye-binding was lower. Specific activity differed with the protein concentration Measurement of crude microbial protein derived from carbohydrate fermentation Measurement of polylysine coating on alginate beads Measurement of IgG concentration in the presence of nonionic surfactants and glycine Manure protein concentration Whey protein concentration
Reference 25
26 27 28 29 30
Table 6: Amino Acid Analysis for Protein Concentration Application
Table 3: Lowry Assay for Protein Concentration Application Determination of salivary protein concentration Determination of protein concentration in aqueous humor Determination of protein concentration in human milk Determination of protein in human lens Protein assay in cell-based toxicity studies
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References 11 12 13 14 15
Use of amino acid analysis as a primary method for the determination of the concentration of poly ADP-ribose polymerase 1 (PARP-1) Determination of the concentration of NADH:ubiquinone oxidoreductase and establishment of coenzyme (FMN) and iron-sulfur cluster stoichiometry Determination of the concentration of immobilized protein Determination of IgG protein concentration Protein concentration of troponin in standard reference preparations
Reference 31 32 33 34 35
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Handbook of Biochemistry and Molecular Biology
158 Table 7: Amido Black (Schwarz) Method for Protein Assay Application
Reference
Measurement of protein concentration by binding to a nitrocellulose membrane by filtration followed by staining with amido black. Protein concentration determined by densitometry Measurement of protein in antibody-polysaccharide complexes Measurement of anchorage-dependent cells Measurement of cell viability in an immortalized keratinocyte cell line Determination of protein concentration after transfer to nitrocellulose from SDS-PAGE gel
36
37 38 39 40
Table 8: Fluorescent Dyes for Determination of Protein Concentration Dye
References
NanoOrange
41-45
References 1. Delanghe, J.R., Hamers, N., Taes, Y.E., and Libeer, J.C., Interference of dextran in biuret-type assays of serum proteins, Clin.Chem.Lab. Med. 43, 71-74, 2005 2. Hortin, G.L. and Meilinger, B., Cross-reactivity of amino acids and other compounds in the biuret reaction: interference with urinary peptide measurements, Clin.Chem. 51, 1411-1419, 2005 3. Carnes, J., Ferrer, A., and Fernandez-Caldas, E., Allergenicity of 10 different apple varieties, Ann.Allergy Asthma Immunol. 96, 564-570, 2006 4. Lipscomb, I.P., Pinchin, H.E., Collin, R., et al., The sensitivity of approved Ninhydrin and Biuret tests in the assessment of protein contamination on surgical steel as an aid to prevent iatrogenic prion transmission, J.Hosp.Infect. 64, 288-292, 2006 5. Popko, J. Marciniak, J., Zalewska, A., et al., Activity of N-acetyl-βhexosaminidase and its isoenzymes in serum and synovial fluid from patients with different arthropathies, Clin.Exp.Rheumatol. 24, 690693, 2006 6. Uehara, J., Kuboki, T, Fujisawa, T., et al., Soluble tumour necrosis factor receptors in synovial fluids from tempromandibular joints with painful anterior disc displacement without reduction and osteoarthritis, Arch.Oral.Biol. 49, 133-142, 2004 7. Were, L.M., Bruce, B., Davidson, P.M., and Weiss, J., Encapsulation of nisin and lysozyme in liposomes enhances efficacy against Listeria monocytogenes, J.Food Prot. 67, 622-627, 2004 8. Cen, L., Neoh, K.G., Li, Y., and Kang, E.T., Assessment of in vitro bioactivity of hyaluronic acid and sulfated hyaluronic acid functionalized electroactive polymer, Biomacromolecules 5, 2238-2246, 2004 9. Yamada, M., Mochizuki, H., Kawai, M., et al., Decreased tear lipocalin concentration in patients with meibomian gland dysfunction, Br.J.Ophthalmol. 89, 803-805, 2005 10. Nicholson, J., Mirtschin, P., Madaras, F., et al., Digestive properties of the venom of the Australian Costal Taipan, Oxyranus scutellatus (Peters, 1867), Toxicon 48, 422-428, 2006 11. Yarat, A., Tunali, T., Pisiriciler, R., et al., Clin.Oral Investig. 8, 36-39, 2004 12. Kawai, K., Sugiyama, K., and Kitazawa, Y., The effect of α2-agonist on IOP rise following Nd-YAG laser iridotomy, Tokai J.Exp.Clin.Med. 29, 23-26, 2004 13. Milnewowicz, H. and Chmarek, M., Influence of smoking on metallothionein level and other proteins binding essential metals in human milk, Acta Pediatr. 94, 402-406, 2005
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14. Raitelaitiene, R., Paunksnis, A., Ivanov, L., and Kurapkiene, S., Ultrasound and biochemical evaluation of human diabetic lens, Medicina (Kaunas) 41, 641-648, 2005 15. Dierickx, P., Prediction of human acute toxicity by the hep G2/24hour/total protein assay, with protein measurement by the CBQCA method, Altern.Lab.Anim. 33, 207-213, 2005 16. Koppelman, S.J., Lakemond, C.M., Vlooswijk, R., and Hefle, S.L., Detection of soy protein in processed foods: literature overview and new experimental work, J.AOAC Int. 87, 1398-1407, 2004 17. Granger, C., Barey, P., Toutain, J., and Cansell, M., Direct quantification of protein partitioning in oil-to-water emulsion by front-face fluorescence: avoiding the need for centrifugation, Colloids Surf. B Biointerfaces 43, 158-162, 2005 18. Wright, S.F., Nichols, K.A., and Schmidt, W.F., Comparison of efficacy of three extractants to solubilize glomalin on hyphae and in soil, Chemosphere 64, 1219-1224, 2006 19. da Silva, M.A. and Arruda, M.A., Mechanization of the Bradford reaction for the spectrophotometric determination of total protein, Anal.Biochem. 351, 1551-157, 2006 20. Feng, S., Pan, Z., and Pan, J., Determination of proteins at nanogram levels with Bordeaux red based on the enhancement of resonance light scattering, Sprectrochim.Acta A Mol.Biomol. Spectrosc. 64, 574579, 2006 21. Faurobert, M., Pelpoir, E., and Chaib, J., Phenol extraction of proteins for proteomic studies of recalcitrant plant tissues, Methods Mol.Biol. 359, 9-14, 2007 22. Changrani, N.R., Chonkar, A., Adeghate, E., and Singh, J., Effects of streptoozotocin-induced type 1 diabetes mellitus on total protein concentrations and cation contents in the isolated pancreas, parotid, submandibular, and lacrimal glands of rats, Ann.N.Y.Acad.Sci. 1084, 503-519, 2006 23. Reis, C.P. ,Ribeiro, A.J., Huong, S., et al., Nanoparticulate delivery system for insulin: design, characterization and in vitro/in vivo bioactivity, Eur.J.Pharm.Sci. 30, 392-397, 2007 24. Campanha, N.H., Pavarina, A.C., Brunetti, I.L., et al., Candida albicans inactivation and cell membrane integrity damage by microwave irradiation, Mycoses 50, 140-147, 2007 25. Lof, A.L., Gustafsson, G., Novak, V., et al., Determination of total protein in highly purified factor IX concentrates, Vox Sang. 63, 172177, 1992 26. Hall, M.B. and Herejk, C., Differences in yields of microbial crude protein from in vitro fermentation of carbohydrates, J.Dairy Sci. 84, 2486-2493, 2001 27. Simsek-Ege, F.A., Bond, G.M., and Stringer, J., Matrix molecular weight cut-off for encapsulation of carbonic anhydrase in polyelectrolyte beads, J.Biomater.Sci.Polym.Ed. 13, 1175-1187, 2002 28. Vidanovic, D., Milic Askrabic, J., Stankovic, M., and Poprzen, V., Effects of nonionic surfactants on the physical stability of immunoglobulin G in aqueous solution during mechanical agitation, Pharmazie 58, 399-404, 2003 29. Leek, A.B., Hayes, E.T., Curran, T.F., et al., The influence of manure composition on emissions of odour and ammonia from finishing pigs fed different concentrations of dietary crude protein, Bioresour. Technol., in press, 2006 30. Cheison S.C., Wang, Z., and Xu, S.Y., Preparation of whey protein hydrolysates using a single- and two-stage enzymatic membrane reactor and their immunological and antioxidant properties: Characterization by multivariate data analysis, J.Agric.Food Chem., in press, 2007 31. Knight, M.I. and Chambers, P.J., Problems associated with determining protein concentration: a comparison of techniques for protein estimations, Mol.Biotechnol. 23, 19-28, 2003 32. Albracht, S.P., van der Linden, E., and Faber, B.W., Quantitative amino acid analysis of bovine NADH:ubiquinone oxidoreductase (Complex I) and related enzymes. Consequences for the number of prosthetic groups, Biochim.Biophys.Acta 1557, 41-49, 2003 33. Salchert, K., Pompe, T., Sperling, C., and Wenner, C., Quantitative analysis of immobilized proteins and protein mixtures by amino acid analysis, J.Chromatog.A 1005, 113-122, 2003
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Assay of Solution Protein Concentration 34 . Schauer, U., Stemberg, F., Rieger, C.H., et al., IgG subclass concentrations in certified reference material 470 and reference values for children and adults determined with the binding site reagents, Clin. Chem. 49, 1924-1929, 2003 35. Bunk, D.M. and Welch, M.J., Characterization of a new certified reference material for human cardiac troponin I, Clin.Chem. 52, 212219, 2006 36. Nakamura, K., Tanaka, T., Kuwahara, A., and Takeo, K., Microassay for proteins on nitrocellulose filter using protein dye-staining procedure, Anal.Biochem. 148, 311-319, 1985 37. Cabrera, M.M. and Lund, F.A., Determination of protein in polysaccharide-antibody complexes, Ann.Inst.Pasteur Immunol. 137C, 51-55, 1986 38. Everitt, E. Wohlfart, C., Spectrophotometric quanitation of anchorage-dependent cell numbers using extraction of naphthol blueblack-stained cellular protein, Anal.Biochem. 162, 122-129, 1987 39. White, P.J., Fogarty, R.D., Werther, G.A., and Wraight, C.J., Antisense inhibition of IGF receptor expression in HaCaT keratinocytes: a model for antisense strategies in keratinocytes, Antisense Nucleic Acid Drug Dev. 10, 195-203, 2000
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159 40. Himmelfarb, J. and McMonagle, E., Albumin is the major plasma protein target of oxidant stress in uremia, Kidney Int. 60, 358-363, 2001 41. Liu, T., Foote, R.S., Jacobson, S.C., et al., Electrophoretic separation of proteins on a microchip with noncovalent, postcolumn labeling, Anal.Chem. 72, 4608-4613, 2000 42. Harvey, M.D., Bablekis, V., Banks, P.R., and Skinner, C.D., Utilization of the non-covalent fluorescent dye, NanoOrange, as a potential clinical diagnostic tool. Nanomolar human serum albumin quantitation, J.Chromatog.B.Biomed.Sci.Appl. 754, 345-356, 2001 43. Jones, L.J, Haugland, R.P., and Singer, V.L., Development and characterization of the NanoOrange protein quantitation assay: a fluorescence-based assay of proteins in solution, BioTechniques 34, 850-854, 2003 44. Stoyanov, A.V., Fan, Z.H., Das, C., et al., On the possibility of applying noncovalent dyes for protein labeling in isoelectric focusing, Anal.Biochem. 350, 263-267, 2006 45. Williams, J.C, Jr., Zarse, C.A., Jackson, M.E., et al., Variability of protein content in calcium oxalate monohydrate stones, J.Endourol. 20, 560-564, 2006
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SPECTROPHOTOMETRIC DETERMINATION OF PROTEIN CONCENTRATION IN THE SHORT-WAVELENGTH ULTRAVIOLET W. B. Gratzer Whereas the extinction coefficients of proteins in the aromatic absorption band at 280 nm vary widely, the spectrum at shorter wavelengths is dominated by the absorption of the peptide bond and, therefore, has only a secondary dependence on amino acid composition and conformation. Measurements in this region can therefore serve for approximate concentration determinations of any protein. The following relations are available:
1. Scopes, Anal. Biochem., 59, 277 (1974):
a. E(1 mg/mL; 1 cm) at 205 nm = 31 with a stated error of 5%. b. This can be improved by applying a correction for the relatively strongly absorbing aromatic residues, by measuring the absorbance also at 280 nm. Two forms of this correction are
E(1 mg/ml; 1 cm) at 205 nm = 27.0 + 120 × (A280/A 205)
or
E(1 mg/ml; 1 cm) at 205 nm =
27.0 1- 3.85( A 280 / A 205 )
where the bracket term is the ratio of the absorbances measured at 280 and 205 nm; stated error, 2%.
2. Tombs, Soutar, and Maclagan, Biochem. J., 73, 167 (1959): E(1 mg/mL; 1 cm) at 210 nm = 20.5 3. Waddell, J. Lab. Clin. Med., 48, 311 (1956): To avoid wavelength-setting error on steeply sloping curves, measurements are made at two wavelengths 10 nm apart and the absorbance difference is used to give the concentration: C(mg/ml) = 0.144(A 215−A 225)
where A 215 and A 225 are the absorbances read in 1 cm at 215 and 225 nm. Note that the longer the wavelength, the lower the sensitivity of the spectrophotometric memethod method, but the hazard of interference from ultraviolet absorbing contaminants is less.
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Section II Lipids
•
A Comprehensive Classification System for Lipids. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 165 Properties of Fatty Acids and Their Methyl Esters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 189 Densities, Specific Volumes, and Temperature Coefficients of Fatty Acids from C8 to C12 . . . . . 191 Composition and Properties of Common Oils and Fats. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 193 Androgens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 199 Bile Acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 201 Corticoids. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 203 Estrogens. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 205 Progestogens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 207 Sterols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 209 Prostaglandins and Related Fatty-Acid Derived Materials. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 221
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A comprehensive classification system for lipids1 Eoin Fahy,* Shankar Subramaniam,† H. Alex Brown,§ Christopher K. Glass,** Alfred H. Merrill, Jr.,†† Robert C. Murphy,§§ Christian R. H. Raetz,*** David W. Russell,††† Yousuke Seyama,§§§ Walter Shaw,**** Takao Shimizu,†††† Friedrich Spener,§§§§ Gerrit van Meer,***** Michael S. VanNieuwenhze,††††† Stephen H. White,§§§§§ Joseph L. Witztum,****** and Edward A. Dennis2,†††††† San Diego Supercomputer Center,* University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0505; Department of Bioengineering,† University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0412; Department of Pharmacology,§ Vanderbilt University Medical Center, Nashville, TN 37232-6600; Department of Cellular and Molecular Medicine,** University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0651; School of Biology,†† Georgia Institute of Technology, Atlanta, GA 30332-0230; Department of Pharmacology,§§ University of Colorado Health Sciences Center, Aurora, CO 80045-0508; Department of Biochemistry,*** Duke University Medical Center, Durham, NC 27710; Department of Molecular Genetics,††† University of Texas Southwestern Medical Center, Dallas, TX 75390-9046; Faculty of Human Life and Environmental Sciences,§§§ Ochanomizu University, Tokyo 112-8610, Japan; Avanti Polar Lipids, Inc.,**** Alabaster, AL 35007; Department of Biochemistry and Molecular Biology,†††† Faculty of Medicine, University of Tokyo, Tokyo 113-0033, Japan; Department of Molecular Biosciences,§§§§ University of Graz, 8010 Graz, Austria; Department of Membrane Enzymology,***** Institute of Biomembranes, Utrecht University, 3584 CH Utrecht, The Netherlands; Department of Chemistry and Biochemistry,††††† University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0358; Department of Physiology and Biophysics,§§§§§ University of California at Irvine, Irvine, CA 92697-4560; Department of Medicine,****** University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0682; and Department of Chemistry and Biochemistry and Department of Pharmacology,†††††† University of California, San Diego, La Jolla, CA 92093-0601
Abstract Lipids are produced, transported, and recognized by the concerted actions of numerous enzymes, binding proteins, and receptors. A comprehensive analysis of lipid molecules, “lipidomics,” in the context of genomics and proteomics is crucial to understanding cellular physiology and pathology; consequently, lipid biology has become a major research target of the postgenomic revolution and systems biology. To facilitate international communication about lipids, a comprehensive classification of lipids with a common platform that is compatible with informatics requirements has been developed to deal with the massive amounts of data that will be generated by our lipid community. As an initial step in this development, we divide lipids into eight categories (fatty acyls, glycerolipids, glycerophospholipids, sphingolipids, sterol lipids, prenol lipids, saccharolipids, and polyketides) containing distinct classes and subclasses of molecules, devise a common manner of representing the chemical structures of individual lipids and their derivatives, and provide a 12 digit identifier for each unique lipid molecule. The lipid classification scheme is chemically based and driven by the distinct hydrophobic and hydrophilic elements that compose the lipid.jlr This structured vocabulary will facilitate the systematization of lipid biology and enable the cataloging of lipids and their properties in a way that is compatible with other macromolecular databases.—Fahy, E., S. Subramaniam, H. A. Brown, C. K. Glass, A. H. Merrill, Jr., R. C. Murphy, C. R. H. Raetz, D. W. Russell, Y. Seyama, W. Shaw, T. Shimizu, F. Spener, G. van Meer,
Manuscript received 22 December 2004 and in revised form 4 February 2005.
Published, JLR Papers in Press, February 16, 2005. DOI 10.1194/jlr. E400004-JLR200
Copyright © 2005 by the American Society for Biochemistry and Molecular Biology, Inc.
Reproduced with permission
This article is available online at http://www.jlr.org
M. S. VanNieuwenhze, S. H. White, J. L. Witztum, and E. A. Dennis. A comprehensive classification system for lipids. J. Lipid Res. 2005. 46: 839–861. Supplementary key words lipidomics • informatics • nomenclature • chemical representation • fatty acyls • glycerolipids • glycerophospholipids • sphingolipids • sterol lipids • prenol lipids • saccharolipids • polyketides The goal of collecting data on lipids using a “systems biology” approach to lipidomics requires the development of a comprehensive classification, nomenclature, and chemical representation system to accommodate the myriad lipids that exist in nature. Lipids have been loosely defined as biological substances that are generally hydrophobic in nature and in many cases soluble in organic solvents (1). These chemical properties cover a broad range of molecules, such as fatty acids, phospholipids, sterols, sphingolipids, terpenes, and others (2). The LIPID MAPS (LIPID Metabolites And Pathways Strategy; http://www.lipidmaps.org), Lipid Library (http://lipidlibrary.co.uk), Lipid Bank (http://lipidbank.jp), LIPIDAT (http://www.lipidat.chemistry.ohiostate.edu), and Cyberlipids (http://www.cyberlipid.org) websites provide useful online resources for an overview of these molecules and their structures. More accurate definitions are possible when lipids are considered from a structural and biosynthetic perspective, and many different classification schemes have been used over the years. However, for the purpose of comprehensive classification, we define lipids as hydrophobic or amphipathic small molecules that may originate entirely or in part by carbanion-based condensations of thioesters (fatty acids, polyketides, etc.) and/or by
The evaluation of this manuscript was handled by the former Editor-inChief Trudy Forte. 2 To whom correspondence should be addressed. E-mail: [email protected] 1
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166
TABLE 1: Lipid Categories and Examples Category Fatty acyls Glycerolipids Glycerophospholipids Sphingolipids Sterol lipids Prenol lipids Saccharolipids Polyketides
Abbreviation
Example
FA GL GP SP ST PR SL PK
dodecanoic acid 1-hexadecanoyl-2-(9 Z-octadecenoyl)-sn-glycerol 1-hexadecanoyl-2-(9 Z-octadecenoyl)-sn-glycero-3-phosphocholine N-(tetradecanoyl)-sphing-4-enine cholest-5-en-3β-ol 2E,6E-farnesol UDP-3-O-(3R-hydroxy-tetradecanoyl)-αd-N-acetylglucosamine aflatoxin B1
carbocation-based condensations of isoprene units (prenols, sterols, etc.). Additionally, lipids have been broadly subdivided into “simple” and “complex” groups, with simple lipids being those yielding at most two types of products on hydrolysis (e.g., fatty acids, sterols, and acylglycerols) and complex lipids (e.g., glycerophospholipids and glycosphingolipids) yielding three or more products on hydrolysis. The classification scheme presented here organizes lipids into well-defined categories that cover eukaryotic and prokaryotic sources and that is equally applicable to archaea and synthetic (man-made) lipids. Lipids may be categorized based on their chemically functional backbone as polyketides, acylglycerols, sphingolipids, prenols, or saccharolipids. However, for historical and bioinformatics advantages, we chose to separate fatty acyls from other polyketides, the glycerophospholipids from the other glycerolipids, and sterol lipids from other prenols, resulting in a total of eight primary categories. An important aspect of this scheme is that it allows for subdivision of the main categories into classes and subclasses to handle the existing and emerging arrays of lipid structures. Although any classification scheme is in part subjective as a result of the structural and biosynthetic complexity of lipids, it is an essential prerequisite for the organization of lipid research and the development of systematic methods of data management. The classification scheme presented here is chemically based and driven by the distinct hydrophobic and hydrophilic elements that constitute the lipid. Biosynthetically related compounds that are not technically lipids because of their water solubility are included for completeness in this classification scheme. The proposed lipid categories listed in Table 1 have names that are, for the most part, well accepted in the literature. The fatty acyls (FA) are a diverse group of molecules synthesized by chain elongation of an acetyl-CoA primer with malonyl-CoA (or methylmalonyl-CoA) groups that may contain a cyclic functionality and/or are substituted with heteroatoms. Structures with a glycerol group are represented by two distinct categories: the glycerolipids (GL), which include acylglycerols but also encompass alkyl and 1 Z-alkenyl variants, and the glycerophospholipids (GP), which are defined by the presence of a phosphate (or phosphonate) group esterified to one of the glycerol hydroxyl groups. The sterol lipids (ST) and prenol lipids (PR) share a common biosynthetic pathway via the polymerization of dimethylallyl pyrophosphate/isopentenyl pyrophosphate but have obvious differences in terms of their eventual structure and function. Another well-defined category is the sphingolipids (SP), which contain a long-chain base as their core structure. This classification does not have a glycolipids category per se but rather places
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glycosylated lipids in appropriate categories based on the identity of their core lipids. It also was necessary to define a category with the term “saccharolipids” (SL) to account for lipids in which fatty acyl groups are linked directly to a sugar backbone. This SL group is distinct from the term “glycolipid” that was defined by the International Union of Pure and Applied Chemists (IUPAC) as a lipid in which the fatty acyl portion of the molecule is present in a glycosidic linkage. The final category is the polyketides (PK), which are a diverse group of metabolites from plant and microbial sources. Protein modification by lipids (e.g., fatty acyl, prenyl, cholesterol) occurs in nature; however, these proteins are not included in this database but are listed in protein databases such as GenBank (http://www.ncbi.nlm.nih.gov) and SwissProt (http://www.ebi.ac.uk/swissprot/).
Lipid nomenclature A naming scheme must unambiguously define a lipid structure in a manner that is amenable to chemists, biologists, and biomedical researchers. The issue of lipid nomenclature was last addressed in detail by the International Union of Pure and Applied Chemists and the International Union of Biochemistry and Molecular Biology (IUPAC-IUBMB) Commission on Biochemical Nomenclature in 1976, which subsequently published its recommendations (3). Since then, a number of additional documents relating to the naming of glycolipids (4), prenols (5), and steroids (6) have been released by this commission and placed on the IUPAC website (http://www. chem.qmul.ac.uk/iupac/). A large number of novel lipid classes have been discovered during the last three decades that have not yet been systematically named. The present classification includes these new lipids and incorporates a consistent nomenclature. In conjunction with our proposed classification scheme, we provide examples of systematic (or semisystematic) names for the various classes and subclasses of lipids. The nomenclature proposal follows existing IUPAC-IUBMB rules closely and should not be viewed as a competing format. The main differences involve a) clarification of the use of core structures to simplify systematic naming of some of the more complex lipids, and b) provision of systematic names for recently discovered lipid classes. Key features of our lipid nomenclature scheme are as follows:
a) The use of the stereospecific numbering (sn) method to describe glycerolipids and glycerophospholipids (3). The glycerol group is typically acylated or alkylated at the sn-1
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A Comprehensive Classification System for Lipids
b)
c)
d)
e)
f)
g)
h)
i)
j)
and/or sn-2 position, with the exception of some lipids that contain more than one glycerol group and archaebacterial lipids in which sn-2 and/or sn-3 modification occurs. Definition of sphinganine and sphing-4-enine as core structures for the sphingolipid category, where the d-erythro or 2S,3R configuration and 4E geometry (in the case of sphing-4-enine) are implied. In molecules containing stereochemistries other than the 2S,3R configuration, the full systematic names are to be used instead (e.g., 2R-amino-1,3R-octadecanediol). The use of core names such as cholestane, androstane, and estrane for sterols. Adherence to the names for fatty acids and acyl chains (formyl, acetyl, propionyl, butyryl, etc.) defined in Appendices A and B of the IUPAC-IUBMB recommendations (3). The adoption of a condensed text nomenclature for the glycan portions of lipids, where sugar residues are represented by standard IUPAC abbreviations and where the anomeric carbon locants and stereochemistry are included but the parentheses are omitted. This system has also been proposed by the Consortium for Functional Glycomics (http:// web.mit.edu/glycomics/consortium/main.shtml). The use of E/Z designations (as opposed to trans/cis) to define double bond geometry. The use of R/S designations (as opposed to α/β or d/l) to define stereochemistries. The exceptions are those describing substituents on glycerol (sn) and sterol core structures and anomeric carbons on sugar residues. In these latter special cases, the α/β format is firmly established. The common term “lyso,” denoting the position lacking a radyl group in glycerolipids and glycerophospholipids, will not be used in systematic names but will be included as a synonym. The proposal for a single nomenclature scheme to cover the prostaglandins, isoprostanes, neuroprostanes, and related compounds, where the carbons participating in the cyclopentane ring closure are defined and where a consistent chain-numbering scheme is used. The “d” and “t” designations used in shorthand notation of sphingolipids refer to 1,3-dihydroxy and 1,3,4-trihydroxy long-chain bases, respectively.
Lipid structure representation In addition to having rules for lipid classification and nomenclature, it is important to establish clear guidelines for drawing lipid structures. Large and complex lipids are difficult to draw, which leads to the use of shorthand and unique formats that often generate more confusion than clarity among lipidologists. We propose a more consistent format for representing lipid structures in which, in the simplest case of the fatty acid derivatives, the acid group (or equivalent) is drawn on the right and the hydrophobic hydrocarbon chain is on the left (Figure 1). Notable exceptions are found in the eicosanoid class, in which the hydrocarbon chain wraps around in a counterclockwise direction to produce a more condensed structure. Similarly, with regard to the glycerolipids and glycerophospholipids, the radyl chains are drawn with the hydrocarbon chains to the left and the glycerol group depicted
9168_Book.indb 167
167 horizontally with stereochemistry at the sn carbons defined (if known). The general term “radyl” is used to denote either acyl, alkyl, or 1-alkenyl substituents (http://www.chem.qmul.ac.uk/ iupac/lipid/lip1n2.html), allowing for coverage of alkyl and 1 Z-alkenylglycerols. The sphingolipids, although they do not contain a glycerol group, have a similar structural relationship to the glycerophospholipids in many cases and may be drawn with the C1 hydroxyl group of the long-chain base to the right and the alkyl portion to the left. This methodology places the head groups of both sphingolipids and glycerophospholipids on the right side. Although the structures of sterols do not conform to these general rules of representation, the sterol esters may conveniently be drawn with the acyl group oriented according to these guidelines. In addition, the linear prenols or isoprenoids are drawn in a manner analogous to the fatty acids, with the terminal functional group on the right side. Inevitably, a number of structurally complex lipids, such as acylaminosugar glycans, polycyclic isoprenoids, and polyketides, do not lend themselves to these simplified drawing rules. Nevertheless, we believe that the adoption of the guidelines proposed here will unify chemical representation and make it more comprehensible.
Databasing lipids, annotation, and function A number of repositories, such as GenBank, SwissProt, and ENSEMBL (http://www.ensembl.org), support nucleic acid and protein databases; however, there are only a few specialized databases [e.g., LIPIDAT (7) and Lipid Bank (8)] that provide a catalog, annotation, and functional classification of lipids. Given the importance of these molecules in cellular function and pathology, there is an imminent need for the creation of a well-organized database of lipids. The first step toward this goal is the establishment of an ontology of lipids that is extensible, flexible, and scalable. Before establishing an ontology, a structured vocabulary is needed, and the IUPAC nomenclature of the 1970s was an initial step in this direction. The ontology of lipids must contain definitions, meanings, and interrelationships of all objects stored in the database. This ontology is then transformed into a well-defined schema that forms the foundation for a relational database of lipids. The LIPID MAPS project is building a robust database of lipids based on the proposed ontology. Our database will provide structural and functional annotations and have links to relevant protein and gene data. In addition, a universal data format (XML) will be provided to facilitate exportation of the data into other repositories. This database will enable the storage of curated information on lipids in a web-accessible format and will provide a community standard for lipids. An important database field will be the LIPID ID, a unique 12 character identifier based on the classification scheme described here. The format of the LIPID ID, outlined in Table 2, provides a systematic means of assigning unique IDs to lipid molecules and allows for the addition of large numbers of new categories, classes, and subclasses in the future, because a maximum of 100 classes/ subclasses (00 to 99) may be specified. The last four characters of the ID constitute a unique identifier within a particular subclass and are randomly assigned. By initially using numeric characters, this allows 9,999 unique IDs per subclass, but with the additional use of 26 uppercase alphabetic characters, a total of 1.68 million possible combinations can be generated, providing ample scalability within each subclass. In cases in which lipid structures are obtained from
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168 O
O OH
O
sn-1 sn-3 sn-2
OH
O H
(a) Fatty Acyls: hexadecanoic acid
O (b) Glycerolipids: 1-hexadecanoyl-2-(9Z-octadecenoyl)-sn-glycerol
O O
sn-1 sn-3 sn-2
O P
H OH
O O O H HO
N+ NH H
O (c) Glycerophospholipids: 1-hexadecanoyl-2(9Z-octadecenoyl)-sn-glycero-3-phosphocholine H
21
22 20
18 12 11 19 1
H
9
2
10
3
8
H
7
5
HO
4
17
13 14
24
O (d) Sphingolipids: N-(tetradecanoyl)-sphing-4-enine 26
25
23
H
27
16 15
H OH
6
(e) Sterol Lipids: cholest-5-en-3β-ol
O O
O
HN O
HO
(f ) Prenol Lipids: 2E, 6E-farnesol
O
OH HO
OH
O
NH
O
P P O O O HO HO
O
HO
N
O
OH
O
O O
H
O O H O (h) Polyketides: aflatoxin B1
(g) Saccharolipids: UDP-3-O-(3R-hydroxy -tetradecanoyl)-αD-N-acetylglucosamine
FigUre 1 Representative structures for each lipid category.
TABLE 2: Format of 12 Character LIPID ID Characters 1–2 3–4 5–6 7–8 9–12
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Description
Example
Fixed database designation Two letter category code Two digit class code Two digit subclass code Unique four character identifier within subclass
LM FA 03 02 7312
other sources such as LipidBank or LIPIDAT, the corresponding IDs for those databases will be included to enable cross-referencing. The first two characters of the ID contain the database identifier (e.g., LM for LIPID MAPS), although other databases may choose to use their own two character identifier (at present, LB for Lipid Bank and LD for LIPIDAT) and assign the last four or more characters uniquely while retaining characters 3 to 8, which pertain to classification. The corresponding IDs of the other databases will always be included to enable cross-referencing. Further details regarding the numbering
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169
TABLE 3: Shorthand Notation for Selected Lipid Categories Category
Abbreviation
GP GP GP GP GP GP GP GP GP GP GP GP GP GP GP GP GP SP SP SP GL GL GL
GPCho GPnCho GPEtn GPnEtn GPSer GPGro GPGroP GPIns GPInsP GPInsP2 GPInsP3 GPA GPP CL CDP-DG [glycan] GP [glycan] GPIns Cer SM [glycan]Cer MG DG TG
a
Class or Subclass
Examplea
Glycerophosphocholines Glycerophosphonocholines Glycerophosphoethanolamines Glycerophosphonoethanolamines Glycerophosphoserines Glycerophosphoglycerols Glycerophosphoglycerophosphates Glycerophosphoinositols Glycerophosphoinositol monophosphates Glycerophosphoinositol bis-phosphates Glycerophosphoinositol tris-phosphates Glycerophosphates Glyceropyrophosphates Glycerophosphoglycerophosphoglycerols CDP-glycerols Glycerophosphoglucose lipids Glycerophosphoinositolglycans Ceramides Phosphosphingolipids Glycosphingolipids Monoradyl glycerols Diradyl glycerols Triradyl glycerols
GPCho (16:0/9Z,12Z-18:2)
EtN-P-6Manα1–2Manα1–6 Manα1–4GlcNα1-6GPIns (14:0/14:0) Cer (d18:1/9E-16:1) SM (d18:1/24:0) NeuAcα2–3Galβ1–4Glcβ-Cer (d18:1/16:0) MG (16:0/0:0/0:0) DG (18:0/16:0/0:0) TG (12:0/14:0/18:0)
Shorthand notation for radyl substituents in categories GP and GL are presented in the order of sn-1 to sn-3. Shorthand notation for category SP is presented in the order of long-chain base and N-acyl substituent. Numbers separated by colons refer to carbon chain length and number of double bonds, respectively.
system will be decided by the International Lipids Classification and Nomenclature Committee (see below). In addition to the LIPID ID, each lipid in the database will be searchable by classification (category, class, subclass), systematic name, synonym(s), molecular formula, molecular weight, and many other parameters that are part of its ontology. An important feature will be the databasing of molecular structures, allowing the user to perform web-based substructure searches and structure retrieval across the database. This aim will be accomplished with a chemistry cartridge software component that will enable structures in formats such as MDL molfile and Chemdraw CDX to be imported directly into Oracle database tables. Furthermore, many lipids, in particular the glycerolipids, glycerophospholipids, and sphingolipids, may be conveniently described in terms of a shorthand name in which abbreviations are used to define backbones, head groups, and sugar units and the radyl substituents are defined by a descriptor indicating carbon chain length and number of double bonds. These shorthand names lend themselves to fast, efficient text-based searches and are used widely in lipid research as compact alternatives to systematic names. The glycerophospholipids in the LIPIDAT database, for example, may be conveniently searched with a shorthand notation that has been extended to handle side chains with acyl, ether, branched-chain, and other functional groups (7). We propose the use of a shorthand notation for selected lipid categories (Table 3) that incorporates a condensed text nomenclature for glycan substituents. The abbreviations for the sugar units follow the current IUPAC-IUBMB recommendations (4).
Lipid classes and subclasses Fatty acyls [FA]
The fatty acyl structure represents the major lipid building block of complex lipids and therefore is one of the most
9168_Book.indb 169
fundamental categories of biological lipids. The fatty acyl group in the fatty acids and conjugates class (Table 4) is characterized by a repeating series of methylene groups that impart hydrophobic character to this category of lipids. The first subclass includes the straight-chain saturated fatty acids containing a terminal carboxylic acid. It could also be considered the most reduced end product of the polyketide pathway. Variants of this structure have one or more methyl substituents and encompass quite complex branched-chain fatty acids, such as the mycolic acids. The longest chain in branched-chain fatty acids defines the chain length of these compounds. A considerable number of variations on this basic structure occur in all kingdoms of life (9–12), including fatty acids with one or more double bonds and even acetylenic (triple) bonds. Heteroatoms of oxygen, halogen, nitrogen, and sulfur are also linked to the carbon chains in specific subclasses. Cyclic fatty acids containing three to six carbon atoms as well as heterocyclic rings containing oxygen or nitrogen are found in nature. The cyclopentenyl fatty acids are an example of this latter subclass. The thia fatty acid subclass contains sulfur atom(s) in the fatty acid structure and is exemplified by lipoic acid and biotin. Thiols and thioethers are in this class, but the thioesters are placed in the ester class because of the involvement of these and similar esters in fatty acid metabolism and synthesis. Separate classes for more complex fatty acids with multiple functional groups (but nonbranched) are designated by the total number of carbon atoms found in the critical biosynthetic precursor. These include octadecanoids and lipids in the jasmonic acid pathway of plant hormone biosynthesis, even though jasmonic acids have lost some of their carbon atoms from the biochemical precursor, 12-oxophytodienoic acid (13). Eicosanoids derived from arachidonic acid include prostaglandins, leukotrienes, and other structural derivatives (14). The docosanoids contain 22 carbon atoms and derive from a common precursor, docosahexaenoic acid (15). Many members of these separate subclasses of more complex fatty acids have distinct biological activities.
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170 TABLE 4: Fatty acyls [FA] Classes and Subclasses Fatty acids and conjugates [FA01] Straight-chain fatty acids [FA0101] Methyl branched fatty acids [FA0102] Unsaturated fatty acids [FA0103] Hydroperoxy fatty acids [FA0104] Hydroxy fatty acids [FA0105] Oxo fatty acids [FA0106] Epoxy fatty acids [FA0107] Methoxy fatty acids [FA0108] Halogenated fatty acids [FA0109] Amino fatty acids [FA0110] Cyano fatty acids [FA0111] Nitro fatty acids [FA0112] Thia fatty acids [FA0113] Carbocyclic fatty acids [FA0114] Heterocyclic fatty acids [FA0115] Mycolic acids [FA0116] Dicarboxylic acids [FA0117] Octadecanoids [FA02] 12-Oxophytodienoic acid metabolites [FA0201] Jasmonic acids [FA0202] Eicosanoids [FA03] Prostaglandins [FA0301] Leukotrienes [FA0302] Thromboxanes [FA0303] Lipoxins [FA0304] Hydroxyeicosatrienoic acids [FA0305] Hydroxyeicosatetraenoic acids [FA0306] Hydroxyeicosapentaenoic acids [FA0307] Epoxyeicosatrienoic acids [FA0308] Hepoxilins [FA0309] Levuglandins [FA0310] Isoprostanes [FA0311] Clavulones [FA0312] Docosanoids [FA04] Fatty alcohols [FA05] Fatty aldehydes [FA06] Fatty esters [FA07] Wax monoesters [FA0701] Wax diesters [FA0702] Cyano esters [FA0703] Lactones [FA0704] Fatty acyl-CoAs [FA0705] Fatty acyl-acyl carrier proteins (ACPs) [FA0706] Fatty acyl carnitines [FA0707] Fatty acyl adenylates [FA0708] Fatty amides [FA08] Primary amides [FA0801] N-Acyl amides [FA0802] Fatty acyl homoserine lactones [FA0803] N-Acyl ethanolamides (endocannabinoids) [FA0804] Fatty nitriles [FA09] Fatty ethers [FA10] Hydrocarbons [FA11] Oxygenated hydrocarbons [FA12] Other [FA00]
Other major lipid classes in the fatty acyl category include fatty acid esters such as wax monoesters and diesters and the lactones. The fatty ester class also has subclasses that include important biochemical intermediates such as fatty acyl thioester-CoA derivatives, fatty acyl thioester-acyl carrier protein (ACP) derivatives, fatty acyl carnitines (esters of carnitine), and fatty adenylates, which are mixed anhydrides. The fatty alcohols and fatty aldehydes are typified by terminal hydroxy and oxo groups, respectively. The fatty amides are also N-fatty acylated amines and unsubstituted
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TABLE 5: Glycerolipids [GL] Classes and Subclasses Monoradylglycerols [GL01] Monoacylglycerols [GL0101] Monoalkylglycerols [GL0102] Mono-(1 Z-alkenyl)-glycerols [GL0103] Monoacylglycerolglycosides [GL0104] Monoalkylglycerolglycosides [GL0105] Diradylglycerols [GL02] Diacylglycerols [GL0201] Alkylacylglycerols [GL0202] Dialkylglycerols [GL0203] 1 Z-Alkenylacylglycerols [GL0204] Diacylglycerolglycosides [GL0205] Alkylacylglycerolglycosides [GL0206] Dialkylglycerolglycosides [GL0207] Di-glycerol tetraethers [GL0208] Di-glycerol tetraether glycans [GL0209] Triradylglycerols [GL03] Triacylglycerols [GL0301] Alkyldiacylglycerols [GL0302] Dialkylmonoacylglycerols [GL0303] 1 Z-Alkenyldiacylglycerols [GL0304] Estolides [GL0305] Other [GL00]
amides, and many simple amides have interesting biological activities in various organisms. Fatty acyl homoserine lactones are fatty amides involved in bacterial quorum sensing (16). Hydrocarbons are included as a class of fatty acid derivatives because they correspond to six electron reduction products of fatty acids that may have been generated by loss of the carboxylic acid from a fatty acid or fatty acyl moiety during the process of diagenesis in geological samples. Long-chain ethers also have been observed in nature. Chemical structures of the fatty acyls are shown in Figure 2.
Glycerolipids [GL]
The glycerolipids essentially encompass all glycerol-containing lipids. We have purposely made glycerophospholipids a separate category because of their abundance and importance as membrane constituents, metabolic fuels, and signaling molecules. The glycerolipid category (Table 5) is dominated by the mono-, di- and tri-substituted glycerols, the most well-known being the fatty acid esters of glycerol (acylglycerols) (17, 18). Additional subclasses are represented by the glycerolglycans, which are characterized by the presence of one or more sugar residues attached to glycerol via a glycosidic linkage (19). Examples of structures in this category are shown in Figure 3. Macrocyclic ether lipids also occur as glycerolipids in the membranes of archaebacteria (20).
Glycerophospholipids [GP]
The glycerophospholipids are ubiquitous in nature and are key components of the lipid bilayer of cells. Phospholipids may be subdivided into distinct classes (Table 6) based on the nature of the polar “head group” at the sn-3 position of the glycerol backbone in eukaryotes and eu-bacteria or the sn-1 position in the case of archaebacteria (21). In the case of the glycerophosphoglycerols and glycerophosphoglycerophosphates, a second glycerol unit constitutes part of the head group, whereas for the glycerophosphoglycerophosphoglycerols (cardiolipins), a third glycerol unit is typically acylated at the sn-1’ and sn-2’ positions to create a pseudosymmetrical molecule. Each head group class is further differentiated on the basis of the sn-1 and sn-2 substituents on
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171 O
O
OH
OH (a) Straight chain fatty acids: hexadecanoic acid
(b) Methyl branched fatty acids: 17-methyl-6Z-octadecenoic acid O OH
O (c) Unsaturated fatty acids: 9Z-octadecenoic acid
OH OH
(d) Hydroxy fatty acids: 2S-hydroxytetradecanoic acid
O
O
OH
OH O (f ) Epoxy fatty acids: 6R,7S-epoxy-octadecanoic acid
O (e) Oxo fatty acids: 2-oxodecanoic acid
O
H
O
OH
OH
S
OCH3 (g) Methoxy fatty acids: 2-methoxy-5Z-hexadecenoic acid
S (h) Thia fatty acids: R-Lipoic acid; 1,2-dithiolane-3R-pentanoic acid
O OH O
H
H
H OOH (i) Hydroperoxy fatty acids: 13S-hydroperoxy-9Z, 11E-octadecadienoic acid
(j) Carbocyclic fatty acids: lactobacillic acid; 11R,12S-methyleneoctadecanoic acid
O
O O (k) Heterocyclic fatty acids: 8-(5-hexylfuran-2-yl)-octanoic acid
OH
OH
NH2 (l) Amino fatty acids: 2S-aminotridecanoic acid Br
O
O2N
OH (m) Nitro fatty acids: 10-nitro, 9Z, 12Z-octadecadienoic acid
O
OH (n) Halogenated fatty acids: 3-bromo-2Z-heptenoic acid O
O
O HO
OH
OH O (o) Dicarboxylic acids: 1,8-octanedioic acid
OH
OH
OH (p) Prostaglandins: Prostaglandin A1; 15S-hydroxy-9-oxo-10Z,13E-prostadienoic acid OH
OH
O OH
(q) Leukotrienes: Leukotriene B4; 5S,12R-dihydroxy-6Z,8E,10E,14Z-eicosatetraenoic acid
O O
O
OH (r) Thromboxanes: Thromboxane A2; 9S,11S-epoxy,15S-hydroxythromboxa-5Z,13E-dien-1-oic acid
FigUre 2 Representative structures for fatty acyls.
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172 OH
O
OH OH
OH
O
OH (s) Lipoxins: Lipoxin A4; 5S,6R,15S-trihydroxy-7E,9E,11Z,13E-eicosatetraenoic acid
O (t) Epoxyeicosatrienoic acids: 14R,15S-epoxy-5Z,8Z,11Z-eicosatrienoic acid
OH
HO
O OH
O O
O
OHC
(u) Hepoxilins: Hepoxilin A3; 8R-hydroxy-11R,12S-epoxy-5Z,9E,14Z-eicosatrienoic acid HO
OH (v) Levuglandins: LGE2; 10,11-seco-9,11-dioxo-15S-hydroxy-5Z,13E-prostadienoic acid
OH
OH
O
O
HO
OH (w) Isoprostanes: 9S,11S,15S-trihydroxy-5Z, 13E-prostadienoic acid-cyclo[8S,12R] OH
O (x) Octadecanoids: 12-oxophytodienoic acid metabolites; (9R,13R)-12-oxo-phyto-10Z,15Z-dienoic acid OH
O
OH
O O (y) Octadecanoids: Jasmonic acids: jasmonic acid; (1R,2R)-3-oxo-2-(pent-2Z-enyl)-cyclopentaneacetic acid
O (z) Docosanoids: Neuroprostanes; 4S-hydroxy-8-oxo(5E,9Z,13Z,16Z,19Z)-neuroprostapentaenoic acid-cyclo[7S,11S] H
(aa) Fatty alcohols: dodecanol
O (ab) Fatty aldehydes: heptanal
OH
O
O N H H
O NH2 (ac) Fatty amides: N-acyl amides: dodecanamide
O O
(ad) Fatty amides: Fatty acyl homoserine lactones: N-(3-oxodecanoyl) homoserine lactone
O N H
OH
(ae) Fatty amides: N-acyl ethanolamides (endocannabinoids): Anandamide; N-(5Z,8Z,11Z,14Z-eicosatetraenoyl)-ethanolamine
C
N
(af ) Fatty nitriles: 4Z,7Z,10Z-octadecatrienenitrile O
(ag) Hydrocarbons: tridecane
(ah) Oxygenated hydrocarbons: nonacosan-2-one
FigUre 2 Representative structures for fatty acyls (Continued).
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A Comprehensive Classification System for Lipids
173 O N
O O O
O O (ai) Wax monoesters: 1-hexadecyl hexadecanoate
(aj) Cyano esters: 1,3-di-(octadec-9Z-enoyl)-1-cyano-2methylene-propane-1,3-diol
NH2 N
N
O
H N
O O
S
H H OH N O
O
O P
O P
O O O HO HO
O O P
HO O HO (al) Fatty acyl CoAs: R-hexanoyl CoA
(ak) Lactones: 11-undecanolactone
N
N
OH
NH2 N
N
O O H
O
O
O P
O HO
OH
N
N O
O
N+
HO (am) Fatty acyl carnitines: O-hexanoyl-R-carnitine
OH
(an) Fatty acyl adenylates: O-hexanoyladenosine monophosphate H
H
O
O
O
O
(ao) Wax diesters: 2S,3R-didecanoyl-docosane-2,3-diol
FigUre 2. Representative structures for fatty acyls (Continued).
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174
O O
O O
OH O H
OH
HO H (a) Monoradylglycerols: Monoacylglycerols: 1-dodecanoyl-sn-glycerol
O (b) Diradylglycerols: Diacylglycerols: 1-hexadecanoyl-2-(9Z-octadecenoyl)-sn-glycerol O
O H O (d) Diradylglycerols: 1Z-alkenylacylglycerols: 1-O-(1Z-tetradecenyl)-2-(9Z-octadecenoyl)-sn-glycerol
O (c) Diradylglycerols: Alkylacylglycerols: 1-O-hexadecyl-2-(9Z-octadecenoyl)-sn-glycerol
O
OH
O
OH O H
O
O O O H
O (e) Triradylglycerols: Triglycerols: 1-dodecanoyl-2-hexadecanoyl-3-octadecanoyl-sn-glycerol
O O
O H
OH O
OH OH O
OH
O (f ) Diradylglycerols: Diacylglycerol glycans: 1,2-di-(9Z,12Z,15Z-octadecatrienoyl)-3-O-β-D-galactosyl-sn-glycerol OH O H
O H O
O
OH
(g) Diradylglycerols: Di-glycerol tetraethers: caldarchaeol OH OH
OH OH
O OH OH OH
O O O
H O
O H O
O
OH (h) Diradylglycerols: Di-glycerol tetraether glycans: gentiobiosylcaldarchaeol; Glcβ1-6Glcβ-caldarchaeol
FigUre 3 Representative structures for glycerolipids.
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A Comprehensive Classification System for Lipids TABLE 6: Glycerophospholipids [GP] Classes and Subclasses Glycerophosphocholines [GP01] Diacylglycerophosphocholines [GP0101] 1-Alkyl,2-acylglycerophosphocholines [GP0102] 1 Z-Alkenyl,2-acylglycerophosphocholines [GP0103] Dialkylglycerophosphocholines [GP0104] Monoacylglycerophosphocholines [GP0105] 1-Alkyl glycerophosphocholines [GP0106] 1 Z-Alkenylglycerophosphocholines [GP0107] Glycerophosphoethanolamines [GP02] Diacylglycerophosphoethanolamines [GP0201] 1-Alkyl,2-acylglycerophosphoethanolamines [GP0202] 1 Z-Alkenyl,2-acylglycerophosphoethanolamines [GP0203] Dialkylglycerophosphoethanolamines [GP0204] Monoacylglycerophosphoethanolamines [GP0205] 1-Alkyl glycerophosphoethanolamines [GP0206] 1 Z-Alkenylglycerophosphoethanolamines [GP0207] Glycerophosphoserines [GP03] Diacylglycerophosphoserines [GP0301] 1-Alkyl,2-acylglycerophosphoserines [GP0302] 1 Z-Alkenyl,2-acylglycerophosphoserines [GP0303] Dialkylglycerophosphoserines [GP0304] Monoacylglycerophosphoserines [GP0305] 1-Alkyl glycerophosphoserines [GP0306] 1 Z-Alkenylglycerophosphoserines [GP0307] Glycerophosphoglycerols [GP04] Diacylglycerophosphoglycerols [GP0401] 1-Alkyl,2-acylglycerophosphoglycerols [GP0402] 1 Z-Alkenyl,2-acylglycerophosphoglycerols [GP0403] Dialkylglycerophosphoglycerols [GP0404] Monoacylglycerophosphoglycerols [GP0405] 1-Alkyl glycerophosphoglycerols [GP0406] 1 Z-Alkenylglycerophosphoglycerols [GP0407] Diacylglycerophosphodiradylglycerols [GP0408] Diacylglycerophosphomonoradylglycerols [GP0409] Monoacylglycerophosphomonoradylglycerols [GP0410] Glycerophosphoglycerophosphates [GP05] Diacylglycerophosphoglycerophosphates [GP0501] 1-Alkyl,2-acylglycerophosphoglycerophosphates [GP0502] 1 Z-Alkenyl,2-acylglycerophosphoglycerophosphates [GP0503] Dialkylglycerophosphoglycerophosphates [GP0504] Monoacylglycerophosphoglycerophosphates [GP0505] 1-Alkyl glycerophosphoglycerophosphates [GP0506] 1 Z-Alkenylglycerophosphoglycerophosphates [GP0507] Glycerophosphoinositols [GP06] Diacylglycerophosphoinositols [GP0601] 1-Alkyl,2-acylglycerophosphoinositols [GP0602] 1 Z-Alkenyl,2-acylglycerophosphoinositols [GP0603] Dialkylglycerophosphoinositols [GP0604] Monoacylglycerophosphoinositols [GP0605] 1-Alkyl glycerophosphoinositols [GP0606] 1 Z-Alkenylglycerophosphoinositols [GP0607] Glycerophosphoinositol monophosphates [GP07] Diacylglycerophosphoinositol monophosphates [GP0701] 1-Alkyl,2-acylglycerophosphoinositol monophosphates [GP0702] 1 Z-Alkenyl,2-acylglycerophosphoinositol monophosphates [GP0703] Dialkylglycerophosphoinositol monophosphates [GP0704] Monoacylglycerophosphoinositol monophosphates [GP0705] 1-Alkyl glycerophosphoinositol monophosphates [GP0706] 1 Z-Alkenylglycerophosphoinositol monophosphates [GP0707] Glycerophosphoinositol bisphosphates [GP08] Diacylglycerophosphoinositol bisphosphates [GP0801] 1-Alkyl,2-acylglycerophosphoinositol bisphosphates [GP0802] 1 Z-Alkenyl,2-acylglycerophosphoinositol bisphosphates [GP0803] Monoacylglycerophosphoinositol bisphosphates [GP0804] 1-Alkyl glycerophosphoinositol bisphosphates [GP0805] 1 Z-Alkenylglycerophosphoinositol bisphosphates [GP0806] Glycerophosphoinositol trisphosphates [GP09] Diacylglycerophosphoinositol trisphosphates [GP0901] 1-Alkyl,2-acylglycerophosphoinositol trisphosphates [GP0902] 1 Z-Alkenyl,2-acylglycerophosphoinositol trisphosphates [GP0903] Monoacylglycerophosphoinositol trisphosphates [GP0904] 1-Alkyl glycerophosphoinositol trisphosphates [GP0905] 1 Z-Alkenylglycerophosphoinositol trisphosphates [GP0906]
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175 TABLE 6: Glycerophospholipids [GP] Classes and Subclasses (Continued) Glycerophosphates [GP10] Diacylglycerophosphates [GP1001] 1-Alkyl,2-acylglycerophosphates [GP 1002] 1 Z-Alkenyl,2-acylglycerophosphates [GP1003] Dialkylglycerophosphates [GP 1004] Monoacylglycerophosphates [GP1005] 1-Alkyl glycerophosphates [GP1006] 1 Z-Alkenylglycerophosphates [GP1007] Glyceropyrophosphates [GP11] Diacylglyceropyrophosphates [GP 1101] Monoacylglyceropyrophosphates [GP1102] Glycerophosphoglycerophosphoglycerols (cardiolipins) [GP12] Diacylglycerophosphoglycerophosphodiradylglycerols [GP1201] Diacylglycerophosphoglycerophosphomonoradylglycerols [GP1202] 1-Alkyl,2-acylglycerophosphoglycerophosphodiradylglycerols [GP1203] 1-Alkyl,2-acylglycerophosphoglycerophosphomonoradylglycerols [GP1204] 1 Z-Alkenyl,2-acylglycerophosphoglycerophosphodiradylglycerols [GP1205] 1 Z-Alkenyl,2-acylglycerophosphoglycerophosphomonoradylglycerols [GP1206] Monoacylglycerophosphoglycerophosphomonoradylglycerols [GP1207] 1-Alkyl glycerophosphoglycerophosphodiradylglycerols [GP1208] 1-Alkyl glycerophosphoglycerophosphomonoradylglycerols [GP1209] 1 Z-Alkenylglycerophosphoglycerophosphodiradylglycerols [GP1210] 1 Z-Alkenylglycerophosphoglycerophosphomonoradylglycerols [GP1211] CDP-glycerols [GP13] CDP-diacylglycerols [GP1301] CDP-1-alkyl,2-acylglycerols [GP1302] CDP-1 Z-alkenyl,2-acylglycerols [GP1303] CDP-dialkylglycerols [GP1304] CDP-monoacylglycerols [GP1305] CDP-1-alkyl glycerols [GP1306] CDP-1 Z-alkenylglycerols [GP1307] Glycerophosphoglucose lipids [GP14] Diacylglycerophosphoglucose lipids [GP1401] 1-Alkyl,2-acylglycerophosphoglucose lipids [GP1402] 1 Z-Alkenyl,2-acylglycerophosphoglucose lipids [GP1403] Monoacylglycerophosphoglucose lipids [GP1404] 1-Alkyl glycerophosphoglucose lipids [GP1405] 1 Z-Alkenylglycerophosphoglucose lipids [GP1406] Glycerophosphoinositolglycans [GP15] Diacylglycerophosphoinositolglycans [GP 1501] 1-Alkyl,2-acylglycerophosphoinositolglycans [GP1502] 1 Z-Alkenyl,2-acylglycerophosphoinositolglycans [GP1503] Monoacylglycerophosphoinositolglycans [GP1504] 1-Alkyl glycerophosphoinositolglycans [GP1505] 1 Z-Alkenylglycerophosphoinositolglycans [GP1506] Glycerophosphonocholines [GP16] Diacylglycerophosphonocholines [GP 1601] 1-Alkyl,2-acylglycerophosphonocholines [GP1602] 1 Z-Alkenyl,2-acylglycerophosphonocholines [GP1603] Dialkylglycerophosphonocholines [GP1604] Monoacylglycerophosphonocholines [GP1605] 1-Alkyl glycerophosphonocholines [GP1606] 1 Z-Alkenylglycerophosphonocholines [GP1607] Glycerophosphonoethanolamines [GP 17] Diacylglycerophosphonoethanolamines [GP1701] 1-Alkyl,2-acylglycerophosphonoethanolamines [GP1702] 1 Z-Alkenyl,2-acylglycerophosphonoethanolamines [GP1703] Dialkylglycerophosphonoethanolamines [GP 1704] Monoacylglycerophosphonoethanolamines [GP1705] 1-Alkyl glycerophosphonoethanolamines [GP1706] 1 Z-Alkenylglycerophosphonoethanolamines [GP1707] Di-glycerol tetraether phospholipids (caldarchaeols) [GP18] Glycerol-nonitol tetraether phospholipids [GP19] Oxidized glycerophospholipids [GP20] Other [GP00]
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176 the glycerol backbone. Although the glycerol backbone is symmetrical, the second carbon becomes a chiral center when the sn-1 and sn-3 carbons have different substituents. A large number of trivial names are associated with phospholipids. In the systematic nomenclature, mono/di-radylglycerophospholipids with different acyl or alkyl substituents are designated by similar conventions for naming of classes (see below) and are grouped according to the common polar moieties (i.e., head groups). Typically, one or both of these hydroxyl groups are acylated with long-chain fatty acids, but there are also alkyl-linked and 1 Z-alkenyl-linked (plasmalogen) glycerophospholipids, as well as dialkylether variants in prokaryotes. The main biosynthetic pathways for the formation of GPCho and GPEtn (see Table 3 for shorthand notation) were elucidated through the efforts of Kennedy and co-workers (22) in the 1950s and 1960s, and more detailed interconversion pathways to form additional classes of phospholipids were described more recently. In addition to serving as a primary component of cellular membranes and binding sites for intracellular and intercellular proteins, some glycerophospholipids in eukaryotic cells are either precursors of, or are themselves, membrane-derived second messengers. A separate class, called oxidized glycerophospholipids, is composed of molecules in which one or more of the side chains have been oxidized. Several overviews are available on the classification, nomenclature, metabolism, and profiling of glycerophospholipids (18, 23–26). Structures from this category are shown in Figure 4.
Sphingolipids [SP]
Sphingolipids are a complex family of compounds that share a common structural feature, a sphingoid base backbone that is synthesized de novo from serine and a long-chain fatty acylCoA, then converted into ceramides, phosphosphingolipids, glycosphingolipids, and other species, including protein adducts (27, 28). A number of organisms also produce sphingoid base analogs that have many of the same features as sphingolipids (such as long-chain alkyl and vicinal amino and hydroxyl groups) but differ in other features. These have been included in this category because some are known to function as inhibitors or antagonists of sphingolipids, and in some organisms, these types of compounds may serve as surrogates for sphingolipids. Sphingolipids can be divided into several major classes (Table 7): the sphingoid bases and their simple derivatives (such as the 1-phosphate), the sphingoid bases with an amide-linked fatty acid (e.g., ceramides), and more complex sphingolipids with head groups that are attached via phosphodiester linkages (the phosphosphingolipids), via glycosidic bonds (the simple and complex glycosphingolipids such as cerebrosides and gangliosides), and other groups (such as phosphono- and arseno-sphingolipids). The IUPAC has recommended a systematic nomenclature for sphingolipids (3). The major sphingoid base of mammals is commonly referred to as “sphingosine,” because that name was affixed by the first scientist to isolate this compound (29). Sphingosine is (2S,3R,4E)2-aminooctadec-4-ene-1,3-diol (it is also called d-erythrosphingosine and sphing-4-enine). This is only one of many sphingoid bases found in nature, which vary in alkyl chain length and branching, the number and positions of double bonds, the presence of additional hydroxyl groups, and other features. The structural variation has functional significance; for example, sphingoid bases in the dermis have additional hydroxyls at position 4 (phytoceramides) and/or 6 that can interact with neighboring molecules, thereby strengthening the permeability barrier of skin.
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Handbook of Biochemistry and Molecular Biology Sphingoid bases are found in a variety of derivatives, including the 1-phosphates, lyso-sphingolipids (such as sphingosine 1-phosphocholine as well as sphingosine 1-glycosides), and N-methyl derivatives (N-methyl, N,N-dimethyl, and N,N,Ntrimethyl). In addition, a large number of organisms, such as fungi and sponges, produce compounds with structural similarity to sphingoid bases, some of which (such as myriocin and the fumonisins) are potent inhibitors of enzymes of sphingolipid metabolism. Ceramides (N-acyl-sphingoid bases) are a major subclass of sphingoid base derivatives with an amide-linked fatty acid. The fatty acids are typically saturated or monounsaturated with chain lengths from 14 to 26 carbon atoms; the presence of a hydroxyl group on carbon 2 is fairly common. Ceramides sometimes have specialized fatty acids, as illustrated by the skin ceramide in Figure 5i, which has a 30 carbon fatty acid with a hydroxyl group on the terminal (ω) carbon. Ceramides are generally precursors of more complex sphingolipids. The major phosphosphingolipids of mammals are sphingomyelins (ceramide phosphocholines), whereas insects contain mainly ceramide phosphoethanolamines and fungi have phytoceramidephosphoinositols and mannose-containing head groups. Glycosphingolipids (4) are classified on the basis of carbohydrate composition: 1) neutral glycosphingolipids contain one or more uncharged sugars such as glucose (Glu), galactose (Gal), N-acetylglucosamine (GlcNAc), N-acetylgalactosamine (GalNAc), and fucose (Fuc), which are grouped into families based on the nature of the glyco-substituents as shown in the listing; 2) acidic glycosphingolipids contain ionized functional groups (phosphate or sulfate) attached to neutral sugars or charged sugar residues such as sialic acid (N-acetyl or N-glycoloyl neuraminic acid). The latter are called gangliosides, and the number of sialic acid residues is usually denoted with a subscript letter (i.e., mono-, di- or tri-) plus a number reflecting the subspecies within that category; 3) basic glycosphingolipids; 4) amphoteric glycosphingolipids. For a few glycosphingolipids, historically assigned names as antigens and blood group structures are still in common use (e.g., Lewis x and sialyl Lewis x). Some aquatic organisms contain sphingolipids in which the phosphate is replaced by a phosphono or arsenate group. The other category includes sphingolipids that are covalently attached to proteins; for example, ω-hydroxyceramides and ω-glucosylceramides are attached to surface proteins of skin, and inositol-phosphoceramides are used as membrane anchors for some fungal proteins in a manner analogous to the glycosylphosphatidylinositol anchors that are attached to proteins in other eukaryotes. Some examples of sphingolipid structures are shown in Figure 5.
Sterol lipids [ST]
The sterol category is subdivided primarily on the basis of biological function. The sterols, of which cholesterol and its derivatives are the most widely studied in mammalian systems, constitute an important component of membrane lipids, along with the glycerophospholipids and sphingomyelins (30). There are many examples of unique sterols from plant, fungal, and marine sources that are designated as distinct subclasses in this schema (Table 8). The steroids, which also contain the same fused four ring core structure, have different biological roles as hormones and signaling molecules (31). These are subdivided on the basis of the number of carbons in the core skeleton. The C18 steroids include the estrogen family, whereas the C19 steroids comprise the androgens such as testosterone and androsterone.
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A Comprehensive Classification System for Lipids O
177 O
O P
O
O O O H HO
O
N+
O
(b) Diacylglycerophosphonocholines: 1-hexadecanoyl-2-(9Z-octadecenoyl)-sn-glycero-3-phosphonocholine
O P
NH2
O
O (c) Diacylglycerophosphoethanolamines: 1-hexadecanoyl-2-(9Z-octadecenoyl) -sn-glycero-3-phosphoethanolamine O O
H COOH
O
NH2
O O O H HO
HO H
O O O H HO
HO H OH
O (f ) Diacylglycerophosphoglycerols: 1-hexadecanoyl-2-(9Z-octadecenoyl)sn-glycero-3-phospho-(1'-sn-glycerol)
O (e) Diacylglycerophosphoserines: 1-hexadecanoyl-2-(9Z-octadecenoyl)-sn-glycero-3-phosphoserine
O
O P
O
O O O H HO
O P
NH2
O HO
O H
O (d) Diacylglycerophosphonoethanolamines: 1-hexadecanoyl-2-(9Z-octadecenoyl -sn-glycero-3-phosphonoethanolamine
O P
O
O P
O
O O O H HO
N+
O HO
O H
O
O (a) Diacylglycerophosphocholines: 1-hexadecanoyl-2-(9Z-octadecenoyl)-sn-glycero-3-phosphocholine O
O P
O P
O
O OH HO
O
O (g) Diacylglycerophosphoglycerophosphates: 1-hexadecanoyl-2-(9Z-octadecenoyl)-sn-glycero3-phospho-(1'-sn-glycerol-3'-phosphate)
O P HO O O HO O H
OH OH
O OH
O (h) Diacylglycerophosphoinositols: 1-hexadecanoyl-2-(9Z-octadecenoyl)sn-glycero-3-phospho-(1'-myo-inositol)
O O O O H
O P HO O O HO
OH OH
O P
O O
O H
O
O OH P O HO OH
O (i) Diacylglycerophosphoinositolmonophosphates: 1-hexadecanoyl-2-(9Z-octadecenoyl)-sn-glycero-3-phospho-(1'-myo-inositol-3'-phosphate)
O H
O HO
OH
O (j) Diacylglycerophosphates (phosphatidic acids): 1-hexadecanoyl-2-(9Z-octadecenoyl)-sn-glycero-3-phosphate NH2
O P
O
O P
O
O O OH HO HO
O (k) Diacylglyceropyrophosphates: 1-hexadecanoyl-2-(9Z-octadecenoyl)-sn-glycero-3-pyrophosphate
O P
O H
O HO
O P
O HO
N O
O
N
O
O HO
OH
(l) CDP-diacylglycerols: 1-hexadecanoyl-2-(9Z-octadecenoyl)-sn-glycero-3-cytidine-5'-diphosphate
FigUre 4 Representative structures for glycerophospholipids. The C21 subclass, containing a two carbon side chain at the C17 position, includes the progestogens as well as the glucocorticoids and mineralocorticoids. The secosteroids, comprising various forms of vitamin D, are characterized by cleavage of the B ring of the core structure, hence the “seco” prefix (32). Additional classes within the sterols category are the bile acids (33), which in mammals are primarily derivatives of cholan-24-oic acid synthesized from cholesterol in the liver and their conjugates (sulfuric acid, taurine, glycine, glucuronic acid, and others). Sterol lipid structures are shown in Figure 6.
9168_Book.indb 177
Prenol lipids [PR]
Prenols are synthesized from the five carbon precursors isopentenyl diphosphate and dimethylallyl diphosphate that are produced mainly via the mevalonic acid pathway (34). In some bacteria (e.g., Escherichia coli) and plants, isoprenoid precursors are made by the methylerythritol phosphate pathway (35). Because the simple isoprenoids (linear alcohols, diphosphates, etc.) are formed by the successive addition of C5 units, it is convenient to classify them in this manner (Table 9), with a polyterpene subclass for those structures containing more than 40
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178
O
O O
O O H
O P O
O
OH
HO O
O
H
O
O P
O O H
O HO
O
OH
HO H O P O O O O H HO O
O (n) Diacylglycerophosphoglycerophosphodiradylglycerols: 1',3'-Bis[1,2-Di-(9Z,12Z-octadecadienoyl)sn-glycero-3-phospho]-sn-glycerol
O
(m) Diacylglycerophosphomonoradylglycerols: 1,2-ditetradecanoyl-sn-glycero-3-phospho(3'-tetradecanoyl-1'-sn-glycerol)
O
O
O H
O P
OR
O O P
R = H2N
O HO
O
OH O
HO HO
OH
HO HO
O
O
O H
O O HO
N+
O (p) 1-alkyl, 2-acylglycerophosphocholines: 1-O-hexadecyl-2-(9Z-octadecenoyl)-sn-glycero-3-phosphocholine
O O
HO HO
OH O
OH O HO H N 2
O
HO HO HO
O P
O
O
O H
O O HO
N+
O
OH
(o) Diacylglycerophosphoinositolglycans: EtN-P-6Manα1-2Manα1-6Manα1-4GlcNα1-6GPIns(14:0/14:0)
(q) 1Z-alkenyl, 2-acylglycerophosphocholines: 1-O-(1Z-tetradecenyl)-2-(9Z-octadecenoyl -sn-glycero-3-phosphocholine
O O
O H
O P
O P
O OH H
NH2
O
O OH
(r) Di-glycerol tetraether phospholipids (caldarchaeols): sn-caldarchaeo-1-phosphoethanolamine
O O
O
OH
OH
H
O P
O OH H
NH2
O
O
OH OH
OH
OH HO (s) Glycerol-nonitol tetraether phospholipids: sn-caldito-1-phosphoethanolamine
FigUre 4 Representative structures for glycerophospholipids (Continued).
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A Comprehensive Classification System for Lipids TABLE 7: Sphingolipids [SP] Classes and Subclasses Sphingoid bases [SP01] Sphing-4-enines (sphingosines) [SP0101] Sphinganines [SP0102] 4-Hydroxysphinganines (phytosphingosines) [SP0103] Sphingoid base homologs and variants [SP0104] Sphingoid base 1-phosphates [SP0105] Lysosphingomyelins and lysoglycosphingolipids [SP0106] N-Methylated sphingoid bases [SP0107] Sphingoid base analogs [SP0108] Ceramides [SP02] N-Acylsphingosines (ceramides) [SP0201] N-Acylsphinganines (dihydroceramides) [SP0202] N-Acyl-4-hydroxysphinganines (phytoceramides) [SP0203] Acylceramides [SP0204] Ceramide 1-phosphates [SP0205] Phosphosphingolipids [SP03] Ceramide phosphocholines (sphingomyelins) [SP0301] Ceramide phosphoethanolamines [SP0302] Ceramide phosphoinositols [SP0303] Phosphonosphingolipids [SP04] Neutral glycosphingolipids [SP05] Simple Glc series (GlcCer, LacCer, etc.) [SP0501] GalNAcβ1-3Galα1-4Galβ1-4Glc- (globo series) [SP0502] GalNAcβ1-4Galβ1-4Glc- (ganglio series) [SP0503] Galβ1-3GlcNAcβ1-3Galβ1-4Glc- (lacto series) [SP0504] Galβ1–4GlcNAcβ1-3Galβ1-4Glc- (neolacto series) [SP0505] GalNAcβ1-3Galα1-3Galβ1-4Glc- (isoglobo series) [SP0506] GlcNAcβ1-2Manα1-3Manβ1-4Glc- (mollu series) [SP0507] GalNAcβ1-4GlcNAcβ1-3Manβ1-4Glc- (arthro series) [SP0508] Gal- (gala series) [SP0509] Other [SP0510] Acidic glycosphingolipids [SP06] Gangliosides [SP0601] Sulfoglycosphingolipids (sulfatides) [SP0602] Glucuronosphingolipids [SP0603] Phosphoglycosphingolipids [SP0604] Other [SP0600] Basic glycosphingolipids [SP07] Amphoteric glycosphingolipids [SP08] Arsenosphingolipids [SP09] Other [SP00]
carbons (i.e., >8 isoprenoid units) (36). Note that vitamin A and its derivatives and phytanic acid and its oxidation product pristanic acid are grouped under C20 isoprenoids. Carotenoids are important simple isoprenoids that function as antioxidants and as precursors of vitamin A (37). Another biologically important class of molecules is exemplified by the quinones and hydroquinones, which contain an isoprenoid tail attached to a quinonoid core of nonisoprenoid origin. Vitamins E and K (38, 39) as well as the ubiquinones (40) are examples of this class. Polyprenols and their phosphorylated derivatives play important roles in the transport of oligosaccharides across membranes. Polyprenol phosphate sugars and polyprenol diphosphate sugars function in extracytoplasmic glycosylation reactions (41), in extracellular polysaccharide biosynthesis [for instance, peptidoglycan polymerization in bacteria (42)], and in eukaryotic protein N-glycosylation (43, 44). The biosynthesis and function of polyprenol phosphate sugars differ significantly from those of the polyprenol diphosphate sugars; therefore, we have placed them in separate subclasses. Bacteria synthesize polyprenols (called bactoprenols) in which the terminal isoprenoid unit attached to oxygen remains unsaturated, whereas in animal polyprenols (dolichols) the terminal isoprenoid is reduced. Bacterial polyprenols are typically 10 to 12 units long (40), whereas dolichols
9168_Book.indb 179
179 usually consist of 18 to 22 isoprene units. In the phytoprenols of plants, the three distal units are reduced. Several examples of prenol lipid structures are shown in Figure 7.
Saccharolipids [SL]
We have avoided the term “glycolipid” in the classification scheme to maintain a focus on lipid structures. In fact, all eight lipid categories in the present scheme include important glycan derivatives, making the term glycolipid incompatible with the overall goal of lipid categorization. We have, in addition, coined the term “saccharolipids” to describe compounds in which fatty acids are linked directly to a sugar backbone, forming structures that are compatible with membrane bilayers. In the saccharolipids (Table 10), a sugar substitutes for the glycerol backbone that is present in glycerolipids and glycerophospholipids. Saccharolipids can occur as glycan or as phosphorylated derivatives. The most familiar saccharolipids are the acylated glucosamine precursors of the lipid A component of the lipopolysaccharides in Gramnegative bacteria (41). Typical lipid A molecules are disaccharides of glucosamine, which are derivatized with as many as seven fatty acyl chains (41, 45). Note that in naming these compounds, the total number of fatty acyl groups are counted regardless of the nature of the linkage (i.e., amide or ester). The minimal lipopolysaccharide required for growth in E. coli is a hexa-acylated lipid A that is glycosylated with two 3-deoxy-d-mannooctulosonic acid residues (see below). In some bacteria, the glucosamine backbone of lipid A is replaced by 2,3-diamino-2,3-dideoxyglucose (46); therefore, the class has been designated “Acylaminosugars.” Included also in this class are the Nod factors of nitrogen-fixing bacteria (47), such as Sinorhizobium meliloti. The Nod factors are oligosaccharides of glucosamine that are usually derivatized with a single fatty acyl chain. Additional saccharolipids include fatty acylated derivatives of glucose, which are best exemplified by the acylated trehalose units of certain mycobacterial lipids (11). Acylated forms of glucose and sucrose also have been reported in plants (48). Some saccharolipid structures are shown in Figure 8.
Polyketides [PK]
Polyketides are synthesized by classic enzymes as well as iterative and multimodular enzymes with semiautonomous active sites that share mechanistic features with the fatty acid synthases, including the involvement of specialized acyl carrier proteins (49, 50); however, polyketide synthases generate a much greater diversity of natural product structures, many of which have the character of lipids. The class I polyketide synthases form constrained macrocyclic lactones, typically ranging in size from 14 to 40 atoms, whereas class II and III polyketide synthases generate complex aromatic ring systems (Table 11). Polyketide backbones are often further modified by glycosylation, methylation, hydroxylation, oxidation, and/or other processes. Some polyketides are linked with nonribosomally synthesized peptides to form hybrid scaffolds. Examples of the three polyketide classes are shown in Figure 9. Many commonly used antimicrobial, antiparasitic, and anticancer agents are polyketides or polyketide derivatives. Important examples of these drugs include erythromycins, tetracylines, nystatins, avermectins, and antitumor epothilones. Other polyketides are potent toxins. The possibility of recombining and reengineering the enzymatic modules that assemble polyketides has recently stimulated the search for novel “unnatural” natural products, especially in the antibiotic arena (51, 52). We consider this minimal classification of polyketides as the first step in a more elaborate scheme. It will be important ultimately to include as many polyketide structures as possible in a
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Handbook of Biochemistry and Molecular Biology
180 H OH
H OH
OH
OH
H2N H
H2N H (b) Sphingosines: sphing-4-enine
(a) Sphinganines: sphinganine
H OH
H OH
OH
OH HO H H2N H
H2N H (d) Sphingoid base homologs and variants: hexadecasphinganine
(c) Phytosphingosines: 4-hydroxysphinganine H OH
O P
H OH OH
N H
H2N H
(e) N-methylated sphingoid bases: N,N-dimethylsphing-4-enine
O HO
OH
(f ) Sphingoid base 1-phosphates: sphing-4-enine-1-phosphate
H OH NH H
O P
H OH
OH
O O NH H HO
O
N+
O (h) Ceramide phosphocholines (sphingomyelins): N-(octadecanoyl)-sphing-4-enine-1-phosphocholine
(g) N-acylsphingosines (ceramides): N-(tetradecanoyl)-sphing-4-enine O O
H OH
H OH NH H
O P
O NH H HO
OH
O
NH2
O
(i) Acylceramides: N-(30-(9Z,12Z-octadecadienoyloxy)-tricontanoyl)-sphing-4-enine
(j) Phosphonosphingolipids: N-(tetradecanoyl)-sphing-4-enine-1-(2-aminoethylphosphonate)
H OH NH H
O
HO
OH O
OH OH
O (k) Neutral Glycosphingolipids: Simple Glc series: Glcβ-Cer(d18:1/12:0)
FigUre 5 Representative structures for sphingolipids.
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A Comprehensive Classification System for Lipids
181
H OH NH H
O
HO
O
O
OH
O HOOC
HO HO
O
OH OH
OH OH
O HO HN
OR
NH
O O
OH O OH
OH =R
O
O HO
OH
HO O
H OH
OH
NH H
O
(l) Acidic Glycosphingolipids: Gangliosides: Galβ1–3GalNAcβ1–4(NeuAcα2–3)Galβ1–4Glcβ-Cer(d18:1/18:0) H OH NH H
O
HO
O
HO
S
O
O OH
O
OH
O (m) Acidic Glycosphingolipids: Sulfosphingolipids: (3'-sulfo)Galβ-Cer(d18:1/18:0)
OH O
OH COOH
O (n) Acidic Glycosphingolipids: Glucuronosphingolipids: GlcUAβ-Cer(d18:1/18:0)
FigUre 5 Representative structures for sphingolipids (Continued)
TABLE 8: Sterol Lipids [ST] Classes and Subclasses Sterols [ST01] Cholesterol and derivatives [ST0101] Cholesteryl esters [ST0102] Phytosterols and derivatives [ST0103] Marine sterols and derivatives [ST0104] Fungal sterols and derivatives [ST0105] Steroids [ST02] C18 steroids (estrogens) and derivatives [ST0201] C19 steroids (androgens) and derivatives [ST0202] C21 steroids (gluco/mineralocorticoids, progestogins) and derivatives [ST0203] Secosteroids [ST03] Vitamin D2 and derivatives [ST0301] Vitamin D3 and derivatives [ST0302] Bile acids and derivatives [ST04] C24 bile acids, alcohols, and derivatives [ST0401] C26 bile acids, alcohols, and derivatives [ST0402] C27 bile acids, alcohols, and derivatives [ST0403] C28 bile acids, alcohols, and derivatives [ST0404] Steroid conjugates [ST05] Glucuronides [ST0501] Sulfates [ST0502] Glycine conjugates [ST0503] Taurine conjugates [ST0504] Hopanoids [ST06] Other [ST00]
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182 H
H H
H
H
H
H
O
H
H
H
O
HO
(b) Cholesteryl esters: cholest-5-en-3β-yl dodecanoate
(a) Cholesterol and derivatives: cholesterol; cholest-5-en-3β-ol
O OH H
H H
H
H
HO (c) C18 steroids (estrogens) and derivatives: β-estradiol; 1,3,5[10]-estratriene-3,17β-diol
H
HO
H (d) C19 steroids (androgens) and derivatives: androsterone; 3α-hydroxy-5α-androstan-17-one H
O
H
OH OH
HO H
H
H
H
HO
O (e) C21 steroids and derivatives: cortisol;11β,17α,21-trihydroxypregn-4-ene-3,20-dione
(f ) Secosteroids: Vitamin D2 and derivatives: vitamin D2; (5Z,7E,22E)-(3S)-9,10-seco-5,7,10(19), 22-ergostatetraen-3-ol O
H
H
HO
OH
H
H
H H
H
HO
HO (g) Secosteroids: Vitamin D3 and derivatives: vitamin D3; (5Z,7E)-(3S)-9,10-seco-5,7,10(19)-cholestatrien-3-ol
HO
H (h) C24 bile acids, alcohols, and derivatives: cholic acid; 3α,7α,12α-trihydroxy-5β-cholan-24-oic acid
O
H
H H
O
H HO
H
H
OH
H
H HO
H OH
H OH
(i) C26 bile acids, alcohols, and derivatives: 3α,7α,12α-trihydroxy-27-nor-5β-cholestan-24-one
H HO
H OH
H (j) C27 bile acids, alcohols, and derivatives: 3α,7α,12α-trihydroxy-5β-cholestan-26-oic acid
FigUre 6 Representative structures for sterol lipids.
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A Comprehensive Classification System for Lipids
183
HO
H
H O HO HO HO
H O
O
O N H
S
OH O
H
H
H OH
H
H
HO
OH (k) Steroid conjugates: Glucuronides: 5α-androstane-3α-ol-17-one glucuronide
H (l) Steroid conjugates: Taurine conjugates: taurocholic acid; N-(3α,7α,12α-trihydroxy-5β-cholan-24-oyl)-taurine
O
H HO
O
H
O
N H
H
OH
O
O
H
H
H H OH H (m) Steroid conjugates: Glycine conjugates: glycocholic acid; N-(3α,7α,12α-trihydroxy-5βcholan-24-oyl)-glycine HO
O
H
H O (n) Steroid conjugates: Sulfates: 5α-androstane-3α-ol-17-one sulfate HO
S
H
O
H H H H (o) Hopanoids: diploptene; hop-22(29)-ene
FigUre 6 Representative structures for sterol lipids (Continued).
TABLE 9: Prenol Lipids [PR] Classes and Subclasses Isoprenoids [PR01] C5 isoprenoids [PR0101] C10 isoprenoids (monoterpenes) [PR0102] C15 isoprenoids (sesquiterpenes) [PR0103] C20 isoprenoids (diterpenes) [PR0104] C25 isoprenoids (sesterterpenes) [PR0105] C30 isoprenoids (triterpenes) [PR0106] C40 isoprenoids (tetraterpenes) [PR0107] Polyterpenes [PR0108] Quinones and hydroquinones [PR02] Ubiquinones [PR0201] Vitamin E [PR0202] Vitamin K [PR0203] Polyprenols [PR03] Bactoprenols [PR0301] Bactoprenol monophosphates [PR0302] Bactoprenol diphosphates [PR0303] Phytoprenols [PR0304] Phytoprenol monophosphates [PR0305] Phytoprenol diphosphates [PR0306] Dolichols [PR0307] Dolichol monophosphates [PR0308] Dolichol diphosphates [PR0309] Other [PR00]
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184 O P
O P
O O OH HO HO (a) C5 isoprenoids: dimethylallyl pyrophosphate; 3-methylbut-2-enyl pyrophosphate
OH (b) C10 isoprenoids; 2E-geraniol
OH OH (c) C15 isoprenoids; 2E,6E-farnesol
HO
(d) C20 isoprenoids; retinol: vitamin A
O HO
O
O
H
(e) C25 isoprenoids: manoalide
O
(f ) C30 isoprenoids: 3S-squalene-2,3-epoxide
O H3CO H
H3CO O (g) C40 isoprenoids: β-carotene
10
(h) Ubiquinones: ubiquinone-10 (Co-Q10); 2-methyl-3-decaprenyl-5,6-dimethoxy-1,4-benzoquinone
O O H 6 O (i) vitamin K: vitamin K2(30): 2-methyl, 3-hexaprenyl-1,4-naphthoquinone; menaquinone-6
HO
H
H
(j) vitamin E: (2R,4'R,8'R)-α-tocopherol
FigUre 7 Representative structures for prenol lipids.
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A Comprehensive Classification System for Lipids
185
15
OH (k) Dolichols: Dol-19; α-dihydrononadecaprenol
OH
OH O
HO HO O
O
NH
O O
O P
O O O HO HO
NH
O
HN O HO
O P
O
NH
O O
HN O
O NH
NH2
O
HN HO
OH
O (l) Bactoprenol diphosphates: The Lipid II peptidoglycan precursor in E. coli; undecaprenyl diphosphate glycan
FigUre 7 Representative structures for prenol lipids (Continued).
TABLE 10: Saccharolipids [SL]Classes and Subclasses Acylaminosugars [SL01] Monoacylaminosugars [SL0101] Diacylaminosugars [SL0102] Triacylaminosugars [SL0103] Tetraacylaminosugars [SL0104] Pentaacylaminosugars [SL0105] Hexaacylaminosugars [SL0106] Heptaacylaminosugars [SL0107] Acylaminosugar glycans [SL02] Acyltrehaloses [SL03] Acyltrehalose glycans [SL04] Other [SL00]
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Handbook of Biochemistry and Molecular Biology
186 OH HO
O
O
H2N
O P
O HO
HO
OH
O
O
O P
O HO
HO
NH O
N
O
HO
O
O
O
O O
HO
HO
OH
(a) Acylaminosugars: Monoacylaminosugars: UDP-3-O-(3R-hydroxy-tetradecanoyl)-GlcN
(b) Acylaminosugars: Diacylaminosugars: lipid X HO HO HO
OH O
O
HO HO OH
OH
HO O O HO O HO
OH O P
OH O O
O HO HO
O
O O
O NH P O OH HO
O
HO O HO O O
O
NH HO
O
O
O
O P
O NH P O OH HO
O O
O
O
NH O O
O
HO O
HO
O
O O
O NH P O OH HO
HO
HO
(c) Acylaminosugars: Tetraacylaminosugars: lipid IVA
(d) Acylaminosugar glycans: Kdo2 lipid A
FigUre 8 Representative structures for saccharolipids. TABLE 11: Polyketides [PK] Classes and Subclasses Macrolide polyketides [PK01] Aromatic polyketides [PK02] Nonribosomal peptide/polyketide hybrids [PK03] Other [PK00]
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187
O O O
OH O
O
OH
O
OH
(a) Macrolide polyketides: 6-deoxyerythronolide B
HO
OH
O
O O
O OH OH O (b) Aromatic polyketides: griseorhodin A
S OH
N O O
OH
O
(c) Polyketide hybrids: epothilone D
FigUre 9 Representative structures for polyketides. lipid database that can be searched for substructure and chemical similarity.
Discussion The goals of the LIPID MAPS initiative are to characterize known lipids and identify new ones, to quantitate temporal and spatial changes in lipids that occur with cellular metabolism, and to develop bioinformatics approaches that establish dynamic lipid networks; the goals of Lipid Bank (Japan) are to annotate and curate lipid structures and the literature associated with them; and the goals of the European Lipidomics Initiative are to coordinate and organize scientific interactions and workshops associated with lipid research. To coordinate the independent efforts from three continents and to facilitate collaborative work, a comprehensive classification of lipids with a common platform that is compatible with informatics requirements must be developed to deal with the massive amounts of data that will be generated by the lipid community. The proposed classification, nomenclature, and chemical representation system was initially designed to accommodate the massive data that will result from the LIPID MAPS effort, but it has been expanded to accommodate as many lipids as possible. We also have attempted to make the system compatible with existing lipid databases and the lipids currently annotated in them. It is designed to be expandable should new categories, classes, or subclasses be required in the future, and updates will be maintained on the LIPID MAPS website. The development of this system has been enriched by interaction with lipidologists across the world in the hopes that this system will be internationally accepted and used.jlr The authors appreciate the agreement of the International Lipids Classification and Nomenclature Committee to advise on future issues involving the maintenance of these recommendations. This committee currently includes Edward A. Dennis (chair), Christian Raetz and Robert Murphy representing LIPID MAPS, Friedrich Spener representing the International Conference on the Biosciences of Lipids, Gerrit van Meer representing the European Lipidomics Initiative, and Yousuke Seyama and Takao Shimizu representing the LipidBank of the Japanese Conference on the Biochemistry of Lipids. The authors are most
9168_Book.indb 187
appreciative of informative discussions and encouragement of this effort with Professor Richard Cammack, King’s College, London, who is the Chairman of the Nomenclature Committee of IUBMB and the IUPAC/IUBMB Joint Commission on Biochemical Nomenclature. The authors thank the Consortium for Functional Glycomics (headed by Ram Sasisekharan at the Massachusetts Institute of Technology) for providing us with their text nomenclature for glycosylated structures. We are grateful to Dr. Jean Chin, Program Director at the National Institutes of General Medical Sciences, for her valuable input to this effort. This work was supported by the LIPID MAPS Large-Scale Collaborative Grant GM-069338 from the National Institutes of Health.
References 1. Smith, A. 2000. Oxford Dictionary of Biochemistry and Molecular Biology. 2nd edition. Oxford University Press, Oxford, UK. 2. Christie, W. W. 2003. Lipid Analysis. 3rd edition. Oily Press, Bridgewater, UK. 3. IUPAC-IUB Commission on Biochemical Nomenclature (CBN). The nomenclature of lipids (recommendations 1976). 1977. Eur. J. Biochem. 79: 11–21; 1977. Hoppe-Seylers Z. Physiol. Chem. 358: 617–631; 1977. Lipids. 12: 455–468; 1977. Mol. Cell. Biochem. 17: 157–171; 1978. Chem. Phys. Lipids. 21: 159–173; 1978. J. Lipid Res. 19: 114–128; 1978. Biochem. J. 171: 21–35 (http://www.chem.qmul. ac.uk/iupac/lipid/). 4. IUPAC-IUB Joint Commission on Biochemical Nomenclature (JCBN). Nomenclature of glycolipids (recommendations 1997). 2000. Adv. Carbohydr. Chem. Biochem. 55: 311–326; 1988. Carbohydr. Res. 312: 167–175; 1998. Eur. J. Biochem. 257: 293–298; 1999. Glycoconjugate J. 16: 1–6; 1999. J. Mol. Biol. 286: 963–970; 1997. Pure Appl. Chem. 69: 2475–2487 (http://www.chem.qmul.ac.uk/iupac/misc/glylp.html). 5. IUPAC-IUB Joint Commission on Biochemical Nomenclature (JCBN). 1987. Nomenclature of prenols (recommendations 1987). Eur. J. Biochem. 167: 181–184 (http://www.chem.qmul.ac.uk/iupac/ misc/prenol.html). 6. IUPAC-IUB Joint Commission on Biochemical Nomenclature (JCBN). 1989. Nomenclature of steroids (recommendations 1989). Eur. J. Biochem. 186: 429–458 (http://www.chem.qmul.ac.uk/iupac/ steroid/).
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188 7. Caffrey, M., and J. Hogan. 1992. LIPIDAT: a database of lipid phase transition temperatures and enthalpy changes. Chem. Phys. Lipids. 61: 1–109 (http://www.lipidat.chemistry.ohio-state.edu). 8. Watanabe, K., E. Yasugi, and M. Ohshima. 2000. How to search the glycolipid data in “Lipidbank for web,” the newly-developed lipid database in Japan. Trends Gycosci. Glycotechnol. 12: 175–184. 9. Vance, D. E., and J. E. Vance, editors. 2002. Biochemistry of Lipids, Lipoproteins and Membranes. 4th edition. Elsevier Science, New York. 10. Small, D. M. 1986. The Physical Chemistry of Lipids. Handbook of Lipid Research. Vol. 4. D. J. Hanahan, editor. Plenum Press, New York. 11. Brennan, P. J., and H. Nikaido. 1995. The envelope of mycobacteria. Annu. Rev. Biochem. 64: 29–63. 12. Ohlrogge, J. B. 1997. Regulation of fatty acid synthesis. Annu. Rev. Plant Physiol. Plant Mol. Biol. 48: 109–136. 13. Agrawal, G. K., S. Tamogami, O. Han, H. Iwahasi, and R. Rakwal. 2004. Rice octadecanoid pathway. Biochem. Biophys. Res. Commun. 317: 1–15. 14. Murphy, R. C., and W. L. Smith. 2002. The eicosanoids: cyclooxygenase, lipoxygenase, and epoxygenase pathways. In Biochemistry of Lipids, Lipoproteins and Membranes. 4th edition. D. E. Vance and J. E. Vance, editors. Elsevier Science, New York. 341–371. 15. Bazan, N. G. 1989. The metabolism of omega-3 polyunsaturated fatty acids in the eye: the possible role of docosahexaenoic acid and docosanoids in retinal physiology and ocular pathology. Prog. Clin. Biol. Res. 312: 95–112. 16. Roche, D. M., J. T. Byers, D. S. Smith, F. G. Glansdorp, D. R. Spring, and M. Welch. 2004. Communications blackout? Do N-acylhomoserinelactone-degrading enzymes have any role in quorum sensing? Microbiology. 150: 2023–2028. 17. Stam, H., K. Schoonderwoerd, and W. C. Hulsmann. 1987. Synthesis, storage and degradation of myocardial triglycerides. Basic Res. Cardiol. 82 (Suppl. 1): 19–28. 18. Coleman, R. A., and D. P. Lee. 2004. Enzymes of triacylglycerol synthesis and their regulation. Prog. Lipid Res. 43: 134–176. 19. Pahlsson, P., S. L. Spitalnik, P. F. Spitalnik, J. Fantini, O. Rakotonirainy, S. Ghardashkhani, J. Lindberg, P. Konradsson, and G. Larson. 1998. Characterization of galactosyl glycerolipids in the HT29 human colon carcinoma cell line. Arch. Biochem. Biophys. 396: 187–198. 20. Koga, Y., M. Nishihara, H. Morii, and M. Akagawa-Matsushita. 1983. Ether polar lipids of methanogenic bacteria: structures, comparative aspects and biosyntheses. Microbiol. Rev. 57: 164–182. 21. Pereto, J., P. Lopez-Garcia, and D. Moreira. 2004. Ancestral lipid biosynthesis and early membrane evolution. Trends Biochem. Sci. 29: 469–477. 22. Kennedy, E. P. 1962. The metabolism and function of complex lipids. Harvey Lecture Series. 57: 143–171. 23. G. Cevc, editor. 1993. Phospholipids Handbook. Marcel Dekker Inc., New York. 24. Forrester, J. S., S. B. Milne, P. T. Ivanova, and H. A. Brown. 2004. Computational lipidomics: a multiplexed analysis of dynamic changes in membrane lipid composition during signal transduction. Mol. Pharmacol. 65: 813–821. 25. Ivanova, P. T., S. B. Milne, J. S. Forrester, and H. A. Brown. 2004. Lipid arrays: new tools in the understanding of membrane dynamics and lipid signaling. Mol. Interventions. 4: 86–96. 26. Cronan, J. E. 2003. Bacterial membrane lipids: where do we stand? Annu. Rev. Microbiol. 57: 203–224. 27. Merrill, A. H., Jr., and K. Sandhoff. 2002. Sphingolipids: metabolism and cell signaling. In New Comprehensive Biochemistry: Biochemistry of Lipids, Lipoproteins, and Membranes. D. E. Vance and J. E. Vance, editors. Elsevier Science, New York. 373–407.
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Handbook of Biochemistry and Molecular Biology 28. Taniguchi, N., K. Honke, and M. Fukuda. 2002. Handbook of Glycosyltransferases and Related Genes. Springer-Verlag, Tokyo. 29. Thudichum, J. L. W. 1884. A Treatise on the Chemical Constitution of Brain. Bailliere, Tindall, and Cox, London. 30. Bach, D., and E. Wachtel. 2003. Phospholipid/cholesterol model membranes: formation of cholesterol crystallites. Biochim. Biophys. Acta. 1610: 187–197. 31. Tsai, M. J., and B. W. O’Malley. 1994. Molecular mechanisms of action of steroid/thyroid receptor superfamily members. Annu. Rev. Biochem. 63: 451–486. 32. Jones, G., S. A. Strugnell, and H. F. DeLuca. 1998. Current understanding of the molecular actions of vitamin D. Physiol. Rev. 78: 1193–1231. 33. Russell, D. W. 2003. The enzymes, regulation, and genetics of bile acid synthesis. Annu. Rev. Biochem. 72: 137–174. 34. Kuzuyama, T., and H. Seto. 2003. Diversity of the biosynthesis of the isoprene units. Nat. Prod. Rep. 20: 171–183. 35. Rodriguez-Concepcion, M. 2004. The MEP pathway: a new target for the development of herbicides, antibiotics and antimalarial drugs. Curr. Pharm. Res. 10: 2391–2400. 36. Porter, J. W., and S. L. Spurgeon. 1981. Biosynthesis of Isoprenoid Compounds. Vol. 1. John Wiley & Sons, New York. 37. Demming-Adams, B., and W. W. Adams. 2002. Antioxidants in photosynthesis and human nutrition. Science. 298: 2149–2153. 38. Ricciarelli, R., J. M. Zingg, and A. AzziI. 2001. Vitamin E: protective role of a Janus molecule. FASEBJ. 15: 2314–2325. 39. Meganathan, R. 2001. Biosynthesis of menaquinone (vitamin K2) and ubiquinone (coenzyme Q): a perspective on enzymatic mechanisms. Vitam. Horm. 61: 173–218. 40. Meganathan, R. 2001. Ubiquinone biosynthesis in microorganisms. FEMS Microbiol. Lett. 203: 131–139. 41. Raetz, C. R. H., and C. Whitfield. 2002. Lipopolysaccharide endotoxins. Annu. Rev. Biochem. 71: 635–700. 42. Lazar, K., and S. Walker. 2002. Substrate analogues to study cell-wall biosynthesis and its inhibition. Curr. Opin. Chem. Biol. 6: 786–793. 43. Schenk, B., F. Fernandez, and C. J. Waechter. 2001. The ins(ide) and out(side) of dolichyl phosphate biosynthesis and recycling in the endoplasmic reticulum. Glycobiology. 11: 61R–71R. 44. Helenius, J., and M. Aebi. 2001. Intracellular functions of N-linked glycans. Science. 291: 2364–2369. 45. Zähringer, U., B. Lindner, and E. T. Rietschel. 1999. Chemical structure of Lipid A: recent advances in structural analysis of biologically active molecules. In Endotoxin in Health and Disease. H. Brade, S. M. Opal, S. N. Vogel, and D. C. Morrison, editors. Marcel Dekker, New York. 93–114. 46. Sweet, C. R., A. A. Ribeiro, and C. R. Raetz. 2004. Oxidation and transamination of the 3’-position of UDP-N-acetylglucosamine by enzymes from Acidithiobacillus ferrooxidans. Role in the formation of lipid A molecules with four amide-linked acyl chains. J. Biol. Chem. 279: 25400–25410. 47. Spaink, H. P. 2000. Root nodulation and infection factors produced by rhizobial bacteria. Annu. Rev. Microbiol. 54: 257–288. 48. Ghangas, G. S., and J. C. Steffens. 1993. UDP glucose: fatty acid transglucosylation and transacylation in triacylglucose biosynthesis. Proc. Natl. Acad. Sci. USA. 90: 9911–9915. 49. Walsh, C. T. 2004. Polyketide and nonribosomal peptide antibiotics: modularity and versatility. Science. 303: 1805–1810. 50. Khosla, C., R. Gokhale, J. R. Jacobsen, and D. E. Cane. 1999. Tolerance and specificity of polyketide synthases. Annu. Rev. Biochem. 68: 219–253. 51. Reeves, C. D. 2003. The enzymology of combinatorial biosynthesis. Crit. Rev. Biotechnol. 23: 95–147. 52. Moore, B. S., and C. Hartweck. 2002. Biosynthesis and attachment of novel bacterial polyketide synthase starter units. Nat. Prod. Rep. 19: 70–99.
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Properties of Fatty Acids and Their Methyl Esters This table gives the names and selected properties of some important fatty acids and their methyl esters. It includes most of the acids that are significant constituents of naturally occurring oils and fats. Compounds are listed first by number of carbon atoms and, secondly, by the degree of unsaturation. Both the systematic name and the common or trivial name are given, as well as the Chemical Abstracts Service Registry Number and the shorthand acid code that is frequently used. The first number in this code gives the number of carbon atoms; the number following the colon is the number of unsaturated centers (mainly double bonds). The location and orientation of the unsaturated centers follow. The symbols used are: c = cis; t = trans; a = acetylenic center; e = ethylenic center at end of chain; ep = epoxy. Thus 9c,11t indicates a double bond with cis orientation at the No. 9 carbon and another with trans orientation at the No. 11 carbon. More details on the codes can be found in Reference 1. The table gives the molecular weight and melting point of the acid and the melting and boiling points of the methyl ester of the acid when available. A superscript on the boiling point indicates the pressure in mmHg (torr); if there is no superscript, the value refers to one atmosphere (760 mmHg). The references cover many
Systematic Name
References 1. Gunstone, F. D., Harwood, J. L., and Dijkstra, A. J., eds., The Lipid Handbook, Third Edition, CRC Press, Boca Raton, FL, 2006. 2. Gunstone, F. D., and Adlof, R. O., Common (non-systematic) Names for Fatty Acids, www.aocs.org/member/division/analytic/fanames. asp, 2003. 3. Firestone, D., Physical and Chemical Characteristics of Oils, Fats, and Waxes, Second Edition, AOCS Press, Urbana, IL, 2006. 4. Dawson, R. M. C., Elliott, D. C., Elliott, W. H., and Jones, K. M., Data for Biochemical Research, Third Edition, Clarendon Press, Oxford, 1986. 5. Altman, P. L., and Dittmer, D. S., eds., Biology Data Book, Second Edition, Vol. 1, Federation of American Societies for Experimental Biology, Bethesda, MD, 1972. 6. Fasman, G. D., Ed., Practical Handbook of Biochemistry and Molecular Biology, CRC Press, Boca Raton, FL, 1989.
Acid Code
CAS RN
Mol. Weight
Palmitic acid Palmitoleic acid Margaric acid Stearic acid Petroselinic acid Oleic acid Elaidic acid cis-Vaccenic acid Vaccenic acid Vernolic acid
C5H10O2 C6H12O2 C7H14O2 C8H16O2 C9H18O2 C10H20O2 C10H18O2 C11H22O2 C12H24O2 C12H22O2 C13H26O2 C14H28O2 C14H26O2 C15H30O2 C16H32O2 C16H30O2 C17H34O2 C18H36O2 C18H34O2 C18H34O2 C18H34O2 C18H34O2 C18H34O2 C18H32O3
4:0 5:0 4:0 3-Me 6:0 7:0 8:0 9:0 10:0 10:1 9e 11:0 12:0 12:1 9c 13:0 14:0 14:1 9c 15:0 16:0 16:1 9c 17:0 18:0 18:1 6c 18:1 9c 18:1 9t 18:1 11c 18:1 11t 18:1 12,13-ep,9c
107-92-6 109-52-4 503-74-2 142-62-1 111-14-8 124-07-2 112-05-0 334-48-5 14436-32-9 112-37-8 143-07-7 2382-40-3 638-53-9 544-63-8 13147-06-3 1002-84-2 57-10-3 373-49-9 506-12-7 57-11-4 593-39-5 112-80-1 112-79-8 506-17-2 693-72-1 503-07-1
88.106 102.132 102.132 116.158 130.185 144.212 158.238 172.265 170.249 186.292 200.318 198.302 214.344 228.371 226.355 242.398 256.424 254.408 270.451 284.478 282.462 282.462 282.462 282.462 282.462 296.445
5.5
Common Name
Mol. Form.
other fatty acids beyond those listed here and give additional properties. We are indebted to Frank D. Gunstone for advice on the content of the table.
Butanoic acid Pentanoic acid
Butyric acid Valeric acid
3-Methylbutanoic acid Hexanoic acid Heptanoic acid Octanoic acid Nonanoic acid Decanoic acid 9-Decenoic acid Undecanoic acid Dodecanoic acid cis-9-Dodecenoic acid Tridecanoic acid Tetradecanoic acid cis-9-Tetradecenoic acid Pentadecanoic acid Hexadecanoic acid cis-9-Hexadecenoic acid Heptadecanoic acid Octadecanoic acid cis-6-Octadecenoic acid cis-9-Octadecenoic acid trans-9-Octadecenoic acid cis-11-Octadecenoic acid trans-11-Octadecenoic acid cis-12,13-Epoxy-cis-9-octadecenoic acid 12-Hydroxy-cis-9-octadecenoic acid
Isovaleric acid Caproic acid Enanthic acid Caprylic acid Pelargonic acid Capric acid Caproleic acid
Ricinoleic acid
C18H34O3
18:1 12-OH,9c
141-22-0
298.461
cis,trans-9,11-Octadecadienoic acid cis,cis-9,12-Octadecadienoic acid
Rumenic (CLA) Linoleic acid
C18H32O2 C18H32O2
18:2 9c,11t 18:2 9c,12c
1839-11-8 60-33-3
280.446 280.446
Lauric acid Lauroleic acid Myristic acid Myristoleic acid
C4H8O2 C5H10O2
mp/°C
Methyl Ester
mp/°C
bp/°C
−5.1 −33.6 −29.3 −3 −7.2 16.5 12.4 31.4 26.5 28.6 43.8
−85.8
5.2
102.8 127.4 116.5 149.5 174 192.9 213.5 224 12020 12310 267
41.5 54.2
6.5 19
921 295
18.5 30
153.5 417 1405 1859 443
−4 52.3 62.5 0.5 61.3 69.3 29.8 13.4 45 15 44 32.5
20 −7
−71 −56 −40 −18
30 39.1 −19.9 13.5
218.520 21824 1630.1 1723 22615
−35
21520
189
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Handbook of Biochemistry and Molecular Biology
190
Properties of Fatty Acids and Their Methyl Esters (Continued) Systematic Name
Common Name
Mol. Form.
Acid Code
CAS RN
Mol. Weight
mp/°C
bp/°C
−12
trans,cis-10,12-Octadecadienoic acid cis-9-Octadecen-12-ynoic acid cis,cis,cis-5,9,12-Octadecatrienoic acid trans,cis,cis-5,9,12-Octadecatrienoic acid cis,cis,cis-6,9,12-Octadecatrienoic acid trans,trans,cis-8,10,12Octadecatrienoic acid cis,trans,cis-9,11,13Octadecatrienoic acid cis,trans,trans-9,11,13Octadecatrienoic acid trans,trans,cis-9,11,13Octadecatrienoic acid trans,trans,trans-9,11,13Octadecatrienoic acid cis,cis,cis-9,12,15-Octadecatrienoic acid 6,9,12,15-Octadecatetraenoic acid, all cis cis,trans,trans,cis-9,11,13,15Octadecatetraenoic acid Nonadecanoic acid Eicosanoic acid 3,7,11,15Tetramethylhexadecanoic acid cis-5-Eicosenoic acid cis-9-Eicosenoic acid cis-11-Eicosenoic acid cis,cis,cis-8,11,14Eicosatrienoic acid 5,8,11,14-Eicosatetraenoic acid, all cis 5,8,11,14,17Eicosapentaenoic acid, all cis Heneicosanoic acid Docosanoic acid cis-11-Docosenoic acid cis-13-Docosenoic acid trans-13-Docosenoic acid cis,cis-5,13-Docosadienoic acid 7,10,13,16,19-Docosapentaenoic acid, all cis 4,7,10,13,16,19Docosahexaenoic acid, all cis Tricosanoic acid Tetracosanoic acid cis-15-Tetracosenoic acid Pentacosanoic acid
(CLA)
C18H32O2
18:2 10t,12c
22880-03-1
280.446
Crepenynic acid Pinolenic acid
C18H30O2 C18H30O2
18:2 9c,12a 18:3 5c,9c,12c
2277-31-8 27213-43-0
278.430 278.430
Columbinic acid
C18H30O2
18:3 5t,9c,12c
2441-53-4
278.430
g-Linolenic acid
C18H30O2
18:3 6c,9c,12c
506-26-3
278.430
Calendic acid
C18H30O2
18:3 8t,10t,12c
28872-28-8
278.430
40
Punicic acid
C18H30O2
18:3 9c,11t,13c
544-72-9
278.430
45
a-Eleostearic acid Catalpic acid
C18H30O2
18:3 9c,11t,13t
506-23-0
278.430
49
C18H30O2
18:3 9t,11t,13c
4337-71-7
278.430
32
b-Eleostearic acid a-Linolenic acid
C18H30O2
18:3 9t,11t,13t
544-73-0
278.430
71.5
13
1621
C18H30O2
18:3 9c,12c,15c
463-40-1
278.430
−11.3
−52
1090.018
Stearidonic acid
C18H28O2
18:4 6c,9c,12c,15c
20290-75-9
276.414
−57
Parinaric acid
C18H28O2
18:4 9c,11t,13t,15c
593-38-4
276.414
86
Arachidic acid Phytanic acid
C19H38O2 C20H40O2 C20H40O2
646-30-0 506-30-9 14721-66-5
298.504 312.531 312.531
69.4 76.5
C20H38O2 C20H38O2 C20H38O2 C20H34O2
19:0 20:0 16:0 3,7,11,15-tetramethyl 20:1 5c 20:1 9c 20:1 11c 20:3 8c,11c,14c
7050-07-9 29204-02-2 2462-94-4 1783-84-2
310.515 310.515 310.515
27 24.5 24
C20H32O2
20:4 5c,8c,11c,14c
506-32-1
304.467
−49.5
20:5 5c,8c,11c,14c,17c 10417-94-4
302.451
−54
2363-71-5 112-85-6 506-36-5 112-86-7 506-33-2 676-39-1
326.557 340.583 338.567 338.567 338.567 336.552
Lignoceric acid Nervonic acid
C23H46O2 C24H48O2 C24H46O2 C25H50O2
21:0 22:0 22:1 11c 22:1 13c 22:1 13t 22:2 5c,13c 22:5 7c,10c,13c,16c, 19c 22:6 4c,7c,10c,13c, 16c,19c 23:0 24:0 24:1 15c 25:0
2433-96-7 557-59-5 506-37-6 506-38-7
368.637 366.621 382.664
79.6 87.5 43 77.5
53.4 60 15 62
Hexacosanoic acid
Cerotic acid
C26H52O2
26:0
506-46-7
396.690
88.5
63.8
C27H54O2
27:0
7138-40-1
87.6
64
C28H56O2
28:0
506-48-9
424.744
90.9
67
C29H58O2
29:0
4250-38-8
438.770
90.3
69
C30H60O2
30:0
506-50-3
452.796
93.6
72
C31H62O2
31:0
38232-01-8
466.823
93.1
C32H64O2
32:0
3625-52-3
480.849
96.2
Gadoleic acid Gondoic acid Dihomo-glinolenic acid Arachidonic acid
Timnodonic acid, C20H30O2 EPA C21H42O2 Behenic acid C22H44O2 Cetolic acid C22H42O2 Erucic acid C22H42O2 Brassidic acid C22H42O2 C22H40O2 C22H34O2 Cervonic acid, DHA
Heptacosanoic acid Octacosanoic acid
Montanic acid
Nonacosanoic acid Triacontanoic acid
Melissic acid
Hentriacontanoic acid Dotriacontanoic acid
9168_Book.indb 190
Lacceric acid
C22H32O2
23
Methyl Ester
mp/°C
1620.5
−65
82 81.5 33 34.7 61.9
−4
1481
41.3 54.5
1904 21510
1950.7
49 54
2074
2215 35
−45
2091-24-9
1650.02 28615
1920.01
4/16/10 1:15 PM
DENSITIES, SPECIFIC VOLUMES, AND TEMPERATURE COEFFICIENTS OF FATTY ACIDS FROM C8 TO C12 Acid Caprylic
Nonanoic
Capric
Hendecanoic
Lauric
Temperature, °C
Density,a g/cc
Specific Volume, l/d
Temp. Coeff. per °C
10.0 15 20 20.02 25 50.27 5.0 10 15 15.00 25 15.0 25 35 35.05 40 0.12 10.0 20 25 30 30.00 35 50.15 35.0 40 45 45.10 50 50.25
1.0326 1.0274 0.9109 0.9101b 0.9090 0.8862b 0.9952 0.9916 0.9097 0.9087b 0.9011 1.0266 1.0176 0.8927 0.8884b 0.8876 1.0431 1.0373 0.9948 0.9905 0.8907 0.8889b 0.8871 0.8741b 1.0099 1.0055 0.8767 0.8744b 0.8713 0.8707b
0.9685 0.9733 1.0979 — 1.1002 — 1.0048 1.0085 1.0993 — 1.1097 0.9741 0.9827 1.1202 — 1.1266 0.9587 0.9640 1.0052 1.0096 1.1227 — 1.1273 — 0.9902 0.9945 1.1406 — 1.1477 —
0.00098 — 0.00046 — — 0.00099 0.00074 — 0.00104 — — 0.00085 — 0.00128 — — 0.00054 — 0.00079 — 0.00093 — — 0.00095 0.00087 — 0.00142 0.00095 — 0.00095
By air thermometer method unless specified otherwise. By pycnometer method.
a
b
From Markley, Klare S., Fatty Acids, 2nd ed., Part 1, Interscience Publishers, Inc., New York, 1960, 535. With permission of copyright owners.
191
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Composition and Properties of Common Oils and Fats This table lists some of the most common naturally occurring oils and fats. The list is separated into those of plant origin, fish and other marine life origin, and land animal origin. The oils and fats consist mainly of esters of glycerol (i.e., triglycerides) with fatty acids of 10 to 22 carbon atoms. The four fatty acids with the highest concentration are given for each oil; concentrations are given in weight percent. Because there is often a wide variation in composition depending on the source of the oil sample, a range (or sometimes an average) is generally given. More complete data on composition, including minor fatty acids, sterols, and tocopherols, can be found in the references. The acids are labeled by the codes described in the previous table, “Properties of Fatty Acids and Their Methyl Esters,” which gives the systematic and common names of the acids. Thus 18:2 9c,12c indicates a C18 acid with two double bonds in the 9 and 12 positions, both with a cis configuration (cis,cis-9,12-octadecadienoic acid, or linoleic acid). The density and refractive index of the oils are typical values; superscripts indicate the temperature in °C. Notes: • The composition figure given for oleic acid (18:1 9c) often includes low levels of other 18:1 isomers.
Type of Oil
• • •
In some oils where a concentration is given for 18:2 9c,12c (linoleic acid), other isomers of 18:2 may be included. Likewise, where a concentration is given for 18:3 9c,12c,15c (α-linolenic acid), other isomers of 18:3 may be included. The acid 20:5 6c,9c,12c,15c,17c, which is prevalent in many fish oils, is often abbreviated as 20:5 ω-3 or 20:5 n-3.
The assistance of Frank D. Gunstone in preparing this table is gratefully acknowledged.
References 1. Firestone, D., Physical and Chemical Characteristics of Oils, Fats, and Waxes, Second Edition, AOCS Press, Urbana, IL, 2006. 2. Gunstone, F. D., Harwood, J. L., and Dijkstra, A. J., eds., The Lipid Handbook, Third Edition, CRC Press, Boca Raton, FL, 2006. 3. Dawson, R. M. C., Elliott, D. C., Elliott, W. H., and Jones, K. M., Data for Biochemical Research, Third Edition, Clarendon Press, Oxford, 1986. 4. Altman, P. L., and Dittmer, D. S., eds., Biology Data Book, Second Edition, Vol. 1, Federation of American Societies for Experimental Biology, Bethesda, MD, 1972.
Principal Fatty Acid Components in Weight %
mp/ Density/ Refractive °C g cm–3 Index
Iodine Value
Saponification Value
Plants Almond kernel oil Apricot kernel oil Argan seed oil Avocado pulp oil Babassu palm oil Blackcurrant oil Borage (star-flower) oil Borneo tallow Cameline oil Canola (rapeseed) oil (low linolenic) Canola (rapeseed) oil (low erucic) Caraway seed oil Cashew nut oil
18:1 9c 16:0 18:1 9c 16:0 18:1 9c 16:0 18:1 9c 16:0 12:0 18:1 9c 18:2 9c,12c 18:3 9c,12c,15c
43–70% 4–13% 58–66% 4.6–6% 42–55% 12–16% 56–74% 9–18% 40–55% 9–20% 45–50% 12–15%
18:2 9c,12c 18:0 18:2 9c,12c 18:0 18:2 9c,12c 18:0 18:2 9c,12c 16:1 9c 14:0 16:0 18:3 6c,9c,12c 18:1 9c
24–30% 1–10% 29–33% 1% 30–34% 2–7% 10–17% 3–9% 11–27% 5.2–11% 14–20% 9–13%
18:2 9c,12c 18:1 9c 18:0 16:0 18:3 9c,12c,15c 20:1 total 18:1 9c 16:0 18:1 9c 18:3 9c,12c,15c 18:1 9c 18:1 6c 18:1 9c 16:0
36–40% 14–21% 39–43% 18–21% 33–38% 14–16% 59–66% 4–5% 52–67% 6–14% 40% 26% 57–80% 4–17%
18:3 6c,9c,12c 16:0 18:1 9c 20:0 18:2 9c,12c 18:1 9c 18:2 9c,12c 18:3 9c,12c,15c 18:2 9c,12c 16:0 18:2 9c,12c 16:0 18:2 9c,12c 18:0
17–25% 9.4–12% 34–37% 1.0% 15–16% 12–24% 24–29% 2–3% 16–25% 3.3–6.0% 30% 3% 16–22% 2–12%
24
38
0.91025
1.46726
89–101
188–200
0.91025
1.46925
97–110
185–199
0.91220
1.46720
92–102
189–195
0.91225
1.46625
85–90
177–198
0.91425
1.45040
10–18
245–256
0.92320
1.48020
173–182
185–195
141–160
189–192
0.855100
1.45640
29–38
189–200
0.92415
1.47720
127–155
180–190
−10 −10
91 0.91520
0.91415
1.46640
110–126
182–193
1.47135
128
178
1.46340
79–89
180–196
193
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194
Composition and Properties of Common Oils and Fats (Continued) Type of Oil
Principal Fatty Acid Components in Weight %
mp/ Density/ Refractive °C g cm–3 Index
Iodine Value
Saponification Value
−18
Plants (Continued) Castor oil Cherry kernel oil Chinese vegetable tallow Cocoa butter Coconut oil Cohune nut oil Coriander seed oil Corn oil Cottonseed oil Crambe oil Cuphea seed oil (caprylic acid rich) Euphorbia lagascae seed oil Evening primrose oil Grape seed oil Hazelnut oil (Chilean) Hazelnut oil (Filbert) Hempseed oil Illipe (mowrah) butter Jojoba oila Kapok seed oilb Kokum butter Kusum oil Linola oil Linseed oil Macadamia nut oil Mango seed oil
9168_Book.indb 194
18:1 12-OH,9c 18:1 9c 18:2 9c,12c 16:0 16:0 18:0 18:0 16:0 12:0 16:0 12:0 18:1 9c 18:1 6c 18:2 9c,12c 18:2 9c,12c 16:0 18:2 9c,12c 18:1 9c 22:1 13c 18:2 9c,12c 8:0 18:2 9c,12c 18:1 12,13-ep,9c 18:2 9c,12c 18:2 9c,12c 16:0 18:2 9c,12c 16:0 18:1 9c 20:1 total 18:1 9c 16:0 18:2 9c,12c 18:1 9c 18:1 9c 18:0 20:1 total 18:1 9c 18:1 9c 18:2 9c,12c 18:0 16:0 18:1 9c 16:0 18:2 9c,12c 16:0 18:3 9c,12c,15c
88% 2.9–6% 42–45% 4–9% 58–72% 1–8% 31–37% 25–27% 45–51% 7.7–10.2% 44–48% 8–10% 53% 7–14% 40–66% 9–16% 47–58% 14–22% 55–60% 8–10% 65–78% 1–4% 64% 9% 65–80% 6–10% 58–78% 5.5–11% 39% 9.7% 72–84% 4.1–7.2% 45–60% 11–16% 34% 23% 66–74% 5–12% 45–65% 7–35% 49–56% 2–5% 57–62% 5–8% 72% 5.6% 52–58%
18:2 9c,12c 22:0 18:1 9c 18:3 9c,11t,13t 18:1 9c 14:0 18:1 9c 18:2 9c,12c 14:0 18:1 9c 14:0 16:0 18:1 9c 16:0 18:1 9c 18:0 16:0 18:0 18:1 9c 18:3 9c,12c,15c 10:0 16:0 18:1 other 16:0 18:3 6c,9c,12c 18:1 9c 18:1 9c 18:0 16:1 11c 22:1 total 18:2 9c,12c 18:0 18:3 9c,12c,15c 16:0 16:0 18:2 9c,12c 22:1 undefined 24:1 15c 16:0 18:0 18:1 9c 18:2 9c,12c 20:0 18:0 18:1 9c 18:0 18:1 9c
3–5% 2.1% 35–49% 3–10% 20–35% 0.5–3.7% 31–35% 2.8–4.0% 17–21% 5.4–9.9% 16–17% 7–10% 32% 3–8% 20–42% 0–3% 18–26% 2.1–3.3% 12–15% 6–7% 19–24% 0.6–3% 19% 4% 8–14% 5–12% 12–28% 3–6% 22.7% 9.5% 5.7–22% 1.5–2.4% 15–30% 6–12% 23% 14% 9–19% 1–5% 10–28% 2–9% 39–49% 1–2% 20–25% 2–6% 16% 4.0% 18–20%
18:2 9c,12c 18:1 9c 16:0 18:1 9c 18:2 9c,12c
17% 56–59% 8–9% 38–50% 3–6%
18:2 9c,12c 16:1 9c 18:0 18:0 20:0
16% 21–22% 2–4% 31–49% 2–6%
0.95225
1.47525
81–91
176–187
0.91825
1.46840
110–118
190–198
44
0.88725
1.45640
16–29
200–218
34
0.97425
1.45740
32–40
192–200
25
0.91340
1.44940
5–13
248–265
0.91425
1.45040
9–14
251–260
0.90825
1.46425
86–100
182–191
−20
0.91920
1.47225
107–135
187–195
−1
0.92020
1.46240
96–115
189–198
0.90625
1.47025
87–113
0.95225
1.47325
102
1.47920
147–155
193–198
0.92320
1.47540
130–138
188–194
0.90925
1.47325
83–90
188–197
0.92125
1.47240
145–166
190–195
27
0.862100
1.46040
53–70
188–207
30
0.92615
1.46925
86–110
189–197
1.45640
33–37
192
1.46140
48–58
220–230
41
142 −24
0.92425
1.48025
170–203
188–196
0.91215
1.46125
39–48
188–195
4/16/10 1:15 PM
Composition and Properties of Common Oils and Fats
195
Composition and Properties of Common Oils and Fats (Continued) Type of Oil Meadowfoam seed oil Melon oil Moringa peregrina seed oil Mustard seed oil Neem oil Niger seed oil Nutmeg butter Oat oil Oiticica oil Olive oil Palm kernel oil Palm oil Palm olein Palm stearin Parsley seed oil Peanut oil Perilla oil Phulwara butter Pine nut oil Poppy seed oil Rice bran oil Safflower seed oil Safflower seed oil (high oleic) Sal fat Sesame seed oil Sheanut butter
9168_Book.indb 195
Principal Fatty Acid Components in Weight % 20:1 5c
58–77%
Plants (Continued) 22:1 total 8–24%
22:2 5c,13c 18:2 9c,12c 16:0 18:1 9c 18:0 22:1 13c 18:3 9c,12c,15c 18:1 9c 16:0 18:2 9c,12c 18:1 9c 14:0 16:0 18:2 9c,12c 16:0 18:3 9c,11t,13t, 4-oxo 18:0 18:1 9c 16:0 12:0 18:1 9c 16:0 18:2 9c,12c 18:1 9c 18:2 9c,12c 16:0 18:0 18:1 6c 18:2 9c,12c 18:1 9c 16:0 18:3 9c,12c,15c 18:1 9c 16:0 18:2 9c,12c 18:2 9c,12c 16:0 18:2 9c,12c 16:0 18:1 9c 16:0 18:2 9c,12c 16:0 18:1 9c 16:0 18:0 16:0 18:2 9c,12c 16:0 18:1 9c 16:0
7–15% 67% (av.) 11% (av.) 70% 3.8% 43% 12% 49–62% 13–18% 52–78% 4–10% 76–83% 4–10% 24–48% 13–39% 70–80%
18:1 9c 18:1 9c 18:0 16:0 22:0 22:1 13c 18:2 9c,12c 18:0 18:2 9c,12c 16:0 18:0 18:1 9c 12:0 18:1 9c 18:0 16:0
1–3% 12% (av.) 9% (av.) 9% 2.4% 22–50% 10–24% 14–24% 7–15% 5–12% 2–12% 5–11% 3–6% 18–53% 0.5–4% 7%
5% 55–83% 7.5–20% 40–55% 12–21% 40–48% 6.5–12% 40–44% 10–13% 48–74% 3.9–5.6% 69–76% 6–14% 36–67% 8.3–14% 59% 11–13% 57–61% 3–4% 47–51% 6–8% 62–73% 7–11% 38–48% 16–28% 68–83% 5.3–8.0% 74–80% 5–6% 33–57% 6–23% 40–51% 7.9–10.2% 45–50% 4–8%
18:1 9c 18:2 9c,12c 18:2 9c,12c 14:0 16:0 18:1 9c 18:0 16:0 18:0 18:1 9c 18:2 9c,12c 18:1 9c 16:0 18:2 9c,12c 22:0 18:2 9c,12c 16:0 18:1 9c 18:0 18:1 9c 18:0 18:1 9c 18:0 18:2 9c,12c 18:0 18:1 9c 18:0 18:2 9c,12c 18:0 18:1 9c 20:0 18:1 9c 18:0 18:0 18:2 9c,12c
4–7% 9% 3.5–21% 14–18% 6.5–10% 36–44% 3.5–6.5% 38–43% 3.7–4.8% 16–36% 3.2–9.8% 12–15% 2% 14–43% 2.1–4.4% 14–18% 6–9% 30–36% 3–4% 36–39% 2–3% 16–30% 1–4% 16–36% 2–4% 8.4–30% 1.9–2.9% 13–18% 1.5–2.0% 31–52% 1–8% 33–44% 4.4–6.7% 36–41% 4–8%
mp/ Density/ Refractive °C g cm–3 Index
Iodine Value
Saponification Value
1.46440
86–91
168
0.90324
1.46040
70
185
0.91320
1.46540
92–125
170–184
0.91230
1.46240
68–71
195–205
0.92415
1.46840
126–135
188–193
1.46840
48–85
170–190
0.91725
1.46740
105–116
190–199
0.97220
1.51425
140–150
188–193
−6
0.91120
1.46920
75–94
184–196
24
0.92215
1.45040
14–21
230–250
35
0.91415
1.45540
49–55
190–209
0.9140
1.45940
>56
194–202
0.88460
1.44940
212°C (decomposes); λmax=278, 361, 550 nm (aqueous solution); E1% 1 cm =115,204, 25 63; [a ]656 =−59±9° (aqueous solution)
HO C63H88N14O14PCo mol wt 1,355.42
N N HO
O
Pteroylglutamic acid Folic acid+ Folacin Vitamin M Lactobacillus casei factor Vitamin Bc, B10 or B11 Norite eluate factor Factor U or R Abbreviation: PGA+ or PteGlua N-[4-([2-Amino-4-hydroxy-6pteridyl)methyl]amino)benzoyl] glutamic acid
N
H 2N N
N N
H N
H N
OH O
C19H19N7O6 mol wt 441.40
COOH COOH
Yellowish-orange crystals; mp > 250°C (decomposes); λmax=256, 283, 265 nm (alkaline solution); E1% 1 cm = 603, 600, 215
Handbook of Biochemistry and Molecular Biology
FOLIC ACIDa OR FOLACINb
4/16/10 1:16 PM
Compound
Formula
Properties
Solubility (g/100 ml)
Stability
FOLIC ACIDa OR FOLACINb (Continued) 5-Formyltetrahydrofolic acid Folinic acid [N-[p-[(2-Amino-5-formyl5,6,7,8-tetrahydro-4-hydroxy6-pteridinyl)methyl] amino] benzoyl]glutamic acid Citrovorum factor Leucovorin Abbreviation: N5-formyl THFA or N5-F-PGAH4 or 5-HCO-H4PteGlu
H2N
N N
H N H N
N OH
H N
CHO O
COOH COOH
Colorless crystals; mp 240 to 250°C (decomposes); λmax=282 nm (alkaline solution); = +14.26°
Very slightly sol in water
More stable at neutral or mild alkaline pH
15, water; insol in absolute alcohol and ether
Relatively stable
Colorless or white needles; mp 235 to 237°C; λmax=261 nm (0.1 N HCl), 263 nm (pH 11); E1% 1 cm =435, 260; optically inactive
1.6, water; 0.73 alcohol; insol in ether
Stable to air, light, and PH
Colorless or white needles; mp129 to 131°C; λmax=261 nm (0.1 N HCI), 262 nm (pH 11); E1% 1 cm =432, 250; optically inactive
100, water; 66.6, alcohol; 10 glycerol; slightly sol in ether
Stable in air and heat, light, and pH (may hydrolyze to nicotinic acid)
Properties of Vitamins
9168_Book.indb 233
PROPERTIES OF VITAMINS (Continued)
C20H23N7O7 mol wt 473.44
Inositol Myo-inositol Inositol meso-Inositol i-Inositol Bios 1 Inosite Mouse antialopecia factor Rat antispectacle eye factor Hexahydroxycyclohexane Cyclohexanehexol, cyclohexitol
OH HO
OH
OH HO
White crystals; mp 225 to 227°C (anhydrous); mp 218°C (dihydrate); optically inactive; sweet taste
OH C6H12O6. mol wt 180.16 Niacin
Nicotinic acid Niacin Antiblacktongue factor Pellegra preventive (PP) factor Vitamin PP Pyridine-3-carboxylic acid Pyridine-β-carboxylic acid Nicotinamide Niacinamide Nicotinic acid amide Vitamin PP Vitamin B3 3-Pyridinecarboxylic acid amide
COOH N C6H5NO2 mol wt 123.11 CONH2 N C6H6N2O mol wt 122.13
233
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Compound
Formula
Properties
234
PROPERTIES OF VITAMINS (Continued) Solubility (g/100 ml)
Stability
Colorless, hydroscopic, viscous oil; [a ]D25 =+37:5° (aqueous) solution); only d-form is biologically active
Freely sol in water and acetic acid; moderately sol in alcohol; insol in benzene and chloroform
Stable at near neutral pH (5 to 7); unstable in acid or alkali; labile to prolonged heat
Colorless or white needles; mp 195 to 196°C (decomposes); [a ]D25 =+28.2° (aqueous solution); 1 g=70,000 to 75,000 chick units
35, water; very sol in glycerol and glacial acetic acid; slightly sol in ether, insol in benzene and chloroform
–
Viscous liquid (d); white crystals (dl); mp 64.5 to 67.5°C (dl); [a ]D20 =+29.7° (in water) (d)
Very sol in water and glycerol
More stable in solution than pantothenic acid or salts
22, water; 1.1, ethanol; slightly sol in acetone; insol in ether
Stable, dry to air, light, and heat; sol in acid solution
Pantothenic Acid Pantothenic acid, d-(+) Chick antidermatitis factor Liver filtrate factor Antidermatosis vitamin Vitamin B5, d(+)-N-(2,4-Dihydroxy-3,3dimethylbutyryl)-β-alanine Pantoyl-β-alanine Calcium salt Calcium pantothenate
O HO OH C9H17NO5 mol wt 219.23 O HO OH C18H32CaN2O10 mol wt 476.55
Ca2
COO
N H
2
OH
HO
H N
O
OH
C9H19NO4 mol wt 205.39
Vitamin B6 Pyridoxine · HCl Pyridoxol hydrochloride Adermine hydrochloride Antiacrodynia factor Yeast eluate factor 5-Hydroxy-6-methyl-3,4pyridinedimethanol hydrochloride 2-Methyl-3-hydroxy-4,5bis(hydroxymethyl)pyridine hydrochloride
HO HO N C8H11NO3·HCl mol wt 205.64
OH HCl
Platelets or rods, white crystals; mp 206°C (decomposes); λmax=291 nm (0.17 N HCl); E1% 1 cm =422; optically inactive
Handbook of Biochemistry and Molecular Biology
Pantothenyl alcohol Panthenol Provitamin for pantothenic acid N-(2,4-Dihydroxy-3,3dimethylbutyryl-βaminopropanol Pantoyl-β-aminopropanol
COOH
N H
4/16/10 1:16 PM
Compound
Formula
Properties
Solubility (g/100 ml)
Stability
–
–
–
–
–
–
–
Yellow to orange-yellow; polymorphic crystals; mp 280 to 290°C (decomposes); λmax=223, 266, 271, 444 nm(0.1HHCl); E1% 1 cm = 800, 870, 288, 310; [a ]D25 =−112 to 122° (dilute alcoholic NaOH); strong green fluorescence when irradiated by UV light
0.013, water; 0.040, alcohol; insol in ether, acetone, chloroform, and benzene; riboflavin phosphate, sodium salt quite water soluble
Unstable in alkali solution especially in light; stable in acid solution dark; reversibly reduced by sodium hydrosulfite and other reducing agents to dehydroriboflavin (leucoflavin); relatively stable in dry form
Orange-yellow crystals; [a ]D20 =+38 to 42° (20% HCl)
4 to 11, water (depending on pH)
Similar to riboflavin
Vitamin B6 (Continued) Pyridoxal 3-Hydroxy-5-(hydroxymethyl)2-methylisonicotinaldehyde
CHO HO
OH
N C8H9NO3 mol wt 167.16 Pyridoxal · HCl
C8H9NO3 HCl mol wt 203.62
Colorless rhombic crystals; mp 165°C (decomposes); λmax=292.5 nm; Emol=7,600
Pyridoxamine Dihydrochloride 2-Methyl-3-hydroxy-4aminomethyl-5hydroxymethylpyridine dihydrochloride
C8H12N2O2 mol wt 168.19 NH2
Colorless crystals; mp 193 to 193.5°C Colorless platelets; mp 226 to 227°C (decomposes); λmax=287.5 nm (at pH 1.94); Emol=9,100
OH
HO N
2HCl
Properties of Vitamins
9168_Book.indb 235
PROPERTIES OF VITAMINS (Continued)
C8H12N2O2·2HCl mol wt 241.12 Riboflavin Riboflavine Vitamin B2 Vitamin G Lactoflavin Ovoflavin Hepatoflavin Lyochrome 7,8-Dimethyl-10-(d-ribo2,3,4,5-tetrahydroxypentyl) isoalloxazine 7,8-Dimethyl-10ribitylisoalloxazine Vitamin B2 phosphate sodium Riboflavin 5′-phosphate sodium Flavin mononucleotide Riboflavin 5′-phosphate ester monosodium salt
Vitamin B2 HO HO HO
CH2OH H H H CH2 N N
O NH
N O
C17H20N4O6 mol wt 376.37 O ONa CH2 OP OH HO H HO H HO H CH2 N O N NH
N
235
O 4/16/10 1:16 PM
C17H20N4O9PNa · 2H2O mol wt 514.37
9168_Book.indb 236
Compound
Formula
Properties
236
PROPERTIES of VITAMINS(Continued) Solubility (g/100 ml)
Stability
Vitamin B1 Thiamine · HCl Thiamine chloride hydrochloride Vitamin B1 hydrochloride Aneurine (hydrochloride) Oryzamin Antiberiberi vitamin 3-(4-Amino-2methylpyrimidyl-5-methyl-4methyl-5-(β-hydroxyethyl) thiazolium chloride hydrochloride
NH2 N
N HO
Cl
N
S
White monoclinic crystals; mp 246 to 250°C (decomposes); λmax= 246 nm (0.1 N HCl); E1% 1 cm = 410; optically inactive; 1 mg=333 IU
100, water; 1, alcohol; insol in organic solvents
Stable when dry, stable in acid, unstable at alkaline pH, to prolonged heating, presence of bisulfite or thiaminase; very hygroscopic
White crystals; mp 196 to 200°C (decomposes); less hydroscopic than chloride hydrochloride; 1 mg=343 IU
2.7, water; insol in organic solvents
More stable than chloride salt in dry products; not hygroscopic
C12HI7ClN4OS HCl mol wt 337.28
Thiamine mononitrate Aneurine mononitrate Vitamin B1 mononitrate
NH2 N HO
S
N N
NO3
C12H7NSO4S mol wt 327.36 Compiled by J. C. Bauernfeind and E. De Ritter.
Approved by IUPAC-IUB. Approved by IUNS.
a
Handbook of Biochemistry and Molecular Biology
b
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9168_Book.indb 237
Compound
Function
Deficiency Symptoms
Hyper-Use Symptoms
Coenzyme and Enzyme Involved
Remarks
Primary role in vision as 11-cis retinal; synthesis of mucopolysaccharides; maintenance of mucous membranes and skin; bone development and growth; maintenance of cerebrospinal fluid pressure; production of corticosterone
Retarded growth, xerophthalmia, nyctalopia, hemeralopia, ataxia, tissue keratinization, cornification, desquamation, emaciation, lachrymation, impaired reproduction or hatchability (eggs), increased susceptibility to infection, optic nerve degeneration, odontoblast atrophy
Weight loss, bone abnormalities, inflammations, exfoliated epithelium, liver enlargement, pain, loss of hair, facial pigmentation
None identified
Other carotenes, α-, β-, γ-, and β-zeacarotene, cryptoxanthin, echinenone, certain apocarotenals, cis isomers of retinol, and dehydroretinol have fractional vitamin A activity, differing for various animal species; retinoic acid may be metabolically active form for certain functions
Vitamin D
Absorption and transport of calcium and phosphorus; synthesis of calcium protein carrier; interrelationship with parathyroid hormone; maintains alkaline phosphatase levels at bone site
Rickets, enlarged joints, softened bones, stilted gait, arched back, thin-shelled eggs, disturbed reproduction and hatchability, osteomalacia, faulty calcification of teeth, tetany, convulsions, raised plasma phosphatase, parturient paresis (dairy cattle)
Abnormal calcium deposition in bones and tissues, brittle or deformed bones, vomiting, abdominal discomfort, renal damage, weight loss
None identified
D3 is the most effective form for the avian species; for man D2 and D3 are fully active; 1α,25 dihydroxycholecalciferol is the active metabolic form of D3 and is considered by some, a hormone
Vitamin E
Biological antioxidant, interrelated with Se; metabolism of nucleic and sulfur amino acids; ubiquinone synthesis; detoxicant and oxidation-reduction action; stabilizes biological membranes against oxidative attack
Muscular dystrophy, encephalomalacia, hepatic necrosis, erythrocyte hemolysis, hock disorders, steatitis, reduced reproduction and hatchability, exudative diathesis, liver dystrophy, anemia, degeneration of testicular germinal epithelium, creatinuria
None identified; relatively nontoxic
None identified
Other tocols (β-, γ-, δ-tocopherol) and trienols (α-, β-, γ-, δ-tocotrienol) exist differing in ring substituents and in the side chain and having fractional vitamin activity; presence of unsaturated fat in the diet increases dietary vitamin E requirements
Vitamin K
Hepatic synthesis of prothrombin; synthesis of thromboplastin; needed in RNA formation and electron transport
Hemorrhage, impaired coagulation (low prothrombin levels), increased blood clotting time
Vomiting, albuminuria, porphyrinuria, polycythemia, splenomegaly, kidney and liver damage
None identified
K1 is plant form of the vitamin; K2 the microbiologically synthesized form; K1 and K2 are metabolically active forms; they also occur with longer or shorter side chain (isoprene) units; coumarin compounds and excess sulfa drugs are dietary stress agents for vitamin K
Ascorbic acid
Required for collagen formation; protects enzymes, hydrogen carriers, and adrenal steroids; functions in incorporation of iron into liver ferritin, folic acid into folinic acid; prevents scurvy, increases phagocytic activity
Scurvy, fragile capillaries, bleeding gums, loose teeth, anemia, follicular keratosis, sore muscles, weak bones, decreased egg shell strength, poor wound healing
None identified; relatively nontoxic
None identified
Dietary essential for man, monkey, guinea pig, fish, Indian pipistrel, Indian fruit bat, and flying fox, most other species synthesize it; glucoascorbic acid is an antagonist
BIOLOGICAL CHARACTERISTICS OF VITAMINS
Vitamin A
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Compound
Function
Deficiency Symptoms
Hyper-Use Symptoms
238
BIOLOGICAL CHARACTERISTICS OF VITAMINS (Continued) Coenzyme and Enzyme Involved
Remarks
Function or need not well understood or accepted; a microbial factor involved in melanin formation and pigmentation; inhibits oxidation of adrenaline; influences activity of tyrosinase; involved in microbial synthesis of folic acid
Nutritional achromotrichia (animals), retarded growth (chicks), disturbed lactation (mice)
Rash, nausea, fever, acidosis, vomiting, pruritis
None identified
Sulfa drugs, carbarzones, and others are antagonists; PAB and sulfa drugs have a common point of attack on certain enzyme systems; PAB has some chemotherapeutic uses
Biotin
As an enzyme component activating carbon dioxide and its transfer in amino acid, carbohydrate, and lipid metabolism; deamination of certain amino acids; synthesis of long-chain fatty acids; potassium metabolism; interrelationship with pyridoxine, cyanocobalamin, pantothenic acid, folic acid, and ascorbic acid
Dermatosis, perosis, scaly skin, loss of hair, spectacle alopecia, cracked foot pads or hoofs, impaired reproduction, retarded growth, anorexia, lassitude, sleeplessness, muscle pain, electrocardial changes
None identified
Carboxybiotin, carboxylases, transcarboxylases, carbamyl phosphate synthetase
Biocytin, a biotin bound form. is(+)-epsilon-N-biotinyl-llysine; mild oxidation converts biotin to sulfoxide, strong oxidation to the sulfone; biotin inactivated by avidin; antagonists are α-dehydrobiotin, biotin sulfone, avidin, and others
Choline
Source of methyl group, a methyl donor; for acetylcholine and phospholipid formation; essential for liver functioning
Fatty liver, hemorrhagic degeneration of kidneys, cirrhosis of liver, involution of thymus, enlarged spleen, retarded growth, impaired production (eggs) lactation and reproduction, perosis, muscle weakness or paralysis
Diarrhea and edema, erythrocyte formation inhibition
None identified
Choline occurs widespread in nature and is synthesized within the body to a limited extent; triethylcholine and others are choline antimetabolites
Cyanocobalamin
Cofactor for methyl malonyl CoA isomerase; involved in isomerizations; dehydrogenations, methylations; interrelated in choline, folic acid, ascorbic acid, pantothenic acid, biotin, and S-amino acid metabolism; synthesis of nucleoproteins
Retarded growth, perosis, poor feathering, megaloblastic anemia, anorexia, degenerative changes in spinal cord, posterior incoordination, impaired hatchability
Polycythemia
Cyanocobalamin coenzyme
Other B12 molecule variations exist wherein cyanide is replaced by chlorine, bromine, hydroxylcyanate, nitrite thiocyanate, etc; 5,6-dichlorobenzimidazole is an antimetabolite
Handbook of Biochemistry and Molecular Biology
p-Aminobenzoic acid
4/16/10 1:16 PM
Compound
Hyper-Use Symptoms
Coenzyme and Enzyme Involved
Deficiency Symptoms
Folic acid
Concerned with single carbon metabolism; for methyl, hydroxyl, and formyl transfers; for purine synthesis and normal histidine metabolism; interconversion of serine and glycine; a growth and hematopoietic factor; interrelationship with cyanocobalamin, ascorbic acid, iron, etc.
Retarded growth, sprue, diarrhea, macrocytic anemia, cervicular paralysis, reduction and abnormalities in white cells, dermatitis, impaired reproduction and lactation, perosis, poor feathering and lowered hatchability (poultry)
Obstruction of renal tubules
Tetrahydrofolic acid enzyme
The compound is a chelate, binding Co; certain molecular modifications of folic and tetrahydrofolic acid yield antagonists such as aminopterin, tetrahydroaminopterin, and others; the active forms of folic acid may have additional group such as formyl or methyl on the nitrogen in the molecule; thus, N5 -formyltetrahydrofolic acid is folinic acid and N5 -methyltetrahydrofolic acid is the form in blood
Remarks
Inositol
Function or need not well understood or accepted; believed to be a lipotropic factor; a supply of methyl group functioning with cyanocobalamin; needed for acetyl choline production, functions in some microbial metabolic role
Fatty liver, hair loss, impaired reproduction and lactation, reduced growth (animal)
None identified; relatively nontoxic
None identified
Hexachlorocyclohexane (lindane) is an antimetabolite; while inositol can exist in eight cis-trans isomeric forms, only the optically inactive, i- or meso-inositol, is active; it occurs in animal tissues in free and phosphate ester form; inositol concentration is high in heart muscle, brain, and skeletal muscle
Nicotinic acid
Involved in enzyme mechanisms in carbohydrate, fat, and protein metabolism; functions as hydrogen transfer agent; interrelated with pyridoxine and tryptophan metabolism
Retarded growth, poor feathering or hair coat, black tongue (dog), pellagra, necrotic enteritis, impaired reproduction and hatchability, bowed legs (ducks and turkeys), enlarged hocks, perosis, stomatitis, diarrhea, headache, depression, paralysis, dermatitis
Vasodilation, flushing, tingling, pruritis, hyperhidrosis, nausea, abdominal cramps
NAD (DPN) NADP (TPN) dehydrogenases, coenzymes such as lactate dehydrogenase
In addition to vitamin need, niacin has pharmacological activity as a vasodilator; possessed to a markedly less degree by the amide; 3-acetylpyridine-6-aminonicotinamide and pyridine-3-sulfonic acid or amide are antagonists
Pantothenic acid
A component of coenzyme A; functions in acetylation (or 2 carbon) reactions in amino acid, carbohydrate, and fat metabolism; involved in biosynthesis of acetyl choline, steroids, triglycerides, phospholipids, and ascorbic acid; interrelationship with cyanocobalamin, folic acid, and biotin mechanisms
Retarded growth, dermatitis, anorexia, weakness, spastic abnormalities or gait, scours, achromotrichia (animals), adrenal hemorrhagic necrosis, burning sensation in hands and feet, impaired reproduction and hatchability
None identified; relatively nontoxic
Coenzyme A, acetylases
d-Form is the one in nature; the dl- and d-forms are also manufactured as calcium salts; ω-methyl pantothenic acid and other compounds are antagonists
239
Function
BIological Characteristics of Vitamins
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BIOLOGICAL CHARACTERISTICS OF VITAMINS (Continued)
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Deficiency Symptoms
Hyper-Use Symptoms
240
BIOLOGICAL CHARACTERISTICS OF VITAMINS (Continued) Coenzyme and Enzyme Involved
Function
Remarks
Pyridoxine
Functions in amino acid metabolism, decarboxylation, transamination, and desulfhydration; oxidation of amines and amino acid transport; phosphorylase activity of muscle; conversion of tryptophan to nicotinic acid and amino acids to biogenic amines
Retarded growth, hyperexcitability, myelin degeneration, convulsions, heart changes, spastic gait, nervousness, anorexia, insomnia, acrodynia, microcytic anemia, impaired production (eggs), reproduction and hatchability, tryptophan metabolites in urine
Convulsions and abnormal encephalograms
Pyridoxal phosphate, pyridoxamine phosphate, transaminases, amino acid decarboxylases
The term vitamin B6 refers to the 3 compounds, pyridoxine (ol), pyridoxal, and pyridoxamine, the latter 2 being important metabolic forms; isonicotinic acid hydrazide, toxopyrimidine, deoxypyridoxine, and 1-amino-d-proline are antagonists; high tryptophan and/or methionine diets increase need for pyridoxine
Riboflavin
Functions as coenzyme; needed in cellular respiration, hydrogen and electron transfer; growth and tissue maintenance; role in visual mechanism
Retarded growth, ocular and orogenital disturbances, greasy scaling of nasolabial folds, cheeks, and chin, angular stomatitis, myelin degeneration, poor feathering or hair growth, impaired reproduction and hatchability, muscle weakness, curled toe paralysis, scours
Itching, paresthesia, anuria
Flavin mono- and dinucleotide, amino acid oxidase, cytochrome c reductase, succinic dehydrogenase, xanthine oxidase, others
Irradiation of alkaline solution produces lumiflavin of acid solution, lumichrome; riboflavin in solution is one of the most photosensitive compounds of the vitamin class; 5-deoxyriboflavin and several other compounds act as antimetabolites
Thiamine
Functions as a coenzyme; activation and transfer of active acetaldehyde, glycoaldehyde, and succinic semialdehyde; functions in carbohydrate metabolism
Polyneuritis, beriberi, convulsions, muscle paralysis, anorexia, bradycardia, heart dilation, myocardial lesions, retarded growth, edema, pyruvic acid accumulation in blood and tissues
Analgesic effect on peripheral nerves, vascular hypertension
Cocarboxylase, transketolase, carboxylases
Like most water-soluble vitamins, there is no significant tissue storage; amprolium, pyrithiamine, oxythiamine, and others are antimetabolites; the two important forms in production are the hydrochloride and the mononitrate
Handbook of Biochemistry and Molecular Biology
Compound
4/16/10 1:16 PM
PROPERTIES FOR ASCORBIC ACID AND ASCORBATE-2-SULFATE Compound
Compound
Ascorbic acid Vitamin C l-Ascorbic acid l-Threoascorbic acid l-Xyloascorbic acid (obsolete) Cevatamic acid (obsolete) Hexuronic acid (obsolete) l-Threo-2,3,4,5,6-pentahydroxy-2-hexene-γ-lactone available as sodium ascorbate) CH2OH OH O
(also
CH2OH OH O
HO O
HO
Ascorbate-2-sulfate (Dipotassium salt) Vitamin C2 l-Ascorbic acid 2-sulfate Potassium 2-O-sulfonato-l-ascorbate
C6H8O6
MW = 176.13
OH
Properties Colorless or white crystals; mp 191°, decomposes λm (pH 5.2–10), 263.5 nm; ε263 1.47 × 104 λm (acid soln), 243.5 nm; ε244, 9,600 λm (isobestic), 252.8 nm Characteristic acid taste
Solubility 33, water; 3.5, alcohol; 1, glycerol; sol, acetonitrile, dimethyl sulfoxide, dimethyl formamide, methanol, formic acid, acetic acid, etc., insol oils, and most organic solvents
O
C6H6O9SK2
MW = 332.37
OSO3K
Properties Colorless or white crystals, pKa, (sulfate group) ~2, pKa 2 (3-OH) ~3. λm(pH 4–10), 254 nm; ε254, 17,700. λm (pH 2), 231 nm; ε231, 11,000.
Solubility Very sol, water; slightly sol, dimethyl formamide, dimethyl sulfoxide, acetonitrile; insoluble most organic solvents. Barium salt slightly sol in water.
Stability Stable in dry solid form; moist solid subject to autocatalytic acid hydrolysis; stable in solution pH 5–9; hydrolyzes in solution < pH 5, rapidly below pH 3; not subject to air oxidation. Compiled by Bert M. Tolbert.
Stability Solid is stable in air in pure or tablet form unless hot and/or humid. Oxidizes autocatalytically in aqueous solution: Dilute (1 mg/ml) moderate rate. Stable in solution if all O2 excluded. Oxidation catalyzed by metals, especially Cu++ or Fe++. Stabilized by acid solutions, especially metaphosphoric acid or trichloroacetic acid. Alkaline solutions unstable. Oxidation intermediates: Monodehydroascorbic acid (a free radical); dehydroascorbic acid; l-2,3-diketogulonate; plus many other products.
241
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242
BIOLOGICAL CHARACTERISTICS FOR ASCORBIC ACID Compound l-Ascorbic acid
Function Prevents scurvy; required for normal collagen formation; involved in transferrition system; required for normal neurological function; a water soluble antioxidant and free radical scavenger; probably the functional cofactor for several hydroxylases; fundamental biochemical role(s) not known in animals nor in plants
Deficiency symptoms Scurvy: Follicular hyperkeratosis, petechiae, ecchymosis, subconjunctional hemorrhage, joint effusions, dyspnea on exertion, swollen gums, neuropathy, edema, psychological impairment; terminal stage, heart blockage
Coenzyme and enzymes involved No proven coenzyme role; Cofactor roles implicated in prolyl hydroxylase, lysyl hydroxylase, p-hydroxyphenylpyruvate hydroxylase, homogentisic acid oxiginase, dopamine-β-hydroxylase and others Subject to active transport through illium (in man), across blood-brain barrier and into the eye
Remarks Probably present in all animals and plants; essential in the diet of man, monkey, baboon (probably all primates), guinea pig, trout, salmon, carp, catfish, several birds (especially insect or fruit eater, as swallows and redwing bulbul), Indian fruit bat, some insects; ascorbate-2-sulfate can substitute for ascorbate in trout and salmon but not in guinea pig; d-erythroascorbic acid (isoascorbic acid, erythorbic acid) is a common food additive, but has little nutritional value. Compiled by Bert M. Tolbert.
Hyper-use symptoms None substantiated; relatively nontoxic in oral form, probably due to limited gut transport and efficient urinary clearance
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Vitamers active component obtained from the irradiation of ergosterol. Later work established vitamin D3 (cholecalciferol) as the major active component. Either must be converted to the 1α-hydroxylderivative (e.g. 1α,25-dihydroxyvitamin D3 (1α,25-dihydroxyvitamin D)10-13. Vitamin E: There are a number of vitamin E (tocopherols/tocotrienols) vitamers14-22 as well as the water-soluble derivative, Trolox23-31 which is used as a standard for antioxidant measurements. Vitamin K: Several vitamers32-41 which have different activities in the support of γ-carboxylation reactions. Thiamin (Vitamin B1): Thiamine and several phosphate esters comprise the vitamers of thiamine42-44 as well as some analogues45-47. Niacin (Vitamin B3): Niacin is also referred to as nicotinic acid. Nicotinic acid and nicotinamide have equivalent activity and are incorporated into a coenzyme, nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP). Nicotinic acid and nicotinamide are the major niacin vitamers48-54. Pyridoxine (Vitamin B6): Multiple vitamers including pyridoxine, pyridoxal, pyridoxamine and the corresponding phosphates and precursors 55-61. Biotin (Vitamin B7; also Vitamin H): Biotin and precursors and precursors including 7-oxo-8-aminopelargonic acid62-68 Folic Acid (Vitamin B9): Several vitamers69-74 which are derivatives of the parent pteroyl glutamate.
A vitamer is one of several chemical compounds, most often of closely related structure, which can fulfill the function of a given vitamin. One of the more prominent examples is Vitamin B6 where the vitamers include pyridoxal, pyridoxine, 4-pyridoxic acid, and pyridoxal 5’-phosphate. Other examples include Vitamin B2 (flavin Vitamers), folic acid, vitamin K, biotin, Vitamin A, Vitamin D and vitamin E. Vitamers are not pseudovitamins. There are also phytonutrients which have been considered to have vitamin-like status. For example, the bioflavonoids are plant derivatives with antioxidant and anti-inflammatory properties; derived from citrus fruit rinds, berries, grains, and wines. Some are considered to have anticancer activity. Bioflavonoids/flavonoids are considered to be polyphenols (Albert, A., Manach, C., Morand, C., Rémésy, C., and Jimémez, L., Dietary polyphenols and the prevention of diseases, Crit.Rev.Food Sci.Nutr. 45, 287-306, 2005). Vitamin A: β-Carotene and retinaldehyde and other retinol derivatives can be considered vitamers of vitamin A. Retinoic acid, although derived from β-Carotene via retinaldehyde or by the oxidation of retinol, is suggested to have a totally different function in growth and development unrelated to vision1-6. In addition, there is considerable interest in the use of vitamin A in cosmeceuticals/skin therapy7-9. Vitamin D: Vitamin D2 and D3 are derived from 7-dehydrocholesterol. Early work identified ergocalciferol (vitamin D2) as an
H3C
CH3
CH3
CH3 H2 C OH
Retinol
CH3
H3C
CH3
CH3
H 3C
CH3
11-cis-Retinol H3C
CH3
CH3
HO
O
CH3
CH2
C H Retinaldehyde CH3 H3C
CH3
CH3
CH3
O C OH
Retinoic acid CH3 H3C
CH3
CH3
CH3 H2 C OH OH
CH3
14-Hydroxyretinol
Figure 1 Structures of Vitamin A vitamers (retinol and retinol derivatives). See Sommer, A.., Vitamin a Deficiency and Clinical Diseases: An Historical Overview, J.Nutr. 138, 1835-1839, 2008; Dragsted, L.O., Biomarkers of Exposure to Vitamins A, C, and E their Relation to Lipid and Protein Oxidation Markers, Eur.J.Nutr. 47 (suppl 2), 3-18, 2008 243
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244
HO
HO Vitamin D2 Ergocalciferol
Vitamin D3 Cholecalciferol
Figure 2 Structures of Vitamin D vitamers (Calciferol derivatives). See Holden, J.M., Lemar, L.E., and Exler, J., Vitamin D in Foods: Development of the US Department of Agriculture Database, Am.J.Clin.Nutr. 87, 1092S-1096S, 2008
CH3 HO CH3 H3C
O CH3 alpha-tocopherol
CH3
CH3
CH3
CH3
CH3 HO CH3 H3C
O CH3
CH3
CH3
CH3
CH3
alpha-tocotrienol CH3 HO
O OH
O
H3C CH3 Trolox
CH3
Figure 3 Structures of Vitamin E vitamers (Tocopherol derivatives). See Yoshida, Y., Saito, Y., Jones, L.S., and Shigeri, Y., Chemical Reactivities and Physical Effects in Comparison between Tocopherols and Tocotrienols: Physiological Significance and Prospects as Antioxidants, J.Biosci.Bioeng. 104, 439-445, 2007; Clarke, M.W., Burnett, J.R., and Croft, K.D., Vitamin E in Human Health and Disease, Crit.Rev.Clin.Lab.Sci. 45, 417-450, 2008
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Vitamers
245 CH3 OH
O CH3
O
CH3
CH3
O
O
OH Menadiol
O
O Menadione
CH3 Menadiol diacetate O CH3
* 3
O Phylloquinone O CH3
* O Menaquinone
n
Figure 4 The structures of Vitamin K vitamers (Naphthoquinone derivatives). The K is derived from the German “koagulationvitamin.” See Doisey, E.A., Brinkley, S.B., Thayer, S.A., and McKee, R.W., Vitamin K, Science 9, 58-62, 1940; Wolff, I.L. and Babior, B.M., Vitamin K and Warfarin. Metabolism, Function and Interaction, Am.J.Med. 53, 261-267, 1972; Sadowski, J.A. and Suttie, J.W., Mechanism of Action of Coumarins. Significance of Vitamin K epoxide, Biochemistry 13, 3696-3699, 1974; Yamada, Y., Inouye, G., Tahara, Y., and Kondo, K., The Structure of the Menaquinones with a Tetrahydrogenated Isoprenoid Side-Chain, Biochim.Biophys.Acta 488, 280-284, 1977; Bell, R.G., Vitamin K Activity and Metabolism of Vitamin K-1 Epoxide-1,4-diol, J.Nutr. 112, 287-292, 1982.
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NH2 H3C N
N HO
N
S
CH3
Thiamine NH2 H3C N
N
O
HO
P
N
S
HO
CH3
Thiamine monophosphate NH2 H3C
O
HO
P
P
HO
N
N
O
O
N
S
HO
CH3
Thiamine Diphosphate NH2 H3C
O
HO
P HO
O
O
P HO
O
N
N
O P
CH3
N
S HO Thiamine Triphosphate
Figure 5 The structures of Vitamin B1 vitamers (Thiamine and derivatives). See Maladrinos, G., Louloudi, M, and Hadjiliadis, N., Thiamine Models and Perspectives on the Mechanism of Action of Thiamine-Dependent Enzymes, Chem.Soc.Rev. 35, 684-692, 2006; Kowalska, E. and Kozik, A., The Genes and Enzymes Involved in the Biosynthesis of Thiamin and Thiamin Diphosphate in Yeasts, Cell Mot.Biol.Lett. 13, 271-282, 2008 O
O
OH
NH2
N Nicotinamide
N Nicotinic acid
Figure 6 The structures of Vitamin B3 (Niacin) vitamers (Nicotinic acid and derivatives). See Skinner, P.J., Cherrier, M.C., Webb, P.J., et al., 3-Nitro-4-Amino Benzoic Acids and 6-Amino Nicotinic Acids are Highly Selective Agonists for GPR109b, Bioorg.Med.Chem. Lett. 17, 6619-6622, 2007; Boovanahalli, S.K., Jin, X., Jin, Y. et al., Synthesis of (Aryloxyacetylamino)-isonicotinic Acid Analogues as Potent Hypoxia-inducible factor (HIF)-1α Inhibitors, Bioorg.Med.Chem.Lett. 17, 6305-6310, 2007; Deng, Q., Frie, J.L., Marley, D.M. et al., Molecular Modeling Aided Design of Nicotinic acid Receptor GPR109A Agonists, Bioorg.Med.Chem.Lett. 18, 4963-4967, 2008
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Vitamers
247 OH OH
HO
O
HO
P
HO
OH
O
CH3
N
N CH3 Pyridoxine Phosphate
Pyridoxine O
OH
HO
O
HO
P
OH
O
HO
N
CH3
N Pyridoxal
CH3
Pyridoxal Phosphate
NH2
OH
H
O
H
NH2 OH
HO HO
N
CH3
Pyridoxamine
O P
OH
O
N CH3 Pyridoxamine Phosphate
Figure 7 The structure of Pyridoxine (Vitamin B6) vitamers. See Snell, E.E., Analogs of Pyridoxal or Pyridoxal Phosphate: Relation of Structure to Binding with Apoenzymes and to Catalytic Activity, Vitam.Horm. 28, 265-290, 1970; Drewke, C. and Leistner, E., Biosynthesis of Vitamin B6 and Structurally Related Derivatives, Vitam.Horm. 61, 121-155, 2001; Garrido-Franco, M., Pyridoxine 5-Phosphate Synthase: De novo Synthesis of Vitamin B6 and Beyond, Biochim.Biophys.Acta 1647, 92-97, 2003
O
O
HO
OH Pimelic acid; heptanedioic acid O
O
H3C
OH
NH2 8-amino-7-oxopelargonic acid; 8-amino-7-oxononanoic acid O
H N
O
OH
HN S Biotin
Figure 8 The structure of Biotin (Vitamin H) vitamers.
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Handbook of Biochemistry and Molecular Biology
248
O
OH
O OH N H
O N
O
HN H2N
N H N
N Folic acid (pteroylglutamic acid) O
OH
O OH O
N H
H N HN H2N
O N H
N
N H Tetrahydrofolic acid
Figure 9 The structure of Folic acid vitamers
References 1. Pitt, G.A., Chemical structure and the changing concept of vitamin A activity, Proc.Nutr.Soc. 42, 43-51, 1983 2. Wolf, G., The regulation of retinoic acid formation, Nutr.Rev. 54, 182184, 1996 3. Clagett-Dame, M. and DeLuca, H.F., The role of vitamin A in mammalian reproduction and embryonic development, Annu.Rev.Nutr. 22, 347-381, 2002 4. Matt, N, Dupe, V., Garnier, J.M., Retinoic acid-dependent eye morphogenesis is orchestrated by neural crest cells, Development 132, 4789-4800, 2005 5. Moise, A.R., Isken, A., Dominguez, M., et al., Specificity of zebrafish retinol saturase: formation of all-trans-13,14-dihydroretinol and alltrans-7,8-dihydroretinol, Biochemistry 46, 1811-1820, 2007 6. Reichrath, J., Lebmann, B., Carlberg, C., et al., Vitamins as hormones, Horm.Metab.Res. 39, 71-84, 2007 7. Mayer, H., Bollag, W., Hanni, R., and Ruegg, R., Retinoids, a new class of compounds with prophylactic and therapeutic activities in oncology and dermatology, Experientia 34, 1105-1119, 1978 8. Zoubloulis, C.C., Retinoids—which dermatological indications will benefit in the near future?, Skin Pharmacol.Appl.Skin Physiol. 14, 303315, 2001 9. Borg, O., Antille, C., Kaya, G., and Saurat, J.H., Retinoids in cosmeceuticals, Dermatol.Ther. 19, 289-296, 2006 10. Holick, M.F., The use and interpretation of assays for vitamin D and its metabolites, J.Nutr. 120, 1464-1469, 1990 11. Coburn, J.W., Tan, A.U., Jr., Levine, B.S., et al., 1α-Hydroxy-vitamin D: a new look at an “old compound”, Nephrol.Dial.Transplant. 11(supp 3), 153-157, 1996 12. Wikvall, K., Cytochrome P450 enzymes in the bioactivation of vitamin D to its hormonal form, Int.J.Mol.Med. 7, 201-209, 2001 13. Wu-Wong, J.R., Tian, J., and Goltzman, D., Vitamin D analogs as therapeutic agents: a clinical study update, Curr.Opin.Investig.Drugs 5, 32-326, 2004 14. Panfili, G., Fratianni, A., and Irano, M., Normal phase high-performance liquid chromatography method for the determination of tocopherols and tocotrienols in cereals, J.Agric.Food Chem. 51, 39403944, 2003
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15. McCormick, C.C. and Parker, R.S., The cytotoxicity of vitamin E is both vitamer- and cell-specific and involves a selectable trait, J.Nutr. 124, 3335-3342, 2004 16. Sontag, T.J. and Parker, R.S., Vitamin E exhibits concentration- and vitamer-dependent impairment of microsomal enzyme activities, Ann.N.Y.Acad.Sci. 1031, 376-377, 2004 17. Amaral, J.S., Casal, S., Torres, D., et al., Simultaneous determinations of tocopherols and tocotrienols in hazelnuts by a normal phase liquid chromatographic method, Anal.Sci. 21, 1545-1548, 2005 18. Cunha, S.C., Amaral, J.S. Ferandes, J.O., and Oliviera, B.P., Quantification of tocopherols and tocotrienols in Portuguese olive oils using HPLC with three different detection systems, J.Agric.Food Chem. 54, 3351-3356, 2006 19. Sookwong, P., Nakagawa, K., Murata, K., et al., Quantitation of tocotrienols and tocopherol in various rice brans, J.Agric.Food Chem. 55, 461-466, 2007 20. Bustamante-Rangel, M. Delgado-Zamrreno, M.M., Sanchez-Perez A., and Carabias-Martinez, R., Determination of tocopherols and tocotrienols in cereals by pressurized liquid extraction-liquid chromatography-mass spectrometry, Anal.Chim.Acta 587, 216-221, 2007 21. Hunter, S.C. and Cahoon, E.B., Enhancing vitamin e in oilseeds: unraveling tocopherol and tocotrienol biosynthesis, Lipids 41, 97-108, 2007 22. Tsuzuki, W., Yunoki, R., and Yoshimura, H., Intestinal epithelial cell absorb γ-tocopherol faster than α-tocopherol, Lipids 42, 163-170, 2007 23. Kralli, A. and Moss, S.H., The sensitivity of an actinic reticuloid cell strain to near-ultraviolet radiation and its modification by trolox-C, a vitamin E analogue, Br.J.Dermatol. 116, 761-772, 1987 24. Nakamura, M., One-electron oxidation of Trolox C and vitamin E by peroxidase, J.Biochem. 110, 595-597, 1991 25. Miura, T., Muraoka, S., and Ogiso, T., Inhibition of hydroxyl radicalinduced protein damages by trolox, Biochem.Mol.Biol.Int. 31, 125133, 1993 26. Forrest, V.J., Kang, Y.H., McClain, D.E., et al., Oxidative stress-induced apoptosis prevented by Trolox, Free Radic.Biol.Med. 16, 675-684, 1994 27. Albertini, R. and Abuja, P.M., Prooxidant and antioxidant properties of Trolox C, analogue of vitamin E, in oxidation of low-density lipoprotein, Free Radic.Res. 30, 181-188, 1999
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Vitamers 28. Wang, C.C., Chu, C.Y., Chu, K.O., et al., Trolox-equivalent antioxidant capacity assay versus oxygen radical absorbance capacity assay in plasma, Clin.Chem. 50, 952-954, 2004 29. Raspor, P., Plesnicar, S., Gazdag, Z., et al., Prevention of intracellular oxidation in yeast: the role of vitamin E analogue, Trolox (6-hydroxy2,5,7.8-tetramethylkorman-2-carboxyl acid), Cell Biol.Int. 29, 57-63, 2005 30. Abudu, N., Miller, J.J., and Levinson, S.S., Fibrinogen is a co-antioxidant that supplements the vitamin E analog trolox in a model system, Free Radic Res. 40, 321-331, 2005 31. Castro, I.A., Rogero, M.M., Junqueira, R.M., and Carrapeiro, M.M., Free radical scavenger and antioxidant capacity correlation of α-tocopherol and Trolox measured by three in vitro methodologies, Int.J.Food Sci.Nutr. 57, 75-82, 2006 32. Lefevere, M.F., De Lennheer, A.P., and Claeys, A.E., Highperformance liquid chromatography assay of vitamin K in human serum, J.Chromatog. 186, 749-762, 1979 33. Lowenthan, J. and Vergel Rivera, G.M., Comparison of the activity of the cis and trans isomer of vitamin K1 in vitamin K-deficient and coumarin anticoagulant-pretreated rats, J.Pharmacol.Exp.Ther. 209, 330333, 1979 34. Preusch, P.C. and Suttie, J.W., Stereospecificity of vitamin K-epoxide reductase, J.Biol.Chem. 258, 714-716, 1983 35. Hwang, S.M., Liquid chromatographic determination of vitamin K1 trans- and cis-isomers in infant formula, J.Assoc.Off.Anal.Chem. 68, 684-689, 1985 36. Will, B.H., Usui, Y., and Suttie, J.W., Comparative metabolism and requirement of vitamin K in chicks and rats, J.Nutr. 122, 2354-2360, 1992 37. Vermeer, C., Gijsbers, B.L., Craciun, A.M. et al., Effects of vitamin K on bone mass and bone metabolism, J.Nutr. 126(4 suppl), 1187S-1191S, 1996 38. Gijsbers, B.L., Jie, K.S., and Vermeer, C., Effect of food composition on vitamin K absorption in human volunteers, Br.J.Nutr. 76, 223-229, 1996 39. Woolard, D.C., Indyk, H.E., Fong, B.Y., and Cook, K.K., Determination of vitamin K1 isomers in food by liquid chromatography with C30 bonded phase column, J. AOAC Int. 85, 682-691, 2002 40. Cook, K.K., Grundel, E., Jenkins, M.Y. and Mitchell, G.V., Measurement of cis and trans isomers of vitamin K1 in rat tissues by liquid chromatography with a C30 column, J.AOAC Int. 85, 832-840, 2002 41. Carrie, I., Pertoukalian, J., Vicaretti, R., et al., Menequinone-4 concentration is correlated with sphingolipid concentration in rat brain, J.Nutr. 134, 167-172, 2004 42. Botticher, B. and Botticher, D., A new HPLC-method for the simultaneous determination of B1-, B2- and B6-vitamers in serum and whole blood, Int.J.Vitam.Nutr.Res. 57, 273-278, 1987 43. Batifoulier, F., Verny, M.A., Bessom, C. et al., Determination of thiamine and its phosphate esters in rat tissues analyzed as thiochromes on a RP-amide C16 column, J.Chromatog.B.Analyt.Technol.Biomed. Life Sci. 816, 67-72, 2005 44. Konings, E.J., Water-soluble vitamins, J.AOAC Int. 89, 285-288, 2006 45. Lowe, P.N., Leeper. F.J., and Perham, R.N., Stereoisomers of tetrahydrothiamine pyrophosphate, potent inhibitors of the pyruvate dehydrogenase multienzyme complex from Escherichia coli, Biochemistry 22, 150-157, 1983. 46. Klein, E., Nghiem, H.O., Valleix, A., et al., Synthesis of stable analogues of thiamine di- and triphosphate as tools for probing a new phosphorylation pathway, Chemistry 8, 4649-4655, 2002 47. Erixon, K.M., Dabalos, C.L., and Leeper, F.J., Inhibition of pyruvate decarboxylase from E.mobilis by novel analogues of thiamine pyrophosphate: investigating pyrophosphate mimics, Chem.Commun. (9), 960-962, 2007 48. Sauberlich, H.E., Newer laboratory methods for assessing nutriculture of selected B-complex vitamins, Annu.Rev.Nutr. 4, 377-407, 1984 49. Stein, J., Hahn, A., and Rehner, G., High-performance liquid chromatographic determination of nicotinic acid and nicotinamide in biological samples applying post-column derivatization resulting in bathochrome absorption shifts, J.Chromatog.B.Biomed.Appl. 665, 71-78, 1995
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249 50. Gillmor, H.A., Bolton, C.H., Hopton, M., et al., Measurement of nicotinamide and N-methyl-2-pyridone-5-carboxamide in plasma by high performance liquid chromatography, Biomed.Chromatog. 13, 360362, 1999 51. Khan, A.R., Khan, K.M, Perveen, S., and Butt, N., Determination of nicotinamide and 4-aminobenzoic acid in pharmaceutical preparations by LC, J.Pharm.Biomed.Anal. 29, 723-727, 2002 52. Chatzimichalakis, P.F., Samanidou, V.F., Verpoorte, R., and Papadoyannis, I.N., Development of a validated HPLC method for the determination of B-complex vitamins in pharmaceuticals and biological fluids after solid phase extraction, J.Sep.Sci. 27, 1181-1188, 2004 53. Hsieh, Y. and Chen, J., Simultaneous determination of nicotinic acid and its metabolites using hydrophilic interaction chromatography with tandem mass spectrometry, Rapid Commun.Mass Spectrom. 19, 3031-3036, 2005 54. Marszall, M.P., Markuszewski, M.J., and Kaliszan, R., Separation of nicotinic acid and its structural isomers using 1-ethyl-3-methylimidazolium ionic liquid as a buffer additive by capillary electrophoresis, J.Pharm.Biomed.Anal. 41, 329-332, 2006 55. Vanderslice, J.T., Maire, C.E., and Beecher, G.R., B6 Vitamer analysis in human plasma by high performance liquid chromatography: a preliminary report, Am.J.Clin.Nutr. 34, 947-950, 1981 56. Hachey, D.L. Coburn, S.P., Brown, L.T., et al., Quantitation of vitamin B6 in biological samples by isotope dilution mass spectrometry, Anal. Biochem. 151, 159-168, 1985 57. Driskell, J.A. and Chrisley, B.M., Plasma B-6 vitamer and plasma and urinary 4-pyridoxic acid concentrations in young women as determined using high performance liquid chromatography, Biomed. Chromatogr. 5, 198-201, 1991 58. Sharma, S.K. and Dakshinamurti, K., Determination of vitamin B6 vitamers and pyridoxic acid in biological samples, J.Chromatogr. 578, 45-51, 1992 59. Schaeffer, M.C., Gretz, D., Mahuren, J.D., and Coburn, S.P., Tissue B-6 vitamer concentrations in rats fed excess vitamin B-6, J.Nutr. 125, 2370-2378, 1995 60. Fu, T.F., di Salvo, M., and Schirch, V., Distribution of B6 vitamers in Escherichia coli as determined by enzymatic assay, Anal.Biochem. 298, 314-321, 2001 61. Bisp, M.R., Bor, M.V., Heinsvig, E.M., et al., Determination of vitamin B6 vitamers and pyridoxic acid in plasma: development and evaluation of a high-performance liquid chromatography assay, Anal. Biochem. 305, 82-89, 2002 62. Eisenberg, M.A., The biosynthesis of biotin in growing yeast cells: The formation of biotin from an early intermediate, Biochem.J. 101, 598-600, 1966 63. Birnbaum, J., Pai, C.H., and Lichstein, H.C., Biosynthesis of biotin in microorganisms. V. Control of vitamer production, J.Bacteriol. 94, 1846-1853, 1967 64. Eisenberg, M.A. and Star, C., Synthesis of 7-oxo-8-aminopelargonic acid, a biotin vitamer, in cell-free extracts of Escherichia coli biotin auxotrophs, J.Bacteriol. 96, 1846-1843, 1967 65. Eisenberg, M.A. and Star, C., Synthesis of 7-oxo-8-aminopelargonic acid, a biotin vitamer, in cell-free extracts of Escherichia coli biotin autotrophs, J.Bacteriol. 96, 1291-1297, 1968 66. Ohsugi, M., Miyauchi, K., and Inoue, Y., Biosynthesis of biotinvitamers from unsaturated higher fatty acids by bacteria, J.Nutr.Sci. Vitaminol. 31, 253-263, 1985 67. Sabatie, J., Speck, D., Reymund, J., et al., Biotin formation by recombinant strains of Escherichia coli; influence of host physiology, J.Biotechnol. 20, 29-49, 1991 68. Phalip, V., Kuhn, I., Lemoine, Y., and Jeltsch, J.M., Characterization of the biotin biosynthesis pathway in Saccharomyces cerevisiae and evidence for a cluster containing B105, a novel gene involved in vitamer uptake, Gene 232, 43-51, 1999 69. Wegner, C., Trotz, M., and Nau, H., Direct determination of folate monoglutamates in plasma by high-performance liquid chromatography using an automatic precolumn-switching system as sample clean up procedure, J.Chromatog. 378, 55-65, 1986 70. Freisleben, A., Schieberle, P., and Rychlik, M., Syntheses of labeled vitamers of folic acid to be used as internal standards in stable isotope dilution assays, J.Agric.Food Chem. 50, 4760-4768, 2002
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250 71. Freisleben, A., Schieberle, P., and Rychlik, M., Specific and sensitive quantification of folate vitamers in food by stable isotope dilution assay high-performance liquid chromatography-tandem mass spectrometry, Anal.Bioanal.Chem. 376, 149-156, 2003 72. Pfeiffer, C.M., Fazili, Z., McCoy, L., et al., Determination of folate vitamers in human serum by stable-isotope-dilution tandem mass spectrometry and comparison with radioassay and microbiological assay, Clin.Chem. 50, 423-432, 2004
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Handbook of Biochemistry and Molecular Biology 73. Smulders, Y.M., Smith, D.E, Kok, E.M., et al., Cellular folate vitamer distribution during and after correction of vitamin B12 deficiency: a case for the methylfolate trap, Br.J.Haematol. 132, 623-629, 2006 74. Smith, D.E., Kok, R.M., Teerlink, T., et al., Quantitative determination of erythrocyte folate vitamer distribution by liquid chromatography-tandem mass spectrometry, Clin.Chem.Lab.Med. 44, 450-459, 2006
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Vitamin names discarded • Vitamins Bc, B10, B11, and Bx (these mostly have been used to refer to folic acid or folic acid precursors such paminobenzoic acid although it would appear that these terms were also used to refer to mixtures of vitamins). • Vitamin M is a term that has recently been used to describe delta(Δ)-1-tetrahydrocannabinol Earlier the term vitamin M was used to describe a mixture of B-complex vitamins • Vitamin B4 (mostly used to describe adenine but occasionally for choline) Lecoq, R., The role of adenine (vitamin B4 in the metabolism of organic compounds and its repercussions on acid-base equilibrium, J.Physiol. 46, 406-410, 1954; Whelan, W.J., Vitamin B4, IUBMB Life 57, 125. 2005; Hartmann, J. and Getoff, N., Radiation-induced effect of adenine (vitamin B4) on mitomycin C activity. In vitro experiments, Anticancer Res. 26, 3005-3010, 2006 • Vitamin L (anthranilic acid; o-aminobenzoic acid) • Vitamin Bc – folate although earlier used to describe the B complex vitamins • Vitamin B10 –folate, precursors of folate such as p-aminobenzoic acid; R factor; also used to refer to vitamin A/retinoic acid; Wang, Y. and Okabe, N., Crystal structures and spectroscopic properties of Zinc(II) ternary complexes of Vitamin L, H’ and their isomer m-aminobenzoic acid with bipyridine, Chem.Pharm.Bull. 53, 645-652, 2005
• Vitamin B11 – folic acid: Getoff, N., Transient absorption spectra and kinetics of folic acid (vitamin B11) and some kinetic data of folinic acid and methotrexate, Oncol.Res. 15, 295-300, 2005 Pseudovitamins (also described as “fake” vitamins; Young, V.R. and Newberne, P.M., Vitamins and cancer prevention: Issues and dilemmas, Cancer 47, 1226-1240, 1981) Vitamin B17 (Laetrile®; amygdalin; 1-mandelonitrile-β-glucuronic acid) Vitamin B15 (pangamic acid; not a chemically defined entity) Vitamin B13 (Orotic acid) H3 (Gerovital) U (methionine sulfonium salts)
General references for vitamins Handbook of Vitamins, 2nd edn., ed. L.J. Machlin, Marcel Dekker, Inc., New York, NY, USA, 1991 Coumbs, G.F., The Vitamins. Fundamental Aspects in Nutrition and Health, 2nd edn., Academic Press, San Diego, CA, USA, 1998 Stipanuk, M.H., Biochemical and Physical Aspects of Human Nutrition, W.B. Saunders, Philadelphia, PA, USA, 2000 Bender, D.A., Nutritional Biochemistry of the Vitamins, 2nd Edn., Cambridge University Press, Cambridge, United Kingdom, 2003
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Section IV Nucleic Acids
•
UV Spectral Characteristics and Acidic Dissociation Constants of 280 Alkyl Bases, Nucleosides, and Nucleotides. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 255 Ultraviolet Absorbance of Oligonucleotides Containing 2¢-O-Methylpentose Residues. . . . . . . 263 Spectrophotometric Constants of Ribonucleotides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 265 Purines, Pyrimidines, Nucleosides, and Nucleotides: Physical Constants and Spectral Properties. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 269 Chemical Modification of Nucleic Acids. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 359 Transfection Technologies. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 373
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Uv Spectral Characteristics and Acidic Dissociation Constants Of 280 Alkyl Bases, Nucleosides, And Nucleotides B. Singer The λmax (nm), in those cases where more than one value has been reported, are either the most frequent value or an average of several values, the range being ± 1 to 2 nm for the λmax. Since the λmin is more sensitive than the λ max to impurities in the sample, the values of λmin in the table are generally the lowest reported. Values in parentheses are shoulders or inflexions. The cationic and anionic forms are either so stated by the authors or are arbitrarily taken at pH 1 and pH 13. Individual values are given for pKa except when there are more than two values. In that case, a range is given. Complete spectra representing a range of derivatives are shown in the figures, and reference to these is made in the table with an asterisk and number preceding the name of the compound.
λmax(nm)
Acidic
λmin(nm)
All spectra were obtained in the author’s laboratory from samples isolated from paper chromatograms. It is recognized that pH 1 or pH 13 is not ideal for obtaining the cationic or anionic forms when these pHs are close to a pK. Nevertheless, these conditions are useful for purposes of identification since the spectra are reproducible. Additional data not quoted here are available in many of the references. These data include spectral characteristics in other solvents than H2O and at other pH values, extinction coefficients, RF values in various paper chromatographic systems, column chromatographic systems, methods of synthesis or preparation of alkyl derivatives, mass spectra, and NMR, optical rotatory dispersion and infrared spectra.
λmax(nm)
Basic
λmin(nm)
pKa
References
234 242
7.2 6.9, 7.0
239 244 247
7.0 7.2 7.2 ∼5.1 6.1, 6.1 6.5
1–7 5, 8, 9 10 11, 12 13 6 3, 4 2, 5, 6, 13–15 5, 8, 16 10 17, 18 13 19 1–4, 7, 15, 20, 21 8, 21 21 72 19 8, 15, 18, 23, 24 8, 16 10 5, 13, 15 5, 13, 25 10 13 19 18
* Adenine 1
Monoalkylated *21-Methyl 1-Ethyl 1-Isopropyl 1-Benzyl 1-(2-Hydroxypropyl) 1-(2-Hydroxyethylthioethyl) 2-Methyl *33-Methyl 3-Ethyl 3-lsopropyl 3-Benzyl 3-(2-Hydroxypropyl) 3-(2-Diethyloaminoethyl) *4N6-Methyl N6-Ethyl N6-ButylN6-(2-Hydroxyethyl)N6-(2-Diethylaminoethyl)*57-Methyl7-Ethyl7-Isopropyl9-Methyl9-Ethyl9-Isopropyl9-(2-Hydroxypropyl)9-(2-Diethylaminoethyl) 9-Benzyl-
259 260 259 260 259 262 267 274 274 274 275 274 275 267 268 270 272 275 273 272 272 261 258 260 259 258 259
Dialkylated 1,N6-Dimethyl1,7-Dimethyl1-Butyl, 7-methyl1,9-Dimethyl1,9-Di(2-hydroxypropyl)1,9-Di(2-diethylaminoethyl)-
261 270 268 260 260 257
228 233
228 235 240
236 231 231 233 233 237 239 230 230 227
270 271 269 271 271 271 270 (280) 273 273 273 272 273 274 273 (280) 274(281) 275 273 274 270(280) 270 (280) 272 262 262 262 261 261 261
245 238 241 236 239 230 234 228 228
4.2, 4.2 3. 7 ∼3.5, 3.6 3.9 4.1
229
230
273
245
235
260(265) 260 261
235
233
5.1 6.0
232
9.08
1, 8, 20 26 26 13, 20, 27 13 19
255
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256
UV SPECTRAL CHARACTERISTICS AND ACIDIC DISSOCIATION CONSTANTS OF ALKYL BASES, NUCLEOSIDES, AND NUCLEOTIDESa (Continued) λmax(nm)
Acidic
λmin(nm) *1
1-Ethyl-9-methyl1-Propyl-9-methyl3,N6-Dimethyl3,N6-Di(2-diethylaminoethyl)3,7-Dimethyl3,7-Dibenzyl3,7-Di(2-hydroxypropyl)3,7-Di(2-diethylaminoethyl)N6,N6-DimethylN6,N6-DiethylN6,7-Dimethyl*6N6-Methyl-7-ethylN6-Propyl-7-methy1N6-Butyl-7-methylN6,9-DimethylN6,9-Di-2-diethylaminoethyl)N6-Ethyl-9-methylN6-Propyl-9-methylN6-Butyl-9-ethylTrialkylated 1,N6,N6-Trimethyl3,N6,N6′-TrimethylN6,N6,7-TrimethylN6,N6,9-TrimethylN6,N6-Dimethyl-9-ethyl3,N6,7-Tribenzyl-
261 261 281 282 276 278 278 276 276 278 279 277 (285) 281 279 265 266 265 266 266 221, 293 290 233, 293 269 270 289
λmax(nm)
9168_Book.indb 256
250 (270) 251 (274) 253 255, 275 251, 279 253 (280) 252 263 (244) 263 (244) 263 263 (243) 286 286 286 285 (230) 286 286 (232) 249 (272) 249 (274) 249 (274) 250 250 250, 272 253, 270 250 249, 276 249, 278 249, 277 251, 276 252, 277 253, 276
261 261 287 282 225, 280 281 281 279 282 282 275 276 277
243 246 237 236 241
268 270 268 270 269
229
246 243 250 234
228 229 228 229 227 233
253
226 233 229 229
230
λmin(nm)
pKa
References
9.16 9.15
27 27 28 19 2, 13, 20 18 13 19 7, 21 21, 29 24, 26 8 24 26 27, 29 19 27, 29 27 27
Adenine (Continued)
232, 301 293 291 276 277 Unstable *7
Monoalkylated 1-Methyl*81-Ethyl1-Isopropyl1-(2-Diethylaminoethyl)N2-Methyl-b *9N2-EthylN2-Isopropyl3-Methyl*103-Ethyl3-Isopropyl3-BenzylO6-Methyl*11O6-EthylO6-PropylO6-IsopropylO6-ButylO6-Isobutenyl7-Methyl*127-Ethyl7-Isopropyl7-Benzyl7-(2-Hydroxyethyl)7-(2-Hydroxypropyl)7-(2-Diethylaminoethyl)7-(β-Hydroxyethylthioethyl)8-Propyl8-lsobutyl8-(3-Methylbutyl)9-Methyl *139-Ethyl9-Isopropyl-
Basic
249 221, 247
11 9.6
245 245 244
233
4.02 4.08 4.14
262 250 246 237 9.4
30 30 30 29, 30 25 18
Guanine 277 (262) 278 (260) 257, 268 245–255, 278 245, 279 277 273 273 273 274 246, 284 284 (246) 246, 283 283 (245) 246, 285 283(245) 281 (240) 280 278 (240) 281 281 280 275 (250) 281 276 276 275 268 (258) 253, 268 256, 258
242 243
3.1
238 263
3.3
246 248 4.00 259
255 258 261 257
238
3.5 3.7 3.2
2.9
4, 7, 15, 23, 31–34 35, 36 10 19 4, 7, 23, 33, 34, 37, 38 37, 39 10 15, 33, 40, 41 39 10 42 43, 44 35, 44 44 10, 45 44 45 4, 23, 31, 32, 46 5, 35 10, 45 11 47 13 19 48 45 45 45 33 35 10, 45
4/16/10 1:16 PM
UV Spectral Characteristics and Acidic Dissociation Constants
257
UV SPECTRAL CHARACTERISTICS AND ACIDIC DISSOCIATION CONSTANTS OF ALKYL BASES, NUCLEOSIDES, AND NUCLEOTIDESa (Continued) λmax(nm)
Acidic
λmin(nm) *7
Dialkylated 1,7-Dimethyl*141,7-Diethyl1,9-DimethylN2,N2-Dimethyl-b 7,8-Dimethyl7,9-Dimethyl 7,9-Di(2-diethylaminoethyl)7-Methyl-9-ethyl8,9-DimethylTrialkylated 1,7,9-Trimethyl-
252 (272) 252 (275) 254 (277) 255 (289) 249, 277 254 (278) 257, 278 254, 281 252, 277 (289)
λmax(nm)
λmin(nm)
pKa
References
4.4
23, 33, 35 49 33 4, 34, 37, 38 50 28, 33 19 14 50
Guanine (Continued)
230 232 229 229
284(251) 285 (250)
262 263
277–283 280 (235) c c c 280(252)
229
254, 280
7.3 4.11
c *15
Monoalkylated 1-MethylO2-Methyl3-Methyl*163-EthylN4-MethylN4-Ethyl5-Methyl6-Methyl-
Basic
33
Cytosine
213, 283 260 273 275 278 277 211, 284
241 241 240 241 240 244 242
274 270 294 296 286 (230) 284 288
250 246 251 257 256 253 254
4.55–4.61 5.41 7.4, 7.49 4.55 4.58 4.6 5.13
51–53 53 4, 53, 54 55 4, 56, 57 55, 57 58 59
Dialkylated 1,3-Dimethyl1,N4-DimethylN4,N4-Dimethyl1,5-Dimethyl-
281 218, 285 283 291
243 244 242 244
272 274(235) 290(235)
247 250 259
9.29–9.4 4.38–4.47 4.15, 4.25 4.76
51, 53, 54 51, 53, 56 56, 57 60
Trialkylated 1,3,N4-Trimethyl1,N4,N4-Trimethyl-
212, 287 220, 288
248 248
280 283
247 242
9.65 4.2
19, 51, 53 56
7.1
7 14
5-Hydroxymethylcytosine Unmodified 279 278
3-Methyl-
284 296 *17
1-Methyl3-Methyl-d *197-Methyl-d 9-Methyl-d 1,3-Dimethyl1,7-Dimethyl1,9-Dimethyl3,7-Dimethyl3,9-Dimethyl7,9-Dimethyl8,9-Dimethyl1,3,7-Trimethyl1,3,9-Trimethyl1.7,9-Trimethyl*18
d
260–265, (235) 266 267 260 266 ∼260 262 265 265 239, 262 238, 265 266 266 232, 262
Xanthine
239
283 (245) 275 (232) 289 (237) 245, 278 275 233, 289 248, 277 234, 274 270 (240) c 245, 278
233
9168_Book.indb 257
208, 268 218, 260 258 267 269 266 208, 268 213, 269 205, 259 267 206, 276
255
241
35, 61, 62 61, 62 32, 35, 61 61, 62 61, 62 61, 62 61, 62 61, 62 61, 62 63 63 61 61 63
Uracil
234 234
265 221, 265 218, 283 276 220, 278 266 266
234 241
231 236 240
281 275 273
245 245 245
230
1.3 0.8 0.8 2.0 0.7 0.5 2.5 0.3 1.0 0.5 0.6
c *20
1-MethylO2-Ethyl*213-MethylO4-MethylO4-Ethyl1,3-Dimethyl1,6-DimethylO2,3-Dimethyl3,6-Dimethyl5,6-Dimethyl1,5,6-Trimethyl-
257
242 245
∼1.8
58, 64 58 4, 58, 65 65a 58 58 65 65 b 65 65 65
4/16/10 1:16 PM
Handbook of Biochemistry and Molecular Biology
258
UV SPECTRAL CHARACTERISTICS AND ACIDIC DISSOCIATION CONSTANTS OF ALKYL BASES, NUCLEOSIDES, AND NUCLEOTIDESa (Continued) λmax(nm)
Acidic
λmin(nm)
λmax(nm) *22
1-Methyl1-(2-Diethylaminoethyl)3-Methyl*233-Ethyl3-(2-Diethylaminoethyl)1,3-Di(2-diethylaminoethyl)1,04-Di(2-diethylaminoethyl)O2,3-Dimethyl-
266 265
Basic
217, 272
pKa
References
Thymine 269 265 290 289 288 269 274
237 237
λmin(nm) 244 248 248 247 244 245 245
66 19 31, 35, 66 35 19 19 19 65b
Hypoxanthine 1-Methyl3-Methyl3-Ethyl3-Benzyl3-(2-Diethylaminoethyl)O6-(3-Methyl-2-butenyl)7-Methyl9-Methyl1,7-Dimethyl1,7-Dibenzyl1,9-Dibenzyl3,7-Dimethyl3,7-DibenzylN6,7-Dimethyl7,9-Dimethyl-
249 253 254 254 260 247 250 250 252 255 263
260 265 266 264(277) 262 262 262 254 267 256 259 267 267 258 c
234
232
256 256 251 *24
1-Methyl1-Methyldeoxy*261-Ethyl1-Ethyldeoxy1-Benzyedeoxy2-Methyl*27N6-MethylN6-MethyldeoxyN6-EthylN6-EthyldeoxyN6-BenzylN6-BenzyldeoxyN6-ButylN6-(2-Hydroxyethyl)*287-Ethyl*291,N6-DimethylN6,N6-Dimethyl*30N6,7-DimethylN6-Methyl-7-ethyl*25
257 257 259 259 259 258 262 262 264 263 265 264 263 263 268 261 268 276 276
231 239 235 231
233 239 234 233 241 242 *31
*321-Methyl1-Methyldeoxy1-Ethyl1-Ethyldeoxy1-Butyldeoxy-
N2-Methyl-b O -MethylO6-Methyldeoxy*33O6-EthylO6-EthyldeoxyO6-Butyldeoxy7-Methyl6
9168_Book.indb 258
258 (280) 257 (278) 261 (272) 256 258 (282) 251–258 (280–290) 284 (243) 284 (230) 244, 286 286 246, 287 257 (275)
230 232 232
222–234 259 252 239, 260 252 260 230
243 2.12 237
15, 66a 15, 66a, 67 68 69 19 45 15 15 70 69 69 20 69 24 28
Adenosine 258 (265) 258 261, (268, 300) 260 (268) 259 264 266 266 268 268 268 268 267 267 c 263 (300) 276 c c
230 231 231 239 237 235
2.61
233 242 237 236 227 223 226 243 241 236
8.8, 8.3
4.0
232
3.1
234 237
4.5
3, 4, 8, 16, 71 8, 72 8 8 11 3, 4, 73 1–4, 8, 74, 74a 4, 8, 72, 74 8 8 69, 75 11 76 22 8 1, 8, 20 3, 4, 30 8 8
Guanosine 255 (270) 255 (270) 258 (270) 257 257 (280) 248–258 (270–275) 243, 277 243, 278 247, 278 248, 280 248, 280 c
228 229 239
2.6 2.8
227–238 239, 261 233, 261 233, 261 261 261
4, 7, 20, 34 20, 31, 77 49 77 77 4, 34, 38
2.4 25 6.7–7.3
49 31, 78 49 77 77 4, 14, 46, 49, 74
4/16/10 1:16 PM
UV Spectral Characteristics and Acidic Dissociation Constants
259
UV SPECTRAL CHARACTERISTICS AND ACIDIC DISSOCIATION CONSTANTS OF ALKYL BASES, NUCLEOSIDES, AND NUCLEOTIDESa (Continued) λmax(nm)
Acidic
λmin(nm) *31
7-Methyldeoxy7-Ethyl7-Isopropyl7-Benzyl7-Butyldeoxy8-Methyl1,7-Dimethyl*351,7-Diethyl1-Methyl-7-ethyl1-Ethyl-7-methylN2,N2-DimethylN2,O6-Dimethyldeoxy*36,N2,O6-DiethylN2,N2,7-Trimethyl*34
256 (275) 258 (277) 256 (275) 258 257 (280) 260 (273) 260 (270) 263 (270) 259 (277) 261 (275) 265 (290) 288 246, 292 267, 300
λmax(nm)
233, 262 233, 262 278 280 280 281 284 217, 281 282 281 279 281
229 238
c c c c c 256 c c c c 262 (283) 249, 284 252, 281 c
236 237 233 235 237 239, 267 239, 286
O -MethylO2-Ethyl3-Methyl*443-Ethyl3-Benzyl3-(2-Hydroxyethyl)*44aO4-Methyl6-Methyl5,6-Dimethyl-
221, 243 221, 243 243 247 245
Unstable Unstable 225, 267 267 268 266 271 237, 273 236, 270 272 272 271
243 207, 243 242 244 247 242
9168_Book.indb 259
7.2, 7.4 7.2 3.01
240 237, 268
212, 244 248 247 250 229, 248 253 253 250
281
242
271
247
288 287
245 246
278 278 286 287 284 219, 285 287 286 287
241 241 249 252 245 245 245 249 246
278 278 278 273 273 277 277
255 255 255 252 252 249 253
218, 287 287
246 252
279 278 276 278 275 (234) 275(235) 289
238 238 249 238 252
229, 251 228, 253 263 262
213, 238 213, 237 233 235
261 271 261 206, 269
235 235 230 236 *45
3-Methyl*463-Ethyl3-(2-Diethylaminoethyl)O4-Methyl-(α) O4-Methyl-(β)
266 269
238 239
279 279
245 241
pKa
References 31, 32, 74, 77 14, 49 45 11 77 50 20, 49 49 49 49 4, 7, 34, 38 77 35, 49 79
Cytidine
*43 2
λmin(nm)
Guanosine (Continued)
*37
O2-Methyl*37aO2-Ethyl3-Methyl*383-Ethyl3-Ethyldeoxy3-Benzyl3-(2-Diethylaminoethyl)deoxyN4-MethylN4-Methyldeoxy*39N4-EthylN4-Ethyldeoxy2′-O N4-Dimethyl-e 2′,3′,5′-Tri-O-methyl-N4methyl-e 5-Methyl5-Methyldeoxy5-Ethyldeoxy6-Methyl 6-Methyldeoxy3,N4-Dimethyl*403,N4-Diethyl3,N 4Di(2-diethylaminoethyl)deoxyN4, N4-DimethylN4, N4-Dimethyldeoxy*41,N4,N4-Diethyl2′-O,N4,N4-Trimethyl-e N4,5-DimethylN4,5-Dimethyldeoxy*423,N4,N4-Triethyl-
Basic
253
>8.6 >8.6 8.3–8.9 8.4 8.6 7.7 3.85, 3.92 4.01 4.2 4.2
35 35 4, 54–56, 71 55 55 11 19 56, 57 57 55 55 80 81
4.28 4.40 4.42
3.7, 3.62 3.79
4.04
60 60, 82 83 59, 84 84 55 55 19 56, 57 57 55 80 60 60 55
Uridine
262 264 264 262 274 264 270
239 242 248
35, 86, 87 87 4, 14, 55, 85 35, 55 83 88 80 65, 84 65
239 240 242 243 243
31, 35 35 19 89 89
233 237 235
Thymidine (deoxy-) 267 270 270 279 280
4/16/10 1:16 PM
Handbook of Biochemistry and Molecular Biology
260
UV SPECTRAL CHARACTERISTICS AND ACIDIC DISSOCIATION CONSTANTS OF ALKYL BASES, NUCLEOSIDES, AND NUCLEOTIDESa (Continued) λmax(nm)
Acidic
λmin(nm)
λmax(nm) *47
1-Methyl1-Methyldeoxy1-Benzyl1-(2-Hydroxyethyl)O6-Methyl*487-Methyl7-Ethyl1,7-Dimethyl-
250 250 251 250 250 252 252 265
Basic
λmin(nm)
pKa
References
Inosine
223
249 250(265) 249 250 250 c c c
226 221
226 6.4
4 31 69 88 66a, 74a 35, 70 68 70
Xanthosine 7-Methyl-
262
237
c
32, 35
Adenylic Acid or ADPe,f 1-Methyl1-(2-Hydroxyethylthioethyl)2-MethylN6-MethylN6-(2-Hydroxyethylthioethyl)N6-(2-Hydroxyethyl)2,N6-Dimethyl
258 261 259 261
232
263 263
233
231
259 (268) 261 263 265 268 266 269
230
6, 35, 90 6 73 6, 90 6 91 92
229 230
Guanylic Acid or GDPe,f 1-Methyl7-MethylO6-MethylN2-MethylN2,N2-DimethylN2,N2,7-Trimethyl-
258 (280) 259, 279 245, 288 263 265 262 (290)
230 230 262 237 232 237
256 (273) c 249, 281 263 263 c
230 263 240 237
340°
–
˜5.1f
–
C10H13N5(203.24)
231–232°
–
–
5.4
C8H8N6O3 (236.19)
233–234° (dec)
–
–
–
N H
N
HO
272
PURINES, PYRIMIDINES, NUCLEOSIDES AND NUCLEOTIDES: PHYSICAL CONSTANTS AND SPECTRAL PROPERTIES (Continued)
NH2 6
2-Methyladenine
N
N
2MeAde
N H
N NH
N
N 3iPeAde 3PeiAde
3-(Δ2-Isopentenyl)adenine Triacanthine
7
N
N H
N H N
COOH
O HN
N6-Glycinocarbonyladenine N-(Purin-6-ylcarbamoyl)glycine
8
6(Gly C O) Ade
N
No.
Acidic Spectral Data pH
λmax
εmax (×10−3)
λmin
Neutral Spectral Data Spectral Ratios
5
2
284*
11.7*
248
230 –
6 7 8
1 1 1.4
266 277 276.5
12.9 18.3 18.6
229 239 235
0.26 – 0.46
240 –
250 0.45
270 –
280 3.16
290 –
0.48 – 0.44
0.79 – 0.61
1.03 – 1.62
0.56 – 1.82
0.04 – 0.92
pH
λmax
7
240 286 263 273 269
7 7 6.2
εmax (×10−3) 7.8 8.0* 12.7 12.5 17.4
λmin 210 255 226 247 231
Alkaline Spectral Data Spectral Ratios
230 – – – 0.40
240 – 0.46 – 0.49
250 –
270 –
280 –
290 –
0.89 – 0.68
0.83 – 1.43
0.16 – 0.98
– – 0.17
pH
λmax
λmin
284
εmax (×10−3) 12.3
12 13 – 12.3
271 – 278
10.7 – 16.2
Spectral Ratios
253
230 –
240 –
250 0.80
270 –
280 3.47
290 –
238 – 240
0.77 – 1.34
0.40 – 0.37
0.61 – 0.54
1.28 – 1.76
0.84 – 2.10
0.04 – 1.23
References No. 5 6 7 8
Origin and Synthesis C: 282,340a,358,111 C: 17g,257a,340,111 R: 18 C: 287,1 C: 408,R: 295
t
[α] D
pK
Spectral Data
– – – –
22 19 1 –
28,283*bc,223*e,282b,284b,340,358* 18b,317bcde,20d,21,257 1 408
Mass Spectra
Rf
– – – 408
340 18,21,291,340 – 408
Handbook of Biochemistry and Molecular Biology
N H
N
4/16/10 1:16 PM
No.
Compound
3-Letter
Symbol
1-Letter
Structure
O
O N6-Threoninocarbonyladenine N-(Purin-6-ylcarbamoyl)threonine
9
N
N6-(Δ2-Isopentenyl)adenine (N6-(γγ-Dimethylallyl)adenine; 6-(3-Methyl-2-butenylamino)purine
N6-(Δ2-Isopentenyl)-2-methylthioadenine 6-(3-Methyl-2-butenylamino)-2-methylthiopurine
215–220°
C10H13N5 (203.24)
213–215°
C11H15N5S (249.32)
C6H7N5 (149.16)
+3025 (0.4, H2O)
pK
Acidic
350°
–
390° (dec)
–
˜0, 3.5
9.9*
C5H4N4O (136.11)
>350° (dec)
–
2.0
8.9*, 12.1
C6H6N4O (150.14)
311–312°
–
˜2
8.9*, ˜13
C6H6N4O (150.14)
>280° (dec)
–
2.6
8.3
C6H6N4O (150.14)
355° (dec)
–
2.1
8.9
N
N
H2N
pK
Formula (Mol Wt)
O 27
Hypoxanthine
N
HN
Hyp
N H
N O 28
1-Methylhypoxanthine
N
N
1MeHyp
N H
N O 29
3-Methylhypoxanthine
N
N
3MeHyp
N
N H
Purines, Pyrimidines, Nucleosides, and Nucleotides
9168_Book.indb 277
PURINES, PYRIMIDINES, NUCLEOSIDES AND NUCLEOTIDES: PHYSICAL CONSTANTS AND SPECTRAL PROPERTIES (Continued)
O 30
7-Methylhypoxanthine
N
N
7MeHyp
N H No.
Acidic Spectral Data pH
λmax
26
1
27
0
250 272 248
28 29 30
1 0 0
249 253 250
εmax (×10−3)
λmin
10.6* 6.9* 10.8 9.4 11.0 10.2
N
Neutral Spectral Data Spectral Ratios
pH
230
240
250
270
280
290
228*
0.55
0.99
1.30
0.84
0.79
0.52
7
215
–
–
1.45
–
0.04
0.00
6
219 – 224
0.59 – –
1.11 – –
1.37 – –
0.43 – –
0.10 – –
0.01 – –
5 5 5
λmax
εmax (×10−3) 283 9.3 248 5.7* 283 7.4* 249.5 10.7 251 264 256
9.4 14.0 9.5
λmin
Alkaline Spectral Data Spectral Ratios
230
240
250
270
280
290
pH
λmax
εmax (×10−3)
λmin
Spectral Ratios 230
240
250
270
280
290
235 261 222
1.54
1.42
1.46
1.35 1.87
1.73
12
280*
7.4*
257
1.92
1.50
1.013
1.50
1.89
1.47
0.53
1.05
1.32
0.57 0.09*
0.01*
223 – 229
0.51 – 0.42
0.95 – 0.81
1.31 – 0.97
0.53 0.16 – – 0.55 0.07
0.02 – 0.00
11 14 11 11 11
259 263 260 265 262
11.1 11.5 9.7 10.9 10.6
232 233 242 – 230
0.48 – – 0.36
0.46 – – 0.49
0.84 0.71 – – 0.76
0.84 – – 0.83
0.12 0.19* – – 0.21
0.01 0.01* – – 0.00
References Origin and Synthesis
26 27 28 29 30
C: 45,10 C: 257,337,358,359,377 C: 33,26,360 C: 26,50a C: 45
[α] D – – – – –
pK 39,27* 22,170,253,51* 33,27* 33 33,51
Spectral Data 46b,47b,42*b,35b,39,41,48 232b,242*e,334*,317b,205,257,358 49,33cd,317bc,27,121,360 33,121 52,33c,d
Mass Spectra
Rf
– – – – –
10,13,24,27,41,42,46,334 258,242,278 24,26,27,49,33,121 26,33,121 10,26,27,33
277
No.
t
4/16/10 1:17 PM
9168_Book.indb 278
No.
Compound
3-Letter
31
Uracil
Ura
Symbol
1-Letter
Structure O
NH N H
278
PURINES, PYRIMIDINES, NUCLEOSIDES AND NUCLEOTIDES: PHYSICAL CONSTANTS AND SPECTRAL PROPERTIES (Continued) pK
Formula (Mol Wt)
Melting Point °C
[α]Dt
Basic
Acidic
C4H4N2O2 (112.09)
315° (dec)
–
–
9.5, >13
C5H6N2O2 (126.11)
232–233°
–
–
9.7
C4H4N2OS (128.15)
340° (dec)
–
–
–
C5H6N2OS (142.18)
196–198°
–
–
–
O
O 32
1-Methyluracil
NH
1MeUra N
O
O 33
2-Thiouracil
NH
2SUra 2Sra
S
N H O 34
2-Methylthiouracil S-Methyl-2-thiouracil
31 32 33 34
NH N
Acidic Spectral Data εmax (×10−3)
λmin
260 259.5 272 270*
7.8 8.2 – 13.9*
229 227 – 241
221 248
10.0 7.9
pH
λmax
0q 4 2 1.5 1 (MeOH)
Neutral Spectral Data Spectral Ratios
230 0.23
240 0.48
–
–
S
250 0.80 0.84 0.60
270 0.68 –
280 0.30 0.17 0.64
290 0.05 0.01 0.08
pH
λmax
εmax (×10−3) 259.5 8.2
λmin
7 7.4
267 268*
9.8 11.8
232 240
(MeOH)
228 286
7.9 7.9
7
227
Alkaline Spectral Data Spectral Ratios
230 240 0.21 0.47 0.17
0.25
250 0.84
270 280 0.68 0.17
290 0.01
0.59
1.12 0.70
0.12
pH
λmax
12 12 11 13 (MeOH)
λmin
284
εmax (×10−3) 6.2
265* 259 307* 222 243
7.0 10.7 6.8 15.9 7.9
241 243* 291*
241
Spectral Ratios 230 1.09
240 0.56
250 0.71
270 1.25
280 1.40
290 1.27
0.92
0.53
0.68
1.00
0.44
0.03
References No.
Origin and Synthesis
31 32 33 34
C: 364,362a,84,395 C: 79,78,362, C: 84 C: 301
[α] D
pK
– – – –
66,80 66,80 – –
t
Spectral Data 66b,242be,232bcde,317b 66b,81 *b,c,e 277b,331bc,86*bd 301
Mass Spectra
Rf
– – – –
258,40,242,402 69,81 – 301
Handbook of Biochemistry and Molecular Biology
No.
2(MeS)Ura 2MeSra
4/16/10 1:17 PM
Symbol
No.
Compound
3-Letter
35
5-Carboxymethyl-2-thiouracil 2-Thiouracil-5-acetic acid
5Cm2SUra 5Cm2Sra 5(CxMe)2SUra
5-Carbamoylmethyl-2-thiouracil 2-Thiouracil-5-acetamide
5Ncm2Sra 5(NcMe)2SUra 5(NcMe)2Sra 5Ncm2SUra
5-(Methoxycarbonylmethyl)-2- thiouracil 2-Thio-5-carboxymethyluracil methyl ester
5(MeCm)2Sura 5MeCm2Sra 5(MeCxMe)2Sra
36
1-Letter
Structure O
HOOC
NH N H
pK
Formula (Mol Wt)
Melting Point °C
[α]Dt
Basic
C6H6N2O3S (186.19)
275–279° (dec)
–
–
4.15, 8.4, ˜13.5
C6H7N3O2S (185.21)
269–270° (dec)
–
–
–
C7H8N2O3S (200.22)
218–220°
–
–
–
S
Acidic
O H2NOC
NH N H
S
O 37
No.
MeOOC N H
Acidic Spectral Data pH
λmax
εmax (×10−3)
λmin
35
1
276
15.6
242
36
1
214 274
13.9 14.9
240
37
1.5
214 276
15.6 16.6
240
0.49
240
0.31
S
Neutral Spectral Data Spectral Ratios
230
NH
250
0.47
270
1.48
280
1.52
290
1.44
pH
7
λmax
215 275
εmax (×10−3)
λmin
– 16.6n
240
Alkaline Spectral Data Spectral Ratios
230
0.47
240
0.32
Purines, Pyrimidines, Nucleosides, and Nucleotides
9168_Book.indb 279
PURINES, PYRIMIDINES, NUCLEOSIDES AND NUCLEOTIDES: PHYSICAL CONSTANTS AND SPECTRAL PROPERTIES (Continued)
250
0.47
270
1.45
280
1.49
290
1.41
pH
λmax
13q
12
λmin
236 259 313
εmax (×10−3) 9.9 11.1 8.3
234 260 313
– 12.0n 8.6n
244 290
Spectral Ratios 230
240
250
270
280
290
0.91
0.83
0.85
0.80
0.59
0.51
244 288
References No.
Origin and Synthesis
35 36 37
C: 302 C: 302 C: 5,302
t
[α] D
pK
Spectral Data
– – –
302 – –
302 302 317b,302cd,5c
Mass Spectra
Rf
– – –
302 302 302
279
4/16/10 1:17 PM
9168_Book.indb 280
Symbol
No.
Compound
3-Letter
38
5-Methoxy-2-thiouracil 4-Hydroxy-2-mercapto-5-methoxypyrimidine
5MeO2SUra 5MeO2Sra
2-Thiothymine 5-Methyl-2-thiouracil
2Sthy 5Me2SUra 5Me2Sra
1-Letter
Structure O
MeO
NH N H
280
PURINES, PYRIMIDINES, NUCLEOSIDES AND NUCLEOTIDES: PHYSICAL CONSTANTS AND SPECTRAL PROPERTIES (Continued) pK
Formula (Mol Wt)
Melting Point °C
[α]Dt
Basic
Acidic
C5H6N2O2S (158.18)
280–281° (dec)
–
–
–
C5H6N2OS (142.18)
265–267°
–
–
–
C5H6N2O2 (126.12)
179°
–
–
10.0
C4H4N2OS (128.15)
289–290° (dec)
–
–
–
S
O 39
NH S
N H O
40
3-Methyluracil
N
3MeUra
O
N H SH 41
4Sura 4Sra
4-Thiouracil
N H
Acidic Spectral Data pH
λmax
εmax (×10−3)
λmin
Neutral Spectral Data Spectral Ratios
230
240
250
270
280
290
pH
λmax
7 7 7
274 259 328
38 39 40 41
1 3 1
276 259 327
19.5 7.3 17.5
– 277
– –
– –
O
0.86 –
– –
0.14 –
0.02 –
εmax (×10−3)
16.9 7.3 16.6
λmin
230 275
Alkaline Spectral Data Spectral Ratios
230
0.29 –
240
0.46 –
250
0.84 –
270
0.66 –
280
0.14 –
290
0.00 –
pH
λmax
εmax (×10−3)
11
260 309 283 335
16.6 7.3 10.7 17.6
12 11
λmin
243 278
Spectral Ratios 230
240
250
270
280
290
1.43 –
0.34 –
0.37 –
2.23 –
3.47 –
3.01 –
References No.
Origin and Synthesis
38 39 40 41
C: 397 C: 301 C: 82,79a,83 C: 85,84
[α]tD
pK
– – – –
– – 66,80 –
Spectral Data – 86 66b,81b,e 86b,317b
Mass Spectra
Rf
– – – –
– – 13,69,81 –
Handbook of Biochemistry and Molecular Biology
No.
N
4/16/10 1:17 PM
Symbol
No.
Compound
3-Letter
42
5-Carboxymethyluracil Uracil-5-acetic acid
5CmUra 5CxMeUra
1-Letter
Structure O
HOOC
NH N H
pK
Melting Point °C
[α]Dt
Basic
C6H6N2O4 (170.12)
316–318° (dec)
–
–
4.3, 10.0
C7H8N2O4 (184.15)
236–237°
–
–
–
C4H4N2O3 (128.09)
>300° (dec)
–
–
8.0
C5H6N2O3 (142.12)
341–345° (dec)
–
–
–
Formula (Mol Wt)
Acidic
O
O 43
5-(Methoxycarbonylmethyl)uracil 5-Carboxymethyluracil methyl ester Uracil-5-acetic acid methyl ester
5MeCmUra 5(MeCxMe)Ura
MeOOC
NH N H
O
O 44
HO
5HOUra
5-Hydroxyuracil
NH N H
O
O 45
5-Methoxyuracil 2,4-Dihydroxy-5-methoxy-pyrimidine
No.
1 1 2 –
λmax 262 262 278*
εmax (×10−3)
λmin
8.1 8.2 6.4
231 232 244
NH
5MeOUra N H
Acidic Spectral Data pH
42 43 44 45
MeO
O
Neutral Spectral Data Spectral Ratios
230 0.23
240 0.37
250 0.71
270 0.89
280 0.42
290 0.05
1.16
0.58
0.56
1.52
1.63
1.26
pH
λmax
7
264
6
278*
εmax (×10−3) 7.6 6.4
λmin 234 2.44
Alkaline Spectral Data Spectral Ratios
230 240 0.33 0.33 1.08
0.53
Purines, Pyrimidines, Nucleosides, and Nucleotides
9168_Book.indb 281
PURINES, PYRIMIDINES, NUCLEOSIDES AND NUCLEOTIDES: PHYSICAL CONSTANTS AND SPECTRAL PROPERTIES (Continued)
250 0.66
270 0.98
280 0.56
290 0.13
0.54
1.45
1.55
1.18
pH
λmax
λmin
290
εmax (×10−3) 5.5
13 12
239 305*
6.5* 5.7
Spectral Ratios
246
230 1.95
240 0.78
250 0.63
270 1.40
280 1.77
290 2.00
270
–
–
1.65
0.69
0.83
1.13
References No.
Origin and Synthesis
42 43 44 45
C: 279,278,302a C: 302 C: 87,88a,89 C: 397
t
[α] D
pK
Spectral Data
Mass Spectra
Rf
– – – –
302 – 90 –
278b,302d 302 87b,317bcde,91*b –
278 – – –
278,302,402 302 – –
281
4/16/10 1:17 PM
9168_Book.indb 282
Symbol
No.
Compound
3-Letter
46
Thymine 5-Methyluracil
Thy 5MeUra
5-(Putrescinomethyl)uracil 5-(4-Aminobutylaminomethyl) uracil; N-Thyminylputrescine
5(PutMe)Ura 5(NH2BuNHMe) Ura 5PutThy*
47
1-Letter
Structure NH N H
pK
Melting Point °C
[α]Dt
Basic
C5H6N2O2 (126.11)
310° (dec)
–
–
9.9 > 13
C9H16N4O2 (212.25)
255° (dec) (HCI salt)
–
–
–
C6H9N3O2 (155.16)
230–232°
–
–
–
C5H6N2O3 (142.11)
260–300° (dec)
–
–
9.4* ˜14
Formula (Mol Wt)
O
282
PURINES, PYRIMIDINES, NUCLEOSIDES AND NUCLEOTIDES: PHYSICAL CONSTANTS AND SPECTRAL PROPERTIES (Continued) Acidic
O O
H2N
N H
NH N H
O
O 48
5-(Methylaminomethyl)uracil
N H
5(MeNHMe)Ura
NH O
N H O 49
HO N H
Acidic Spectral Data pH 4 1 1 2
λmax 264.5 261 262 261 206
εmax (×10−3)
λmin
7.9 7.8 – 8.0 9.55
233 230 230 231
240 – – 0.07 –
O
Neutral Spectral Data Spectral Ratios
230 – – 0.01 –
NH
250 0.67 – 0.70 0.77
270 0.97 – 0.85 –
280 0.53 – 0.39 0.32
290 0.09 – 0.13 –
pH 7 7 7.5 7 H2O
λmax
εmax (×10−3) 264.5 7.9 262 – 262 – 261 8.1 207 9.5
λmin
230 240 233 0.28 0.35 230.5 – – 230 0.01 0.07 231 0.27 0.43
Alkaline Spectral Data Spectral Ratios 250 0.67 – 0.70 0.77
270 0.96 – 0.85 0.80
280 0.53 – 0.39 0.33
290 0.09 – 0.13 0.05
pH
λmax
12 13 10.1 12
εmax (×10−3) 291 5.4 288.5 – 287 – 286 7.4
λmin 244 246 244 245
Spectral Ratios 230 1.53 – 3.88 1.77
240 0.68 – 0.42 0.75
250 0.65 – 0.39 0.67
270 1.24 – 1.69 1.39
References No.
Origin and Synthesis
46 47 48 49
C: 263,361,254,101,348,356,395 C: 402 D: 402 C: 270 C: 69,92,93 D: 96
[α] D
pK
– – – –
66,80 – – 72,94*
t
Spectral Data 66b,232b,317b 402 270 94b,69cde,317bd,72b,95b,74
Mass Spectra – 402 270 –
Rf 258,21,18,402 402 270 69,96,97,74,402
280 1.31 – 2.18 1.80
290 1.41 – 2.32 1.75
Handbook of Biochemistry and Molecular Biology
No.
46 47 48 49
5HmUra 5HOMeUra
5-Hydroxymethyluracil
4/16/10 1:17 PM
Symbol
No.
Compound
3-Letter
50
S(+)5-(4,5-Dihydroxypentyl)uracil
5(HO)2PeUra
1-Letter
Structure O
HO
NH OH
N H
pK
Formula (Mol Wt)
Melting Point °C
[α]Dt
Basic
Acidic
C9H14N2O4 (214.22)
255–226°
–
–
9.7
C4H6N2O2 (114.10)
275–276°
–
–
–
C5H8N2O2 (128.13)
264–265°
–
–
–
C5H4N2O4 (156.10)
345° (dec)
–
–
2.4, 9.5, >13
O
O 51
Dihydrouracil 5,6-Dihydrouracil
NH
H2 Ura
O
N H O
52
5,6-Dihydrothymine 5,6-Dihydro-5-methyluracil
NH
H2Thy
O
N H O
53
Orotic acid Uracil-6-carboxylic acid; 6-Carboxyuracil
No.
NH HOOC
Acidic Spectral Data pH
50 51 52 53
Oro 6CxUra
λmax
εmax (×10−3)
λmin
240
O
Neutral Spectral Data Spectral Ratios
230
N H
250
270
280
290
pH
λmax
2.8 –
264 –
7.05 –
–
–
–
–
–
–
–
–
265 –
1
280
7.5
241
0.61
0.41
0.54
1.54
1.82
1.56
7
279
227
36.0
230
εmax (×10−3) 7.7 –
λmin
Alkaline Spectral Data Spectral Ratios
230
240
Purines, Pyrimidines, Nucleosides, and Nucleotides
9168_Book.indb 283
PURINES, PYRIMIDINES, NUCLEOSIDES AND NUCLEOTIDES: PHYSICAL CONSTANTS AND SPECTRAL PROPERTIES (Continued)
250
270
280
290
–
–
–
–
–
–
–
7.7
241
0.68
0.43
0.57
1.49
1.71
1.36
31.5
249
pH
λmax
12 13o 13o 12
291 230 230 286 240 231
εmax (×10−3) 5.9 8.2 8.1 6.0 8.9 33.4
λmin
Spectral Ratios 230
240
250
270
280
290
224*
–
–
–
–
–
–
244 222 252
1.36
0.80
0.80
1.38
1.71
1.72
8.11
3.64
0.80
1.39
1.36
1.44
References Origin and Synthesis
50 51 52 53
C: 326 D: 325,326 C: 98,85,99-102 C: 101,99 C: 104–106
[α] D
pK
– – – –
325 – – 66,105
Spectral Data 326,325 103b,317bc,383*b 103b,383 66b,104b
Mass Spectra
Rf
326,325 – – –
401,325 383 383 69
283
No.
t
4/16/10 1:17 PM
9168_Book.indb 284
No.
Compound
3-Letter
54
Wyej (Formerly “Yt base” or “Yt+”) 4,9-Dihydro-4, 6-dimethyl-9-oxo-1H-imidazo[1,2-a]purine; 1,N2-isopropeno-3-methylguanine
Wye ImGua*
55
56
Wybutinej 7-[3-(Methoxycarbonyl)-3-(methoxyformamido)propyl] wyej (Formerly “Y base” or “Y‡”); α-(Carboxyamino)-4,9-dihydro-4,6-dimethyl-9-oxo-1Himidazo[1,2-a]purine-7-butyric acid dimethyl ester
Peroxywybutinej 7-[2-(Hydroperoxy)-3-(methoxycarbonyl)-3(methoxyformamide)propyl] wyej (Formerly “Yw base, Yr base, Peroxy Y base” or “Yw+”) α-(Carboxyamino)-4,9dihydro-β-hydroperoxy-4,6-dimethyl-9-oxo-1Himidazo[1,2-a]purine-7-butyric acid dimethyl ester
1-Letter
λmax
εmax (×10−3)
λmin
N
54
1.5
284
8.7
244
55
2
233 286
35.6 7.6
254*
–
N
O
Y-Wye (MeO)2 FnBtoWyej Y-imGua*
N N
[α]Dt
Basic
N
–
–
3.66
8.52
C16H20N6O5 (376.38)
204–206°
–
3.7*
8.6*
C16H20N6O7 (408.37)
–
–
˜3.3p
˜9p
C9H9N5O (203.20)
N
H N N
O
HOO
N N
N
H N N
Neutral Spectral Data
240 1.17
250 0.89
270 1.34
280 1.96
290 1.87
3.45
1.04*
1.03
1.12*
1.12
56
pH
λmax
6.0
264 307 235 263 313 236 260*
6.5
H2O
εmax (×10−3) 5.3 5.9 32.0 5.8 5.0 32.0 6.0
λmin 282
258* 288* 211 256
Alkaline Spectral Data Spectral Ratios
230 240 6.73 2.30
250 0.67
270 1.01
280 0.54
290 0.74
–
5.35
1.25
0.85
0.54*
0.50
5.02
4.07
1.16
0.81
0.56
0.69
pH
λmax
11.0
275 301 236 265* 304
10
εmax (×10−3) 6.6 8.05 32.8 6.8 7.2
λmin
Spectral Ratios 230
240
250
270
280
290
–
4.95
1.35
1.01
0.91
0.86
284 262* 287
References No. 54 55 56
Origin and Synthesis C: 312 R: 312 C: 320 R: 316,310 R: 327,321,324
[α]tD – – –
Acidic
NHCOOMe
MeOOC O2 Y-Wye O2 (MeO)2 FnBto Wyej O2 Y-imGua*
H N
pK
Melting Point °C
Formula (Mol Wt)
NHCOOMe
MeOOC
Spectral Ratios 230 7.55
O
N
Acidic Spectral Data pH
Structure
pK
Spectral Data
Mass Spectra
312 320,310* 324
312b,403b 320,310,316*b 327b,321*b
312 310,311 321,327
Rf 312 324,316,310,311 324,321,327
Handbook of Biochemistry and Molecular Biology
No.
Symbol
284
PURINES, PYRIMIDINES, NUCLEOSIDES AND NUCLEOTIDES: PHYSICAL CONSTANTS AND SPECTRAL PROPERTIES (Continued)
4/16/10 1:17 PM
No.
Compound
3-Letter
57
Xanthine
Xan
Symbol
1-Letter
Structure O
N
HN
N H
N H
O
pK
Melting Point °C
[α]Dt
Basic
C5H4N4O2 (152.11)
None (dec)
–
˜0.8
C6H6N4O2 (166.14)
˜380° (dec)
–
–
8.4, ˜13
C10H13N5O (219.24)
207–208°
–
–
–
Formula (Mol Wt)
Acidic 7.5*, 11.1*
O 58
7-Methylxanthine
59
No. pH
λmax
λmin
240
N H
Neutral Spectral Data 250
270
280
290
pH
λmax 308 267
57
0q
260
9.15
242
–
–
0.77
–
0.15
0.01
6
58 59
1 2 1
– 268 207 275
– 9.3 14.5 14.65
– 241 235
0.53 – –
0.42 0.38 –
0.66 – –
0.94 – –
0.40 0.75 –
0.04 – –
7 6 7
q
N
N N
Spectral Ratios 230
OH
HN
Zea 6(tr HoiPe) Ade
Acidic Spectral Data εmax (×10−3)
N
N H
O
Zeatin 6-(trans-4-Hydroxy-3-methyl-2-butenylamino)purine; N6-(trans-4-Hydroxy isopentenyl)adenine
N
HN
7MeXan
q
– 269 212 270
εmax (×10−3) 5.2 10.25 – 10.0 17.1 16.2
λmin
Purines, Pyrimidines, Nucleosides, and Nucleotides
9168_Book.indb 285
PURINES, PYRIMIDINES, NUCLEOSIDES AND NUCLEOTIDES: PHYSICAL CONSTANTS AND SPECTRAL PROPERTIES (Continued)
Alkaline Spectral Data Spectral Ratios
pH
λmax
εmax (×10−3)
λmin
0.07
10
0.14 – –
13 14 13
240 277 – 284 220 276
8.9 9.3 – 9.4 15.9 14.65
222 257 – 257 242
230
240
250
270
280
290
281 239
–
–
0.57
–
0.61
– 240 233
0.45 – –
0.42 – –
0.62 – –
1.16 – –
0.69 – –
Spectral Ratios 230
240
250
270
280
290
–
–
1.29
–
1.71
0.92
1.81 – –
1.33 – –
1.12 1.11 –
1.63 – –
2.28 2.39 –
2.07 2.27 –
References No.
Origin and Synthesis
57 58 59
C: 231,377 C: 45 C: 227 N: 228
[α] D t
– – –
pK
Spectral Data
22,248,170*,53*,55*,253* 232 ,242 ,205 53–56 53b,41c,d,27e,54* – 227 b
e
Mass Spectra – – –
Rf 258,242 10,27 32,227
285
4/16/10 1:19 PM
9168_Book.indb 286
No.
Compound
Symbol
3-Letter
1-Letter
Structure
Formula (Mol Wt)
Melting Point °C
286
PURINES, PYRIMIDINES, NUCLEOSIDES AND NUCLEOTIDES: PHYSICAL CONSTANTS AND SPECTRAL PROPERTIES (Continued) [α]Dt
Basic
pK
Acidic
RIBONUCLEOSIDES NH2 N
N 60
Adenosine
Ado
A
N
N
HO
O
C10H13N5O4 (267.24)
235–236°
−6125 (1.0, H2O)
3.5*
12.5
C15H21N5O4 (355.36)
131–133° (HBr salt)
–
8.5
–
C11H15N5O4 (281.27)
214–217° (dec)
−5926 (2.0, H2O)
8.8*
–
OH OH NH N
N 61
1-(Δ2-Isopentenyl)adenosine 1-(γγ-Dimethylallyl)adenosine 1-(3-Methyl-2-butenyl)adenosine
1PeiAdo 1PeiA
i1 A
N
N
HO
O
OH OH NH N
N 1-Methyladenosine
m1 A
1MeAdo
HO
N
N O
OH OH
No.
Acidic Spectral Data pH
60 61 62
1 1 2
λmax 257 257.5 258
εmax (×10−3)
λmin
14.6 13.9 13.7
230 235 232*
Neutral Spectral Data Spectral Ratios
230 0.23
240 0.44
250 0.84
270 –
280 0.22
290 0.03
0.28*
0.41
0.81
0.70
0.23*
0.04
pH
λmax
6 7 7
260 258 258
εmax (×10−3) 14.9 13.6 13.9*
λmin 227 235 232*
Alkaline Spectral Data Spectral Ratios
230 240 0.18 0.42 0.29
0.41
250 0.78
270 –
280 0.14
290 0.00
0.81
0.70
0.23
0.05
pH
λmax
11 13o 10.5o
259 259* 259*
εmax (×10−3) 15.4 14.25* 14.6
λmin 227 236 231*
Spectral Ratios 230 0.24
240 0.40
250 0.79
270 –
280 0.15
0.25
0.38
0.76
0.76
0.35
References No.
Origin and Synthesis
60 61 62
C: 111,235,363,350,351,379 C: 319,15a,14 C: 10,30
[α] D t
pK
Spectral Data
363,235,350 250,255*,336,371,51,220 232b,212cde,242e,183b,317b,205 – 15 319,15* 10 15,318* 90,10*d,317*bd,318*b,109,12b
Mass Spectra 315,341,317,405 15 314,317
Rf 258,110,18,242,291,292 15,32,319 10,12,314,291
290 0.00 0.31 0.30
Handbook of Biochemistry and Molecular Biology
62
4/16/10 1:19 PM
No.
Compound
Symbol
3-Letter
1-Letter
Structure
Formula (Mol Wt)
Melting Point °C
C12H17N5O4 (295.30)
206°
Basic
[α]Dt
pK
Acidic
N N
N
63
1,N6-Dimethyladenosine 1-Methyl-6-methylamino-9-β-D-ribofuranosylpurine
1,6Me2, Ado
m1 2 , 6A m1 m6 A
N
N
HO
O
–
–
–
−7126 (1.06, 0.1N NaOH)
–
–
−66.625 (1.0, H2 O)
–
–
OH OH NH2 N
N
64
2-Hydroxyadenosine Crotonoside; Isoguanosine
o2A isoG*
2HOAdo isoGuo*
N
N
HO HO
O
C10H13N5O5 (283.25)
237–252° (dec)
C11H15N5O4(281.27)
>200° (dec) (picrate)
OH OH NH2 N
N
65
2-Methyladenosine
2MeAdo
N
N
m2 A
Purines, Pyrimidines, Nucleosides, and Nucleotides
9168_Book.indb 287
PURINES, PYRIMIDINES, NUCLEOSIDES AND NUCLEOTIDES: PHYSICAL CONSTANTS AND SPECTRAL PROPERTIES (Continued)
HO
O
OH OH
No.
Acidic Spectral Data pH
63 64
1 1.2
65
1
λmax 261 235 283* 258*
εmax (×10−3)
λmin
14.2 6.14 12.7* 14.0
Neutral Spectral Data Spectral Ratios
pH
234 –
230 0.26 –
240 0.31 –
250 0.68 –
270 0.81 –
280 0.47 –
290 0.18 –
H2O
230
0.22
0.44
0.84
0.86
0.40
0.05
6
λmax
247* 293* 264
εmax (×10−3) 8.9 11.1* 14.5
λmin
Alkaline Spectral Data Spectral Ratios
230
240
250
270
280
290
pH
λmax 262 251* 285* 263
–
–
–
–
–
–
–
14 12.8
228
0.24
0.41
0.75
0.84
0.17
0.01
13
εmax (×10−3) 14.9 6.9* 10.55* 15.2
λmin
Spectral Ratios 230
240
250
270
280
290
234 –
–
–
–
–
–
–
230
0.24
0.41
0.75
0.84
0.17
0.01
References Origin and Synthesis
[α]tD
pK
63 64 65
C: 24,12 C: 269,N: 268 E: 18 C: 111,342 R: 18,291
– 269 342
– – –
Spectral Data 24,90e 12 269b,268*b 18b,342*cd,317bce,291b
Mass Spectra
Rf
– – 317
24,12 269 18,291
287
No.
4/16/10 1:19 PM
9168_Book.indb 288
No.
Compound
Symbol
3-Letter
1-Letter
Structure NH2
2-Methylthioadenosine
ms A
2MeSAdo
N
N
S HO
2
Melting Point °C
Basic
[α]Dt
pK
Acidic
N
N 66
Formula (Mol Wt)
288
PURINES, PYRIMIDINES, NUCLEOSIDES AND NUCLEOTIDES: PHYSICAL CONSTANTS AND SPECTRAL PROPERTIES (Continued)
C11H15N5O4 S (313.33)
O
+429 (1.0, 0.1N HCl)
227°
–
–
OH OH O N H N
HN
67
N6-Glycinocarbonyladenosine N-[(9-β-D-Ribofuranosylpurin-6-yl)carbamoyl]glycine
N
6(GlyCO)Ado
gc6A
N
N HO
COOH
C13H14N6O7 (368.31)
214–216°
–
–
–
C14H18N6O7 (382.33)
173–174°
33.0923 (0.55 H2O)
–
–
O
OH OH O N H N
N N
N6-Methyl-N6-glycinocarbonyladenosine N-[(9-β-D-Ribofuranosylpurin-6-yl)-Nmethylcarbamoyl]glycine
6Me6(GlyCO) Ado
m6 gc6 A
N
N HO
O
OH OH No.
Acidic Spectral Data pH
λmax
εmax (×10−3)
λmin
Neutral Spectral Data Spectral Ratios
230
240
250
270
280
290
pH
λmax
7
235 274 269 276
66
1
270
15.2
67
1.2
271 276
18.2 19.1
238
0.58
0.37
0.59
1.58
1.58
0.57
5.0
68
1.0
283
16.7
239
0.75
0.44
0.62
1.70
2.39
1.83
5.5
277 284
εmax (×10−3) 17.7 13.5 20.9 17.1 17.1 16.7
λmin
Alkaline Spectral Data Spectral Ratios
230
240
250
270
280
290
230
0.12
0.28
0.59
1.37
0.78
0.00
236
0.49
0.35
0.60
1.68
2.14
1.28
pH
λmax
13
235 274* 270m 277 298 277 284
12.1o 13o
εmax (×10−3) 17.8* 13.5* 13.5m 13.8 12.8 15.4 15.8
λmin
Spectral Ratios 230
240
250
270
280
290
236
0.37
0.32
0.57
1.41
1.00
0.60
245
1.36
0.79
0.76
1.38
1.85
1.33
References
4/16/10 1:19 PM
No.
Origin and Synthesis
[α] D
pK
Spectral Data
Mass Spectra
66 67 68
C: 399,111a C: 408,295 R: 295 C: 330
111 – 330
– – –
399,111*,299 408,295b,330 330
– 295,408,330 –
t
Rf 299 295,408,330 330
Handbook of Biochemistry and Molecular Biology
68
COOH
No.
Compound
3-Letter
Symbol
1-Letter
Structure O HN
N -Threoninocarbonyladenosine N-[(9-β-D-Ribofuranosylpurin-6-yl)carbamoyl]-Lthreonine; N-(Nebularin-6-ylcarbamoyl)-L-threonine 6
69
6(ThrCO)Ado
N H N
N
tc6 A
[α]Dt
Basic
Acidic
C15H20N6O8 (412.36)
204–207°
−13.925 (1.0, Me2 SO)
180° (dec)
˜0.6
9.6
[α]Dt
N 89
O2′-Methylcytidine 2′-O-Methylcytidine
N
O
HO
Cm
2′MeCyd
O
HO
OMe HN N
90
m4Cm
2′,4Me2Cyd
N4,O2′-Dimethylcytidine
N
O
HO
O
HO
OMe
OH N
N 5-N-Methylformamido-6-ribosylamino isocytosinet 2-Amino-4-hydroxy-5-N-methylformamido-6ribosylaminopyrimidine
N
H2N
5MeFn6(RibNH) isoCyt
HO
O
HO No.
Acidic Spectral Data pH
λmax
εmax (×10−3)
λmin
89 90
1 1
281 281
12.9* –
241 243
91
0.1o
270
25.1n
239
0.49
240 0.03 0.37
250 0.43 0.49
270 1.75 1.65
280 2.13 1.99
290 1.61 1.66
pH
λmax
(EtOH) 7
0.44
0.54
1.41
0.85
0.14
7
273 238 270 273
εmax (×10−3) 8.2 – –
λmin 252 227 250 247
Alkaline Spectral Data Spectral Ratios
230 240 – – 0.87 0.88 0.96
0.51
+32.525 (1, H2O
OH
Neutral Spectral Data Spectral Ratios
230 0.62 0.79
NH
250 – 0.83
270 – 1.15
280 – 0.92
290 – 0.34
0.46
1.76
1.27
0.24
pH
λmax
λmin
272 –
εmax (×10−3) 8.9 –
11 – 12
265
16.3
Spectral Ratios
251 –
230 1.46 –
240 0.96 –
250 0.85 –
270 1.18 –
280 0.94 –
290 0.31 –
244
0.74
0.57
0.59
0.99
0.24
0.02
References No.
Origin and Synthesis
[α]tD
pK
Spectral Data
Mass Spectra
Rf
89 90 91
C: 124,369a R: 131 C: 137,41
124 – 137
124 131 52
124,369*cd,317b 131b 52,137d,13b,41
315,317 – –
109,110,124,369 131 41
Handbook of Biochemistry and Molecular Biology
91
CHO
4/16/10 1:19 PM
No.
Compound
3-Letter
Symbol
1-Letter
Structure O
Guanosine
Guo
N
N
H2N HO
G
Melting Point °C
Basic
[α]Dt
pK
Acidic
N
HN 92
Formula (Mol Wt)
O
HO
C10H13N5O5 (283.24)
>235° (dec)
C11H15N5O5 (297.27)
225–227° (dec)
−7226 (1.4, 0.1N NaOH)
1.6*
9.2*, 12.4
–
˜2.41
–
−34.626 (1.0 Me2SO4/ EtOH)
2.3p
9.7p
OH
O N
N 93
1-Methylguanosine
H2N HO
m1G
1MeGuo
N
N O
HO
OH
Purines, Pyrimidines, Nucleosides, and Nucleotides
9168_Book.indb 297
PURINES, PYRIMIDINES, NUCLEOSIDES AND NUCLEOTIDES: PHYSICAL CONSTANTS AND SPECTRAL PROPERTIES (Continued)
O N
HN 94
N2-Methylguanosine 2-Methylamino-9-β-ribofuranosyl-purin-6-one
2MeGuo
N H HO
mG 2
N
N
HO
No.
Acidic Spectral Data pH
λmax
εmax (×10−3)
λmin
1o,q
271
22.3
246
92 93 94
0.7q 1 1
256 258 259
12.3 11.4* 14.2
228 232* 231
No.
Origin and Synthesis
92 93 94
C: 237,231,351,372,373,111 E: 40 C: 24,400 E: 40 C: 44,372,381
0.26 0.28 0.52
240
0.56 0.45 0.49
>200° (dec)
OH
Neutral Spectral Data Spectral Ratios
230
C11H15N5O5 (297.27)
O
250
0.94 0.85 0.85
270
0.75 0.77 0.73
280
0.70 0.71 0.56
290
0.50 0.53 0.52
pH
6 6 7
λmax
εmax (×10−3)
λmin
13.6 13.1* 15.9
223 225 225
253 256 253
Alkaline Spectral Data Spectral Ratios
230
0.36 0.28 0.35
240
0.79 0.61 0.70
250
1.15 1.00 1.18
270
0.83 0.84 0.68
280
0.67 0.63 0.58
290
0.28* 0.21 0.52
pH
λmax
11.3
256– 266 – 256* 258*
13 13 13
εmax (×10−3) 11.3
λmin
– 12.9* 12.0*
Spectral Ratios
230
230 –
240 –
250 0.89
270 –
280 0.61
290 0.13
– 231 237*
0.43 0.39 0.98
0.56 0.61 0.70
0.88 0.99 0.93
0.97 0.83 0.91
0.60 0.63 0.82
0.09 0.22* 0.48
References pK
237,351 – 44,381
231,170*,334* – 40
Spectral Data
Mass Spectra
232b,242*e,183b,317b,212,205,111 40b,375cd,317*bce,24*,265*b,400 40b,375cd,317*bcde,381,44*
314,315,374,317 314,317 314,317
Rf 258,110,40,189,314,242 24,40,110,314 40,44,110,314
297
[α] D t
4/16/10 1:19 PM
9168_Book.indb 298
No.
Compound
3-Letter
Symbol
1-Letter
Structure O
95
2Me2Guo
N
m2 G 2
Melting Point °C
Basic
[α]Dt
pK
Acidic
N
HN
N2,N2-2-Dimethylguanosine 2-Dimethylamino-9-β-ribofuranosyl-purin-6-one
Formula (Mol Wt)
298
PURINES, PYRIMIDINES, NUCLEOSIDES AND NUCLEOTIDES: PHYSICAL CONSTANTS AND SPECTRAL PROPERTIES (Continued)
N
N
HO
O
HO
C12H17N5O5 (311.30)
242° (dec)
−35.626 (1.1 Me2SO4/ EtOH)
C11H15N5O5 (297.27)
165° (hemihydrate)
−35.527 (0.4, H2O)
C13H19N5O5 (325.33)
–
2.5p
9.7p
r
7.0*
r
–
OH
O N
N
96
7-Methylguanosine
7MeGuo
H2N
m7G
N
N
HO
O
HO
OH
O N
N
97
2
2,2,7Me3Guo
N
M2 3, 7G m2 2 m7G
HO
O
HO
No.
Acidic Spectral Data pH
λmax
εmax (×10−3)
λmin
N
N
OH
Neutral Spectral Data Spectral Ratios
240 0.32
250 0.63
270 0.92
280 0.56
290 0.48
pH
λmax
7
260 258 281 266
95
1
265
17.7*
236*
230 0.45
96
3
257
10.7*
230
0.27
0.47
0.88
0.73
0.68
0.53
7q
97
1
266 295
–
240
0.67
0.32
0.57
1.06
0.59
0.49
5
εmax (×10−3) 18.9*n 8.5 7.4 10.3
λmin 228
Alkaline Spectral Data Spectral Ratios
230 240 0.18 0.40
–
250 0.78
270 0.76
280 0.57
290 0.50
pH
λmax 262 263 282 234 302
238*
–
–
0.89
–
1.04
0.90
11 13 9o
239
0.67
0.32
0.57
1.08
0.63
0.51
10o
εmax (×10−3) 12.2 14.3* 8.0 –
λmin
Spectral Ratios
240 242* 242
230 – 1.37 2.10
240 – 0.75 0.84
250 – 0.85 0.90
270 – 0.88 1.16
280 – 0.78 1.46
290 – 0.65 1.30
280
2.04
1.93
1.31
0.84
0.62
0.73
References
4/16/10 1:19 PM
No.
Origin and Synthesis
95 96 97
E: 40 C: 44,372,381 C: 13,41,10 C: 7,376
[α]tD
pK
44,381 10 –
40 41,13,334* –
Spectral Data 40b,375cd,317*bcde,44*cd,265*b,381* 52,13bd,41*cd,10*s,334 7b
Mass Spectra
Rf
314,317 314,317 376
40,44,110,314 10,41,314,334 –
Handbook of Biochemistry and Molecular Biology
N ,N ,7-Trimethylguanosine 2-Dimethylamino-7-methyl-9-β-d-ribofuranosylpurin6-one 2
No.
Compound
3-Letter
Symbol
1-Letter
Structure O
O2′-Methylguanosine 2′-O-Methyl guanosine
N
N
H2N HO
Gm
2′MeGuo
pK
Acidic
Melting Point °C
C11H15N5O5 (297.27)
218–220°
−38.422 (0.6, H2O
–
–
C10H12N4O5 (268.23)
218°
−58.8 (2.5, H2O)
1.2
8.8,12.
C11H14N4O5 (282.25)
210–212°
−49.228 (0.5, H2O)
–
–
[α]Dt
N
HN 98
Basic
Formula (Mol Wt)
O
HO
OMe
O N
HN 99
Inosine
Ino
1
N
N
HO
O
HO
OH
O N
N 100
1-Methylinosine
1MeIno
m1I
O
HO No.
Acidic Spectral Data pH
98 99 100
1 0 3 2q
λmax 256 251 248 250
εmax (×10−3)
λmin
10.7 10.9 12.2 10.4
– 221 223 223
240 – – – 1.14
OH
Neutral Spectral Data Spectral Ratios
230 – – – 0.59
N
N
HO
250 – 1.21 1.68 1.42
270 – – – 0.57
280 – 0.11 0.25 0.23
290 – 0.00 0.03 0.03
pH – 6 6
λmax
εmax (×10−3) – – 248.5 12.3 249
10.4
λmin – 223 223*
Alkaline Spectral Data Spectral Ratios
230 – – 0.64
240 – – 1.35
Purines, Pyrimidines, Nucleosides, and Nucleotides
9168_Book.indb 299
PURINES, PYRIMIDINES, NUCLEOSIDES AND NUCLEOTIDES: PHYSICAL CONSTANTS AND SPECTRAL PROPERTIES (Continued)
250 – 1.68
270 – –
280 – 0.25
290 – 0.03
1.69
0.71
0.40
0.07
pH
λmax
11 11
258 253
εmax (×10−3) 9.8 13.1
12
249
10.7
λmin – 224
Spectral Ratios 230 – –
240 – –
250 – 1.05
270 – –
280 – 0.18
290 – 0.01
0.86
1.28
1.6
0.67
0.35
0.07
References No.
Origin and Synthesis
[α]tD
pK
98 99 100
C: 108 R: 231a C: 377,378 C: 10,122
108 255 10
– 170,231,51,250 –
Spectral Data 108 232b,205,317b 49,10*d,410ce,122
Mass Spectra 315 314,317 314,317
Rf 109,110 110,314,378 10,49,110,122,314
299
4/16/10 1:19 PM
9168_Book.indb 300
No.
Compound
3-Letter
Symbol
1-Letter
Structure O
2-Methylinosine
2MeIno
m2I
N
N
HO
O
HO
102
Acidic
C11H14N4O5 (282.25)
165–166°
−50.026 (1.0, H2O
–
–
C10H12N4O4 (252.33)
181–182°
−48.625 (1.0, H2O)
2.1
–
C9H12N2O6 (244.20)
165–166°
+9.616 (2.0, H2O)
–
9.2, 12.5
[α]Dt
N N
N Neb
pK
Melting Point °C
OH
N Nebularine 9-β-Ribofuranosylpurine
Basic
Formula (Mol Wt) N
HN 101
300
PURINES, PYRIMIDINES, NUCLEOSIDES AND NUCLEOTIDES: PHYSICAL CONSTANTS AND SPECTRAL PROPERTIES (Continued)
O
HOH2C
HO
OH O
HN 103
Uridine
Urd
U
HO
N
O O
No.
Acidic Spectral Data pH
101 102 103
1 1 1
λmax 253 262 262
εmax (×10−3) 11.9 5.9 10.1
λmin
235 230
Neutral Spectral Data Spectral Ratios
230 0.51 –
240 0.49 –
250 0.74 0.74
270 0.85 –
280 0.29 0.35
290 0.07 0.03
pH 7 H2O 7
λmax
εmax (×10−3) 251.5 12.7 262 7.1 262 10.1
λmin
222 230
Alkaline Spectral Data Spectral Ratios
230 0.35 –
240 0.54 –
250 0.71 0.74
270 0.68 –
280 0.11 0.35
290 0.04 0.03
pH
λmax
13 13 12
258 262 262
εmax (×10−3) 13.1 7.1 7.45
λmin
234 243
Spectral Ratios 230
240
250
270
280
290
0.45 –
0.48 –
0.71 0.83
0.75 –
0.22 0.29
0.11 0.02
References No.
Origin and Synthesis
101 102 103
C: 377 C: 123,363,391 R: 236,181 C: 379,276a,380
[α]tD 377 123,363 236,276
pK – 123 163,231
Spectral Data
Mass Spectra
377 123b,391 163b,232*be,181b,212,183b,
– 405 341,315,317
Rf 377
– 258,110,40
Handbook of Biochemistry and Molecular Biology
OH OH
4/16/10 1:19 PM
No.
Compound
3-Letter
Symbol
1-Letter
Structure
O
Formula (Mol Wt)
Melting Point °C
C9H12N2O5S (260.26)
214°
Basic
[α]Dt
pK
Acidic
HN 104
2Srd 2SUrd
2-Thiouridine
s2U
N
S
HO
O
+3920 (1.2, H2O)
–
8.8
OH OH S HN 105
s U ssU
2,4Srd 2,4S2 Urd
2,4-Dithiouridine
2,4 2 2 4
N
S
HO
O
C9H12N2O4S2 (276.33)
166–167°
–
–
7.4
C11H14N2O7S (318.31)
–
–
–
–
OH OH O
106
5Cm2SUrd 5Cm2Srd 5(CxMe)2SUrd
5-Carboxymethyl-2-thiouridine 2-Thiouridine-5-acetic acid
COOH
HN
cm5s2U cm5S
HO
N
S
Purines, Pyrimidines, Nucleosides, and Nucleotides
9168_Book.indb 301
PURINES, PYRIMIDINES, NUCLEOSIDES AND NUCLEOTIDES: PHYSICAL CONSTANTS AND SPECTRAL PROPERTIES (Continued)
O
OH OH
No.
Acidic Spectral Data pH
λmax
εmax (×10−3)
λmin
Neutral Spectral Data Spectral Ratios
230
240
250
270
280
290
pH
λmax
εmax (×10−3)
λmin
Alkaline Spectral Data Spectral Ratios
230
240
250
270
280
290
pH
104
2
279
16.4
247
1.32
0.63
0.53
1.66
1.86
1.66
7
218* 275*
16.2 13.6*
247
1.16
0.70
0.62
1.47
1.57
1.40
14 9q 12
105
–
–
–
–
–
–
–
–
–
–
5.8
283
22.5
–
–
–
–
–
–
–
9
106
1
274.5
7
277.5
λmax
εmax (×10−3) 264.5 7.5 241 21.8 239 21.0 271 13.4 280 16.9 320 24.8
λmin
Spectral Ratios 230
240
250
270
280
290
243 261
1.07
1.43
1.22
1.02
0.88
0.59
–
–
–
–
–
–
–
References Origin and Synthesis
[α] D
pK
Spectral Data
104 105 106
C: 276,285,301 E: 277 C: 271 R: 303
276,301 – –
285 271 –
276*b,277b,301*cd,306b,305b 271 303
Mass Spectra – – –
Rf 276,306,307,305 271 303
301
4/16/10 1:19 PM
No.
t
9168_Book.indb 302
No.
Compound
107
5-Carbamoylmethyl-2-thiouridine 2-Thiouridine-5-acetamide
Symbol
3-Letter
1-Letter
Structure O
ncm5s2U ncm5S
HO
Melting Point °C
Basic
[α]Dt
pK
Acidic
CONH2
HN
5Ncm2Srd 5Ncm2SUrd 5(NcMe)2Srd
Formula (Mol Wt)
302
PURINES, PYRIMIDINES, NUCLEOSIDES AND NUCLEOTIDES: PHYSICAL CONSTANTS AND SPECTRAL PROPERTIES (Continued)
N
S O
C11H15N3O6S (317.32)
217–218°
C12H16N2O7S (332.33)
C10H14N2O6S (290.30)
–
–
–
199°
+19.820 (0.5, H2O)
–
–
221–222°
+18.224 (0.5, H2O)
–
–
OH OH O COOMe
HN
108
5-(Methoxycarbonylmethyl)-2-thiouridine 5-Carboxymethyl-2-thiouridine methyl ester
5(MeCm)2SUrd 5(MeCm)2Srd
mcm5s2U mcm5S
HO
N
S O
OH OH
O OMe
HN 109
5MeO2SUrd 5MeO2Srd
5-Methoxy-2-thiouridine
mo5S mo5s2U
N
S
HO
O
OH OH
Acidic Spectral Data pH
λmax
εmax (×10−3)
λmin
Neutral Spectral Data Spectral Ratios
230
240
250
270
280
290
107 108
1
277*
15.6
244
1.22
0.73
0.68
1.49
1.54
109
1.41
pH
λmax
(MeOH)
221 276 220 277* 227 285
7 (MeOH)
εmax (×10−3) 12.9 13.7 15.3 15.8 10.0 12.6
λmin
244
Alkaline Spectral Data Spectral Ratios
230
1.18
240
0.84
250
0.76
270
1.42
280
1.45
290
1.24
pH
λmax
13 (MeOH) 12
242 272 242 271 248 272
13 (MeOH)
εmax (×10−3) 18.2 15.6 22.4 15.8 20.0 12.6
λmin
261
Spectral Ratios 230
240
250
270
280
290
1.06
1.37
1.16
1.02
0.86
0.46
References No.
Origin and Synthesis
[α] D
pK
107 108 109
C: 301 C: 5,310 R: 275,306 C: 301
– 301 301
– – –
t
Spectral Data
Mass Spectra
Rf
301 5,301cd,317bc,275*b 301
– 317,5,301,275 –
301 275,301,303,306,307 –
Handbook of Biochemistry and Molecular Biology
No.
4/16/10 1:19 PM
No.
Compound
3-Letter
Symbol
1-Letter
Structure
Formula (Mol Wt)
O
Melting Point °C
Basic
[α]Dt
pK
Acidic
HN 110
5-Methyl-2-thiouridine 2-Thio-1-ribosylthymine
m5s2U s2T
5Me2Surd 5MeSrd
N
S
HO
O
C10H14N2O5S (274.30)
217°
+3128 (1.23, H2O)
–
–
C11H17N3O5S (303.33)
–
–
–
–
C10H14N2O6 (258.23)
119–120°
+20.126 (H2O)
–
–
OH OH O N H
HN
111
5-(Methylaminomethyl)-2-thiouridine 5-(N-Methylaminomethyl)-2-thiouridine
5(MeNHMe)Srd 5(MeNHMe)2Surd
mnm s U mnm5S 5 2
N
S
HO
O
OH OH
O N 112
3-Methyluridine
3MeUrd
m3U
HO
N
O O
Purines, Pyrimidines, Nucleosides, and Nucleotides
9168_Book.indb 303
PURINES, PYRIMIDINES, NUCLEOSIDES AND NUCLEOTIDES: PHYSICAL CONSTANTS AND SPECTRAL PROPERTIES (Continued)
OH OH
No.
Acidic Spectral Data pH
110
2
111
1
112
2
v
λmax 218 273 220 273 262*
εmax (×10−3)
λmin
17.4 14.8 – 9.5
Neutral Spectral Data Spectral Ratios
pH
λmax
7
219 272 220 273 262
243
230 1.05
240 0.53
250 0.58
270 1.41
280 1.43
290 1.22
242
0.84
0.56
0.66
1.31
1.29
1.20
7
232
0.31
0.39
0.74
0.90
0.35*
0.04*
7
εmax (×10−3) 16.2 14.1 – 9.5*
λmin 247
Alkaline Spectral Data Spectral Ratios
230 240 1.06 0.77
250 0.69
270 1.40
280 1.39
290 1.15
pH
λmax
λmin
239
εmax (×10−3) 21.0
9q
Spectral Ratios
259
230 1.35
240 1.64
250 1.20
270 1.10
280 1.04
290 0.60
242
0.84
0.56
0.66
1.33
1.31
1.22
13
243
–
227
1.00
1.33
1.21
1.01
0.87
0.49
232
0.31
0.39
0.74
0.90
0.35
0.04
12
263*
9.4
232
0.32
0.38
0.70
0.91
0.46*
0.10
References No.
Origin and Synthesis
[α] D
pK
110 111 112
C: 276,307 R: 307 R: 270,305 C: 139,125,140–142,81
276 – 139
– – –
t
Spectral Data 276b,307b 270,317b,305b 317b,142*,13*,81*,139,126*
Mass Spectra – 270,317 314,317
Rf 276,307,306 270,305 13,57,81,110,125,126,142,143,173,314
303
4/16/10 1:19 PM
9168_Book.indb 304
No.
Compound
3-Letter
Symbol
1-Letter
Structure NH2
HOOC
113
3-(3-Amino-3-carboxypropyl)uridine Uridine-3-(α-aminobutyric acid)
3NH2 BtoUrd 3(NH2 CxPr)Urd
O
304
PURINES, PYRIMIDINES, NUCLEOSIDES AND NUCLEOTIDES: PHYSICAL CONSTANTS AND SPECTRAL PROPERTIES (Continued) Basic
pK
Acidic
Formula (Mol Wt)
Melting Point °C
C13H19N3O8 (345.31)
161–163° (·2HCl salt)
–
–
–
C9H12N2O5S (260.26)
135–138° (dec)
–
–
8.2
–
–
–
[α]Dt
N
nbt3U
N
O
HO
O
OH OH
SH N 114
4Srd 4SUrd
4-Thiouridine
s4U
N
O
HO
O
OH OH S
S
N
115
4-Thiouridine disulfide bis(4-4′-Dithiouridine)
(4SUrd)2
HO
N N
O
HO
O
No.
Acidic Spectral Data pH
113 114
2 2
115
–
λmax 263 245 331 –
εmax (×10−3)
λmin
8.5 5.2* 17.0* –
O
188–190°
OH OH
Neutral Spectral Data Spectral Ratios
233 275*
230 0.41 –
240 0.45 –
250 0.76 –
270 0.89 –
280 0.43 –
290 0.05 –
–
–
–
–
–
–
–
pH
λmax
6 6.5
263 245 331 261 309
7
εmax (×10−3) 8.5 4.0* 21.2* –
λmin 233 225 274 236 278
Alkaline Spectral Data Spectral Ratios
230 240 0.41 0.45 – – –
–
250 0.76 –
270 0.89 –
280 0.43 –
290 0.05 –
–
–
–
–
pH
λmax 263 316
εmax (×10−3) 8.5 19.7*
12 12 –
–
–
λmin
Spectral Ratios
234 268*
230 0.46 –
240 0.52 –
250 0.77 –
270 0.89 –
280 0.44 –
290 0.08 –
–
–
–
–
–
–
–
References No.
Origin and Synthesis
113 114 115
C: 300 R: 300,345 C: 344,128 R: 144 C: 128
[α]tD
pK
Spectral Data
– – –
– 144 –
300b 344*,144*bcd,317*b,128 128,208b
Mass Spectra
Rf
300 – –
300,345 144,344 144
Handbook of Biochemistry and Molecular Biology
OH OH
C18H22N4O10S2 (518.51)
N
O
4/16/10 1:19 PM
No.
Compound
3-Letter
Symbol
1-Letter
Structure O
116
5-Carboxymethyluridine Uridine-5-acetic acid
5CmUrd 5(CxMe)Urd
cm5U
HO
N
O
Melting Point °C
[α]Dt
Basic
C11H14N2O8 (302.24)
242–244°
−24.325 (1N NaOH)
–
4.2, 9.8
C11H15N3O7 (301.26)
227–230°
–
–
–
C12H16N2O8 (316.27)
163–165°
–
–
–
C9H12N2O7 (260.20)
242–245°
–
–
7.8
COOH
HN O
pK
Formula (Mol Wt)
Acidic
OH OH O CONH2
HN
117
5-Carbamoylmethyluridine Uridine-5-acetamide
5NcmUrd 5(NcMe)Urd
ncm5U
HO
N
O O
OH OH O
118
COOMe
HN
5-(Methoxycarbonylmethyl)uridine 5-Carboxymethyluridine methyl ester Uridine-5-acetic acid methyl ester
5MeCmUrd 5(MeCxMe)Urd
mcm U 5
HO
N
O O
Purines, Pyrimidines, Nucleosides, and Nucleotides
9168_Book.indb 305
PURINES, PYRIMIDINES, NUCLEOSIDES AND NUCLEOTIDES: PHYSICAL CONSTANTS AND SPECTRAL PROPERTIES (Continued)
OH OH
O OH
HN 119
5-Hydroxyuridine
5HOUrd
o5U
HO
N
O O
OH OH No.
Acidic Spectral Data pH
116 117 118 119
1 1 1 2
λmax 265 265 265 280
εmax (×10−3)
λmin
9.7 10.0 5.2 –
234* 232* 232 245
Neutral Spectral Data Spectral Ratios
pH
230 0.39 –
240 0.40 –
250 0.69 –
270 1.02 –
280 0.64 –
290 0.19 –
7 H2O
0.92
0.50
0.56
1.52
1.76
1.46
7q
λmax
εmax (×10−3) 266.5 – 266 – 280*
8.2
λmin 236 233 –
Alkaline Spectral Data Spectral Ratios
230 240 0.50 0.41 0.24 0.34 –
–
250 0.68 0.67
270 1.07 1.05
280 0.75 0.66
290 0.27 0.17
–
–
–
–
pH 13 13 12
λmax
εmax (×10−3) 266.5* 7.1 267* 7.0 306
–
λmin
Spectral Ratios
245 244
230 1.29 1.10
240 0.84 0.78
250 0.80 0.79
270 1.05 0.98
280 0.71 0.54
290 0.25 0.11
267
–
1.60
1.29
0.70
0.89
1.16
References Origin and Synthesis
[α] D
pK
Spectral Data
Mass Spectra
116 117 118 119
R: 278 C: 302,322a C: 302 R: 398 C: 302,304 R: 304 C: 145-147 R: 148
322 – – –
302 – – 382
278b,302*d,317b 302*,398*ce 302,414b 148b,145cd,146*
– – 414 317
Rf 278,302,303,304 302 302,303,304 148
305
4/16/10 1:19 PM
No.
t
9168_Book.indb 306
No.
Compound
3-Letter
Symbol
1-Letter
Structure O
O
HN
120
5-Carboxymethoxyuridine Uridine-5-oxyacetic acid
5CmOUrd 5(CxMeO)Urd
cmo5U
pK
Formula (Mol Wt)
Melting Point °C
[α]Dt
Basic
Acidic
C11H14N2O9 (318.24)
–
–
–
2.9
C10H14N2O6 (258.23)
183–185°
–
9.7
C10H14N2O7 (274.23)
167–168°
–
–
COOH
N
O
HO
306
PURINES, PYRIMIDINES, NUCLEOSIDES AND NUCLEOTIDES: PHYSICAL CONSTANTS AND SPECTRAL PROPERTIES (Continued)
O
OH OH
O HN 121
5-Methyluridine 1-β-Ribofuranosylthymine Ribosylthymine
5MeUrd rThd* Thd
m5U T
N
O
HO
O
−1031 (2.0, H2O)
OH OH
O OH
HN 122
om5U hm5U*
5HmUrd 5HOMeUrd
5-Hydroxymethyluridine
HO
N
O O
–
No.
Acidic Spectral Data pH
120 121 122
2q 1 2
λmax 277 267 264
εmax (×10−3)
λmin
8.4 9.9* 9.5
243 235* 233
Neutral Spectral Data Spectral Ratios
230 0.44 –
240 0.39 –
250 0.67 0.70
270 1.07 –
280 0.74 0.52
290 0.27 –
pH
λmax
7.5 7 7
280 267 263
εmax (×10−3) 7.6 9.8 –
λmin 247 236 –
Alkaline Spectral Data Spectral Ratios
230 0.44 –
240 0.39 –
250 0.67 –
270 1.07 –
280 0.74 0.53
290 0.27 –
pH
λmax
13 13 12
278 268 263
εmax (×10−3) 6.7 7.5 7.0
λmin 252 246 243
Spectral Ratios 230
240
250
270
280
290
1.31 –
0.91 –
0.83 0.79
1.08 –
0.75 0.45
0.31* –
References No.
Origin and Synthesis
120 121 122
C: 308 R: 308,309 E: 18,149 C: 150,69,380,386,379 C: 69,151
[α] D
pK
Spectral Data
– 150,322,379 –
308 150 –
308b,309b,313b 18b,150bd,149bcd,317be,69*,265*b,379 69,94ce
t
Mass Spectra 308 314,317 –
Rf 308,309 18,69,110,142,149,314,304 69
Handbook of Biochemistry and Molecular Biology
OH OH
4/16/10 1:19 PM
No.
Compound
Symbol
3-Letter
1-Letter
Structure
OH
Formula (Mol Wt)
Melting Point °C
C9H14N2O6 (246.22)
106–108°
C10H16N2O6 (260.25)
–
C10H14N2O6 (258.23)
159°
Basic
[α]Dt
pK
Acidic
N 123
Dihydrouridine 5,6-Dihydrouridine
hU D
H2Urd
N
O
HO
O
−36.820 (2.1, H2O)
–
–
–
–
–
˜9.3P
OH OH OH N 124
5-Methyl-5,6-dihydrouridine 5,6-Dihydroribosylthymine
mD m5hU 5
5MeH2 Urd
N
O
HO
O
–
OH OH O
Purines, Pyrimidines, Nucleosides, and Nucleotides
9168_Book.indb 307
PURINES, PYRIMIDINES, NUCLEOSIDES AND NUCLEOTIDES: PHYSICAL CONSTANTS AND SPECTRAL PROPERTIES (Continued)
HN 125
O2′-Methyluridine 2′-O-Methyluridine
Um
2′MeUrd
HO
N
O O
+4120 (1.6, H2O)
OH OMe
No.
Acidic Spectral Data pH
123 124 125
λmax
εmax (×10−3)
λmin
Neutral Spectral Data Spectral Ratios
–
–
–
–
230 –
2
263*
10.0
231
0.23
pH
λmax
240 –
250 –
270 –
280 –
290 –
H2O
208
0.39
0.75
0.86
0.38
0.04
7
263
εmax (×10−3) 6.6 10.1
λmin – 231
Alkaline Spectral Data Spectral Ratios
230 – 0.23
240 – 0.39
pH
λmax
250 –
270 –
280 –
290 –
13o
235*
εmax (×10−3) 10.1
0.75
0.86
0.38
0.04
12
262
7.4
λmin
Spectral Ratios
–
230 –
240 –
250 –
270 –
280 –
290 –
243
0.98
0.76
0.83
0.82
0.3
0.03
References No.
Origin and Synthesis
123 124 125
C: 266,384,99,147 C: 333 R: 333 C: 124,369
[α] D
pK
384,412 – 124
– – 124,138
t
Spectral Data 266,283cd,375*,265b – 124*,317bcde,369
Mass Spectra 314,317 – 315,317
Rf 314,383,333 333 109,110,124,369
307
4/16/10 1:19 PM
9168_Book.indb 308
No.
Compound
Symbol
3-Letter
1-Letter
Structure O
308
PURINES, PYRIMIDINES, NUCLEOSIDES AND NUCLEOTIDES: PHYSICAL CONSTANTS AND SPECTRAL PROPERTIES (Continued) pK
Formula (Mol Wt)
Melting Point °C
[α]Dt
Basic
Acidic
C11H16N2O6 (272.26)
–
–
–
–
C10H12N2O8 (288.21)
183–184° (CHA salty)
–
–
–
–
9.3
HN 126
5,O2′-Dimethyluridine O2′-Methylribothymidine
m5Um Tm
2′,5Me2 Urd
N
O
HO
O
OH OMe O HN 127
Orotidine Uridine-6-carboxylic acid 6-Carboxyuridine
Ord 6CxUrd
O
HO
N
O
COOH
O
OH OH O HN 128
Spongouridine 1-β-D-Arabinofuranosyluracil
AraUrd aUrd
aU
HO
N
O
C9H12N2O6 (244.20)
222–224°
OH
No.
Acidic Spectral Data pH
126 127 128
1 1 1
λmax 268 267 264
εmax (×10−3)
λmin
– 9.8 9.3
237 234 232
Neutral Spectral Data Spectral Ratios
230 0.52 0.39 –
240 0.37 0.41 –
250 0.66 0.66 –
270 1.10 1.12 –
280 0.76 0.81 –
290 0.26 0.37 –
pH
λmax
7 – H2O
267 – 263
εmax (×10−3) – – 10.5*
λmin 237 – 231
Alkaline Spectral Data Spectral Ratios
230 240 0.51 0.36 – – – –
250 0.64 – –
270 1.09 – –
280 0.74 – –
290 0.26 – –
pH
λmax
13 13 11.5 14
267 266 263 265
εmax (×10−3) – 7.8 7.2 7.9
λmin 247 245 242 241
Spectral Ratios 230 – 1.07 –
240 0.88 0.83 –
250 0.78 0.83 –
270 1.05 1.04 –
References No.
Origin and Synthesis
[α] D
pK
Spectral Data
126 127 128
C: 323 R: 323 N: 160 N: 393 C: 394,392
– – 392
– – 392
323 160b 393*b,392cd
t
Mass Spectra
Rf
– – –
323 – 392,393
280 0.67 0.71 –
290 0.15 0.29 –
Handbook of Biochemistry and Molecular Biology
O HO
+13120 (0.63, H2O)
4/16/10 1:19 PM
No.
Compound
3-Letter
Spongothymidine 1-β-D-Arabinofuranosylthymine
AraThd aThd 5MeaUrd
Symbol
1-Letter
Structure O
Basic
pK
Acidic
Formula (Mol Wt)
Melting Point °C
C10H14N2O6 (258.23)
238–242°
9324 589 (0.5, H2 O);
–
9.8
C9H12N2O6 (244.20)
223–224°
−3.0 (1.0, H2O)
–
9.0*, >13
[α]Dt
HN 129
aT m5 aU
N
O
HO
O HO OH O
130
HN
Pseudouridine β f-Pseudouridine Pseudouridine C; 5-β-D-Ribofuranosyluracil 5-Ribosyluracil
Ψrd βfΨrd ΨrdC*
Ψ
NH O
HO
O
OH OH
No.
Acidic Spectral Data pH
129 130
1 2v
λmax 268 262
εmax (×10−3)
λmin
10.0 7.9*
236 233
Neutral Spectral Data Spectral Ratios
230 0.36 –
240 0.31 –
250 0.61 0.74
270 1.15 –
280 0.85 0.42*
290 0.36 0.06*
pH
λmax
7 7
268 263
εmax (×10−3) 10.0 8.1*
λmin 236 233
Alkaline Spectral Data Spectral Ratios
230 240 0.36 0.31 0.33 0.42
Purines, Pyrimidines, Nucleosides, and Nucleotides
9168_Book.indb 309
PURINES, PYRIMIDINES, NUCLEOSIDES AND NUCLEOTIDES: PHYSICAL CONSTANTS AND SPECTRAL PROPERTIES (Continued)
250 0.61 0.74
270 1.15 0.90
280 0.85 0.44
290 0.36 0.08
pH
λmax
12 12 14q
269 286 279
εmax (×10−3) 7.9 7.7* 5.7n
λmin 245 245 248
Spectral Ratios 230 1.17 2.06 2.31
240 0.70 0.73 1.11
250 0.69 0.62 0.61
270 1.15 1.51 1.67
280 0.77 2.06 2.09
290 0.22 2.16 1.51
References No.
Origin and Synthesis
[α] D
pK
129 130
N: 161 C: 162 N: 153 C: 154,387 R: 388
162 95
163 155,156,94*,95*
t
Spectral Data 163b,161b 155,94*bcde,154cd,95*be,156b,157,265*b,317b
Mass Spectra – 314,317
Rf 393 110,157–159,314
309
4/16/10 1:19 PM
9168_Book.indb 310
No.
131
Compound
3-Letter
α-f-Pseudouridine Pseudouridine B5-α-D-Ribofuranosyluracil
Symbol
1-Letter
Structure
HO
pK
Formula (Mol Wt)
Melting Point °C
[α]Dt
Basic
Acidic
C9H12N2O6 (244.20)
–
–
–
9.2*, >13
C9H12N2O6 (244.20)
–
–
–
9.6, >13
O
OH OH
ΨB*
αfΨrd Ψrd B*
310
PURINES, PYRIMIDINES, NUCLEOSIDES AND NUCLEOTIDES: PHYSICAL CONSTANTS AND SPECTRAL PROPERTIES (Continued)
O HN
NH O
132
βpΨrd Ψrd As*
β-p-Pseudouridine Pseudouridine As;5-β-D-Ribopyranosyluracil
ΨAS*
O HN
NH
O O HO HO
Acidic Spectral Data pH
λmax
εmax (×10−3)
λmin
Neutral Spectral Data Spectral Ratios
131
v
–
–
–
230 –
132
v
–
–
–
–
240 –
250 –
270 –
280 –
290 –
–
–
–
–
–
pH
λmax
7
264
7
262
εmax (×10−3) – 8.3
λmin 234 231
Alkaline Spectral Data Spectral Ratios
230 240 0.33 0.38 0.25
0.41
250 0.70
270 0.95
280 0.51
290 0.09
0.75
0.83
0.34
0.04
pH
λmax
12 14q 12 14q
288 279 286 281
εmax (×10−3) – – 9.2 7.5n
λmin 245 248 244 247
Spectral Ratios 230 1.88 2.20 2.10 2.24
240 0.76 1.02 0.60 0.81
250 0.70 0.61 0.56 0.54
References No.
Origin and Synthesis
131 132
C: 154 R: 94,388 R: 94,388 C: 154
[α] D t
– –
pK
Spectral Data
Mass Spectra
Rf
156,94* 94
155,94bce,156b 155,94bcde
– –
158 97
270 1.26 1.56 1.69 1.73
280 1.46 1.81 2.50 2.28
290 1.56 1.37 2.66 1.93
Handbook of Biochemistry and Molecular Biology
No.
OH
4/16/10 1:19 PM
No.
Compound
3-Letter
Symbol
1-Letter
Structure O
pK
Formula (Mol Wt)
Melting Point °C
[α]Dt
Basic
Acidic
C9H12N2O6 (244.20)
–
–
–
9.6, >13
C10H14N2O6 (258.23)
–
–
–
–
C10H14N2O6 (258.23)
–
–
–
–
HO 133
αpΨrd Ψrd AF*
α-p-Pseudouridine Pseudouridine AF; 5-α-D-Ribopyranosyluracil
HO
ΨAF*
OH O HN
NH O O
N 134
1-Methylpseudouridine 1-Methyl-5-ribosyluracil
1Meψrd
m1ψ
NH O
HO
O
OH OH O HN 135
O2′-Methylpseudouridine 2′-O-Methylpseudouridine
ψm
2′Meψrd
NH O
HO
Purines, Pyrimidines, Nucleosides, and Nucleotides
9168_Book.indb 311
PURINES, PYRIMIDINES, NUCLEOSIDES AND NUCLEOTIDES: PHYSICAL CONSTANTS AND SPECTRAL PROPERTIES (Continued)
O
OH OMe
No.
Acidic Spectral Data pH
λmax
εmax (×10−3)
λmin
Neutral Spectral Data Spectral Ratios
240 –
250 –
270 –
280 –
290 –
pH
λmax
7
263
εmax (×10−3) –
λmin
Alkaline Spectral Data Spectral Ratios
133
v
–
–
–
230 –
134
2
–
–
–
–
–
0.54
–
1.03*
0.38
7
265
–
–
–
–
–
–
0.66
–
135
1
261
–
–
–
–
–
–
–
–
7
261
–
–
–
–
–
–
–
–
233
230 240 0.27 0.37
250 0.71
270 0.90
280 0.42*
290 0.05
pH
λmax
12 14q 12 14 13
287 278 269 272 281
εmax (×10−3) – – – –
λmin
Spectral Ratios
244* 248 246
230 1.59 2.09 –
240 0.62 1.04 –
250 0.67 0.65 0.62
270 1.26 1.52 –
–
–
–
–
–
280 1.49 1.75 0.69* 1.05 –
290 1.58 1.32 0.08 –
References No.
Origin and Synthesis
133 134 135
R: 94,388 C: 154 C: 94,81 R: 118,415
t
[α] D
pK
Spectral Data
Mass Spectra
Rf
– – –
94 – –
155,94bce 94*b,81be,345c 118b
– – –
97 81,345 118,415
311
4/16/10 1:19 PM
9168_Book.indb 312
No.
Compound
Symbol
3-Letter
1-Letter
Structure O
136
N Wyo imGuo*
pK
Formula (Mol Wt)
Melting Point °C
[α]Dt
Basic
Acidic
C14H17N5O5 (335.32)
–
–
–
–
C21H28N6O9 (508.49)
–
–
–
–
N
N Wyosinej (Formerly “Yt”) 1-N2-Isopropeno-3-methylguanosine; 4,9-Dihydro-4,6-dimethyl-9-oxo-3-β-D-ribofuranosyl 1H-imidazo[1,2-a]purine
312
PURINES, PYRIMIDINES, NUCLEOSIDES AND NUCLEOTIDES: PHYSICAL CONSTANTS AND SPECTRAL PROPERTIES (Continued)
N
N
W HO
O OH OH
MeOOC
137
Wybutosinej (Formerly “Y”) 7-[3-(Methoxycarbonyl)-3(methoxyformamido)propyl] wyosinej, α-(Carboxyamino)-4,9-dihydro-4,6-dimethyl-9-oxo-3-βD-ribofuranosyl-1H-imidazo[1,2-a]purine-7-butyric acid dimethyl ester
NHCOOMe O
Y-Wyo (MeO)2 FnBtoWyoj Y-imGuo*
N
N
yW m2 fnbtW
N HO
N
N O
OH OH
Acidic Spectral Data pH
λmax
εmax (×10−3)
λmin
Neutral Spectral Data Spectral Ratios
pH
λmax
136
8.5
137
7
236 295 240
230
240
250
270
280
290
εmax (×10−3)
λmin 257 213 283
Alkaline Spectral Data Spectral Ratios
230 240 4.14 4.63
3.58
4.70
250 1.31
270 1.01
280 1.14
290 1.37
2.40
0.93
0.88
0.89
pH
λmax
12
236 295 236
13o
εmax (×10−3)
269
λmin 257
Spectral Ratios 230 4.14
240 4.63
250 1.31
255
References No.
Origin and Synthesis
136 137
R: 403 R: 346
[α] D
pK
– –
– –
t
Spectral Data 403b 346b
Mass Spectra
Rf
– –
– –
270 1.01
280 1.14
290 1.37
Handbook of Biochemistry and Molecular Biology
No.
4/16/10 1:19 PM
No.
Compound
3-Letter
Symbol
1-Letter
Structure O
Xanthosine
Xao
N H
O HO
X
Melting Point °C
[α]Dt
Basic
C10H12N4O6 (284.23)
–
−51.230 (8, 0.3N NaOH)
13
C10H15N3O5 (257.24)
203° (dec)
+5120 (H2O)
3.5
–
[α]Dt
N 144
5MedCyd d5MeCyd
5-Methyldeoxycytidine
m5dC
O
HO
N
O
OH NH2 OH
N
145
5HmdCyd d5HmCyd 5(HOMe)dCyd
5-Hydroxymethyldeoxycytidine
om5dC hm5dC*
HO
O
N
O
OH
No.
Acidic Spectral Data pH
144 145
1 1
λmax 287 283
εmax (×10−3)
λmin
12.4* 12.6
245 243
Neutral Spectral Data Spectral Ratios
230 1.34 0.16
240 0.43 0.21*
250 0.42 0.64*
270 1.93 1.88*
280 2.93 2.47*
290 3.12 2.27*
pH
λmax
7 7
277 272
εmax (×10−3) 8.5 –
λmin 255 247
Alkaline Spectral Data Spectral Ratios
230 240 1.47 1.29 – 0.88
Purines, Pyrimidines, Nucleosides, and Nucleotides
9168_Book.indb 315
PURINES, PYRIMIDINES, NUCLEOSIDES AND NUCLEOTIDES: PHYSICAL CONSTANTS AND SPECTRAL PROPERTIES (Continued)
250 1.00 0.97
270 1.37 1.19
280 1.54 1.17
290 1.01 0.58*
pH
λmax
14 13
279 274
εmax (×10−3) 8.8 –
λmin 255 252
Spectral Ratios 230 1.57 –
240 1.27 1.21
250 0.98 0.97
270 1.43 1.26
280 1.67 1.17*
290 1.13 0.68*
References No.
Origin and Synthesis
[α] D
pK
Spectral Data
144 145
C: 128,391a D: 133,134 D: 75,135 C: 136,385
391 136
128 75
128b,133*b,134b,391,317b 75b,135*bce136*d,317b
t
Mass Spectra
Rf
– –
70 70,75
315
4/16/10 1:19 PM
9168_Book.indb 316
No.
Compound
Symbol
3-Letter
1-Letter
Structure O
H2N Deoxyguanosine
dGuo
dG
pK
Formula (Mol Wt)
Melting Point °C
[α]Dt
Basic
Acidic
C10H13N5O4 (267.24)
250°
−30.223.5 (0.2, H2 O)
2.5
–
C11H15N5O4 (281.27)
249–250° (dec)
–
–
N
HN
146
316
PURINES, PYRIMIDINES, NUCLEOSIDES AND NUCLEOTIDES: PHYSICAL CONSTANTS AND SPECTRAL PROPERTIES (Continued)
N
N
HO
O
OH O N
N H2N 147
1MedGuo d1MeGuo
1-Methyldeoxyguanosine
m1dG
N
HO
N –
O
OH
Acidic Spectral Data pH
146 147
1o 1o
λmax
εmax (×10−3)
λmin
12.1 12.1
232 –
255 257
Neutral Spectral Data Spectral Ratios
230 0.26 –
240 0.60 –
250 1.0 –
270 0.84 –
280 0.69 –
290 0.47 –
pH
λmax
H2O –
254 –
εmax (×10−3) 13.0 –
λmin 223 –
Alkaline Spectral Data Spectral Ratios
230 240 0.38 0.81 – –
250 1.16 –
270 0.75 –
280 0.68 –
290 0.27 –
pH
λmax
12 11
260 254
εmax (×10−3) 9.2 13.6
λmin 230 –
Spectral Ratios 230 0.40 –
240 0.55 –
250 0.87* –
References No.
Origin and Synthesis
[α]tD
pK
Spectral Data
146 147
C: 267 D: 238 C: 24
267 –
246 –
317b,242e,267*bcd 24
Mass Spectra
Rf
317 –
242 24
270 0.98 –
280 0.61 –
290 0.09 –
Handbook of Biochemistry and Molecular Biology
No.
4/16/10 1:19 PM
No.
Compound
Symbol
3-Letter
1-Letter
Structure
pK
Formula (Mol Wt)
Melting Point °C
[α]Dt
Basic
Acidic
C11H15N5O4 (281.27)
None (dec)
–
–
–
C9H12N2O5 (228.20)
163°
+50.022 (1.1 1N NaOH)
–
9.3, >13
C10H14N2O5 (242.23)
183–184°
+32.816 (1.04, 1N NaOH)
–
9.8, >13
O N
N H2N 148
7MedGuo d7MeGuo
7-Methyldeoxyguanosine
m7dG
N
N
HO
O
OH O HN 149
Deoxyuridine
dUrd
N
O O
HO
dU
Purines, Pyrimidines, Nucleosides, and Nucleotides
9168_Book.indb 317
PURINES, PYRIMIDINES, NUCLEOSIDES AND NUCLEOTIDES: PHYSICAL CONSTANTS AND SPECTRAL PROPERTIES (Continued)
OH O HN 150
Thymidine 5-Methyldeoxyuridine
dThd
O O
HO
dT
N
OH
No.
Acidic Spectral Data pH
148 149 150
1o 1 1
λmax 256 262 267
εmax (×10−3)
λmin
10.8 10.2 9.65
229 231 235
Neutral Spectral Data Spectral Ratios
230 – 0.20 0.33
240 – 0.40 0.34
250 – 0.74 0.65
270 – 0.83 1.06
280 – 0.32* 0.70
290 – – 0.22
pH
λmax
6 7 7
257 262 267
εmax (×10−3) – 10.2 9.65
λmin 235 231 235
Alkaline Spectral Data Spectral Ratios
230 240 – – 0.20 0.40 0.32 0.33
250 – 0.74 0.65
270 – 0.83 1.06
280 – 0.32* 0.70
290 – – 0.21*
pH
λmax
9o 12 13
– 262 267
εmax (×10−3) – 7.6 7.4
λmin – 242 246
Spectral Ratios 230 – 0.95 1.18
240 – 0.70 0.76
250 – 0.80 0.74
270 – 0.80 1.05
280 – 0.27* 0.65
290 – – 0.16
References Origin and Synthesis
[α]tD
pK
Spectral Data
148 149 150
C: 10,41 D: 238,243a D: 238 C: 128,356,69
– 243 241
– 163 163
10z,41c 163b,317be,232* 163b,242e,232*b,317b
Mass Spectra – 341,317 317
Rf 10 243 21,18,69,242
317
No.
4/16/10 1:19 PM
9168_Book.indb 318
No.
Symbol
Compound
3-Letter
5-Hydroxymethyldeoxyuridine
5HmdUrd 5(HOMe)dUrd d5HmUrd
1-Letter
Structure O
om5dU hm5dU*
N
O O
HO
pK
Formula (Mol Wt)
Melting Point °C
[α]Dt
Basic
Acidic
C10H14N2O6 (258.23)
180–182°
+1920 (H2O)
–
–
C10H14N2O4S (258.30)
182–183°
+1620 (0.5 MeOH)
–
–
OH
HN
151
318
PURINES, PYRIMIDINES, NUCLEOSIDES AND NUCLEOTIDES: PHYSICAL CONSTANTS AND SPECTRAL PROPERTIES (Continued)
OH
O HN 152
2-Thiothymidine 5-Methyl-2-thiodeoxyuridine
S O
HO
s2dT
2SdThd
N
OH
No.
Acidic Spectral Data pH 2
264
λmin
9.6
233
Neutral Spectral Data Spectral Ratios
230 0.27
240 0.37
250 0.70
270 0.97
280 0.51
290 0.10
pH
λmax
7 (MeOH)
264 221
εmax (×10−3) 9.6n 15.2
λmin 233
Alkaline Spectral Data Spectral Ratios
230 240 0.27 0.37
250 0.70
270 0.97
280 0.51
290 0.10
pH
λmax
12 13
264 242
εmax (×10−3) 7.0 22.8
λmin 243
Spectral Ratios 230 1.13
240 0.72
250 0.75
References No.
Origin and Synthesis
[α]tD
pK
151 152
C: 69,152 C: 301
152 301
– –
a
Spectral Data 317 ,69* 301 b
cd
Mass Spectra
Rf
– –
69 –
270 0.95
280 0.54*
290 0.18
Handbook of Biochemistry and Molecular Biology
151 152
λmax
εmax (×10−3)
4/16/10 1:19 PM
No.
Compound
3-Letter
Symbol
1-Letter
Structure
pK
Melting Point °C
[α]Dt
Basic
C10H14N5O7P (347.22)
183° (dec)
−65.422 (0.5, 0.5M, Na2HPO4)
3.8
–
C10H14N5O7P (347.22)
195° (dec)
−45.422 (0.5, 0.5M Na2HPO4)
3.65
–
C10H14N5O7P (347.22)
192° (dec)
−46.324 (H2O)
3.8
–
Formula (Mol Wt)
Acidic
RIBONUCLEOTIDES NH2 N
N 153
Adenosine 2′-phosphate
N
N
Ado-2′-P 2′-AMP
HO
O OH OH O P O OH NH2 N
N
154
Adenosine 3′-phosphate
N
N
Ap A-
Ado-3′-P 3′-AMP
HO
O
O HO
P
OH
O
Purines, Pyrimidines, Nucleosides, and Nucleotides
9168_Book.indb 319
PURINES, PYRIMIDINES, NUCLEOSIDES AND NUCLEOTIDES: PHYSICAL CONSTANTS AND SPECTRAL PROPERTIES (Continued)
OH NH2 N
N 155
O
PA -A
Ado-5′-P AMP
Adenosine 5′-phosphate
HO
P
N
N O
O
OH HO
No.
Acidic Spectral Data pH
153 154 155
2 1 2
λmax
257z 257 257
εmax (×10−3) 14.4z 15.1 15.0
λmin
229z 230 230
Neutral Spectral Data Spectral Ratios
230 – – 0.23
240 – – 0.43
OH
250 0.85 0.85 0.84
270 – 0.71 0.68
280 0.23 0.22* 0.22
290 0.04 0.04* 0.44
pH
λmax
7 7 7
276 259z – 259
εmax (×10−3) 16.3 15.4z – 15.4
λmin
– – 227*
Alkaline Spectral Data Spectral Ratios
230 – – 0.18
240 – – 0.39
250 0.80 0.80 0.79
270 – – 0.66
280 0.15 0.15 0.16
290 0.01 0.01 0.01
pH
λmax
(MeOH) 12 13 11
264 259z 259 259
εmax (×10−3) 16.6 15.4z 15.4 15.4
λmin
– 227 227
Spectral Ratios 230
240
250
270
280
290
– – –
– – –
0.80 0.78 0.79
– 0.73 –
0.15 0.22 0.15
– 0.05 –
References Origin and Synthesis
[α]tD
pK
153 154 155
C: 191,193,219 R: 182,221 C: 193,190,219 R: 182,221,194z C: 191,197,368 R: 195 N: 198
221 221 368
220,170,218 220,170,218 220,170,212
Spectral Data 179e,223z 265*b,179e 212,179e,184b,183b,368*
Mass Spectra – – 374
Rf 258,219,263 258,219,263,292 188,219,263,368
319
4/16/10 1:19 PM
No.
9168_Book.indb 320
No.
Compound
3-Letter
Symbol
1-Letter
Structure
NH2
ppA
Ado-5′-P2 ADP
Adenosine 5′-diphosphate
HO
O
O
P O
P
OH
OH
pK
Formula (Mol Wt)
Melting Point °C
[α]Dt
Basic
Acidic
C10H15N5O10P2 (427.21)
–
–
3.9
–
C10H16N5O13P3 (507.19)
–
–
4.1
–
C11H16N5O7P (361.25)
–
–
8.81
–
N
N
156
320
PURINES, PYRIMIDINES, NUCLEOSIDES AND NUCLEOTIDES: PHYSICAL CONSTANTS AND SPECTRAL PROPERTIES (Continued)
N
N O
O
OH
HO
NH2 N
N
157
Ado-5′-P3 ATP
Adenosine 5′-triphosphate
O
pppA
HO
P
O
OH
O
O
P O
P
OH
OH
N
N O
O
HO
OH
NH N
N
N
N 158
1-Methyladenosine 3′(2′)-phosphate
1MeAdo-3′ (2′)-P
m1 Ap m1 Afor 3′
HO
O
O
O
No.
Acidic Spectral Data pH
156 157 158
2 2 2
λmax 257 257 258
εmax (×10−3)
λmin
15.0 14.7 13.2w
230 230 230
Neutral Spectral Data Spectral Ratios
230 – – 0.24
240 – – 0.44
250 0.85 0.85 0.83
270 – – 0.67
280 0.21 0.22 0.26
290 – – 0.07
pH
λmax
7 7 –
259 259 –
εmax (×10−3) 15.4 15.4 –
λmin 227 227 –
Alkaline Spectral Data Spectral Ratios
230 – – –
240 – – –
250 0.78 0.80 –
270 – – –
280 0.16 0.15 –
290 – – –
pH
λmax
11 11 13o
259 259 259
εmax (×10−3) 15.4 15.4 12.9w
λmin 227 227 –
Spectral Ratios 230 – – –
240 – – –
250 0.78 0.80 0.77
270 – – 0.76
References No.
Origin and Synthesis
156 157 158
C: 255,211,188,196 E: 226 N: 198 C: 211,214,188 N: 198 C: 90,292 R: 77
t
[α] D
pK
Spectral Data
Mass Spectra
Rf
– – –
212 212 –
212,183b,206 212b,206,183b 90
– – –
188 188 143,291,292
280 0.15 0.15 0.4
290 – – 0.32
Handbook of Biochemistry and Molecular Biology
O P OH OH
4/16/10 1:19 PM
No.
Compound
3-Letter
Symbol
1-Letter
Structure
Formula (Mol Wt)
NH
1MeAdo-5′-P 1MeAMP
1-Methyladenosine 5′-phosphate
O
pm1 A -m1 A
HO
N
N
P
[α]Dt
Basic
pK
Acidic
N
N 159
Melting Point °C
O
O
C11H16N5O7P (361.25)
–
–
8.81
–
C11H17N5O10P2 (441.23)
–
–
–
–
C11H16N5O7P (361.25)
–
–
–
–
OH OH
HO NH
N
N
160
1MeAdo-5′-P 1MeADP 2
1-Methyladenosine 5′-diphosphate
ppm1 A
HO
O
O
P O
P
OH
OH
N
N O
O
OH
HO
NH2 N
N
N
N 161
2-Methyladenosine 3′-phosphate
2MeAdo-3′-P
HO
m2 Ap
Purines, Pyrimidines, Nucleosides, and Nucleotides
9168_Book.indb 321
PURINES, PYRIMIDINES, NUCLEOSIDES AND NUCLEOTIDES: PHYSICAL CONSTANTS AND SPECTRAL PROPERTIES (Continued)
O
O HO
P
O
OH
OH
No.
Acidic Spectral Data pH
159 160 161
2 2 1
λmax
εmax (×10−3)
λmin
– 11.9 10.8
232 234 –
258 257 259
Neutral Spectral Data Spectral Ratios
230 0.34 0.37 –
240 0.46 0.44 –
250 0.81 0.84 –
270 0.74 0.66 –
280 0.32 0.23 –
290 0.10 0.04 –
pH
λmax
H2Oq – H2O
259 – 264
εmax (×10−3) – – 12.9
Alkaline Spectral Data Spectral Ratios
λmin
230 240 0.22 0.34 – – – –
233 – –
250 0.77 – –
270 0.74 – –
280 0.27 – –
290 – – –
pH
λmax
12o 12o 13
259 259 263
εmax (×10−3) 13.1w 12.5 13.1
λmin 230* 232 –
Spectral Ratios 230 0.22* 0.32 –
240 0.36* 0.40 –
250 0.75 0.75 –
270 0.71 0.74 –
280 0.36 0.36 –
290 0.3 0.31 –
References No.
Origin and Synthesis
159 160 161
C: 164,8 C: 164 C: 291 R: 291
[α]tD
pK
– – –
– – –
Spectral Data 164,8* 164b 291b
bd
Mass Spectra
Rf
– – –
8,164 164 291
321
4/16/10 1:19 PM
9168_Book.indb 322
No.
Compound
3-Letter
Symbol
1-Letter
Structure
Formula (Mol Wt)
NH2
2MeAdo-5′-P 2MeAMP
2-Methyladenosine 5′-phosphate
pm2 A
O HO
N
N
P
pK
[α]Dt
Basic
Acidic
–
–
–
N
N 162
Melting Point °C
322
PURINES, PYRIMIDINES, NUCLEOSIDES AND NUCLEOTIDES: PHYSICAL CONSTANTS AND SPECTRAL PROPERTIES (Continued)
O
O
C11H16N5O7P (361.25)
260° (dec) (Ba salt)
OH OH
HO HN
163
N6-(Δ2-Isopentenyl)adenosine 5′-phosphate: 6-(γγ-Dimethylallylamino)-9-β-D-ribofuranosylpurine 5′-phosphate
6Pe1Ado-5′-P 6iPeAdo-5′-P 6iPeAMP
N
N O
pi6 A
HO
P
N
N O
O
C15H22N5O7P (415.35)
–
–
–
–
C11H16N5O7P (361.25)
–
–
–
–
OH HO
OH
HN N
N
N6-Methyladenosine 3′(2′)-phosphate
6MeAdo-3′ (2′)-P
HO
O
O
O
O P OH OH
No.
Acidic Spectral Data pH
162 163 164
1 1 1
λmax 259 264 262
εmax (×10−3) 10.9 20.9 18.3
λmin
232.5 231*
Neutral Spectral Data Spectral Ratios
230
240
250
0.64
270
0.91
280
0.45
290
0.14
pH
λmax
6 7
264 267
εmax (×10−3) 13.2 19.2
λmin
Alkaline Spectral Data Spectral Ratios
230
240
250
233
270
280
290
pH
λmax
13 13 12
264 268 266*
εmax (×10−3) 13.4 19.0
λmin
232 230*
Spectral Ratios 230
240
250
270
280
290
0.58
1.05
0.67
0.22
References
4/16/10 1:19 PM
No.
Origin and Synthesis
162 163 164
C: 342 C: 319 R: 165,292 C: 292
[α]tD
pK
– – –
– – –
Spectral Data 342 319 265*b,292*c,165*c,90e
Mass Spectra – – –
Rf – 319 143,165,291,292
Handbook of Biochemistry and Molecular Biology
164
N
N
m6 Ap m6 A- for 3′
No.
Compound
3-Letter
Symbol
1-Letter
Structure
pK
Melting Point °C
[α]Dt
Basic
C11H16N5O7P (361.25)
–
–
˜3.7p
–
C11H17N5O10P2 (441.23)
–
–
˜3.7p
–
–
–
Formula (Mol Wt)
Acidic
HN N
N 165
6MeAdo-5′-P 6MeAMP
N6-Methyladenosine 5′-phosphate
pm6 A -m6 A
O HO
P
N
N O
O
OH OH
HO HN
N
N
166
6MeAdo-5′-P2 6MeADP
N6-Methyladenosine 5′-diphosphate
O
O
OH
N
N
ppm6 A HO P O P O
O
OH HO
OH
N N
N 167
6Me2Ado-5′-P 6Me2AMP
N6,N6-Dimethyladenosine 5′-phosphate
pm26A -m26A
O HO
P
N
N O
O
C12H18N5O7P (375.28)
−5120 (2.0, H2O)
225° (dec)
OH HO
No.
Acidic Spectral Data pH
165 166 167
2 2 H2O
λmax 261 262 268
εmax (×10−3)
λmin
16.3 15.7 18.3
231 231 –
230 0.28 0.18 –
240 0.39 0.32 –
OH
Neutral Spectral Data Spectral Ratios 250 0.73 0.69 –
270 0.85 0.84 –
280 0.36* 0.29 –
290 0.13 0.08 –
pH
λmax
H2O – 7
264 – 274
εmax (×10−3) 13.4 – –
λmin 229 – –
Alkaline Spectral Data Spectral Ratios
230 240 0.17 0.29 – – –
Purines, Pyrimidines, Nucleosides, and Nucleotides
9168_Book.indb 323
PURINES, PYRIMIDINES, NUCLEOSIDES AND NUCLEOTIDES: PHYSICAL CONSTANTS AND SPECTRAL PROPERTIES (Continued)
250 0.63 – –
270 0.97 – –
280 0.56 – –
290 0.17 – –
pH
λmax
12 12 –
266* 265 –
εmax (×10−3) 15.2n 15.4 –
λmin 231* 229 –
Spectral Ratios 230 0.20 0.14 –
240 0.32* 0.26 –
250 0.60 0.60 –
270 1.08 0.99 –
280 0.66* 0.57 –
290 0.26* 0.18 –
References No.
Origin and Synthesis
[α]tD
pK
Spectral Data
165 166 167
C: 164,166 C: 164 C: 115,166
– – 115
164 164 –
164*,8*bcde 164b 115,166c
Mass Spectra – – –
Rf 164,166,407 164 115,166,167
323
4/16/10 1:19 PM
9168_Book.indb 324
No.
Compound
3-Letter
Symbol
1-Letter
Structure
Formula (Mol Wt)
Melting Point °C
324
PURINES, PYRIMIDINES, NUCLEOSIDES AND NUCLEOTIDES: PHYSICAL CONSTANTS AND SPECTRAL PROPERTIES (Continued) Basic
[α]Dt
pK
Acidic
N N
N
168
6Me, Ado-5′P2 6Me2 ADP
N6,N6-Dimethyladenosine 5′-diphosphate
ppm6 A
HO
O
O
P O
P
OH
OH
O
HO
N H N
N N -Threoninocarbonyladenosine 3′(2′)-phosphate N-[(9-β-D-Ribofuranosylpurin-6-yl)- N-carbamoyl] threonine 3′(2′)-phosphate 6
169
6(ThrCO) Ado3′(2′)-P
HO
–
–
–
C15N21N6O11P (492.34)
–
–
2.1p
–
COOH
O
O
–
OH
N
N
tc6 AP for 3′
C12H19N5O10P2 (455.26)
OH
O HN
N
N O
O
O P OH OH
Acidic Spectral Data pH
168 169
– –
λmax – –
εmax (×10−3)
λmin
– –
– –
Neutral Spectral Data Spectral Ratios
230 – –
240 – –
250 – –
270 – –
280 – –
290 – –
pH
λmax
– 5
– 269
εmax (×10−3) – –
λmin – 231
Alkaline Spectral Data Spectral Ratios
230 – 0.2
240 – 0.33
250 – 0.62
270 – 1.38
280 – 0.87
290 – 0.03
pH
λmax
– –
– –
εmax (×10−3) – –
λmin – –
Spectral Ratios 230 – –
240 – –
250 – –
References No.
Origin and Synthesis
168 169
C: 167 R: 90,410
t
[α] D
pK
Spectral Data
– –
– 90
– 90
Mass Spectra
Rf
– –
167 –
270 – –
280 – –
290 – –
Handbook of Biochemistry and Molecular Biology
No.
4/16/10 1:19 PM
No.
Compound
3-Letter
Symbol
1-Letter
Structure O N H N
HN
N -Threoninocarbonyladenosine 5′-phosphate N-[(9-β-D-Ribofuranosylpurin-6-yl)- N-carbamoyl] threonine 3′(2′)-phosphate 6
170
6(ThrCO)Ado-5′-P 6(ThrCO) AMP
ptc6 A
N O HO P O OH
OH
Melting Point °C
Basic
[α]Dt
pK
Acidic
COOH
N
N
Formula (Mol Wt)
C15H21N6O11P (492.34)
–
–
–
˜3.0p
C16H23N6O11P (506.37)
–
–
–
˜3.0p
O
OH OH OH
O
N -Methyl-N -threoninocarbonyladenosine 5′-phosphate N-[(9-β-D-Ribofuranosylpurin-6-yl)N-methylcarbamoyl] threonine 5′-phosphate 6
171
6
6Me6(ThrCO) Ado-5′-P 6Me6(ThrCO) AMP
pm6tc6A
N
N H N
N
N
N O HO P O OH
COOH
O
OH OH
No.
Acidic Spectral Data pH
λmax
εmax (×10−3)
λmin
Neutral Spectral Data Spectral Ratios
230
240
250
270
280
290
pH
λmax
170
1
276
237
0.49
0.41
0.61
1.53
1.45
0.48
6.8
275 269 276
171
1
283
240
0.82
0.58
0.68
1.41
1.89
1.68
6.8
278
εmax (×10−3)
λmin
Alkaline Spectral Data Spectral Ratios
230
240
Purines, Pyrimidines, Nucleosides, and Nucleotides
9168_Book.indb 325
PURINES, PYRIMIDINES, NUCLEOSIDES AND NUCLEOTIDES: PHYSICAL CONSTANTS AND SPECTRAL PROPERTIES (Continued)
250
270
280
290
231
0.28
0.37
0.63
1.29
0.69
0.03
239
0.83
0.63
0.73
1.47
1.87
1.25
pH
13o 13
λmax
εmax (×10−3)
269 277 297 278
λmin
239 273 287 238
Spectral Ratios 230
240
250
270
280
290
0.73
0.48
0.65
1.37
1.07
0.71
0.73
0.54
0.68
1.47
1.89
1.29
References No.
Origin and Synthesis
170 171
R: 407 R: 407
t
[α] D
pK
– –
407 407
Spectral Data 407b 407b
Mass Spectra
Rf
– –
407 407
325
4/16/10 1:19 PM
9168_Book.indb 326
No.
Compound
3-Letter
Symbol
1-Letter
Structure
NH2
326
PURINES, PYRIMIDINES, NUCLEOSIDES AND NUCLEOTIDES: PHYSICAL CONSTANTS AND SPECTRAL PROPERTIES (Continued) Basic
pK
Acidic
Formula (Mol Wt)
Melting Point °C
C9H14N3O8P (323.21)
238–240° (dec)
+20.720 (1.0, H2O)
4.4
–
C9H14N3O8P (323.21)
232–234° (dec)
+49.420 (1.0, H2O)
4.3
–
C9H14N3O8P (323.21)
233° (dec)
+27.114 (0.54, H2O)
4.5
–
[α]Dt
N 172
N
O
HO
Cyd-2′-P 2′CMP
Cytidine 2′-phosphate
O O O P OH O
HO
NH2 N 173
HO
Cp C-
Cyd-3′-P 3′-CMP
Cytidine 3′-phosphate
O
N O
HO HO P O O
OH NH2 N
pC -C
Cyd-5′-P CMP
Cytidine 5′-phosphate
N
O O
HO
No.
Acidic Spectral Data pH
172 173 174
2 2 2
λmax
278 279 280*
εmax (×10−3) 12.7 13.0 13.2
λmin
240 240 241*
Neutral Spectral Data Spectral Ratios
230 – – 0.56
240 – – 0.25
OH
250 0.48 0.45* 0.44
270 – 1.51 1.73
280 1.80 2.00* 2.09
290 1.22 1.43* 1.55
pH
λmax
7 7 7
285 – 270z 271
εmax (×10−3) – 9.0z 9.1
λmin
– 250z 249
Alkaline Spectral Data Spectral Ratios
230 – – 1.07
240 – – 0.92
250
270
280
290
0.90 0.86 0.84
– – 1.21
0.85 0.93 0.98
0.26 0.30 0.33
pH
λmax
εmax (×10−3)
λmin
12 12 11
272 272 271
8.6 8.9 9.1
250 250 249
Spectral Ratios 230
240
250
270
280
290
– – –
– – –
0.9 0.86 0.84
– 1.16 –
0.85 0.93 0.98
0.26 0.30* 0.33
References
4/16/10 1:19 PM
No.
Origin and Synthesis
[α] D
172 173 174
R:215a,192,178 R: 215a,192,178 C: 190,196,368 R: 195 N: 198
215 215 190
t
pK 218,170,192 218,170,192 212
Spectral Data 223,179be 223,179be,181zbcd,251,265*be 212,183bc,184b,179,205,368*
Mass Spectra
Rf
– – –
258 258 74
Handbook of Biochemistry and Molecular Biology
174
HO HO P O O
No.
Compound
3-Letter
Symbol
1-Letter
Structure
NH2
Basic
pK
Acidic
Formula (Mol Wt)
Melting Point °C
C9H15N3O11P2 (403.18)
–
–
4.6
–
C9H16N3O14P3 (483.16)
–
–
4.8
–
C9H14N3O7SP (339.27)
–
–
3.6p
–
[α]Dt
N
175
Cyd-5′-P2 CDP
Cytidine 5′-diphosphate
OH HO HO P O P O O O
ppC
N
O O
HO
OH NH2 N
176
Cyd-5′-P3 CTP
Cytidine 5′-triphosphate
OH OH HO HO P O P O P O O O O
pppC
N
O O
HO
OH
NH2 N S
HO 177
2Syd-3′(2′)-P 2SCyd-3′(2′)-P
2-Thiocytidine 3′(2′)-phosphate
N O
S Cp for 3′ 2
O
Purines, Pyrimidines, Nucleosides, and Nucleotides
9168_Book.indb 327
PURINES, PYRIMIDINES, NUCLEOSIDES AND NUCLEOTIDES: PHYSICAL CONSTANTS AND SPECTRAL PROPERTIES (Continued)
O
O P OH OH
No.
Acidic Spectral Data pH
175 176 177
2 2 1
λmax 280 280 227 276
εmax (×10−3)
λmin
12.8 12.8 –
241 241 247
Neutral Spectral Data Spectral Ratios
230 – – 1.24
240 – – 0.91
250 0.46 0.45 0.75
270 – – 1.24
280 2.07 2.12 1.28
290 1.48 – 0.83
pH
λmax
7 7 H2O
271 271 248
εmax (×10−3) 9.1 9.0 –
λmin 249 249 220
Alkaline Spectral Data Spectral Ratios
230 240 – – – – 0.64 0.94
250 0.83 0.84 1.08
270 – – 0.88
280 0.98 0.97 0.69
290 0.32 – 0.39
pH
λmax
11 11 13
271 271 249
εmax (×10−3) 9.1 9.0 –
λmin 249 249 228
Spectral Ratios 230 – – 0.71
240 – – 0.93
250 0.83 0.84 1.08
270 – – 0.89
280 0.98 0.97 0.71
290 – – 0.4
References Origin and Synthesis
175 176 177
C: 196 N: 198 N: 198 R: 270,329 C: 329
[α]tD
pK
Spectral Data
Mass Spectra
Rf
– – –
212 212 329
212,179e,183b 212b,183b 270b,329b
– – –
– – 270,329
327
No.
4/16/10 1:19 PM
9168_Book.indb 328
No.
Compound
3-Letter
Symbol
1-Letter
Structure
pK
Melting Point °C
[α]Dt
Basic
C10H16N3O8P (337.22)
–
–
˜9.0p
–
C10H16N3O8P (337.22)
–
–
–
–
C10H17N3O11P2 (417.21)
–
–
˜9.0p
–
Formula (Mol Wt)
NH
328
PURINES, PYRIMIDINES, NUCLEOSIDES AND NUCLEOTIDES: PHYSICAL CONSTANTS AND SPECTRAL PROPERTIES (Continued) Acidic
N
178
3-Methylcytidine 3′(2′)-phosphate
3MeCyd-3′(2′)-P
N
O
HO
m3CP m3C - for 3′
O
O
O
O P OH OH NH N 179
3MeCyd-5′-P 3MeCMP
3-Methylcytidine 5′-phosphate
HO HO P O O
pm3C -m3C
O
N O
HO
OH NH N
3-Methylcytidine 5′-diphosphate
ppm3C
OH HO HO P O P O O O
O
HO
No.
Acidic Spectral Data pH
178 179 180
1 – 1
λmax
εmax (×10−3)
λmin
11.5 – 11.0
242 – 241
276 – 278
240 – – –
OH
Neutral Spectral Data Spectral Ratios
230 – – –
N
O
250 – – –
270 – – –
280 – – –
290 – – –
pH
λmax
7 – 7
276 – 277
εmax (×10−3) 11.2 – 11.0
λmin 242 – 241
Alkaline Spectral Data Spectral Ratios
230 240 0.66 0.34 – – – –
250 0.50 – –
270 1.56 – –
280 1.64 – –
290 0.98 – –
pH
λmax
– – –
– – –
εmax (×10−3) – – –
λmin – – –
Spectral Ratios 230 – – –
240 – – –
250 – – –
270 – – –
References
4/16/10 1:20 PM
No.
Origin and Synthesis
178 179 180
C: 130 R: 413 C: 125 C: 130
t
[α] D
pK
Spectral Data
– – –
130 – 130
130,413e – 130
Mass Spectra – – –
Rf 13,130,143 125 130
280 – – –
290 – – –
Handbook of Biochemistry and Molecular Biology
180
3MeCyd-5′-P2 3MeCDP
No.
Compound
3-Letter
Symbol
1-Letter
Structure
pK
Melting Point °C
[α]Dt
Basic
C10H16N3O8P (337.22)
–
–
–
–
C10H16N3O8P (337.22)
–
–
–
–
–
–
Formula (Mol Wt)
Acidic
NH N
181
N4-Methylcytidine 3′(2′)-phosphate
4MeCyd-3′(2′)-P
O
HO
m4Cp m4C- for 3′
N O
O
O
O P OH OH HN N 182
4MeCyd-5′-P 4MeCMP
N4-Methylcytidine 5′-phosphate
HO HO P O O
pm C -m4C 4
O
N O
HO
Purines, Pyrimidines, Nucleosides, and Nucleotides
9168_Book.indb 329
PURINES, PYRIMIDINES, NUCLEOSIDES AND NUCLEOTIDES: PHYSICAL CONSTANTS AND SPECTRAL PROPERTIES (Continued)
OH HN N
183
4MeCyd-5′-P2 4MeCDP
N4-Methylcytidine 5′-diphosphate
ppm4C
OH HO HO P O P O O O
O
HO
No.
Acidic Spectral Data pH
λmax
εmax (×10−3)
λmin
181 182
1 1
281 280
12.9 14.8
242 242
183
1
280
12.9
241
–
C10H17N3O11P2 (417.21)
–
–
OH
Neutral Spectral Data Spectral Ratios
230 – –
N
O
pH
λmax – 237 272 –
240 – –
250 – –
270 – –
280 – –
290 – –
– H2O
–
–
–
–
–
–
εmax (×10−3) – – –
λmin – 227 248 –
Alkaline Spectral Data Spectral Ratios
230 – –
240 – –
–
–
250 – –
270 – –
280 – –
290 – –
–
–
–
–
pH
λmax
λmin
– –
εmax (×10−3) – –
– – –
–
–
Spectral Ratios
– –
230 – –
240 – –
250 – –
270 – –
280 – –
290 – –
–
–
–
–
–
–
–
References Origin and Synthesis
181 182 183
C: 130 C: 130,59,168 C: 130,59
[α] D
pK
– – –
– – –
Spectral Data 130 130,168c 130
Mass Spectra
Rf
– – –
130 130,59 130,59
329
4/16/10 1:20 PM
No.
t
9168_Book.indb 330
No.
Compound
3-Letter
Symbol
1-Letter
Structure
NH2
Formula (Mol Wt)
Melting Point °C
330
PURINES, PYRIMIDINES, NUCLEOSIDES AND NUCLEOTIDES: PHYSICAL CONSTANTS AND SPECTRAL PROPERTIES (Continued) Basic
[α]Dt
pK
Acidic
N
184
pm5C -m5C
5MeCyd-5′-P 5MeCMP
5-Methylcytidine 5′-phosphate
HO HO P O O
N
O O
HO
C10H16N3O8P (337.22)
–
–
–
–
C10H17N3O11P2 (417.21)
–
–
–
–
C10H14N5O8P (363.22)
175–180° (dec)z (dihydrate)
−57.025z (1.0, 2% NaOH)
–
–
OH NH2 N
185
5MeCyd-5′-P2 5MeCDP
5-Methylcytidine 5′-diphosphate
ppm5C
OH HO HO P O P O O O
O
N O
HO
OH
O N
HN 186
N
N
H2N HO
Guo-2′-P 2′-GMP
Guanosine 2′-phosphate
O
No.
Acidic Spectral Data pH
184 185 186
4q – 1
λmax
εmax (×10−3)
λmin
10.7 – –
– – –
284 – –
Neutral Spectral Data Spectral Ratios
230 – – –
240 – – –
250 – – 0.9
270 – – –
280 – – 0.68
290 – – 0.48
pH
λmax
– – 7
8 – –
εmax (×10−3) 278 – –
λmin 8.8 – –
Alkaline Spectral Data Spectral Ratios
230 – – –
240 – – –
250 – – 1.15
270 – – –
280 – – 0.68
290 – – 0.29
pH
λmax
– – 12
– – –
εmax (×10−3) – – –
λmin – – –
Spectral Ratios 230 – – –
240 – – –
250 – – 0.89
References No.
Origin and Synthesis
[α] D
pK
184 185 186
C: 59 C: 59 R: 222az,170
– – 222z
– – –
t
Spectral Data 169 – 179
Mass Spectra
Rf
– – –
59 59 334
270 – – –
280 – – 0.60
290 – – 0.11
Handbook of Biochemistry and Molecular Biology
O OH O P OH OH
4/16/10 1:20 PM
No.
Compound
Symbol
3-Letter
1-Letter
Structure O
187
Guanosine 3′-phosphate
N
N
H2N HO
Gp G-
Melting Point °C
[α]Dt
Basic
pK
Acidic
N
HN Guo-3′-P 3′-GMP
Formula (Mol Wt)
O
C10H14N5O8P (363.22)
175–180° (dec)z (dihydrate)
−57.025z (1.0, 2% NaOH)
2.3
9.7
C10H14N5O8P (363.22)
190-200° (dec)
–
2.4
9.4
C10H15N5O11P2 (443.21)
–
–
2.9
9.6
O OH HO P O OH O N
HN 188
N
N
H2N O HO P O OH
pG -G
Guo-5′-P GMP
Guanosine 5′-phosphate
O
HO
Purines, Pyrimidines, Nucleosides, and Nucleotides
9168_Book.indb 331
PURINES, PYRIMIDINES, NUCLEOSIDES AND NUCLEOTIDES: PHYSICAL CONSTANTS AND SPECTRAL PROPERTIES (Continued)
OH O N
HN
189
Guo-5′-P2 GDP
Guanosine 5′-diphosphate
O
HO
No.
Acidic Spectral Data pH
187 188 189
1 1 1
λmax 257 256 256
εmax (×10−3)
λmin
12.2 12.2 12.3
228* 228 228
240 – 0.55 –
OH
Neutral Spectral Data Spectral Ratios
230 – 0.22 –
N
N
H2N O O HO P O P O OH OH
ppG
250 0.93 0.96 0.95
270 0.77 0.74 –
280 0.69 0.67 0.67
290 0.49 0.29 –
pH
λmax
7 7 7
252 252 253
εmax (×10−3) 13.4* 13.7 13.7
λmin 227* 224 224
Alkaline Spectral Data Spectral Ratios
230 240 – – 0.36 0.81 – –
250 1.15 1.16 1.15
270 0.86 0.81 –
280 0.68 0.66 0.66
290 0.29 0.29 –
pH
λmax
10.8q 11 11
257 258 258
εmax (×10−3) 11.25 11.6 11.7
λmin 230* 230 230
Spectral Ratios 230 – 0.38 –
240 – 0.82 –
250 0.92 0.90 0.91
270 1.00 0.97 –
280 0.64 0.61 0.61
290 0.15 0.29 –
References Origin and Synthesis
[α] D
pK
Spectral Data
187 188 189
R: 222az,170 C: 190,199,189 N: 198 R: 195 C: 213,196 N: 198
222z – –
231 212 212
232b,179e,265be,400*be 212,183b,184b,198 212,183b
Mass Spectra
Rf
– – –
258,334 189,213,334 213,334
331
No.
t
4/16/10 1:20 PM
9168_Book.indb 332
No.
Compound
3-Letter
Symbol
1-Letter
Structure
190
Guo-5′-P3 GTP
Guanosine 5′-triphosphate
pppG
[α]Dt
Basic
Acidic
C10H16N5O14P3 (523.19)
–
–
3.3
9.3
C11H16N5O8P (377.25)
–
–
2.4p
–
N
HN
N
N
H2N O O O HO P O P O P O OH OH OH
pK
Melting Point °C
Formula (Mol Wt)
O
332
PURINES, PYRIMIDINES, NUCLEOSIDES AND NUCLEOTIDES: PHYSICAL CONSTANTS AND SPECTRAL PROPERTIES (Continued)
O
HO
OH
O N
N
191
1-Methylguanosine 3′(2′)-phosphate
1MeGuo-3′(2′)-P
m1 GP m1 Gfor 3′
H2N HO
N
N O
O
O
O P OH OH
Acidic Spectral Data pH
190 191
1 1
λmax 256 258
εmax (×10−3)
λmin
12.4 11.4n
228 230*
Neutral Spectral Data Spectral Ratios
230 – 0.21
240 – 0.45
250 0.96 0.86
270 – 0.8
280 0.67 0.72
290 – 0.51*
pH
λmax
7 H2O
253 255
εmax (×10−3) 13.7 12.4n
λmin 223 222
Alkaline Spectral Data Spectral Ratios
230 240 – – 0.23 0.67
250 1.17 1.04
270 – 0.86
280 0.66 0.63
290 – 0.20
pH
λmax
11 13
257 256
εmax (×10−3) 11.9 13.0n
λmin 230 227*
Spectral Ratios 230 – 0.3
240 – 0.66
250 0.92 1.02
References No.
Origin and Synthesis
190 191
C: 213 N: 198 R: 165,40
[α]tD
pK
Spectral Data
– –
212 40
212b,183b 90,265*b,165
Mass Spectra – –
Rf 213,334 165
270 – 0.84
280 0.59 0.63
290 – 0.20*
Handbook of Biochemistry and Molecular Biology
No.
4/16/10 1:20 PM
No.
Compound
3-Letter
Symbol
1-Letter
Structure O
192
N2-Methylguanosine 3′(2′)-phosphate
2MeGuo-3′(2′)-P
[α]Dt
Basic
Acidic
C11H16N5O8P (377.25)
–
–
2.4p
–
C12H18N5O8P (391.28)
–
–
2.6p
–
N
HN m2 Gp m2 Gfor 3′
pK
Melting Point °C
Formula (Mol Wt)
N H HO
N
N O
O
O
O P OH OH O N
HN N 193
N2,N2-Dimethylguanosine 3′(2′)-phosphate
2Me2 Guo-3′(2′)-P
m2 2Gp m2 2G-; for 3′
N
N O
HO
O
Purines, Pyrimidines, Nucleosides, and Nucleotides
9168_Book.indb 333
PURINES, PYRIMIDINES, NUCLEOSIDES AND NUCLEOTIDES: PHYSICAL CONSTANTS AND SPECTRAL PROPERTIES (Continued)
O
O P OH OH
No.
Acidic Spectral Data pH
192 193
1 1
λmax 259 265
εmax (×10−3)
λmin
14.2n 17.7n
232 237*
Neutral Spectral Data Spectral Ratios
230 0.29 0.42
240 0.44 0.29
250 0.81 0.62
270 0.77 0.97
280 0.60 0.58*
290 0.53 0.57
pH
λmax
H2O H2O
253 259
εmax (×10−3) 15.7n 19.2n
λmin 224 228
Alkaline Spectral Data Spectral Ratios
230 240 0.4 0.78 0.25 0.47
250 1.12 0.84
270 0.71 0.72
280 0.69 0.58
290 0.45 0.50
pH
λmax
13 13
258 263
εmax (×10−3) 13.3n 14.9n
λmin 236 241*
Spectral Ratios 230 0.72 1.18
240 0.59 0.54
250 0.89 0.77
270 0.91 0.93
280 0.78 0.83
290 0.41 0.60*
References No.
Origin and Synthesis
192 193
R: 40 R: 165,40
[α]tD
pK
Spectral Data
– –
40 40
90 90,265*b,165
Mass Spectra
Rf
– –
– 165
333
4/16/10 1:20 PM
9168_Book.indb 334
No.
Compound
3-Letter
Symbol
1-Letter
Structure
Formula (Mol Wt)
334
PURINES, PYRIMIDINES, NUCLEOSIDES AND NUCLEOTIDES: PHYSICAL CONSTANTS AND SPECTRAL PROPERTIES (Continued) pK
Melting Point °C
[α]Dt
Basic
Acidic
–
–
r
7.0
–
r
6.9
O N
N 194
7-Methylguanosine 2′-phosphate
H2N HO
7MeGuo-2′-P
N
N
C11H16N5O8P (377.25)
O O OH O P OH OH O N
N
195
7-Methylguanosine 3′-phosphate
7MeGuo-3′-P
H2N HO
m7 Gp m7 G-
N
N O
C11H16N5O8P (377.25)
–
O OH HO P O OH
Acidic Spectral Data pH
λmax
εmax (×10−3)
λmin
Neutral Spectral Data Spectral Ratios
194
2
257
12.6
230
230 –
195
2
257
13.2
230
0.26
pH
λmax 258 280 258
240 –
250 –
270 –
280 –
290 –
7.4q
0.51
0.89
0.74
0.68
0.52
7.4q
282
εmax (×10−3) 9.6 9.0 9.8 9.6
λmin 239 271 240
Alkaline Spectral Data Spectral Ratios
230 –
240 –
–
–
270
pH
λmax
250 –
270 –
280 –
290 –
12ot
268
εmax (×10−3) 9.6
–
–
–
–
8.9o
258
- 9.9
12
282
ot
λmin
241 245
245
Spectral Ratios 230 –
240 –
250 –
270 –
280 –
290 –
–
0.84
0.92
1.09
1.4
1.26
References No.
Origin and Synthesis
194 195
C: 334 R: 77 C: 334,90 R: 77
[α]tD
pK
Spectral Data
– –
334 334
334 334b,90e
Mass Spectra
Rf
– –
334 334
Handbook of Biochemistry and Molecular Biology
No.
4/16/10 1:20 PM
No.
Compound
3-Letter
Symbol
1-Letter
Structure O
7MeGuo-5′-P 7MeGMP
7-Methylguanosine 5′-phosphate
pm7 G -m7 G
Melting Point °C
[α]Dt
Basic
Acidic
C11H16N5O8P (377.25)
–
–
r
7.1
C11H17N5O11P2 (457.23)
–
–
r
7.2
C11H18N5O14P3 (537.21)
–
–
r
7.5
N
N 196
pK
Formula (Mol Wt)
H2N (HO)2(O)PO
N
N O
OH OH O N
N
197
7MeGuo-5′-P2 7MeGDP
7-Methylguanosine 5′-diphosphate
ppm7 G
H2N O O HO P O P O OH OH
N
N O
OH OH O N
N
198
7MeGuo-5′-P3 7MeGTP
7-Methylguanosine 5′-triphosphate
pppm7 G
H2N O O O HO P O P O P O OH OH OH
N
N O
Purines, Pyrimidines, Nucleosides, and Nucleotides
9168_Book.indb 335
PURINES, PYRIMIDINES, NUCLEOSIDES AND NUCLEOTIDES: PHYSICAL CONSTANTS AND SPECTRAL PROPERTIES (Continued)
OH OH
No.
Acidic Spectral Data pH
λmax
εmax (×10−3)
λmin
Neutral Spectral Data Spectral Ratios
230
240
250
270
280
290
pH
196
2
257
12
230
–
–
–
–
–
–
7.4q
197
2
257
11.0
230
–
–
–
–
–
–
7.4q
198
2
257
11.7
230
–
–
–
–
–
–
7.4q
λmax
258 280 258 280 258
εmax (×10−3) 10.3 8.6 8.9 7.3 9.8
λmin
236 271 236 271 236
Alkaline Spectral Data Spectral Ratios
230
240
250
270
280
290
pH
λmax 266 282 268 272 281
–
–
–
–
–
–
–
–
–
–
–
–
8.9o 12ot 12os
–
–
–
–
–
–
12o
εmax (×10−3) 9.9 8.3
λmin
7.0
245 242 244 242
8.55
243
Spectral Ratios 230
240
250
270
280
290
–
0.78
0.93
1.11
1.43
1.29
–
–
–
–
–
–
–
–
–
–
–
–
References No.
Origin and Synthesis
196 197 198
C: 334,125 C: 334 C: 334
t
[α] D
pK
– – –
334 334 334
Spectral Data 334b 334 334
Mass Spectra
Rf
– – –
125,334 334 334
335
4/16/10 1:20 PM
9168_Book.indb 336
No.
Compound
Symbol
3-Letter
1-Letter
Structure O
Ino-3′(2′)-P
Melting Point °C
[α]Dt
Basic
Acidic
C10H13N4O8P (348.21)
–
–
–
–
–
−18.424 (0.9, 0.2 NHCl)
–
–
–
–
–
–
N
N
HO Inosine 3′(2′)-phosphate
pK
Formula (Mol Wt) N
HN
199
336
PURINES, PYRIMIDINES, NUCLEOSIDES AND NUCLEOTIDES: PHYSICAL CONSTANTS AND SPECTRAL PROPERTIES (Continued)
O
Ip for 3′ O
O
O P OH OH 200
IMP Ino-5′-P
Inosine 5′-phosphate
pI
C10H13N4O8P (348.21)
O N
HN O N HO P O O OH HO
N
OH O
IDP Ino-5′-P2
Inosine 5′-diphosphate Inosinic acid
ppI
HO
No.
Acidic Spectral Data pH
199 200 201
λmax
– – –
– – –
εmax (×10−3)
λmin
– – –
– – –
– – –
240 – – –
N
C10H14N4O11P2 (428.19)
OH
Neutral Spectral Data Spectral Ratios
230
N
250 – – –
270 – – –
280 – – –
290 – – –
pH
5 6 6
λmax
εmax (×10−3) 280 8.0 248 – 248 12.2 248.5 12.2
λmin 271 222 22.5 –
Alkaline Spectral Data Spectral Ratios
230 0.63 – –
240 1.32 – –
250
270
280
290
1.59 1.68 1.68
0.63 – –
0.29 0.25 0.25
0.04 – –
pH
λmax
εmax (×10−3)
λmin
– – –
– – –
– – –
– – –
Spectral Ratios 230
240
250
270
280
290
– – –
– – –
– – –
– – –
– – –
– – –
References
4/16/10 1:20 PM
No.
Origin and Synthesis
[α] D
pK
199 200 201
R: 90 C: 368 C: 411
– 368 –
– – –
t
Spectral Data 90 368,411e 411
Mass Spectra – – –
Rf – 368,411 411
Handbook of Biochemistry and Molecular Biology
201
HN O O N HO P O P O O OH OH
No.
Compound
3-Letter
Symbol
1-Letter
Structure O
1Melno-3′(2′)-P
C11H15N4O8P (362.24)
–
Basic
[α]Dt
pK
Acidic
N
N
HO 1-Methylinosine 3′(2′)-phosphate
Melting Point °C
N
N
202
Formula (Mol Wt)
O
M1 Ip for 3′ O
–
–
–
+22.322z (2.0, H2O)
–
–
O
O P OH OH O HN 203
O
HO
Urd-2′-P 2′-UMP
Uridine 2′-phosphate
O
HO
No.
Acidic Spectral Data pH
202 203
– 2
λmax – 260*,z
εmax (×10−3)
λmin
– 9.9z
– 230z
240 – –
C9H13N2O9P (324.18)
O O P OH OH
190–191° (dec)z (Diammonium salt)
Neutral Spectral Data Spectral Ratios
230 – –
N
250 – 0.8
270 – –
280 – 0.28
290 – 0.03
pH
λmax
4 7
249 260*,z
εmax (×10−3) – 10.0z
λmin 233 230z
Alkaline Spectral Data Spectral Ratios
230 240 0.66 1.35 – –
Purines, Pyrimidines, Nucleosides, and Nucleotides
9168_Book.indb 337
PURINES, PYRIMIDINES, NUCLEOSIDES AND NUCLEOTIDES: PHYSICAL CONSTANTS AND SPECTRAL PROPERTIES (Continued)
250 1.65 0.78
270 0.72 –
280 0.41 0.30
290 0.07 0.03
pH 9.5 12
λmax
εmax (×10−3) 249.5 – 261z 7.3z
λmin 224 242z
Spectral Ratios 230 0.67 –
240 1.3 –
250 1.67 0.85
270 0.73 –
280 0.43 0.25
290 0.09 0.02
References No.
Origin and Synthesis
[α] D
pK
Spectral Data
Mass Spectra
Rf
202 203
R: 410 R: 178,170,216az
– 216z
– –
410 181*zb,179e,90zc
– –
– 258
t
337
4/16/10 1:20 PM
9168_Book.indb 338
No.
Compound
3-Letter
Symbol
1-Letter
Structure
O
338
PURINES, PYRIMIDINES, NUCLEOSIDES AND NUCLEOTIDES: PHYSICAL CONSTANTS AND SPECTRAL PROPERTIES (Continued) pK
Formula (Mol Wt)
Melting Point °C
[α]Dt
Basic
Acidic
C9H13N2O9P (324.18)
192°
+22.322z (2.0, H2O)
–
9.4
190–202° (dibrucine salt)
+3.4428 (1.02, 10% HCl)
–
9.5
–
–
–
9.4
HN 204
Up U-
Urd-3′-P 3′-UMP
Uridine 3′-phosphate
HO
N
O O
O HO P O
OH
OH O HN 205
Uridine 5′-phosphate 5′-Uridylic acid
pU -U
Urd-5′-P UMP
O HO P O OH
N
O
C9H13N2O9P (324.18)
O
OH OH O HN
206
ppU
O O HO P O P O OH OH
N
O O
C9H14N2O12P2 (404.16)
OH OH
No.
Acidic Spectral Data pH
204 205 206
1 2 2
λmax 262 262 262
εmax (×10−3)
λmin
10 10.0 10.0
230 230 230
Neutral Spectral Data Spectral Ratios
230 – – –
240 – – –
250 0.76 0.73 0.73
270 0.82 – –
280 0.32* 0.39 0.39*
290 0.03* 0.03 0.04
pH
λmax
7 7 7
262z 262 262
εmax (×10−3) 10.0z 10.0 10.0
λmin 230z 230 230
Alkaline Spectral Data Spectral Ratios
230 240 – – 0.21 0.38 – –
250 0.73 0.73 0.73
270 – 0.87 –
280 0.35 0.39 0.39
290 0.03 0.03 –
pH
λmax
13 11 11
261 261 261
εmax (×10−3) 7.8 7.8 7.9
λmin 241 241 241
Spectral Ratios 230 – 0.79 –
240 – 0.5 –
250 0.83 0.8 0.8
References No.
Origin and Synthesis
[α]tD
pK
Spectral Data
204 205 206
C: 190 R: 178,170,216z C: 264,190a,368 R: 195 N: 198 C: 210 N: 198
216z 217 –
223 212 212
265*b,181zbcd,179e 21,183be,184b,179e 212,183b,179*e
Mass Spectra
Rf
– – –
258 210,74 210
270 0.85 – –
280 0.28* 0.31 0.32
290 0.02* 0.02 –
Handbook of Biochemistry and Molecular Biology
Urd-5′-P2 UDP
Uridine 5′-diphosphate
4/16/10 1:20 PM
No.
Compound
3-Letter
Symbol
1-Letter
Structure
O
pK
Formula (Mol Wt)
Melting Point °C
[α]Dt
Basic
Acidic
C9H15N2O15P3 (484.15)
–
–
–
C9H13N2O8PS (340.25)
–
–
–
–
C12H17N2O10PS (412.31)
–
–
–
–
HN
207
Urd-5′-P3 UTP
Uridine 5′-triphosphate
pppU
O O O HO P O P O P O OH OH OH
N
O O
9.6
OH OH
O HN 208
2SUrd-5′-P 2Srd-5′-P 2SUMP
2-Thiouridine 5′-phosphate
ps2U p2S
O HO P O OH
N
S O
OH OH O COOMe
HN
209
5-(Methoxycarbonylmethyl)-2-thiouridine 3′-phosphate 5-Carboxymethyl-2-thiouridine methyl ester 3′-phosphate
No.
mcm5s2Up mcm5Sp
λmax
2
262
1
275
εmax (×10−3)
λmin
10.0
230
240 0.37
N
S O
Neutral Spectral Data Spectral Ratios
230 0.21
HO
O OH HO P O OH
Acidic Spectral Data pH
207 208 209
5MeCm2SUrd -3′-P 5MeCm2Srd -3′-P
250 0.75
270 0.88
280 0.38
243
290 –
Purines, Pyrimidines, Nucleosides, and Nucleotides
9168_Book.indb 339
PURINES, PYRIMIDINES, NUCLEOSIDES AND NUCLEOTIDES: PHYSICAL CONSTANTS AND SPECTRAL PROPERTIES (Continued)
pH
λmax
7 H2O 7
262 272 275
εmax (×10−3) 10.0
λmin 230 243 243
Alkaline Spectral Data Spectral Ratios
230 240 0.21 0.38
250 0.75
270 0.86
280 0.38
290 –
pH
λmax
11
261
12o
241
εmax (×10−3) 8.1
λmin 239
Spectral Ratios 230 0.79
240 0.65
250 0.81
270 0.78
280 0.31*
290 –
213
References Origin and Synthesis
207 208 209
C: 210 N: 198,204 C: 344 R: 306
[α] D
pK
Spectral Data
Mass Spectra
Rf
– – –
204,212 – –
212b,204*e,183b 344 306b
– – –
210 344 –
339
No.
t
4/16/10 1:20 PM
9168_Book.indb 340
No.
Compound
3-Letter
Symbol
1-Letter
Structure O
210
5(MeNHMe)2Srd -3′-P 5(MeNHMe)2SUrd -3′-P
mnm5s2Up mnm5Sp
HO
Basic
pK
Acidic
Formula (Mol Wt)
Melting Point °C
C11H18N3O8PS (383.32)
–
–
–
–
C10H15N2O9P (338.21)
–
–
–
–
[α]Dt
N H
HN
5-(Methylaminomethyl)-2-thiouridine 3′-phosphate
340
PURINES, PYRIMIDINES, NUCLEOSIDES AND NUCLEOTIDES: PHYSICAL CONSTANTS AND SPECTRAL PROPERTIES (Continued)
N
S O
O OH HO P O OH
O N
211
3-Methyluridine 3′(2′)-phosphate
3MeUrd-3′(2′)-P
N
O
HO
m3UP m3Ufor 3′
O
O
O
O P OH OH
Acidic Spectral Data pH
210 211
2v
λmax
εmax (×10−3)
λmin
–
233
258
Neutral Spectral Data Spectral Ratios
230
240
250
270
280
290
–
–
–
–
–
–
pH
λmax
H2O
262
εmax (×10−3) 8.8*
λmin
–
Alkaline Spectral Data Spectral Ratios
230
240
250
270
280
290
–
–
0.77
–
0.45
–
pH
λmax
εmax (×10−3)
λmin
11.6
260
9.3
233
Spectral Ratios 230
240
250
270
280
290
–
–
–
–
–
–
References No.
Origin and Synthesis
210 211
R: 305 C: 173,142,174
[α]tD
pK
– –
– –
Spectral Data – 173,174c,142*cd
Mass Spectra – –
Rf 305 13,142,143,173
Handbook of Biochemistry and Molecular Biology
No.
4/16/10 1:20 PM
No.
Compound
3-Letter
Symbol
1-Letter
Structure
O
pK
Formula (Mol Wt)
Melting Point °C
[α]Dt
Basic
Acidic
C10H15N2O9P (338.21)
–
–
–
–
C9H13N2O8PS (340.25)
–
–
–
–
N 212
3MeUrd-5′-P 3MeUMP
3-Methyluridine 5′-phosphate
O HO P O OH
Pm3U -m3U
N
O O
OH OH S HN
213
4Srd-3′(2′)-P 4SUrd-3′(2′)-P
4-Thiouridine 3′(2′)-phosphate
N
O
HO
s4Up 4SP s4U- for 3′
O
O
O
O P OH OH
No.
Acidic Spectral Data pH
212 213
– 1
λmax
εmax (×10−3)
λmin
– 4.0 20.6*
– 225 276
– 245 331
Neutral Spectral Data Spectral Ratios
230 –
240 –
250 –
270 –
280 –
290 –
pH
λmax
H2O 5.6
262 245 331
εmax (×10−3) 8.8 4.0 20.6
λmin 232 225 276
Alkaline Spectral Data Spectral Ratios
230 –
240 –
Purines, Pyrimidines, Nucleosides, and Nucleotides
9168_Book.indb 341
PURINES, PYRIMIDINES, NUCLEOSIDES AND NUCLEOTIDES: PHYSICAL CONSTANTS AND SPECTRAL PROPERTIES (Continued)
250 –
270 –
280 –
290 –
pH
λmax
– 13
– 315*
εmax (×10−3) – 18.3*
λmin – 257
Spectral Ratios 230 –
240 –
250 –
270 –
280 –
290 –
References No.
Origin and Synthesis
212 213
C: 344 C: 343 R: 144
[α] D
pK
– –
– –
t
Spectral Data 344 343,144*b
Mass Spectra
Rf
– –
125,344 343
341
4/16/10 1:20 PM
9168_Book.indb 342
No.
Compound
Symbol
3-Letter
1-Letter
Structure
S
342
PURINES, PYRIMIDINES, NUCLEOSIDES AND NUCLEOTIDES: PHYSICAL CONSTANTS AND SPECTRAL PROPERTIES (Continued) pK
Melting Point °C
[α]Dt
Basic
C9H13N2O8PS (340.25)
–
–
–
–
C11H15N2O11P (382.22)
–
–
–
∼4P
Formula (Mol Wt)
Acidic
HN 214
4-Thiouridine 5′-phosphate
O HO P O OH
ps4U -s4U p4S
4Srd-5′-P 4Surd-5′-P 4SUMP
N
O O
OH OH O COOH
HN
215
5CmUrd-3′(2′)-P 5CxMeUrd3′(2′)-P
5-Carboxymethyluridine 3′(2′)-phosphate Uridine-5-acetic acid 3′(2′)-phosphate
cm5Up for 3′
N
O
HO
O
O
O
O P OH OH
Acidic Spectral Data pH
λmax
εmax (×10−3)
λmin
Neutral Spectral Data Spectral Ratios
230
240
250
270
280
290
214 215
2
265
9.7n
232
0.26
0.37
0.71
0.97
0.55
0.11
pH
λmax
H2O
245 331 267
7
εmax (×10−3) – 20.6 9.8n
λmin 225 274 232
Alkaline Spectral Data Spectral Ratios
230
0.23
240
0.28
250
270
280
290
0.62
1.06
0.69
0.14
pH
λmax
εmax (×10−3)
λmin
12.3
266
7.0n
242
Spectral Ratios 230
240
250
270
280
290
1.13
0.74
0.77
0.98
0.55
0.07
References No.
Origin and Synthesis
214 215
C: 344,343 R: 278,410
[α]tD
pK
Spectral Data
– –
– 278
344 410
Mass Spectra – –
Rf 343,344 278
Handbook of Biochemistry and Molecular Biology
No.
4/16/10 1:20 PM
No.
Compound
3-Letter
Symbol
1-Letter
Structure O
216
5NcmUrd-3′(2′)-P 5NcMeUrd3′(2′)-P
ncm5Up for 3′
HO
Melting Point °C
Basic
Acidic
C11H16N3O10P (381.24)
–
–
–
–
C9H13N2O10P (340.18)
–
–
–
–
CONH2
HN
5-Carbamoylmethyluridine 3′(2′)-phosphate Uridine-5-acetamide 3′(2′)-phosphate
pK
[α]Dt
Formula (Mol Wt)
N
O O
O
O
O P OH OH O OH
HN
217
5HOUrd-5′-P 5(HO)UMP
5-Hydroxyuridine 5′-phosphate
O HO P O OH
po5U o5U
N
O O
OH OH
No.
Acidic Spectral Data pH
216 217
2 –
λmax 266 –
εmax (×10−3)
λmin
10.0n –
232 –
Neutral Spectral Data Spectral Ratios
230 0.31 –
240 0.38 –
250 0.71 –
270 0.98 –
280 0.55 –
290 0.11 –
pH
λmax
6 6
266 278
εmax (×10−3) 10.2n –
λmin 232 245
Alkaline Spectral Data Spectral Ratios
230 240 0.30 0.39 – –
Purines, Pyrimidines, Nucleosides, and Nucleotides
9168_Book.indb 343
PURINES, PYRIMIDINES, NUCLEOSIDES AND NUCLEOTIDES: PHYSICAL CONSTANTS AND SPECTRAL PROPERTIES (Continued)
250 0.68 –
270 1.02 –
280 0.62 –
290 0.16 –
pH
λmax
11.5 9
265 236 300
εmax (×10−3) 6.9n –
λmin 244 268
Spectral Ratios 230 1.08 –
240 0.76 –
250 0.79 –
270 0.97 –
280 0.55 –
290 0.13 –
References No.
Origin and Synthesis
216 217
R: 398,410 C: 146,74
[α] D
pK
– –
– –
t
Spectral Data 410 146
Mass Spectra
Rf
– –
– 74
343
4/16/10 1:20 PM
9168_Book.indb 344
No.
Compound
3-Letter
Symbol
1-Letter
Structure
Formula (Mol Wt)
O
Melting Point °C
344
PURINES, PYRIMIDINES, NUCLEOSIDES AND NUCLEOTIDES: PHYSICAL CONSTANTS AND SPECTRAL PROPERTIES (Continued) Basic
[α]Dt
pK
Acidic
HN
218
5-Methyluridine 3′(2′)-phosphate Ribosylthymine 3′(2′)-phosphate
5MeUrd-3′(2′)-P Thd-3′(2′)-P
M5Upm5UTp T-for 3′
N
O
HO
O
O
C10H15N2O9P (338.21)
–
–
–
–
–
−12.326 (2.0, 0.1N HCl)
–
–
O
O P OH OH O
219
5-Methyluridine 5′-phosphate Ribosylthymine 5′-phosphate
HN
PT -T pm5U -m5U
Thd-5′-P TMP 5MeUMP
O HO P O OH
C10H15N2O9P (338.21)
N
O O
OH OH
Acidic Spectral Data pH
218 219
1 2
λmax
εmax (×10−3)
λmin
9.8 8.8
235 –
267* 267
Neutral Spectral Data Spectral Ratios
230 – –
240 – –
250 0.68 –
270 1.05 –
280 0.66 –
290 0.23 –
pH
λmax
– –
– –
εmax (×10−3) – –
λmin – –
Alkaline Spectral Data Spectral Ratios
230 – –
240 – –
250 – –
270 – –
280 – –
290 – –
pH
λmax
13 –
268 –
εmax (×10−3) – –
λmin 247 –
Spectral Ratios 230 – –
240 – –
250 0.79 –
270 1.04 –
References No.
Origin and Synthesis
[α]tD
pK
218 219
R: 165 C: 142 C: 368
– 368
– –
Spectral Data 265b,165* 368
Mass Spectra
Rf
– –
165,142 142
280 0.69 –
290 0.23 –
Handbook of Biochemistry and Molecular Biology
No.
4/16/10 1:20 PM
No.
220
Compound
Symbol
3-Letter
ppm5U ppT ppT
Thd-5′-P2 TDP 5MeUDP
5-Methyluridine 5′-diphosphate Ribosylthymine 5′-diphosphate
1-Letter
Structure
Formula (Mol Wt)
O
pK
Melting Point °C
[α]Dt
Basic
Acidic
–
–
–
–
–
–
–
9.6
–
–
–
–
HN O O HO P O P O OH OH
C10H16N2O12P2 (418.18)
N
O O
OH OH
O N
221
Pseudouridine 3′(2′) phosphate β-f-Pseudouridine 3′(2′)-phosphate; 5-Ribosyluracil 3′(2′)-phosphate
ψrd-3′(2′)-P
O
HO
Ψp ψFor 3′
NH
O
O
C9H13N2O9P (324.18) O
O P OH OH O HN
222
Pseudouridine 5′-phosphate β-f-Pseudouridine 5′-phosphate; 5-Ribosyluracil 5′-phosphate
O HO P O OH
Pψ -ψ
ψrd-5′-P ψMP
O
Purines, Pyrimidines, Nucleosides, and Nucleotides
9168_Book.indb 345
PURINES, PYRIMIDINES, NUCLEOSIDES AND NUCLEOTIDES: PHYSICAL CONSTANTS AND SPECTRAL PROPERTIES (Continued)
NH O C9H13N2O9P
(324.18)
OH OH
No.
Acidic Spectral Data pH
220 221 222
2 2v –
λmax 268 263 –
εmax (×10−3)
λmin
10.0 8.4 –
234 233 –
Neutral Spectral Data Spectral Ratios
230 0.37 0.30 –
240 0.34 0.41 –
250 0.64 0.75 –
270 1.10 0.86 –
280 0.77 0.40* –
290 0.27 0.07* –
pH
λmax
– 7 –
– 263 –
εmax (×10−3) – – –
λmin – 233 –
Alkaline Spectral Data Spectral Ratios
230 240 – – 0.28 0.39 – –
250 – 0.74 –
270 – 0.85 –
280 – 0.40 –
290 – 0.07 –
pH
λmax
– 12 12
– 286 –
εmax (×10−3) – 8.4 –
λmin – 246 –
Spectral Ratios 230 – 2.13 –
240 – 0.75 –
250 – 0.64* –
270 – 1.54* –
280 – 2.06* 1.40
290 – 2.14* –
References Origin and Synthesis
220 221 222
C: 175 R: 157,94 C: 158,176
[α] D
pK
Spectral Data
– – –
– 157 –
175 157b,94,158,265*b 158
Mass Spectra
Rf
– – –
175 165 158
345
No.
t
4/16/10 1:20 PM
9168_Book.indb 346
No.
223
Compound
3-Letter
Symbol
1-Letter
Structure
O O HO P O P O OH OH
ψrd-5′-P2 ψDP
pK
Formula (Mol Wt)
Melting Point °C
[α]Dt
Basic
O
C9H14N2O12P2 (404.16)
–
–
–
–
COOH
C10H13N2O11P (368.19)
–
–
–
–
C11H18N5O9P (395.27)
–
–
–
–
O
HN
Pseudouridine 5′-diphosphate β-f-Pseudouridine 5′-diphosphate; 5-Ribosyluracil 5′-diphosphate
346
PURINES, PYRIMIDINES, NUCLEOSIDES AND NUCLEOTIDES: PHYSICAL CONSTANTS AND SPECTRAL PROPERTIES (Continued) Acidic
NH
O
OH OH
O
224
HN
Ord-5′-P OMP 6CxUMP
Orotidine 5′-phosphate 6-Carboxyuridine 5′-phosphate
O HO P O OH
pO -O
N
O O
OH OH
OH N
N N
H2N 225
5-N-Methyl formamido-6-ribosylamino iso cystosine 3′(2′)-phosphatet 2-Amino-4-hydroxy-5-N-methylformamido-6ribosylaminopyrimidine 3′(2′)-phosphate
HO
5MeFn6(RibNH) isoCyt-3′(2′)-P
CHO
NH
O
O
O P OH OH
No.
Acidic Spectral Data pH
223 224 225
λmax
– – 2
– – 273
εmax (×10−3)
λmin
– – 14.0x
– – 247
Neutral Spectral Data Spectral Ratios
230 – – 1.00
240 – – 0.50
250 – – 0.47
270 – – 1.81
280 – – 1.41
290 – – 0.31
pH
λmax
– 7 –
– 266 –
εmax (×10−3) – – –
λmin – – –
Alkaline Spectral Data Spectral Ratios
230 – – –
240 – – –
250 – – –
270 – – –
280 – 0.66 –
290 – – –
pH
λmax
12 – 13
– – 265
εmax (×10−3) – – 10.5x
λmin – – 244
Spectral Ratios 230 – – 0.77
240 – – 0.51
250 – – 0.56
References
4/16/10 1:20 PM
No.
Origin and Synthesis
223 224 225
C: 158 E: 177 C: 90
[α] D
pK
– – –
– – –
t
Spectral Data 158 177 90,334b
Mass Spectra
Rf
– – –
158 – 334
270 – – 1.03
280 1.30 – 0.26
290 – – 0.05
Handbook of Biochemistry and Molecular Biology
O
No.
Compound
Symbol
3-Letter
1-Letter
Structure
Formula (Mol Wt)
Melting Point °C
Basic
[α]Dt
pK
Acidic
DEOXYRIBONUCLEOTIDES NH2 N
N 226
dAp dA-
dAdo-3′P 3′dAMP
Deoxyadenosine 3′-phosphate
N
N
HO
O
C10H14N5O6P (331.22)
–
C10H14N5O6P (331.22)
142°
–
–
–
˜4.4
–
–
–
O H
HO P O OH
NH2 N
N 227
O
pdA -dA
dAdo-5′-P dAMP
Deoxyadenosine 5′-phosphate
HO
N
N
P
O
O
−38.019 (0.23, H2O)
OH
Purines, Pyrimidines, Nucleosides, and Nucleotides
9168_Book.indb 347
PURINES, PYRIMIDINES, NUCLEOSIDES AND NUCLEOTIDES: PHYSICAL CONSTANTS AND SPECTRAL PROPERTIES (Continued)
H
HO
NH2 N
N
228
dado-5′-P3 dATP
Deoxyadenosine 5′-triphosphate
pppdA
O HO
P
O
OH
O
O
P O
P
OH
OH
O
O
HO
No.
Acidic Spectral Data pH
226 227 228
– 2 –
λmax – 258 –
εmax (×10−3)
λmin
– 14.3* –
– 230 –
240 – – –
C10H16N5O12P3 (491.19)
–
250 – 0.82 –
270 – – –
280 – 0.23 –
290 – 0.04 –
pH
λmax
7 7 7
– – –
εmax (×10−3) – 15.3 –
λmin – – –
Alkaline Spectral Data Spectral Ratios
230 – – –
240 – 0.42 –
–
H
Neutral Spectral Data Spectral Ratios
230 – – –
N
N
250 0.79 0.8 0.77
270 0.68 0.66 –
280 0.14 0.14 0.14
290 – 0.01 –
pH
λmax
– – –
– – –
εmax (×10−3) – – –
λmin – – –
Spectral Ratios 230 – – –
240 – – –
250 – – –
270 – – –
280 – – –
290 – – –
References No.
Origin and Synthesis
[α] D
pK
Spectral Data
226 227 228
D: 263 C: 203 D: 200a,260 C: 202 E: 209
– 260 –
– 180 –
263 186,185* e,200,223cd 209
t
Mass Spectra
Rf
– – –
263 263 –
347
4/16/10 1:20 PM
9168_Book.indb 348
No.
Compound
Symbol
3-Letter
1-Letter
Structure
Formula (Mol Wt)
pK
348
PURINES, PYRIMIDINES, NUCLEOSIDES AND NUCLEOTIDES: PHYSICAL CONSTANTS AND SPECTRAL PROPERTIES (Continued) Melting Point °C
[α]Dt
Basic
Acidic
–
–
3.6p
–
HN N
N 229
6MedAdo-5′ -P 6MedAMP d6MeAdo-5′-P
N6-Methyldeoxyadenosine 5′-phosphate
Pm6dA -m6dA
O HO
P
N C H NOP 11 16 5 6 (345.25)
N O
O
OH H
HO
NH2 N 230
dCyd-3’-P 3′-dCMP
Deoxycytidine 3′-phosphate
HO
dCp dC-
N
O O
HO HO P O O
C9H14N3O7P (307.20)
196–197° (dec)
+57.017 (1.35, H2O)
–
–
C9H14N3O7P (307.20)
183–184° (dec)
+35.021 (0.2, H2O)
4.6
–
H
NH2
231
pdC -dC
dCyd-5′-P dCMP
Deoxycytidine 5′-phosphate
O
N O
HO
No.
Acidic Spectral Data pH
229 230 231
4q 3 2
λmax 266 – 280
εmax (×10−3)
λmin
– – 13.5
– – 239
Neutral Spectral Data Spectral Ratios
230 0.18 – –
240 0.28 – –
H
250 0.63 – 0.43
270 1.09 – –
280 0.66 2.0 2.12
290 0.25 – 1.55
pH
λmax
– 7 7
– – 271
εmax (×10−3) – – 9.3
λmin – – 249
Alkaline Spectral Data Spectral Ratios
230 – – –
240 – – 0.91
250 – 0.84 0.82
270 – 1.19 1.25
280 – 0.93 0.99
290 – – 0.30
pH
λmax
13 – 12
266 – –
εmax (×10−3) – – –
λmin 234 – –
Spectral Ratios 230 0.18 – –
240 0.24 – –
250 0.57 – 0.82
270 1.08 – –
References
4/16/10 1:20 PM
No.
Origin and Synthesis
[α] D
pK
Spectral Data
229 230 231
D: 21 D: 263 C: 201,203,202 C: 201 D: 200
– 201 207
21 – 180
21b 263,201e 185b,179e,186e,201
t
Mass Spectra
Rf
– – –
– 203,201,263 185,201,263,74
280 0.63 – 0.99
290 0.21 – 0.30
Handbook of Biochemistry and Molecular Biology
N HO HO P O O
No.
Compound
Symbol
3-Letter
1-Letter
Structure
NH2
pK
Melting Point °C
[α]Dt
Basic
C9H16N3O13P3 (467.17)
–
–
–
–
C10H16N3O7P (321.22)
–
–
4.4
–
C10H16N3O8P (337.22)
–
–
–
–
Formula (Mol Wt)
Acidic
N
232
dCyd-5′-P3 dCTP
Deoxycytidine 5′-triphosphate
pppdC
OH OH HO HO P O P O P O O O O
N
O O
HO
H
NH2 N 233
5MedCyd-5′-P 5MedCMP d5MeCyd-5′-P
5-Methyldeoxycytidine 5′-phosphate
HO pm5 dC HO P O 5 -m dC O
O
N O
H
HO
NH2
234
5HmdCyd-5′-P 5HmdCMP 5HOMedCMP
5-Hydroxymethyldeoxycytidine 5′-phosphate
pom5 dC HO -om5 dC HO P O phm5 O dC* -hm5 dC*
N O
HO
No.
Acidic Spectral Data pH
232 233 234
2 2 1
λmax – 287 284
εmax (×10−3)
λmin
– – 12.5
– 244 245
240 – 0.43 0.39
H
Neutral Spectral Data Spectral Ratios
230 – 1.51 1.12*
OH N
O
250 0.44 0.36 0.44
270 – 2.10 1.89
280 2.14 3.14 2.68
290 – 3.44 2.53
pH
λmax
– 7 7
– 278 275
εmax (×10−3) – – 7.7
λmin – 254 254
Alkaline Spectral Data Spectral Ratios
230 240 – – 1.52 1.29 1.50 1.10
Purines, Pyrimidines, Nucleosides, and Nucleotides
9168_Book.indb 349
PURINES, PYRIMIDINES, NUCLEOSIDES AND NUCLEOTIDES: PHYSICAL CONSTANTS AND SPECTRAL PROPERTIES (Continued)
250 – 0.95 0.90
270 – 1.40 1.35
280 – 1.52 1.33
290 – 1.01 0.71
pH
λmax
– 12 12
– 278 275
εmax (×10−3) – – 7.7
λmin – – 254
Spectral Ratios 230 – – 1.40*
240 – – 1.08
250 – – 0.93
270 – – 1.33
280 – – 1.31
290 – – 0.65
References Origin and Synthesis
[α]tD
pK
Spectral Data
232 233 234
E: 209 C: 214 D: 68 C: 74 E: 70 D: 171,172
– – –
– 170 –
209 68b,186 74,70*bcde,171d,172*
Mass Spectra
Rf
– – –
– – 74
349
No.
4/16/10 1:20 PM
9168_Book.indb 350
No.
Compound
Symbol
3-Letter
1-Letter
Structure O
235
Deoxyguanosine 3′-phosphate
Basic
[α]Dt
pK
Acidic
N
N
H2N HO
dGp dG-
Melting Point °C
N
HN dGuo-3′-P 3′-dGMP
Formula (Mol Wt)
350
PURINES, PYRIMIDINES, NUCLEOSIDES AND NUCLEOTIDES: PHYSICAL CONSTANTS AND SPECTRAL PROPERTIES (Continued)
O
C10H14N5O7P (347.23)
–
C10H14N8O7P (347.23)
180–182°
–
–
–
2.9
9.7
O H HO P O OH O N
HN 236
H2N O HO P O OH
pdG -dG
dGuo-5′-P dGMP
Deoxyguanosine 5′-phosphate
O
HO
Acidic Spectral Data pH
235 236
– 1o 2q
λmax – 255 –
εmax (×10−3)
λmin
– 11.8 –
– 228 –
230 – – –
240 – – –
H
Neutral Spectral Data Spectral Ratios 250 – 1.02 1.03
270 – – –
280 – 0.70 0.70
290 – – 0.46
pH
λmax
7 7
– –
εmax (×10−3) – –
λmin – –
Alkaline Spectral Data Spectral Ratios
230 – –
240 – 0.79
−3119 (0.43, H2O)
250 1.20 1.13
270 0.82 0.81
280 0.67 0.67
290 – 0.27
pH
λmax
– –
– –
εmax (×10−3) – –
λmin – –
Spectral Ratios 230 – –
240 – –
250 – –
270 – –
References No.
Origin and Synthesis
[α] D
pK
Spectral Data
235 236
D: 263 C: 203 D: 200,180a,259
– 259
– 180
263 186,185e,200,223cde
t
Mass Spectra
Rf
– –
263 185,263
280 – –
290 – –
Handbook of Biochemistry and Molecular Biology
No.
N
N
4/16/10 1:20 PM
No.
Compound
3-Letter
Symbol
1-Letter
Structure
O
237
dGuo-5′-P3 dGTP
Deoxyguanosine 5′-triphosphate
pppdG
[α]Dt
Basic
Acidic
C10H16N5O13P3 (507.20)
–
–
–
–
C11H16N5O7P (361.24)
–
–
r
–
C11H18N5O13P3 (521.21)
–
–
r
7.5
N
HN
N
N
H2N O O O HO P O P O P O OH OH OH
pK
Melting Point °C
Formula (Mol Wt)
O
HO
H
O N
N 238
7MedGuo-5′-P 7MedGMP d7MeGuo-5′-P
7-Methyldeoxyguanosine 5′-phosphate
pm7 dG -m7 dG
H2N (HO)2(O)PO
N
N O
OH H O N
N
239
7MedGuo-5′-P3 7MedGTP
7-Methyldeoxyguanosine 5′-triphosphate
pppm dG
7
H2N O O O HO P O P O P O OH OH OH
N
N O
Purines, Pyrimidines, Nucleosides, and Nucleotides
9168_Book.indb 351
PURINES, PYRIMIDINES, NUCLEOSIDES AND NUCLEOTIDES: PHYSICAL CONSTANTS AND SPECTRAL PROPERTIES (Continued)
OH H
No.
Acidic Spectral Data pH
λmax
εmax (×10−3)
λmin
237 238
– –
– –
– –
– –
239
2
257
10.6
230
Neutral Spectral Data Spectral Ratios
230 – –
240 – –
250 – –
270 – –
280 – –
290 – –
pH
λmax
7 7oq
– 256 283 258 280
7.4q
εmax (×10−3) – 9.8 7.8 8.9 7.25
λmin – –
Alkaline Spectral Data Spectral Ratios
230 – –
240 – –
250 1.14 –
236 271
270 – –
280 0.66 –
290 – –
pH
λmax
λmin
– –
εmax (×10−3) – –
– – 12o
281
7.9
243
– –
Spectral Ratios 230 – –
240 – –
250 – –
270 – –
280 – –
290 – –
References No.
Origin and Synthesis
237 238 239
E: 209 C: 214 C: 46 C: 334
[α]tD
pK
– – –
– – 334
Spectral Data 209 46 334
Mass Spectra
Rf
– – –
– 46 334
351
4/16/10 1:20 PM
9168_Book.indb 352
No.
Compound
Symbol
3-Letter
1-Letter
Structure
O
352
PURINES, PYRIMIDINES, NUCLEOSIDES AND NUCLEOTIDES: PHYSICAL CONSTANTS AND SPECTRAL PROPERTIES (Continued) pK
Formula (Mol Wt)
Melting Point °C
[α]Dt
Basic
Acidic
C9H13N2O8P (308.18)
–
–
–
–
C8H13N2O8P (308.18)
–
–
–
–
–
–
–
–
HN 240
HO
dUp dU-
dUrd-3′-P 3′-dUMP
Deoxyuridine 3′-phosphate
N
O O
O HO P O
H
OH O HN 241
pdU -dU
dUrd-5′P dUMP
Deoxyuridine 5′-phosphate
O HO P O OH
N
O O
OH H O HN
242
dUrd-5′-P3 dUTP
Deoxyuridine 5′-triphosphate
pppdU
O O O HO P O P O P O OH OH OH
N
O O
C9H15N2O14P3 (468.15)
No.
Acidic Spectral Data pH
240 241 242
λmax
– 2 1
– 260 262
εmax (×10−3)
λmin
– 9.8 –
– 231 –
Neutral Spectral Data Spectral Ratios
230 – – –
240 – – –
250 – – 0.72
270 – – –
280 – – 0.45
290 – – –
pH
λmax
– 7 –
– 260 –
εmax (×10−3) – – –
λmin – 230 –
Alkaline Spectral Data Spectral Ratios
230 – – –
240 – – –
250 – – –
270 – – –
280 – – –
290 – – –
pH
λmax
– 12 –
– 261 –
εmax (×10−3) – 7.6n –
λmin – 241 –
Spectral Ratios 230 – – –
240 – – –
250 – – –
References No.
Origin and Synthesis
240 241 242
– E: 229 C: 289
[α] D
pK
– – –
– – –
t
Spectral Data – 230b 289
Mass Spectra
Rf
– – –
– 74 –
270 – – –
280 – – –
290 – – –
Handbook of Biochemistry and Molecular Biology
OH H
4/16/10 1:20 PM
No.
Compound
Symbol
3-Letter
1-Letter
Structure
Formula (Mol Wt)
O
Basic
pK
Acidic
Melting Point °C
[α]Dt
C10H15N2O8P (322.21)
178°q (dibrucine salt)
+7.320 (1.5, H2O)
–
–
C10H15N2O8P (322.21)
175° (dibrucine salt)
−4.421 (0.4, H2O)
–
10.0
C10H17N2O14P3 (482.18)
–
–
–
–
HN 243
HO
dTp dT-
dThd-3′-P 3′dTMP
Thymidine 3′-phosphate
N
O O
O HO P O
H
OH O HN
244
pdT -dT
dThd-5′-P dTMP
Thymidine 5′-phosphate
O HO P O OH
N
O O
OH H O
Purines, Pyrimidines, Nucleosides, and Nucleotides
9168_Book.indb 353
PURINES, PYRIMIDINES, NUCLEOSIDES AND NUCLEOTIDES: PHYSICAL CONSTANTS AND SPECTRAL PROPERTIES (Continued)
HN
245
dThd-5′-P3 dTTP
Thymidine 5′-triphosphate
pppdT
O O O HO P O P O P O OH OH OH
N
O O
OH H
No.
Acidic Spectral Data pH
243 244 245
2 2 2
λmax – 267 –
εmax (×10−3)
λmin
– 102 –
– – –
Neutral Spectral Data Spectral Ratios
230 – – –
240 – – –
250 – 0.64 0.64
270 – – –
280 0.69 0.72 0.72
290 – 0.23 –
pH
λmax
7 7 –
267 267 –
εmax (×10−3) 9.5 10.2 –
λmin – – –
Alkaline Spectral Data Spectral Ratios
230 – – –
240 – 0.34 –
250 0.65 0.65 –
270 1.08 1.10 –
280 0.71 0.73 –
290 – 0.24 –
pH
λmax
– 12 –
– – –
εmax (×10−3) – – –
λmin – – –
Spectral Ratios 230 – – –
240 – – –
250 – 0.74 –
270 – – –
280 – 0.67 –
290 – 0.17 –
References No.
Origin and Synthesis
[α] D
pK
Spectral Data
243 244 245
C: 262,202,187a,203 D: 263 C: 187,202 D: 200 E: 209 C: 209
187 207 –
– 180 –
262,187e,263e 185b,186e,179e,223 209
t
Mass Spectra
Rf
– – –
187,262,263 187,185,263 –
353
4/16/10 1:20 PM
9168_Book.indb 354
No.
246
Compound
Symbol
3-Letter
5HmdUrd-5′P 5HmdUMP 5(HOMe) dUMP
5-Hydroxymethyldeoxyuridine 5′-phosphate
1-Letter pom5 dU -om5 dU phm5 dU*
Structure
Formula (Mol Wt)
O
C10H15N2O9P (338.21)
N
O
Basic
[α]
pK
Acidic
OH
HN O HO P O OH
Melting Point °C
t D
354
PURINES, PYRIMIDINES, NUCLEOSIDES AND NUCLEOTIDES: PHYSICAL CONSTANTS AND SPECTRAL PROPERTIES (Continued)
O
–
–
–
–
OH H
No.
Acidic Spectral Data pH
246
2
λmax 264
εmax (×10−3)
λmin
10.2
234
Neutral Spectral Data Spectral Ratios
230 0.32
240 0.37
250 0.69
270 0.97
280 0.56
290 0.11
pH
λmax
–
–
εmax (×10−3) –
λmin –
Alkaline Spectral Data Spectral Ratios
230 –
240 –
250 –
270 –
280 –
290 –
pH
λmax
12
264
εmax (×10−3) –
λmin 244*
Spectral Ratios 230 1.15
240 0.75
250 0.80
270 0.95
280 0.48
290 0.09
References No.
Origin and Synthesis
246
C: 74 D: 96,171,349
[α]tD
pK
–
–
Spectral Data 74,171* ,96 d
Mass Spectra
Rf
–
74
b
4/16/10 1:20 PM
The authors are indebted to a number of authors who supplied unpublished data, provided original spectra for calculations of the values or gave advice on the selection of the most reliable data. They also wish particularly to thank Mr. I. H. Flack, Mr. R. Thedford and Miss L. Csonka for their assistance in the preparation of the table.
Handbook of Biochemistry and Molecular Biology
Melting point from this reference. Full spectrum given. c λ max and/or λmin from this reference. d ε max from this reference. e Spectral ratios from this reference. f pK of 2-methyl-6-methylaminoadenine. Compare the similar pK of adenine and N6-methyladenine.15 g Spectral data taken on material synthesized this way then further purified by paper chromatography. h In 50% dimethylformamide (HCONMe ). 2 i In 50% dimethylsulfoxide/ethanol (Me SO/EtOH). 2 j For an explanation of this nomenclature and abbreviations, see General Remarks on Wyosine in Natural Occurrence of Modified Nucleosides. k pK of 6-amino-5-formamidoisocytosine. l pK of nucleotide. m λ max and εmax due to adenosine. n ε calculated from spectral data using ε max acid of nucleoside. o Decomposes at this pH. p Determined from electrophoretic mobility. q Values very dependent on pH (near pK). r Basic ionization at all pH values. s Spectral data in water and pH 11 indicate decomposition. t Alkaline degradation product of 7-methylguanosine or nucleotide. v Spectra in acid and neutral similar. w ε estimated from conversion to N6-methyladenosine 3′ (2′)- or 5′-phosphate using ε of 15.2 × 103 in alkali, for the N6-isomers. x Based on ε of 7-methylguanosine 3′(2′)-phosphate assuming quantitative conversion in alkali. For a possible error in this value see General Remarks on 7-Methylguanosine in Natural Occurrence of Modified Nucleosides. y Cyclohexylamine salt. z Data on mixed 2′ and 3′ phosphates. a
Purines, Pyrimidines, Nucleosides, and Nucleotides
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Chemical Modification of Nucleic Acids The chemical modification of nucleic acids is not as complex as that of proteins since there are fewer monomer units and, for all practical purposes, only nitrogen as a nucleophilic reactive group; the nitrogen is reactive as a primary and secondary amine. Reaction at the primary amine groups
of, for example, adenine, is referred to an exocyclic modification whereas reaction at the imine nitrogens of pyrimidines and purine rings is referred to as an endocyclic modification. There are also ring-opening reactions and cross-linking reactions.
TABLE 1: Chemical Modification of Nucleic Acids Reagent
Base Modified
Product
Reference
Aldehydes
Most data on pyrimidines with less on purines Various adducts
1-10
Alkylation
Purine and pyrimidines
11-20
Diethylpyrocarbonate
Purine and pyrimidines
a
Dimethyl Sulfate (Methylation) Purines and Pyrimidines
Hydrazine
Pyrimidines
Hydroxylamine
Purines and pyrimidines
Nitrous Acid (HNO2)
Purines and pyrimidines
Potassium Permanganate
Purines and Pyrmidines
Sodium Bisulfate
Cytosine
Various product resulting from environmental agents such as ethylene oxides and nitrogen mustards Carboxethylation of the N-7 site on the purine ring followed by ring opening; pyrimidines react at primary amino groups to yield carboethoxy derivativesb N 7-Methylguanidine with minor reaction at the N1 and N3-positions; reaction also occurs at N3 in adenine and at N3-position in cytidine; reactivity is controlled by polynucleotide structurec,d Ring cleavage yielding pyrazole derivativese and the ribosyl backbone ; reaction used sequence analaysis and footprinting Hydroxamate formation at “exocyclic” nitrogen; conversion of guanine to isoxazolone. Deamination; crosslinking at guanine, cytosine bases Oxidation at double bonds; preferential reaction with thymidine Uracil (5-methylcytosine is converted to thymine)f
21-28
29-41
42-47
48-55 56-64 65-75 76-84
e.g. formaldehyde, acetaldehyde, acrolein, crotonaldehyde; 4-hydroxy-2-nonenal (4-HNE) Diethylpyrocarbonate modification is used for DNA footprinting (Fox, K.R., Webster, R., Phelps, R.J., et al., Sequence selective binding of bis-daunorubicin WP631 to DNA, Eur.J.Biochem. 271, 3556-3566, 2004) c The N7-position of guanine is always reactive for methylation (the N7-position of guanine is the most reactive site in nucleic acids); methylation of guanine also occurs at the N1-position of guanine at high concentrations of methylating agents. Methylation of the N3-position of cytidine occurs in single-stranded DNA but is blocked in double-stranded DNA. Methylation (alkylation) also occurs at the N1 and N3 position of adenine but methylation is restricted in at the N1 position in double-stranded DNA. In vivo methylation of DNA occurs at cytidine residues largely in CpG islands (Ehrlich, M. and Wang, R.Y., 5-Methylcytosine in eukaryotic DNA, Science 212, 1350-1357, 1981; Lewis, J. and Bird, A., DNA methylation and chromatin structure, FEBS Lett. 285, 155-159, 1991; Cheng, X., Structure and function of DNA methyltransferases, Annu.Rev.Biophys.Biomol.Struct. 24, 293-318, 1995; Scheule, R.K., The role of CpG motifs in immunostimulation and gene therapy, Adv.Drug Deliv.Rev. 44, 119-134, 2000). d Base treatment of N7-methylguanine results in opening of the imidazolium ring while N3-methylcytidine undergoes base-catalyzed deamination to give N3-methyluridine. Methylation of cytidine blocks conversion to thymidine by sodium bisulfite e 4-methyl-5-pyrazolone with thymidylic acid; 3(5)-aminopyrazole with deoxycytidine f Conversion to uracil does not occur with 5-methylcytosine; 5-methylcytosine is converted to thymine. The rate of reaction of 5-methylcytosine with sodium bisulfite is much slower than reaction of cytosine. a
b
References for Table 1 1. Alegria, A.H., Hydroxymethylation of pyrimidine mononucleotides with formaldehyde, Biochim.Biophys.Acta 149, 317-324, 1967 2. Feldman, M.Y., Reactions of nucleic acids and nucleoproteins with formaldehyde, Prog.Nucleic Acid Res.Mol.Biol. 13, 1-49, 1973 3. McGhee, J.D. and von Hippel, P.H., Formaldehyde as a probe of DNA structure. I. Reaction with exocyclic amine groups of DNA bases, Biochemistry 25, 1281-1296, 1975
4. McGhee, J.D. and von Hippel, P.H., Formaldehyde as a probe of DNA structure. II. Reaction with endocyclic imine groups of DNA bases, Biochemistry 25, 1297-1303, 1975 5. Yamazaki, Y. and Suzuki, H., A new method of chemical modification of N6-amino group in adenine nucleotides with formaldehyde and a thiol and its application to preparing immobilized ADP and ATP, Eur.J.Biochem. 92, 197-207, 1978 6. Chung, F.L., Young, R., and Hecht, S.S., Formation of cyclic 1, N2-propanodeoxyguanosine adducts in DNA upon reaction with acrolein or crotonaldehyde, Cancer Res. 44, 990-995, 1984
359
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360 7. Winter, C.K., Segall, H.J., and Haddon, W.F., Formation of cyclic adducts of deoxyguanosine with the aldehydes trans-4-hydroxy-2hexenal and trans-4-hydroxynonenal in vitro, Cancer Res. 46, 56825686, 1986 8. Kennedy, G., Slaich, P.K., Golding, B.T., and Watson, W.P., Structure and mechanism of formation of a new adduct from formaldehyde and guanosine, Chem.Biol.Interact. 102, 93-100, 1996 9. Hecht, S.S., McIntee, E.J., and Wang, M., New DNA adducts of crotonaldehyde and acetaldehyde, Toxicology 166, 31-36, 2001 10. Kurtz, A.J. 1, N2-deoxyguanosine adducts of acrolein, crotonaldehyde, and trans-4-hydroxynonenal cross-link to peptides via Schiff base linkage, J.Biol.Chem. 278, 5970-5976, 2003 11. Singer, B., The chemical effects of nucleic acid alkylation and their relation to mutagenesis and carcinogenesis, Prog.Nucleic Res.Mol. Biol. 15, 219-284, 1975 12. Lawley, P.D., DNA as a target of alkylating carcinogenesis, Br.Med. Bull. 36, 19-24, 1980 13. Coles, B., Effects of modifying structure on electrophilic reactions with biological nucleophiles, Drug Metab.Rev. 15, 1307-1334, 1984-1985 14. Wild, C.P., Antibodies to DNA alklylation adducts as analytical tools its chemical carcinogenesis, Mutat.Res. 233, 219-233, 1990 15. Lawley, P.D., Alkylation of DNA and its aftermath, Bioessays 17, 561-568, 1995 16. Bolt, H.M., Quantification of endogenous carcinogens. The ethylene oxide paradox, Biochem.Pharmacol. 52, 1-5, 1996 17. Rios-Blanco, M.N., Plna, K., Faller, T., et al., Propylene oxide: mutatgenesis, carcinogenesis and molecular dose, Mutat.Res. 380, 179-197, 1997 18. Wilson, D.S. and Szostak, J.W., In vitro selection of functional nucleic acids, Annu.Rev.Biochem. 68, 611-647, 1999 19. Denny, W.A., DNA minor groove alkylating agents, Curr.Med.Chem. 8, 533-544, 2001 20. Mishina, Y. and He, C., Oxidative dealkylation DNA repair mediated by the mononuclear non-heme iron AlkB proteins, J.Inorg.Biochem. 100, 670-678, 2006 21. Leonard, N.J., McDonald, J.J., Henderson, R.E.I., and Reichmann, M.E., Reaction of diethyl pyrocarbonate with nucleic acid components. Adenosine. Biochemistry 10, 335-3342, 1971 22. Solymosy, F., Hüvös, P., Gulyás, A., et al., Diethyl pyrocarbonate, a new tool in the chemical modification of nucleic acids, Biochim. Biophys.Acta 238, 406-426, 1971 23. Vincze, A., Henderson, R.E.I., McDonald, J.J., and Leonard, N.J., Reaction of diethyl pyrocarbonate with nucleic acid components. Bases and nucleosides derived from guanine, cytosine, and uracil, J.Amer.Chem.Soc. 95, 2677-2683, 1973 24. Ehrenfeld, E., Interaction of Diethylpyrocarbonate with poliovirus double-stranded RNA, Biochem.Biophys.Res.Commun. 56, 214-219, 1974 25. Herr, W., Diethyl pyrocarbonate: A chemical probe for secondary structure in negatively supercoiled DNA, Proc.Nat.Acad.Sci.USA 82, 8009-8013, 1985 26. Johnston, B.H. and Rich, A., Chemical probes of DNA conformation: Detection of Z-DNA at nucleotide resolution, Cell 42, 713-724, 1985 27. Runkel, L. and Nordheim, A., Chemical footprinting of the interaction between left-handed Z-DNA and anti-Z-DNA antibodies by Diethylpyrocarbonate carboethoxylation, J.Mol.Biol. 189, 487-501, 1986 28. Buckle, M. and Buc, H., Fine mapping of DNA single-stranded regions using base-specific chemical probes: Study of an open complex formed between RNA polymerase and the lac UV5 promoter, Biochemistry 28, 4388-4396, 1989 29. Jordan, D.O., The physical properties of nucleic acids, in The Nucleic Acids. Chemistry and Biology, Vol. 1, ed. E. Chargaff and J.N. Davidson, Academic Press, New York, NY, USA, Chapter 13, pps. 447-492, 1955 30. Kanduc, D., tRNA chemical modification In vitro and in vivo formation of 1,7-dimethylguanosine at high concentrations of methylating agents, Biochem.Biophys.Acta 653, 9-17, 1981 31. Singer, B., The chemical effects of nucleic acid alkylation and their relation to mutagenesis and carcinogenesis, Prog.Nucl.Acids Res.Mol. Biol. 15, 219-284, 1975
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Handbook of Biochemistry and Molecular Biology 32. Behmoaras, T., Toulme, J.-J., and Relene, C., Specific recognition of apurinic sites in DNA by a tryptophan-containing peptide, Proc.Nat. Acad.Sci.USA 78, 926-930, 1981 33. Mhaskar, D.N., Chang, M.J.W., Hart, R.W., and D’Ambrosio, S.M., Analysis of alkylated sites at N-3 and N-7 positions of purines as an indicator for chemical carcinogens, Cancer Res. 41, 223-229, 1981 34. Kirkegaard, K., Buc, H., Spassky, A., and Wang, J.C., Mapping of single-stranded regions in duplex DNA at the sequence level: Singlestrand-specific cytosine Methylation in RNA polymerase-promoter complexes, Proc.Nat.Acad.Sci.USA 80, 2544-2548, 1983 35. Potaman, V.N. and Sinden, R., Stabilization of triple-helical nucleic acids by basic oligopeptides, Biochemistry 34, 14885-14892, 1995 36. Lawley, P.D., Effects of some chemical mitogens and carcinogens on nucleic acids, Prog.Nucl.Acid Res.Mol.Biol. 5, 89-131, 1996 37. Dobner, T., Buchner, D., Zeller, T., et al., Specific nucleoprotein complexes within adenovirus capsids, Biol.Chem. 382, 1373-1377, 2001 38. Hock, T.D., Nick, H.S., and Agarwal, A. Upstream stimulatory factors, USF1 and USF2, bind to the human haem oxygenase-1 proximal promoter in vivo and regulates its transcription, Biochem.J. 383, 209-218, 2004 39. Lagor, W.R., de Groh, E.D., and Ness, G.C., Diabetes alters the occupancy of the hepatic 5-hydroxy-3-methylglutaryl-CoA reductase promoter, J.Biol.Chem. 280, 36601-36608, 2005 40. Haugen, S.P., Berkmen, M.B., Ross, W., et al., tRNA promoter regulates by nonoptimal binding of sigma region 1.2: an additional recognition element for RNA polymerase, Cell 125, 1069-1082, 2006 41. Temperli, A., Türler, H., Rüst, P. et al., Studies on the nucleotide arrangement in deoxyribonucleic acids. IX. Selective degradation of pyrimidines deoxyribonucleotides, Biochim.Biophys.Acta 91, 462-476, 1964 42. Cashmore, A.R. and Peterson, G.B., The degradation of DNA by hydrazine: a critical study of the suitability of the reaction for the quantitative determination of purine nucleotide sequences, Biochim. Biophys.Acta 174, 591-603, 1969 43. Türler, H. and Chargaff, E., Studies on the nucleotide arrangement in deoxyribonucleic acids. XII. Apyrimidinic acid from calf-thymus deoxyribonucleic acid: preparation and properties, Biochim.Biophys. Acta 195, 446-455, 1969 44. Maxam, A.M. and Gilbert, W., A new method for sequencing DNA, Proc.Nat.Acad.Sci.USA 74, 560-564, 1977 45. Cashmore, A.R. and Petersen, G.B., The degradation of DNA by hydrazine: identification of 3-ureidopyrazole as a product of the hydrazinolysis of deoxycytidylic acid residues, Nucleic Acids Res. 5, 2485-2891, 1978 46. Peattie, D.A., Direct chemical method for sequencing RNA, Proc.Nat. Acad.Sci.USA 76, 1760-1764, 1979 47. Tolson, D.A. and Nicholson, N.H., Sequencing RNA by a combination of exonuclease digestion and uridine-specific chemical cleavage using MALDI-TOF, Nucleic Acids Res. 26, 446-451, 1998 48. Small, G.D. and Gordon, M.P., Reaction of hydroxylamine and methoxyamine with the ultraviolet-induced hydrate of cytidine, J.Mol. Biol. 14, 281-291, 1968 49. Brown, D.M. and Osborne, M.R., The reaction of adenosine with hydroxylamine, Biochim.Biophys.Acta 247, 514-518, 1971 50. Fraenkel-Conrat, H., and Singer, B., The chemical basis for the mutagenicity of hydroxylamine and methoxyamine, Biochim.Biophys.Acta 262, 264-268, 1972 51. Iida, S., Chung, K.C., and Hayatsu, H., The reaction of hydroxylamine with 4-thiouridine, Biochim.Biophys.Acta 308, 198-204, 1973 52. Kasai, H. and Nishimura, S., Hydroxylation of deoxyguanosine at the C-8 position by ascorbic acid and other reducing agents, Nucleic Acids Res. 12, 2137-2145, 1984 53. Johnston, B.H., Hydroxylamine and methoxyamine as probes of DNA structure, Methods Enzymol. 212, 180-194, 1992 54. Simandan, T., Sun, J., and Dix, T.A., Oxidation of DNA bases, deoxyribonucleosides and homopolymers by peroxyl radicals, Biochem.J. 335, 233-240, 1998. 55. Tessman, I., Poddar, R.K., and Kumar, S., Identification of the altered bases in mutated single-stranded DNA. I. In vitro mutagenesis by hydroxylamine, ethyl methanesulfonate and nitrous acid, J.Mol.Biol. 93, 352-363, 1964
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Chemical Modification of Nucleic Acids 56. Stuy, J.H., Inactivation of transforming deoxyribonucleic acid by nitrous acid, Biochem.Biophys.Res.Commun. 6, 328-333, 1961 57. Horn, E.E. and Herriott, R.M., The mutagenic action of nitrous acid on “single-stranded” (denatured) Hemophilus transforming DNA, Proc.Nat.Acad.Sci.USA 48, 1409-1416, 1962 58. Kotaka, T. and Baldwin, R.L., Effects of nitrous acid on the DAT copolymer as a template for DNA polymerase, J.Mol.Biol. 93, 323-329, 1964 59. Carbon, J. and Curry, J.B., A change in the specificity of transfer RNA after partial deamination with nitrous acid, Proc.Nat.Acad.Sci.USA 59, 467-474, 1968 60. Shapiro, R. and Yamaguchi, H., Nucleic acid reactivity and conformation I. Deamination of cytosine by nitrous acid, Biochim.Biophys. Acta. 281, 501-506, 1972 61. Verly, W.G. and Lacroix, M. DNA and nitrous acid, Biochim.Biophys. Acta 414, 185-192, 1975 62. Dubelman, S. and Shapiro, R, A method for the isolation of crosslinked nucleosides from DNA: application to cross-links induced by nitrous acid, Nucleic Acids Res. 4, 1815-1827, 1977 63. Shapiro, R. Dubelman, S., Feinberg, A.M., et al., Isolation and identification of cross-linked nucleosides from nitrous acid treated deoxyribonucleic acid, J.Amer.Chem.Soc. 99, 302-303, 1977 64. Edfeldt, N.B., Harwood, E.A., Sigurdsson, S.T. et al., Solution structure of a nitrous acid induced DNA interstrand cross-link, Nuc.Acids Res. 32, 2785-2794, 2004 65. Darby, C.K., Jones, A.S., Tittensor, J.R., and Walker, R.T., Chemical degradation of DNA oxidized by permanganate, Nature 216, 793-794, 1967 66. Hayatsu, H. and Ukita, T., The selective degradation of pyrimidines in nucleic acids by permanganate oxidation, Biochem.Biophys.Res. Commun. 29, 556-561. 1967 67. Rubin, C.M. and Schmid, C.W., Pyrimidine-specific chemical reactions useful for DNA sequencing, Nucleic Acids Res. 8, 4613-4619, 1980 68. Fritzsche, E., Hayatsu, H., Igloi, G.L., et al., The use of permanganate as a sequencing reagent for identification of 5-methylcytosine residues in DNA, Nucleic Acisd Res. 15, 5517-5528, 1987 69. Sasse-Dwight, S. and Gralla, J.D., KMnO4 as a probe for lac promoter DNA melting and mechanism in vivo, J.Biol.Chem. 264, 8074-8081, 1989 70. Klysik, J., Rippe, K., and Jovin, T.M., Reactivity of parallel-stranded DNA to chemical modification reagents, Biochemistry 29, 9831-9839, 1990 71. Jiang, H., Zacharias, W., and Amirhaeri, S., Potassium permanganate as an in situ probe for B-Z and Z-Z junctions, Nucleic Acids Res. 19, 6943-6948, 1991 72. Nawamura, T., Negishi, K., and Hayatsu, H., 8-Hydroxyguanine is not produced by permanganate oxidation of DNA, Arch.Biochem. Biophys. 311, 523-524, 1994 73. Bailly, C. and Waring, M.J., Comparison of different footprinting methodologies for detecting binding sites for a small ligand on DNA, J.Biomol.Struct.Dyn. 12, 869-898, 1995 74. Kahl, B.F. and Paule, M.R., The use of Diethylpyrocarbonate and potassium permanganate as probes for strand separation and structural distortions in DNA, Methods Mol.Biol. 148, 63-75, 2001 75. Spicuglia, S., Kumar, S., Chasson, L., Potassium permanganate as a probe to map DNA-protein interactions in vivo, J.Biochem.Biophys. Methods 59, 189-194, 2004 76. Hayatsu, H., Wataya, Y., Kai, K., and Iida, S., Reaction of sodium bisulfite with uracil, cytosine, and their derivatives, Biochemistry 9, 2858-2865, 1970 77. Shapiro, R., Braverma, B., Louis, J.B., and Servis, R.E., Nucleic-acid reactivity and conformation 2. Reaction of cytosine and uracil with sodium bisulfite, J.Biol.Chem. 248, 4060-4064, 1973
9168_Book.indb 361
361 78. Wang, R.Y.-H., Gehrke, C.W., and Ehrlich, M., Comparison of bisulfite of 5-methyldeoxycytidine and deoxycytidine residues, Nucleic Acids Res. 8, 4777-4790, 1980 79. Frommer, M., McDonald, L.E, Millar, D.S., et al., A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands, Proc.Nat.Acad.Sci.USA 89, 1827-1831, 1992 80. Chen, H. and Shaw, B.R., Kinetics of bisulfite-induced cytosine deamination in single-stranded DNA, Biochemistry 32, 3535-3539, 1993 81. Herman, J.G., Graff, J.R., Myöhänen, S., et al., Methylation-specific PCR: A novel PCR assay for methylation status of CpG islands, Proc. Nat.Acad.Sci.USA 93, 9821-9826, 1996 82. Hong, K.-M., Yang, S.-H., Guo, M., et al., Semiautomatic detection of DNA methylation at CpG islands, BioTechniques 38, 354-358, 2005 83. Ordway, J.M., Bedell, J.A, Citek, R.W. et al., MethylMapper: a method for high-throughput, multilocus bisulfite sequence analysis and reporting, BioTechniques 39, 464-470, 2005 84. Zhou, D., Qiao, W., Yang, L., and Lu, Z., Bisulfite-modified target DNA array for aberrant methylation analysis, Anal.Biochem. 351, 26-35, 2006
There are modifications of ribose moiety in ribonucleotides such as periodate oxidation which was used in early structural analysis. Oxidation of the ribose ring has also been used to couple RNA to protein amino groups and to amino-containing matrices for affinity chromatography. TABLE 2: Reaction of Periodate with Nucleic Acids Reaction
Conditions
Coupling of periodate-oxidized RNA to hydrazide-agarose
140 µL 0.2 M NaIO4 added to 500 – 3000 µg RNA in a volume of 1 mL 0.1 M sodium acetate, pH 5.0 for one hours at 23oC in the dark; reaction terminated with 80 µL ethylene glycol; after removal of reactants, the modified RNA was coupled to hydrazide matrix in the same solvent 12 µL 0.1 M NaIO4 (sodium meta-periodate) /10 A260 units of RNA(40 A260 units/mL) in 0.1 M sodium acetate, pH 5.0, and incubated for 1 hr at 23oC. The reaction is terminated by precipitation of the RNA with ethanol. Coupling to agarose was accomplished in 0.1 M sodium acetate, pH 5.0
Coupling of double-stranded RNA to agarose
Reference 1
2
References for Table 2 1. Robberson, D.L. and Davidson, N., Covalent coupling of ribonucleic acid to agarose, Biochemistry 11, 533-537. 1972 2. Langland, J.O., Pettiford, S.M., and Jacobs, B.L., Nucleic acid affinity chromatography: Preparation and characterization of doublestranded RNA agarose, Protein Exp.Purif. 6, 25-32, 1995
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Handbook of Biochemistry and Molecular Biology
362 H
NH2 N
N
N
H
HN
O Formaldehyde
N
OH
N
N
N
R
H2 C
N
R 6-(hydroxymethylamino)purine
Adenine
R N N N HN N
N
N H
N
N N
R Crosslinking with heterogeneous products O O R= H
H
H
O
H OH
Figure 1 The reaction of formaldehyde with adenine in ribonucleic acid. (Adapted from Nucleic Acids in Chemistry and Biology, ed. G.M. Blackburn and M.J. Galt, Oxford University Press, Oxford, UK, 1996.)
9168_Book.indb 362
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Chemical Modification of Nucleic Acids
363 Cl
Cl N H3C
N HO
Cl
Cl
O
Chlormethine
Chlorambucil OH
Cl
S
S
Cl
Cl bis(2-chloroethyl) sulfide Mustard Gas; Sulfur Mustard
2-chloroethyl-2-hydroxyethyl sulfide Hemisulfur Mustard
HO O
O
H3C
S N
+
N O
N
NH
N
N
NH2
NH
+
N
NH2
O O
H
O
H
H
O
H
H
Deoxyguanyl; Monosubstitution product with bis(2-chloroethyl) sulfide or product with hemisulfur mustard
H
H
O
H
H
H
Reaction product of deoxyguanyl residue with methyl iodide
Figure 2 Some alkylating agents for the modification of DNA. Nitrogen mustards are described as a group of bis(2-chloroalkylamines). The original mustards were chloroalkyl disulfides. Also shown is the methylation of purine with methyl iodide. The N7-position on the guanine ring is the most susceptible site for alkylation. See Nucleic Acids in Chemistry and Biology, ed. G.M. Blackburn and M.J. Galt, Oxford University Press, Oxford, UK, 1996; Denny, W.A., DNA minor groove alkylating agents, Curr.Med. Chem. 8, 533-544, 2001.
9168_Book.indb 363
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Handbook of Biochemistry and Molecular Biology
364
CO2Et HN
NH2 EtO2C
N
H N
N
N
N
HN
N
N
O
O
O
O
H
H
H
O
OH
H
H
H
H
O
OH
H
Adenosyl Diethylpyrocarbonate O N
N O
O EtO2C
NH
N
H N
NH2
HN
NH
N
NH2
O O H H
O
H
O
OH Guanyl
H
H
H
H
O
OH
H
Figure 3 The reaction of diethylpyrocarbonate with adenyl and guanyl residues in ribonucleic acid.
9168_Book.indb 364
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Chemical Modification of Nucleic Acids
365 O
O
NH2
S
Me N
N
Me O
NH2
O
Me N
Dimethyl Sulfate
+
N
O
O
R
R Cytidine O
O O N
NH
Me
S
Me
O
O
Me
O N
N
NH
Dimethyl Sulfate N
NH2 N
R Guanidine
N
NH2
R
O O R=
H
H
O
H
H
H
Figure 4 The modification of purines and pyrimidines with dimethyl sulfate.
9168_Book.indb 365
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Handbook of Biochemistry and Molecular Biology
366
H2N O N N
NH2
O O NH2
H NH2
H
O
H
H
H2N
N
N
H
O
Hydrazine H2N
O O
O H H
O
N
H H
N
H
NH2
O
Deoxycytidine
O
H
H
H
O
H
H
Further Degradation
Figure 5 The degradation of cytidine with hydrazine. (Adapted from Cashmore, A.R. and Petersen, G.B., The degradation of DNA by hydrazine: identification of 3-ureidopyrazole as a product of the hydrazinolysis of deoxycytidylic acid residues, Nucleic Acids Res. 5, 2485-2491, 1978.)
9168_Book.indb 366
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Chemical Modification of Nucleic Acids
367 OH HN
N
O
N O NH2
O H N
H OH
N
O
H H
O
H2N
OH N
Hydroxylamine
O O H H
NH
H
O H Deoxycytidine
H
N
O
O O H
H
H
H O H N4-hydroxy-2'-deoxycytidine
Figure 6 The reaction of hydroxylamine with cytosine. (Adapted from Blackburn, G.M., Jarvis, S., Ryder, M.C., et al., Kinetics and mechanism of reaction of hydroxylamine with cytosine and its derivatives, J.Chem.Soc.Perkins Trans 1, 370-375, 1975 and Nucleic Acids in Chemistry and Biology, ed. G.M. Blackburn and M.J. Galt, Oxford University Press, Oxford, UK, 1996.)
9168_Book.indb 367
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Handbook of Biochemistry and Molecular Biology
368 NH2 N
O N
N
N H
NH
HNO2
N H
N
N
Hypoxanthine
Adenine
O
O N
N H
N
NH
NH
HNO2
N H
NH2
N Guanine NH2
N H Xanthine
O
O N
N H Cytosine
NH
HNO2
O
N H Uracil
O
Figure 7 The reaction of purines and pyrimidines with nitrous acid resulting in deamination. The riboside derivative of hypoxanthine is inosine.
9168_Book.indb 368
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Chemical Modification of Nucleic Acids
369
O
O NH
O
KMnO4
N
O
NH
Mn
O
O
HO
N
R Thymidine
R Aqueous base
O
O
O
O R= H
H
H
O
H
HN
H
Me O
H2N
CHO
N R
O
O
O
NH +
Me
R OH + CO2
Figure 8 The oxidation of thymidine with potassium permanganate. Adapted from Bui, C.T., Rees, K., and Cotton, R.G.H., Permanganate oxidation reactions of DNA: Perspective in biological studies, Nucleosides, Nucleotides, and Nucleic Acids 22, 18351855, 2003.
NH2
NH2 NaHSO3 pH 6.0
N
N H Cytosine
O
pH 9.0
O N
–O
3S
N H
NH
pH 5.6 O
–
O3S
O
N H
NaHSO3 pH 6.0
pH 9.0
O
NH
N H
O
Uracil
Figure 9 The modification of cytosine with sodum bisulfite results in the formation of uracil.
9168_Book.indb 369
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Handbook of Biochemistry and Molecular Biology
370
HN
Base
O O HO
RNA =
Agarose
H CNBr H2 C H 2N
H2 C C H2
H2 C
H
H
OH
OH
periodate
O
C H2
H
CH3
HN
O 6-aminohexanoic acid methyl ester
Base
O O RNA = H
NH H2 C
Agarose O
N H
H2 C C H2
H2 C
CHO
CHO
H
O
C H2
CH3 O
Hydrazine hydrate H2NNH2 . H2O
NH H2 C
Agarose N H
O
H2 C C H2
H2 C
H N
C H2
NH2 O
NH
O H2 C
Agarose O
N H
H2 C C H2
H2 C
N
C H2
N
RNA C H2
O
Figure 10 The covalent coupling of RNA to an agarose matrix. The RNA is oxidized with periodate and then coupled to an alkyl hydrazide derivative of agarose.
General references for the chemical modification of nucleic acids Jordan, D.O., The reaction of nucleic acids with mustard gas, Biochem.J. 42, 308-316, 1948 Jordan, D.O., Nucleic acids, purines, and pyrimidines, Annu.Rev.Biochem. 21, 209-244, 1952 Jordan, D.O., The physical properties of nucleic acids, in The Nucleic Acids. Chemistry and Biology, Vol. 1., ed. E.Chargaff and J.N. Davidson, Academic Press, New York, New York, Chapter 13, pps 447-492, 1955. Lawley, P.D., Effects of some chemical mutagens and carcinogens on nucleic acids, Prog.Nucl.Acid.Res.Mol.Biol. 5, 89-131, 1966 Lawley, P.D., Effects of some chemical mutagens and carcinogens on nucleic acids, Prog.Nucleic Acid Res.Mol.Biol. 5, 89-131, 1966 Singer, B. and Fraenkel-Conrat, H., The role of conformation in chemical modification, Prog.Nucl.Acid Res.Mol.Biol. 9, 1-29. 1969
9168_Book.indb 370
Kochetkov, N.K. and Budowsky, E.T., The chemical modification of nucleic acids, Prog.Nucl.Acid Res.Mol.Biol. 9, 403-438, 1969 Steinschneider, A., Effect of methylamine on periodate-oxidized adenosine 5’-phosphate, Biochemistry 10,173-178, 1971 Solymosy, S., Hüvös, P., Gulyás, A., et al., Diethyl pyrocarbonate, a new tool in the chemical modification of nucleic acids, Biochim.Biophys. Acta. 238, 406-416, 1971. Lawley, P.D., Orr, D.J., and and Shah, S.A., Reaction of alkylating mutagens and carcinogens with nucleic acids: N-3 of guanine as a site of alkylation by N-methyl-N-nitrosourea and dimethyl sulphate, Chem.Biol. Interact. 4, 431-434, 1972 Uziel, M., Periodate oxidation and amine-catalyzed elimination of the terminal nucleoside from adenylate or ribonucleic acid. Products of overoxidation, Biochemistry 12, 938-942, 1973. Singer, B., The chemical effects of nucleic acid alkylation and their relation to mutgenesis and carcinogensis, Prog.Nucl.Acid Res.Mol.Biol. 15, 219-284, 1975
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Chemical Modification of Nucleic Acids Maxam, A.M. and Gilbert, W., A new method for sequencing DNA, Proc. Nat.Acad.Sci.USA 74, 560-564, 1977 Swenson, D.H. and Lawley, P.D., Alkylation of deoxyribonucleic acid by carcinogens dimethyl sulphate, ethylmethanesulphonate, N-ethyl- N-nitrosourea and N-methyl-N-nitrosourea. Relative reactivity of the phosphodiester site thymidylyl(3’-5’)thymidine, Biochem.J. 171, 575-587, 1978 Erhesamann, C., Baudin, F., Mougel, M., et al., Probing the structure of DNA in solution, Nucleic Acids Res. 15, 9109-9128 Chemistry of Nucleosides and Nucleotides, Volume 1, ed. L.B. Townsend, Plenum Press, New York, New York, 1988 Chemistry of Nucleosides and Nucleotides, Volume 2, ed. L.B. Townsend, Plenum Press, New York, New York, 1991 Oakley, E.J., DNA methylation analysis: A review of current methodologies, Pharmacol.Therapeut. 84, 389-400, 1991 Glennon, R.A. and Tejon-Butl, S., Mesoionic nucleosides and heterobases, in Chemistry of Nucleosides and Nucleotides, Volume 2, ed. L.B. Townsend, Plenum Press, New York, New York, Chapter 1, pps. 1-21, 1991 Adams, R.L.P., Knowler, J.T. and Leader, D.P., The Biochemistry of the Nucleic Acids, 11th Edn., Chapman & Hall, London, 1992
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371 Brown, D.J.. Evans, R.E., Cowder, W.B., and Fenn, M.D., The Pyrimidines, Interscience/John Wiley, New York, New York, 1994 Chemistry of Nucleosides and Nucleotides, Vol. 3, ed. L.B. Townsend, Plenum Press, New York, New York, 1994 Shaw, G., The synthesis and chemistry of imidazole and benzimidazole nucleosides and nucleotides, in Chemistry of Nucleosides and Nucleotides, Vol. 3, ed. L.B. Townsend, Plenum Press, New York, New York, Chaper 4, pps. 263-420, 1994 Nucleic Acids in Chemistry and Biology, 2nd Edn., ed. G.M. Blackburn and M.J. Gait, Oxford University Press, Oxford, UK, 1996 Oakeley, E.J., DNA methylation analysis: a review of current methodologies, Pharmacol. Therapeut. 84, 389-400, 1999 Ordway, J.M., Bedell, J.A., Citek, R.W., et al., MethylMapper: A method for high-throughput, multilocus bisulfite sequence analysis and reporting, BioTechniques 39, 464-470, 2005 Chen, X, Dudgeon, N., Shen, L., and Wang, J.H., Chemical modification of gene silencing oligonucleotides for drug discovery and development, Drug Discov.Today 10, 587-593. 2005 Zhang, W.-Y., Du, Q., Wahlestedt, C., and Liang, Z., RNA interference with chemically modified siRNA, Curr.Top.Med.Chem. 6, 893-900, 2006
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Transfection Technologies 1 2 3 4 5 6
Overview of Transfection Technologies Calcium Phosphate Transfection in Eukaryotic Cell Culture DEAE-Dextran and Transfection in Eukaryotic Cell Culture Electroporation for the Transfection of Eukaryotic Cells Lipofection for Transfection of Eukaryotic Cells in Culture Polyethyleneimine and Transfection in Eukaryotic Cell Culture
1 Overview of transfection technologies Transfection refers to the process by which foreign DNA (transgene) is incorporated into and expressed by a eukaryotic cell1. Stable transfection describes the incorporation of the transgene into the host cellular genome such that it is transferred with other host genetic material to daughter cells. In general, transfected cells must be “selected” from transiently transfected cells to obtain a stable transfected type. Selection is accomplished by inclusion of a “selectable” trait such as drug resistance such as the inclusion of dihydrofolate reductase (DHFR) gene to provide for growth in the presence of methotrexate when a DHFR-deficient cell is used as substrate2-6. The work of Robert Schimke and colleagues on the development of this system was one the critical factors in the development of the current biotechnology industry. In transient transfection, the transgene persists for several days with a peak period of expression after the first day. Transformation is the term used to describe the incorporation of naked DNA into prokaryocytes while conjugation refers to bacterial-bacterial exchange of DNA and transduction refers to the phage-mediated transfer of DNA. The use of naked DNA is usually ineffective with eukaryotic cells and a carrier or process is required7. Electroporation appears to be the most popular method with cationic lipids used to a less extent. Calcium phosphate and polyethyleneimine are some less frequently used. DEAE-dextran is used the least. Other technologies such as the gene gun (biolistic labeling)8-11 are also being developed.
References 1. Baum, C., Transfection, in Encyclopedia of Molecular Biology, ed. T.E. Creighton, John Wiley and Sons, Inc., New York, NY, USA, 1999 2. Schimke, R.T., Gene amplification and methotrexate resistance in cultured animal cells, Harvey Lect. 76, 1-25, 1980-1981. 3. Schimke, R.T., Gene amplification in cultured animal cells, Cell 37, 705-713, 1984 4. Assaraf, Y.G., Molina, A., and Schimke, R.T., Sequential amplification of dihydrofolate reductase and multidrug resistance genes in Chinese hamster ovary cells selected for stepwise resistance to the lipid-soluble antifolate trimetrexate, J.Biol.Chem. 264, 19326-18234, 1989 5. Sharma, R.C., and Schimke, R.T., The propensity for gene amplification: a comparison of protocols, cell lines, and selection agents, Mutat.Res. 304, 243-260, 1994 6. Jun, S.C., Kim, M.N., Baik, J.Y., Hwang, S.O. and Lee, G.M., Selection strategies for the establishment for the establishment of recombinant Chinese hamster ovary cell line with dihydrofolate reductase-mediated gene amplification, Appl.Microbiol.Biotechnol. 69, 162-169, 2005 7. Primrose, S.B. and Twyman, R.M., Principles of Gene Manipulation and Genomics, Blackwell, Malden, MA, USA, 2006 8. O’Brien, J.A., and Loomis, S.C., Biolistic transfection of neuronal cultures using a hand-held gene gun, Nat.Protoc. 1, 9787-981, 2006 9. O’Brien, J.A., and Loomis, S.C., Biolistic labeling of neuronal culture and intact tissue using a hand-held gene gun, Nat.Protoc. 1, 15171521, 2006
10. Zhang, M., Tao, W., and Pianetta, P.A., Dynamics modeling of biolistic gene guns, Phys.Med.Biol. 52, 1485-1493, 2007 11. Lain, W.H., Chang, C.H., Chen, Y.J., et al., Intracellular delivery can be achieved by bombarding cells of tissues with accelerated molecules or bacteria without the need for carrier particles, Exp.Cell Res. 313, 53-64, 2007
2 Calcium phosphate transfection in eukaryotic cell culture Calcium phosphate is used to transfect mammalian cells. It was first used by Graham and Van Der Eb in 19731 and despite considerable use over the past three decades, this technique is still poorly understood2,3. It seems likely the use of calcium phosphate evolved from the earlier use of calcium for in bacterial transformation4-9. Graham and Van der Eb1 separated the calcium phosphate-mediated transfection into three steps: (1) Calcium phosphate + DNA → DNA-CaPO4 (2) DNA-CaPO4 + Cells → DNA transport into Cell (DNA-Cells) (3) DNA-Cells → Cell Growth Each of these steps has been subsequently demonstrated to be critical and has critical process attributes and critical process parameters (See Table 1). Other observations which are important to calcium phosphate-mediated transfection include: • Inclusion of a “carrier” DNA appears to enhance the efficiency of transfection10-12. • pH is critical with a very narrow pH optima between 7.0-7.21,3,13. Tris buffer was used in the first study on calcium phosphate transfection1 but was replaced by HEPES1,14 to provide stronger buffering in the critical pH range. HEPES is a problematic buffer15 and it is of interest that one of the basic studies on the use of calcium phosphate13 used BES instead of HEPES.
References 1. Graham, F.L. and van der Eb, A.J., A new technique for the assay of infectivity of human adenovirus 5 DNA, Virology 52, 456-467, 1973; Graham, F.L. and van der Eb, Virology 54, 536-539, 1973. 2. Yang, Y.-W. and Yang, J.-C., Calcium phosphate as a gene carrier: electron microscopy, Biomaterials 18, 213-217, 1997 3. Jordan, M. and Wurm, F., Transfection of adherent and suspended cells by calcium phosphate, Methods 33, 136-143, 2004 3a. Chang, P.L., Calcium phosphate-mediated DNA transfection, in Gene Therapeutics: Methods and Applications of Direct Gene Transfer, ed. J.W. Wolff, Birkhauser, Boston, MA, USA, pps 157-179, 1994 3ab. Conn, K.J., Degterev, A., Fontanilla, M.R., et al., Calcium phosphate transfection, DNA Transfer to Cultured Cells, ed. K. Ravid and R.I. Freshney, Wiley-Liss, New York, NY, USA, Chapter 6, pps. 111-124, 1996
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Table 1: Critical Process Attributes and Critical Process Parameters for Calcium Phosphate Transfection Calcium phosphate + DNA → DNA-CaPO4
1. Source of Calcium ions3ab. 16, 17(See note 1) 2. DNA – Calcium -phosphate(order of mixing)17 3. Source of orthophosphate17(See note 1) 4. pH of mixing and choice of buffer1.2.3.13 5. Method of mixing16,17,20 6. Temperature of mixing1,2,13,18 7. DNA concentration1,3,13,14,17,18 8. Mixing container17
DNA-CaPO4 + Cells → DNA transport into Cell (DNA-Cells) 1. Cells growth status (S Stage)1,20 2. Time of “storage” of DNA-CaPO4 before addition to cells1,313,18,19,20 3. Presence or absence of media during addition of DNA-CaPO42 4. Presence or absence of serum during addition of DNA-CaPO42 (See Note 2) 5. Time of contact with cells1,3ab,17,20 6. Volume of transfectant solution added to medium21 7. Use of EDTA wash to remove reagents (See Note 3) 1. Growth time DNA-Cells → Cell Growth 2. Growth temperature Note 1: Most investigators use a sterile source of calcium ions and phosphate solutions. Also, filtration immediately prior to mixing is a frequently cited practice. Note 2: Fetal Calf Serum (FCS, 10% final concentration in medium, usually Dulbecco’s minimal essential medium; DMEM) is usually included in the medium for culturing the cells. Protein has an effect on the organization of calcium phosphate crystals. An additional complication is provided by CO2 which, as HCO31-/CO32- will incorporate into calcium phosphate crystals16. The cell cultures are usually maintained under CO2 and CO2 levels have been shown to be critical during the incubation of DNA with cells3,13. Note 3: Calcium phosphate is toxic to some cells. Individual protocols differ on the washing procedure for the cells following the calcium phosphate step.
4. Tyeryar, F.J. and Lawton, W.D., Factors affecting transformation of Pasturella novicida, J.Bacteriol. 104, 1312-1317, 1970 5. Osowiecki H. and Skalinska, B.A., The conditions of transfection of Escherichia coli cells untreated with lysozyme. I. The effect of some factors on the efficiency of transfection with lambda phage DNA, Mol. Gen.Genet. 133, 335-343 1974. 6. Erhlich, M., Sarafyan, L.P., and Myers, D.J., Interaction of microbial DNA with cultured mammalian cells. Binding of the donor DNA to the cell surface, Biochim.Biophys.Acta 454 397-409, 1976 7. Kahmann, R., Kamp, D., and Zipser, D., Transfection of Escherichia coli by Mu DNA, Mol.Gen.Genet. 149, 323-328, 1976. 8. Norgard, M.V. and Imaeda, T., Physiological factors involved in the transformation of Mycobacterium smegmatis J.Bacteriol. 133 12541262, 1978 9. Dagert, M. and Ehrlich, S.D., Prolonged incubation in calcium chloride improves the competence of Escherichia coli cells, Gene 6 23-28, 1979. 10. Graham, F.L., van der Eb, A.J., and Heijneker, H.L., Size and location of the transforming region in human adenovirus type 5 DNA, Nature 251, 687-691, 1974. 11. Bacchetti, S. and Graham, F.L., Transfer of the gene for thymidine kinase to thymidine kinase-deficient human cells by purified herpes simplex viral DNA, Proc.Nat.Acad.Sci.USA 74, 1590-1594, 1977. 12. Wigler, M., Pellicer, A., Silverstein, S., and Axel, R., Biochemical transfer of single-copy eukaryotic genes using total cellular DNA as donor, Cell 14, 725-731, 1978 13. Chen, C. and Okayama, H., High-efficiency transformation of mammalian cells by plasmid DNA, Mol.Cell.Biol. 7, 2745-2752, 1987. 14. Graham, F.L. and van der Eb, A.J., Transformation of rat cells by DNA of human adenovirus 5, Virology 54, 536-539, 1974 15. Chirpich, T.P., The effect of different buffers on terminal deoxynucleotidyl transferase activity, Biochim.Biophys.Acta 518, 535-538, 1978; Tadolini, B., Iron autoxidation in Mops and Hepes buffers, Free Radic. Res. Commun. 4, 149-160, 1987; Simpson, J.A., Cheeseman, K.H., Smith, S.E., and Dean, R.T., Free-radical generation by copper ions and hydrogen peroxide. Stimulation by Hepes buffer, Biochem.J. 254, 519523, 1988; Abas, L. and Guppy M., Acetate: a contaminant in Hepes buffer, Anal.Biochem. 229, 131-140, 1995; Schmidt, K., Pfeiffer, S., and Mayer, B., Reaction of peroxynitrite with HEPES or MOPS results in
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16. 17. 18. 19. 20.
21.
the formation of nitric oxide donors, Free Radic.Biol.Med. 24, 859862, 1998; Fulop, L., Szigeti, G., Magyar, J., et al., Differences in electrophysiological and contractile properties of mammalian cardiac tissues bathed in bicarbonate – and HEPES-buffered solutions, Acta Physiol.Scand.178, 11-18, 2003; Mash, H.E., Chin, Y.P., Sigg, L., et al., Complexation of copper by zwitterionic aminosulfonic (good) buffers, Anal.Chem. 75, 671-677, 2003 Sokolowska, M., and Bal, W., Cu(II) complexation by “non-coordinating” N-2-hydroxyethylpiperazine-N’ethanesulfonic acid (HEPES buffer), J.Inorg.Biochem. 99, 1653-1660, 2005; Zhao, G. and Chasteen, N.D., Oxidation of Good’s buffers by hydrogen peroxide, Anal.Biochem. 349, 262-267, 2006; Hartman, R.F. and Rose, S.D., Kinetics and mechanism of the addition of nucleophiles to alpha,beta-unsaturated thiol esters, J.Org.Chem. 71, 6342-6350, 2006. Röszler, S., Sewing, A., Stözel, M., et al., Electrochemically assisted deposition of thin calcium phosphate coating at near-physiological pH and temperature, J.Biomed.Mater.Res. 64A, 655-663, 2002. Cosaro, C.M. and Pearson, M.L., Enhancing the efficiency of DNAmediated gene transfer in mammalian cells, Somat.Cell Genet. 7, 603-616, 1981. Jordan, M., Schallhorn, A., and Wurn, F.M., Transfecting mammalian cells: optimization of critical parameters affecting calcium-phosphate precipitate formation Nucleic Acids Res. 24, 596-601, 1996. Coonrod, A., Li, F.-Q., and Horwitz, M., On the mechanism of DNA transfection: efficient gene transfer without viruses, Gene Therapy 4 1313-1321 1997 Watanabe, S.Y., Albsoul-Younes, A.M., Kawano, T., et al., Calcium phosphate-mediated transfection of primary cultured brain neurons using GFP expression as a marker: application to single neuron physiology, Gene 270 61-68, 2001. Fu, H., Hu, Y., McNelis, T., and Hollinger J.O., A calcium phosphatebased gene delivery system, J.Biomed.Mater.Res. 74A 40-48, 2005.
3 DEAE-Dextran and transfection in eukaryotic cell culture The ability of DEAE-Dextran to promote nucleic acid uptake into eukaryotic cells was first noted by Vaheri and Pagano1-2. Dimethylsulfoxide (DMSO) was also observed to enhance
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Transfection Technologies nucleic acid uptake3,4. DMSO was subsequently demonstrated to enhance calcium phosphate transfection5. DMSO “osmotic shock” was later shown to markedly enhance DEAE-dextran transfection efficiency6-8. DEAE-dextran is used less than polyethyleneimine. It would appear that DEAE-dextran is used more with Cos cells or BHK cells than with CHO cells. It is also noted that polyamines such as DEAE-dextran have been demonstrated to enhance nucleic acid transfer in Escherichia coli spheroblast9.
References 1. Vaheri, A. and Pagano, J.S., Infectious poliovirus RNA: A sensitive method of assay, Virology 27, 434-436, 1965 2. Pagano, J.S., McCutchan, J.H., and Vaheri, A., Factors influencing the enhancement of the infectivity of poliovirus ribonucleic acid by diethylaminoethyl-dextran, J.Virol. 1, 891-897, 1967 3. Tovell, D.R. and Colter, J.S., Observations on the assay of infectious viral ribonucleic acid: effects of DMSO and DEAE-dextran, Virology 32, 84-92, 1967
375 4. Tovell, D.R. and Colter, J.R., The interaction of tritium-labelled Mengo virus RNA and L cells: the effects of DMSO and DEAE-dextran, Virology 37, 624-631, 1969 5. Stow, N.D. and Wilkie, N.M., An improved technique for obtaining enhanced infectivity with herpes simplex virus type 1 DNA, J.Gen. Virol. 33, 447-458, 1976 6. Lopata, M.A., Cleveland, D.W., and Sollner-Webb, B., High level transient expression of a chloramphenicol acetyl transferase gene by DEAE-dextran mediated DNA transfection coupled with a dimethyl sulfoxide or glycerol shock treatment, Nucleic Acids Res. 12, 57075117, 1984 7. Kluxen, F.W. and Lubbert, H., Maximal expression of recombinant cDNAs in COS cells for use in expression cloning, Anal.Biochem. 208, 352-356, 1993 8. Schwartz, J.J. and Rosenberg, R., DEAE-dextran transfection, in DNA Transfer to Cultured Cells, ed. K. Ravid and R.I. Freshney, Wiley-Liss, New York, NY, USA, 1998 9. Henner, W.D., Kleber, I., and Benzinger, R., Transfection of Escherichia coli spheroblasts. 3. Facilitation of transfection and stabilization of spheroblasts by different basic polymers, J.Virol. 12, 741-747, 1973
Table 2: Some Examples of DEAE-Dextran Transfection in Cultured Eukaryotic Cells Ehrlich, M., Sarafyan, L.P., and Myers, D.J., Interaction of microbial DNA with cultured mammalian cells. Binding of the donor DNA to the cell surface, Biochim.Biophys.Acta 454, 397-409, 1976 Evaluated the effect of various polyamines on the uptake of microbial DNA by cultured fibroblasts. In the presence of a polycation such as DEAEdextran, 10-30% of DNA was bound to the cell; with DNA alone, 0.5-5% of the DNA was cell associated. Gopal, T.V., Gene transfer method for transient gene expression, stable transformation, and cotransformation of suspension cell culture, Mol.Cell Biol. 5, 1188-1190, 1985 Cells (mouse myeloma, erythroleukemia) were bound to concanavalin A-coated tissue culture plates and treated with DEAE-dextran and plasmid DNA. Subsequent treatment with 40% polyethylene glycol facilitated the update of DNA by the substrate cells. Takai, T. and Ohmori, H., DNA transfection of mouse lymphoid cells by the combination of DEAE-dextran-mediated DNA uptake and osmotic shock procedure, Biochim.Biophys.Acta 1048, 105-109. 1990 Mouse lymphoid cells are first treated with DEAE-dextran followed by osmotic shock (hypertonic Tris hydrochloride buffer with 0.5 M sucrose and 10% PEG followed by hypertonic RPMI 1640) Pazzagli, M., Devine, J.H., Peterson, D.O., and Baldwin, T.O., Use of bacterial and firefly luciferase as reporter genes in DEAE-dextran-mediated transfection of mammalian cells, Anal.Biochem. 204, 315-323, 1992 Development of luciferase transgenes as reporter genes. Gauss, G.H. and Lieber, M.R, DEAE-dextran enhances electroporation of mammalian cells, Nuc.Acids Res. 20, 6739-6740, 1992 DEAE-dextran increases transfection efficiency in several mammalian cell lines including human lymphoid cells and hamster fibroblast cells. Kluxen, F.W. and Lubbert, H., Maximal expression of recombinant cDNAs in COS cells for use in expression cloning, Anal.Biochem. 208, 352-356, 1993 Β-Galactosidase was used as reporter gene in COS cells. DEAE-dextran was found to be superior to electroporation or lipofection in transfection efficiency. COS-1 cells expressed more protein than COS-7 cells. Gonzalez, A.L. and Joly, E., A simple procedure to increase efficiency of DEAE-dextran transfection of COS cells, Trends Genet. 11, 216-217, 1995 Technical improvements in DEAE-dextran transfection technology Yang, Y.W. and Yang, J.C., Studies of DEAE-dextran-mediated gene transfer, Biotechnol.Appl.Biochem. 25, 47-51, 1997 Gene transfer into FR3T3 cells. Optimal ratio of DEAE-dextran to DNA of 50 to 1. It is suggested that the binding of the DEAE-dextran/DNA to the cell surface determines DNA transfection efficiency. Mack, K.D., Wei, R., Elbagarri, A., et al., A novel method of DEAE-dextran mediated transfection of adherent primary cultured human macrophages, J.Immunol.Methods 211, 79-86, 1998 DEAE-dextran concentration, DNA quantity, and incubation time were the three critical factors in the transfection of adherent human macrophages. Schenborn, E.T. and Goiffon, V. DEAE-dextran transfection of mammalian cultured cells, Methods Mol.Biol. 130, 147-153, 2000 Technical review Pari, G.S. and Xu, Y., Gene transfer into mammalian cells using calcium phosphate and DEAE-dextran, Methods Mol.Biol. 245, 25-32, 2004 Technical review comparing transfection technologies (calcium phosphate and DEAE-dextran) Hermans, E., Generation of model cell lines expressing recombinant G-protein-coupled receptors, Methods Mol.Biol. 259, 137-153, 2004 Technical review comparing transfection technologies (calcium phosphate, DEAE-dextran, cationic lipids, and electroporation) Escher, G., Hoang, A., Georges, S., et al., Demethylation using epigenetic modifier, 5-azacytidine, increases the efficiency of transient transfection of macrophages, J.Lipid Res. 46, 356-365, 2005 Several transfection technologies were evaluated using a CMV-LacZ plasmid containing a bacterial β-galactosidase as a reporter gene. DEAEdextran(with DMSO osmotic shock) was slightly more effective than a variety of cationic lipids.
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4 Electroporation for the transfection of eukaryotic cells Electroporation is a process for transfection where cells are suspended at relatively high density (106-107mL) and subjected to a short (milliseconds to microseconds) electric pulses1-8. While electroporation tends to be challenging to cells with cell losses up to 50%, it does see extensive use in commercial biotechnology where it is viewed as a robust process. Iontophoresis is an alternative drug delivery related to electroporation when a low-level electrical current is used to administer ionic drug to the skin9-12. Electroporation has been reported to promote protein uptake by cells13-14
References 1. Neumann, E. and Sowers, A. E., Electroporation and Electrofusion in Cell Biology, Plenum Press, New York, NY, USA, 1989 2. Chang, D.C., Guide to Electroporation and Electrofusion, Academic Press, San Diego, CA, USA, 1992 3. Nickoloff, J.A., Animal Cell Electroporation and Electrofusion protocols, Humana Press, Totowa, NJ, USA, 1995 4. Nickoloff, J.A., Plant Cell Electroporation and Electrofusion Protocols, Humana Press, Totowa, NJ, USA, 1995
5. Nickoloff, J.A., Electroporation Protocols for Microorganisms, Humana Press, Totowa, NJ, USA, 1995 6. Lynch, P.T. and Davey, M.R., Electrical Manipulation of Cells, Chapman & Hall, New York, NY, USA, 1996 7. Polk, C. and Postow, E., Handbook of Biological Effects of Electromagnetic Fields, CRC Press, Boca Raton, FL, USA, 1996 8. Peña, L., Transgenic Plants: Methods and Protocols, Human Press, Totowa, NJ, USA, 2005 9. Kanikkannan, N., Iontophoresis-based transdermal delivery systems, BioDrugs 16, 339-347, 2002 10. Gehl, J., Electroporation: theory and methods, perspectives for drug delivery, gene therapy and research, Acta Physiol.Scand. 177, 437-447, 2003 11. Nanda, A., Nanda, S. and Ghilzai, N.M., Current developments using emerging transdermal technologies in physical enhancement methods, Curr.Drug Deliv. 3, 233-242, 2006 12. Mayes, S. and Ferrone, M., Fentanyl HCl patient-controlled iontophoretic transdermal system for the management of acute postoperative pain, Ann.Pharmacother. 40, 2178-2186, 2006 13. Lambert, H, Pankov, R., Gauthier, J., and Hancock, R., Electroporationmediated uptake of proteins into mammalian cells, Biochem.Cell Biol. 68, 729-734, 1990 14. Mohr, J.C., de Pablo, J.J., and Palecek, S.P., Electroporation of human embryonic stem cells: small and macromolecule loading and DNA transfection, Biotechnol.Prog. 22, 825-834, 2006
Table 3: Some Examples of Electroporation for Transfection of Eukaryotic Cells Maxwell, I.H. and Maxwell, F., Electroporation of mammalian cells with a firefly luciferase expression plasmid: kinetics of transient expression differ markedly among cells types, DNA 7, 557-5562, 1988 Transfection by electroporation was examined in a several mammalian cell types. In some cell lines, expression of the transgene was maximal at 12 hours followed by a rapid decline. It was concluded that the time course of transgene expression following electroporation must be determined for each cell line. Andreason, G.L. and Evans, G.A., Optimization of electroporation for transfection of mammalian cell lines, Anal.Biochem. 180, 269-275, 1989 Evaluated transfection technologies (electroporation, calcium phosphate, and DEAE-dextran) in several rodent cells lines including PC12 and B50 (rat neuroblastoma) with firefly luciferase as the reporter gene. The relative effective of a specific transfection technology varied with the cell line. It was observed that electroporation was more effective at 23°C than at 4°C. Gauss, G.H. and Lieber, M.R., DEAE-dextran enhances electroporation of mammalian cells, Nucleic Acids Res. 20, 6739-6740, 1992 The presence of DEAE-dextran increase transfection efficiency with electroporation. Rols, M.P., Delteil, C, Serin, G., and Teissie, J. ,Temperature effects on electroporation of mammalian cells, Nucleic Acids Res. 22, 540, 1994 Experiments with CHO cells in 10 mm phosphate, pH 7.2, containing 250 mM sucrose and 1 mM MgCl2showed that there are different temperature optima for the steps in the transfection process. Incubation at 4°C prior to electroporation yielded the best results while incubation at 37°C after electroporation increased transfection efficiency. Teifel, M., Heine, L.T. ,Milbredt, S., and Friedl, P., Optimization of transfection of human endothelial cells, Endothelium 5, 21-35, 1997 Electroporation is suggested to more useful than lipofection, DEAE-dextran, or calcium phosphate for the transfection of endothelial cells. Nickoloff, J.A. and Reynolds, J.A., Electroporation-mediated gene transfer efficiency is reduced by linear plasmid carrier DNAs, Anal.Biochem. 205, 237-243, 1992 Linear DNA inhibited transfection by electroporation in CHO cells while circular plasmids enhanced transfection efficiency with electroporation. Melkonyan, H., Sorg, C. and Klempt, M., Electroporation efficiency in mammalian cells is increased by dimethyl sulfoxide (DMSO), Nucleic Acids Res. 24, 4356-4367, 1996 DMSO improved transfection efficiency by electroporation in four cells lines (HL60, TR146, Cos-7, and L132). DMSO was present during electric pulse and in the following incubation. Yang, T.A., Heiser, W.C., and Sedivy, J.M., Efficient in situ electroporation of mammalian cells grown on microporous membranes, Nucleic Acids Res. 23, 2803-2810, 1995 Electroporation with cells grown on microporous membranes such as polyethylene terephthalate or polyester. Baum, C., Forster, P., Hegewisch-Becker, S. and Harbers, K., An optimized electroporation protocol applicable to a wide range of cell lines, BioTechniques 17, 1058-1062, 1994 Optimization of voltage for the electroporation step. With optimization, electroporation is superior to other methods of Transfection Delteil, C., Teissie, J., and Rols, M.P., Effect of serum on in vitro electrically mediated gene delivery and expression in mammalian cells, Biochim. Biophys.Acta 1467, 362-368, 2000 Serum increased transfection efficiency mediated by electroporation in CHO cells. Bodwell, J., Swiff, F., and Richardson, J., Long duration electroporation for achieving high level expression of glucocorticoid receptor in mammalian cell lines, J.Steroid Biochem.Mol.Biol. 68, 77-82, 1999 Long duration electroporation (LDE) uses a lower voltage (440-500 V/cm) for a longer time (140 milliseconds). LDE allowed transient expression in Cos-7 cells at high levels usually seen only in stable cell lines. It was critical that the cells be log phase and less than 70% confluent in culture.
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Table 3: Some Examples of Electroporation for Transfection of Eukaryotic Cells (Continued) Golzio, M., Mora, M.P., Raynaud, C., et al., Control by osmotic pressure of voltage-induced permeabilization and gene transfer in mammalian cells, Biophys.J. 74, 3015-3022, 1998 Electric pulsing at low osmolarity (10 mM phosphate-125 mM sucrose-1 mM MgCl2, pH 7.4) increased electroporation efficiency Muller, K.J., Horbaschek, M., Lucas, K., et al., Electrotransfection of anchorage-dependent mammalian cells, Exp. Cell Res. 288, 344-353, 2003 Methods are developed for the in situ electroporation of anchorage-dependent cells. Distler, J.H.W., Jüngel, A., Kurowska-Stolarska, M., et al., Nucleofection: a new, highly efficient transfection method for primary human keratinocytes, Exptl.Dermatol. 14, 315-320, 2005 Nucleofection is an electroporation technique where DNA directly enters the nucleus. Leclerre, P.G., Panjwani, A., Docherty, R., et al., Effective gene delivery to adult neurons by a modified form of electroporation, J.Neurosci.Methods 142, 137-143, 2005 Used nucleofection for transfection of adult neurons Barry, P.A., Efficient electroporation of mammalian cells in culture, Methods Mol.Biol. 245, 207-214, 2004 Review of electroporation technology Buchser, W.J., Pardinas, J.R., Shi, Y., et al., 96-Well electroporation method for transfection of mammalian central neurons, BioTechniques 41, 619-624, 2006 A 96-well electroporation platform has been developed.
5 Lipofection for transfection of eukaryotic cells in culture Cationic or neutral lipids can be used to prepare liposomes to condense nucleic acids for transfer into cells1-9. These supramolecular structures are known as a lipoplexes. Lipoplexes are sometimes referred to as polyplexes but it would seem that the term polyplex is used more often to describe polyamine complexes with DNA such a PEI-DNA or DNA-DEAE-dextran complexes. The cationic lipids include N-[1-(2,3-dioleyl)propyl]-N,N,Ntrimethylammonium chloride (DOTAP) and N-[1-(2,3-dioleyloxy) propyl]-N,N,N-trimethylammonium chloride (DOTMA)10,11. The inclusion of neutral lipids such as cholesterol or 1,2-dioleoylsn-glycero-3-phosphoethanolamine (DOPE) has been shown to enhance transfection12-14. It is thought that cationic lipids bind to the DNA condensing into a liposomal structure which can then fuse with the cell wall and undergo membrane fusion/endocytosis forming an intracellular endocytotic vesicle which is destabilized in the cytoplasm with the release of DNA. The DNA then enters the nucleus as postulated for the other polyplexes where the transgene may or may not be expressed in, most likely, a transient manner.
References 1. Clark, P.R. and Hersh, E.M., Cationic lipid-mediated gene transfer: current concepts, Curr.Opin.Mol.Ther. 1, 158-176, 1999 2. Ulrich, A.S., Biophysical aspects of using liposomes as delivery vehicles, Biosci.Rep. 22, 1291-150, 2002 3. Pedroso de Lima, M.C., Neves, S., Filipe, A., Duzgunes, N., and Simoes, S., Cationic liposomes for gene delivery: from biophysics to biological applications, Curr.Med.Chem. 10, 1221-1231, 2003 4. May, E. and Ben-Shaul, A., Modeling of cationic lipid-DNA complexes, Curr.Med.Chem. 11, 151-167, 2004 5. Zhang, S., Xu, Y., Wang, B., et al., Cationic compounds used in lipoplexes and polyplexes for gene delivery, J.Control.Release. 100, 165-180, 2004 6. Simoes, S., Filipe, A., Faneca, H., et al., Cationic liposomes for gene delivery, Expert Opin.Drug Deliv. 2, 237-254, 2005 7. Khalil, I.A., Kogure, K., Akita, S. and Harashima, H., Uptake pathways and subsequent intracellular trafficking in nonviral gene delivery, Pharmacol.Rev. 58, 32-45, 2006 8. Wasungu, L. and Hookstra, D., Cationic lipids, lipoplexes and intracellular delivery of genes, J.Control.Release 116, 2555-265, 2006 9. Huang, L. and Hung, M.C., Non-viral vectors for Gene Therapy, Elsevier/Academic Press, Amsterdam, Netherlands, 2005 10. Zhang, S., Xu, Y., Wang, B., et al., Cationic compounds used in lipoplexes and polyplexes for gene delivery, J.Control.Release 100, 165-180, 2004 11. Wasungu, L. and Hookstra, D., Cationic lipids, lipoplexes and intracellular delivery of genes, J.Control.Release 116, 255-264, 2006
Table 4: Some Examples of the Use of Lipofection for Transfection of Mammalian Cell Culture Liu, F., Yang, J., Huang, L., and Liu, D., New cationic lipid formulations for gene transfer, Pharm.Res. 13, 1856-1860, 1996 Tween was combined with lipid components for transfection studies Keogh, M.C., Chen, D., Lupu, F., et al., High efficiency reporter gene transfection of vascular tissue in vitro and in vivo using a cationic lipid-DNA complex, Gene Ther. 4, 162-171, 1997 The most important factors in cationic lipid transfection of a plasmid containing a luciferase reporter gene to rabbit or human arterial tissue was the ratio of lipid reagent to DNA, DNA concentration, transfection time and the presence or absence of serum. However, there was variation with cell line. Hep2 was the only cell line showing a positive effect of serum. The study did show the necessity of establishing optimal conditions for each cell line. Zelphati, O., Nguyen, C., Ferrari, M., et al., Stable and monodisperse lipoplex formulations for gene therapy, Gene Ther. 5, 1272-1282, 1998 A lipoplex formulation was developed which could be stored frozen without losing biological activity or physical stability. The critical parameters for formulation success are size of the cationic liposome, the rate and method of DNA and cationic lipid mixing, and ionic strength of suspension vehicle. Ferrari, M.E, Ngyen, C.M., Zelphati, O., et al., Analytical methods for the characterization of cationic lipid-nucleic acid complexes, Hum.Gene Ther. 9, 341-351, 1998
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Table 4: Some Examples of the Use of Lipofection for Transfection of Mammalian Cell Culture (Continued) Lipid recovery, total DNA, free DNA, nuclease sensitivity, and physical stability by filtration are proposed for the evaluation of formulation variables on the physical properties of lipoplexes. Xu, Y., Hui, S.W., Frederik, P. and Szoka, F.C., Jr., Physicochemical characterization and purification of cationic lipoplexes, Biophys.J. 77, 341-353, 1999 Lipoplexes were prepared with varying excesses of DOTAP or DNA and separated into positively charged lipoplexes or negatively charged lipoplexes. Positively charged lipoplexes had high transfection activity and reduced toxicity. There were also structural differences between the two classes of lipoplexes. Lin, A.J, Slack, N.L., Ahmad, A., et al., Structure and structure-function studies of lipid/plasmid DNA complexes, J.Drug.Target. 8, 13-27, 2000 There appears to be a relationship between physical structure of lipoplexes and transfection efficiency in mouse fibroblast L-cells. One structure consisted of DNA between two ordered multilamellar structures. Lipids used included DOTAP, DOPE, and DOPC. Nchinda, G., Uberla, K., and Zschornig, O., Characterization of cationic lipid DNA transfection complexes differing in susceptibility to serum inhibition, BMC Biotechnol. 2, 12, 2002 Evaluated the effect of serum on transfection efficiency. Simberg, D., Weisman, S., Talmon, Y., and Barenholz, Y., DOTAP (and other cationic lipids): chemistry, biophysics, and transfection, Crit.Rev.Ther. Drug Carrier Syst. 21, 257-317, 2004 Evaluation of the properties of DOTAP in cell transfection. Bengali, E., Pannier, A.K., Segura, T., et al., Gene delivery through cell culture substrate adsorbed DNA complexes, Biotechnol.Bioeng. 90, 290-302, 2005 Pretreatment of tissue culture plates with serum enhanced transfection by lipoplexes by increasing the number of transfected cells with similar level of expression. Decastro, M., Saijoh, Y., and Schoenwolf, O.C., Optimized cationic lipid-based gene delivery reagents for use in developing vertebrate embryos, Dev. Dyn. 235, 2210-2219, 2006 This study evaluated Lipofectamine, Lipofectamine 2000, and Lipofectamine with a disulfide-linked pegylated lipid for GFP transgene expression. Significant levels of ectopic gene expression were observed.
12. Rose, J.K., Buonocore, L., and Whitt, M.A., A new cationic liposome reagent mediating nearly quantitative transfection of animal cells, Biotechniques 10, 520-525, 1991 13. Felgner, J.H., Kumar, R., Sridhar, C.N., et al., Enhanced gene delivery and mechanism studies with a novel series of cationic lipid formulations, J.Biol.Chem. 269, 2550-2561, 1994 14. Teifel, M., Heine, L.T., Milbredt, S., and Friedl, P., Optimization of transfection of human endothelial cells, Endothelium 5, 21-35, 1997
6 Polyethyleneimine and transfection in eukaryotic cell culture Polyethyleneimine (PEI) is one of several vehicles used for non-viral transfection of cells and results in transient rather than stable transfection. PEI appears to be used more frequently that DEAE-dextran. Polyamines such as PEI function in transfection by condensing of DNA with the formation of a polyplex (the term polyplex refers to the DNA/carrier condensate). Branched forms and linear forms of PEI are used. The polyplex binds to the cell membrane, passes into the cell via endocytosis, is released from the endosome and passes into the nucleus1-12 . Early studies by Pollard and coworkers1 showed the polycation was not only required for effective endocytosis of DNA, polycation also stimulated nuclear uptake of the DNA1. These early studies have been extended by a number of workers including Breung and coworkers2 . There have been a number studies on the endocytotic process but the mechanism remains elusive 8,12,. Recent studies13 have suggested that process efficiency is limited by passage of DNA into the nucleus and expression of all inserted DNA. PEI is also useful for transferring RNAi derivatives to the cytoplasm14-18. There is one report on the use of PEI as a transmembrane carrier for fluorescently labeled proteins19.
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References 1. Pollard, R., Remy, J.S., Loussouarn, G., et al., Polyethyleneimine but not cationic lipids promote transgene delivery to the nucleus in mammalian cells, J.Biol.Chem. 273, 7507-511, 1998 2. Breunig, M., Lungwitz, U., Liebl, R. et al., Mechanistic insights into linear polyethyleneimine-mediated gene transfer, Biochim.Biophys. Acta 1770, 196-205, 2007 3. Wagner, E., Effects of membrane-active agents in gene delivery, J.Control.Release 53, 155-158, 1998 4. Choosakookriang, S., Lobo, B.A., Koe, G.S., et al., Biophysical characterization of PEI/DNA complexes, J.Pharm.Sci. 92, 1710-1722, 2003 5. Sonawane, N.D., Szoka, F.C., Jr., and Verkman, A.S., Chloride accumulation and swelling in endosomes enhances DNA transfer by polyamine-DNA polyplexes, J.Biol.Chem. 278, 33826-44832, 2003 6. Akinc, A., Thomas, M., Klibanov, A.M., and Langer, R., Exploring polyethyleneimine–mediated DNA transfection and the proton sponge hypothesis, J.Gene Med. 7, 657-663, 2005 7. Demeneix, B. and Behr, J.P., Polyethyleneimine (PEI), Adv.Genet. 53, 217-230, 2005 8. Heidel, J., Mishra, S., and Davis, M.E., Molecular conjugates, in Gene Therapy and Gene Delivery Systems, ed. D.V. Schoffer and W. Zhou, Springer-Verlag, Berlin, Germany. pps. 7-39, 2005 9. Nimesh, S., Goyal, A., Pawar, V., et al., Polyethyleneimine nanoparticles as efficient transfecting agents for mammalian cells, J.Control Release 110, 457-468, 2006 10. Brissault, B., Leborgne, C., Guis, C., et al., Linear topology confers in vivo gene transfer activity to polyethyleneimine, Bioconjug.Chem. 17, 759-765, 2006 11. Kodaoma, K., Katayama, Y., Shoji, Y., and Nakashima, H., The features and shortcomings for gene delivery of current non-viral carriers, Curr.Med.Chem. 13, 2155-2161, 2006 12. Rejman, J., Conese, M., and Hoekstra, D., Gene transfer by means of lipo- and polyplexes: role of clathrin and caveolae-mediated endocytosis, J.Liposome Res. 16, 237-247, 2006 13. Carpentier, E., Paris, S., Kamen, A.A., and Durocher, Y., Limiting factors governing protein expression following polyethyleneiminemediated gene transfer in HEK293-EBNA1 cells, J.Biotechnol. 128, 268-280, 2007
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Table 5: Some Examples of the Use of Polyethyleneimine for Gene Transfection in Cell Culture Vancha, A.R., Govindaraju, S., Parsa, K.V., et al., Use of polyethyleneimine polymer in cell culture as attachment factor and lipofection enhancer, BMC Biotechnol. 4, 23, 2004 PEI as attachment factor for weakly anchoring cell lines. PEI also appears to enhance lipofection efficiency. Experimental results with PC-12 and HEK-293 cells. Schlatter, S., Stansfield, S.H., Dinnis, D.M., et al., On the optimal ratio of heavy to light chain genes for efficient recombinant antibody production by CHO cells, Biotechnol.Prog. 21, 122-133, 2005 PEI transfection in CHO cells. It was determined that a low heavy chain/light chain ratio was optimal for transient expression Mennesson, E., Erbacher, P., Piller, V., et al., Transfection efficiency and uptake process of polyplexes in human lung endothelial cells: a comparative study non-polarized and polarized cells, J.Gene Med. 7, 729-738, 2005 Compared transfection efficiency of PEI polyplexes with histidylated polylysine (polylysine where approximately 50% of the ε-amino groups are substituted with a histidyl-residue-see Midoux, P. and Monsigny, M., Efficient gene transfer by histidylated polylysine/pDNA complexes, Bioconjug. Chem. 10, 406-411, 1999) with human lung microvascular endothelial cells in monolayer culture. Transfection was more effective with PEI polyplexes as judged with YOYO-labelled plasmids and luciferase activity. Bengali, Z., Pannier, A.K., Segura, T., et al., Gene delivery through cell culture substrate adsorbed DNA complexes, Biotechnol.Bioeng. 90, 290-302, 2005 Transfection efficiency increased when serum-coated tissue culture plate used. The polyplex complexes were immobilized on the serum-coated plate prior to the transfection step. Transfection efficiency was measured by luciferase(transgene) expression NIH/3T3 cells. Breunig, M., Lungwitz, U., Liebl, R., et al., Gen delivery with low molecular weight linear polyethyleneimines, J.Gene Med. 7, 1287-1298, 2005 Low molecular weight PEI polymers (5-9 kDa) may be better than higher molecular weight materials as a result of higher transfection efficiency and lower cytotoxicity. Transfection was measure by green fluorescent protein expression in either CHO cells or HeLa cells. Banerjee, P., Weissleder, R. and Bogdanov, A., Jr., Linear polyethyleneimine grafted to a hyperbranched poly(ethylene glycol)-like core: a copolymer for gene delivery, Bioconjug.Chem. 17, 125-131, 2006 The block copolymer of branched PEI and hyperbranched PEG provided DNA condensates with longer life stability and much higher transfection efficiency that a block copolymer of branched PEI and PEG. Arnold, A.S., Laporte, V., Dumont, S., et al., Comparing reagents for efficient transfection of human primary myoblasts: FuGENE 6, Effectene and ExGen 500, Fundam.Clin.Pharmacol. 20, 81-89, 2006 Fugene 6 (a cationic lipid), Effectene (a cationic lipid), and ExGen500 (a linear PEI polymer) were compared with respect to their transfection efficiency in primary myoblast cells. Transfection was measured by cell viability (mitochondrial dehydrogenase activity). Fugene 6 was the most effective but was more dependent on the presence of serum than ExGen. Braga, D., Laize, V, Tiago, D.M., and Cancela, M.L., Enhanced DNA transfer into fish bone cells using polyethyleneimine, Mol.Biotechnol. 34, 51-54, 2006 Transfection efficiency measured with green fluorescent protein and luciferase. Galbraith, D.J., Tait, A.S., Racher, A.J., et al., Control of culture environment for improved polyethyleneimine-mediated transient production of recombinant monoclonal antibodies by CHO cells, Biotechnol.Prod. 22, 753-762, 2006 Used a branched 25 kDa PEI with suspension-adapted CHO cells. The production of recombinant MABs was followed by ELISA and with alkaline phosphatase reporter gene in static culture and green fluorescent protein in shake cultures. The addition of growth factors to media improved production. Breunig, M., Langwitz, U., Liebl, R., et al., Mechanistic insights into linear polyethyleneimine-mediated gene transfer, Biochim.Biophys.Acta 1770, 196-205, 2007 Linear PEI (6.6 kDa) demonstrated high transfection efficiency (44%) with relatively low cytotoxicity. CHO cells were used in adherent culture. A plasmid with green fluorescent protein transgene was used as the reported gene.
14. Bologna, J.C., Dorn, G., Natt, F., and Weiler, J., Linear polyethyleneimine as a tool for comparative studies of antisense and short doublestranded RNA oliogonucleotides, Nucleosides, Nucleotides Nucleic Acids 22, 1729-1731, 2003 15. Urban-Klein, B,. Werth, S., Abuharbeid, S., et al., RNAi-mediated gene targeting through systemic application of polyethyleneimine (PEI)-complexed siRNA in vivo, Gene Ther. 12, 461-466, 2005 16. Grayson, A.C., Doody, A.M., and Putman, D., Biophysical and structural characterization of polyethyleneimine-mediated siRNA delivery in vitro, Pharm.Res. 23, 1868-1876, 2006 17. Werth, S., Urban-Klein, G., Dai, L, et al., A low molecular weight fraction of polyethyleneimine (PEI) displays increased transfection efficiency of DNA and siRNA in fresh or lyophilized complexes, J.Control.Delivery 112, 257-270, 2006 18. Putnam, D. and Doody, A., RNA-interference effectors and their delivery, Crit.Rev.Ther.Drug.Carrier Syst. 23, 137-164, 2006 19. Didenko, V.V., Ngo, R. and Baskin, D.S., Polyethyleneimine as a transmembrane carrier of fluorescently labeled proteins and antibodies, Anal.Biochem. 344, 168-173, 2005
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General references for transfection Gene Therapy Technologies, Applications and Regulatory. From Laboratory to Clinic, ed. A. Meager, John Wiley & Sons, Ltd., Chichester, UK, 1995 DNA Transfer to Cultured Cells, ed. K.Ravid and R.I. Freshney, Wiley-Liss, New York, NY, USA, 1998 Understanding Gene Therapy, ed. N.R. Lemoine, Bios/Springer, Oxford, UK, 1999 Gene Therapy. Therapeutic Mechanisms and Strategies, ed. N.S. Templeton and D.D. Losic, Marcel Dekker, Inc., New York, NY, USA, 2000 Freshney, I.A., Culture of Animal Cells. A Manual of Basic Techniques, 4th edn., Wiley-Liss, New York, NY, 2000 Gene Therapy and Gene Delivery Systems, ed. D.V. Schoffer and W. Zhou, Springer-Verlag, Berlin, Germany, 2005
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Section V Carbohydrates
•
Introduction to Carbohydrates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 383 Natural Alditols, Inositols, Inososes, and Amino Alditols and Inosamines . . . . . . . . . . . . . . . . . . 407 Natural Acids of Carbohydrate Derivation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 417 Natural Aldoses. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 431 Natural Ketoses. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 457 Carbohydrate Phosphate Esters. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 473 The Naturally Occurring Amino Sugars. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 491 Oligosaccharides (Including Disaccharides). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 499 Mucopolysaccharides (Glycosaminoglycans). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 533
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Introduction to Carbohydrates Nomenclature for carbohydrates The reader is directed to recommendations from the International Union of Pure and Applied Chemistry (IUPAC) (1,2). The IUPAC recommendations are contained in the excellent work on carbohydrate structure by Collins (3). There are also excellent discussions of nomenclature in several reference texts (4,5). The reader is also directed to several recent articles concerning specific nomenclature issues (6-17). There are also IUPAC guidelines for glycolipids (18) as well as a separate article on the nomenclature of inositol derivatives (19). The following are some simple definitions which might be useful. The reader is directed to the formal IUPAC documents for detail. • Aldaric acids are aldoses where the aldehyde and terminal primary alcohol function are replaced by carboxylic acid residues. • Alditols are polyhydric alcohols where the aldehyde function in an aldolose or the ketone function in a ketose has been replaced by a hydroxyl function. • Aldonic acids are monosaccharides which contain an aldehyde and a carboxylic acid function. • Aldoses are monosaccharides with a terminal aldehyde function. • Aldosuloses are monosaccharides which contain an aldehyde and a ketone function. • An Amino sugar is a monosaccharide where a hydroxyl group has been replaced by an amino group such as with glucosamine. • Carbohydrates includes monosaccharides, disaccharides, oligosaccharides, and oligosaccharides or glycans. The term sugar is usually confined to carbohydrates with lower molecular weights such as monosaccharides and disaccharides. • Deoxy is a term describing a monosaccharide where an hydroxyl group has been replaced by a hydrogen. • Dialdoses are dialdehyde monosaccharides. • Disaccharide refers to a compound composed of two monosaccharide units connected by a glycosidic bond. • Furanose (from furan) designates a five-membered ring. • The term glycan is used to describe a oligosaccharide covalently bound to a protein. The N-linked glycans are frequently branched and are described a monoantennary, biantennary, triantennary, etc. • A glycerolipid is a glycolipid with one or more glycerol groups. • Glycodiuloses are diketo monosaccharides (5-keto-fructose). • A glycolipid is a compound composed of one or more monosaccharide units bound by a glycosidic linkage to a hydrophobic group such as sphingoid or ceramide. • A glycoside (glycosidic) bond joins monosaccharide units into disaccharides and oligosaccharide/polysaccharides. The glycoside bond joins the hydroxyl group on the anomeric carbon of a monosaccharide with any hydroxyl group on another monosaccharide through an acetal linkage.
• A glycoside is an acetal derivative of the cyclic form of a sugar where the hydrogen in the hemiacetal is replaced by an alkyl, aryl, or similar group. • Glycosonic acids (ketoaldonic acids) are monosaccharides which contain a ketone function and a carboxylic acid function. • A glycosphingolipid is a lipid containing at least one monosaccharide residue and either a sphingoid or ceramide. • Monosaccharide denotes a single or monomer unit without glycosidic connection to another monosaccharide. The suffix–ose is used to designate a carbohydrate (aldose) such a glucose, pentose, hexose, galactose, etc. The suffix– ulose is used to designate a ketose, which is a carbohydrate containing a ketone function. • The term oligosaccharide describes a compound composed of 10-20 monosaccharide units connected by glycosidic bonds. The length quoted here is arbitrary. Oligosaccharide is used to describe a oligomer of defined length rather than one, such as with starch or glycogen, of undefined length. • The term polysaccharide is usually used to describe a large amorphous polymer consisting of monosaccharide units connected by glycosidic bonds. • Pyranose (from pyran) designates a six-membered ring • A reducing sugar contains an aldehyde or ketone function which reacts with alkaline ferricyanide or alkaline cupric tartrate (Fehling’s solution). • Trisaccharide describes a compound composed of three monosaccharides connected by glycosidic bonds.
1. McNaught, A.D., Nomenclature of carbohydrates, Pure & Appl.Chem.
68, 1919-2006, 1996 2. McNaught, A.D., Nomenclature of carbohydrates (recommendations 1996), Adv.Carbohydr.Chem.Biochem. 52, 43-177, 1996 3. Dictionary of Carbohydrates, 2nd edn., ed. P.M. Collins, Chapman and Hall/CRC, Boca Raton, FL, 2006 4. Collins, P.M. and Ferrier, R.J., Monosaccharides Their Chemistry and Their Roles in Natural Products, Wiley, Chichester, United Kingdom, 1995 5. Glycoscience, Chemistry and Chemical Biology, ed. B.O. Fraser-Reid, K. Tatsuta, and J. Thiem, Spinger-Verlag, Berlin, Germany, 2001 6. Hitchcock, P.J., Leive, L., Makela, P.H., et al., Lipopolysaccharide nomenclature–past, present, and future, J.Bacteriol. 166, 699-705, 1986 7. Moynihan, P.J., Update on the nomenclature of carbohydrates and their dental effects, J.Dent. 26, 209-218, 1998 8. Berreau, U. and Stenutz, R., Web resources of the carbohydrate chemist, Carbohydr.Res. 339, 929-936, 2004 9. Murthy, P.P., Structure and nomenclature of inositol phosphates, phosphoinositides, and glycosylphosphatidylinositols, Subcell. Biochem. 39, 1-19, 2006 10. Chester, M.A., IUPAC-IUB joint commission on biochemical nomenclature (JCBN). Nomenclature of glycolipids–recommendations on 1997, Eur.J.Biochem. 257, 293-298, 1998 11. Roberts, M.C., Sutcliffe, J., Courvalin, P., et al., Nomenclature for macrolide and macrolide-lincosamide-streptogramin B resistance determinants, Antimicrob.Agents Chemother. 43, 2823-2830, 1999 12. Henry, S. and Moulds, J.J., Preview 2000: Proposal for a new terminology to describe carbohydrate histo-blood group antigens/glycoproteins within the ISBT terminology framework, Immunohematol. 16, 49-56, 2000
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384 13. Chai, W., Piskarev, V., and Lawson, A.M., Branching patter and sequence analysis of underivatized oligosaccharides by combined MS/MS of singly and doubly charged molecular ions in negative-ion, J.Amer.Soc.Mass Spectrom. 13, 670-679, 2002 14. Lohmann, K.K., and von der Lieth, C.W., GLYCO-FRAGMENT: A web tool to support the interpretation of mass spectra of complex carbohydrates, Proteomics 3, 2028-2035, 2003 15 Hilden, L. and Johansson, G., Recent developments on cellulases and carbohydrate-bindnig modules with cellulose affinity, Biotechnol.Lett. 26, 1683-1693, 2004 16. Morelle, W. and Michalski, J.C., Glycomics and mass spectrometry, Curr.Pharm.Des. 11, 2615-2645, 2005 17. Meitei, N.S., and Banerjee, S., Interpretation support for multistage MS: a mathematical method for theoretical generation of glycan fragments and calculation of their masses, Proteomics 7, 2530-2540, 2007 18. Chester, M.A., Nomenclature for glycolipids, Adv.Carbohydr.Chem. Biochem. 55, 312-326, 1999 19. Murthy, P.P., Structure and nomenclature of inositol phosphates, phosphoinositides, and glycosylphosphatidylinositols, Subcell. Biochem. 39, 1-19, 2006
General references to carbohydrate chemistry • The Amino Sugars. The Chemistry and Biology of Compounds Containing Amino Sugars, ed. R.W. Jeanloz, Academic Press, New York, NY, USA, 1969 • The Carbohydrates Chemistry and Biochemistry, 2nd edn., ed. W. Pigman and D. Horton, Academic Press, New York, NY, USA, 1970 • Carbohydrates, ed. P.M. Collins, Chapman & Hall, London, United Kingdom, 1987 • Carbohydrate Chemistry, ed. J.F. Kennedy, Oxford, Clarendon Press, Oxford, United Kingdom, 1988 • CRC Handbook of Chromatography, Vol II, Carbohydrates, ed. S.C. Churms and J. Sherma, CRC Press, Boca Raton, FL, USA, 1991 • Collins, P.M. and Ferrier, R.J., Monosaccharides. Their Chemistry and Their Role in Natural Products, Wiley, Chichester, United Kingdom, 1995 • McNaught, A.D., Nomenclature of carbohydrates, Pure & Appl.Chem. 68, 1919-2006, 1996 • Lehman, J., Carbohydrates Structure and Biology, Thieme, Stuttgart, Germany, 1998 • Carbohydrate Biotechnology Protocols, ed. C. Burke, Humana Press, Totowa, NJ, USA, 1999 • Carbohydrates Structure, Syntheses, and Dynamics, ed. D. Finch, Kluwer Academic, Dordrecht, Netherlands, 1999 • Osborn, H. and Khan, T., Oligosaccharides Their Synthesis and Biological Roles, Oxford University Press, Oxford, UK, 2000 • Carbohydrates in Chemistry and Biology, ed. B. Ernst, G.A. Hart, and P. Sinay, Wiley-VCH, Weinheim, Germany, 2000 • Glycoscience, Chemistry and Chemical Biology, ed. B.O. Fraser-Reid, K. Tatsuta, and J. Thiem, Springer-Verlag, Berlin, Germany, 2001 • Vogel, P., De novo synthesis of monosaccharides, in Glycoscience, Chemistry and Chemical Biology, ed.B.O. Fraser-Reid, K. Tatsuta, and J. Thiem, Springer-Verlag, Berlin, Germany, 2001 • Fernandez-Bolaños, J.G. Al-Masaudi, N.A., and Mann, I., Sugar derivatives having a sulfur in the ring, Adv. Carbohydr.Chem.Biochem. 57, 21-98, 2001
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• Casu, B. and Lindahl, U., Structure and biological interactions of heparin and heparin sulfate, Adv.Carbohydr.Chem. Biochem. 57, 159-206, 2001 • Ferrier, R.J. and Hoberg, J.O., Synthesis and reactions of unsaturated sugars, Adv.Carbohydr.Chem.Biochem. 58, 55-119, 2003 • Černý, M., Chemistry of anhydro sugars, Adv.Carbohydr. Chem.Biochem. 58, 121-198, 2003 • NMR Spectroscopy of Glycoconjugates, ed. J. JiménezBarbero and T. Peters, Wiley-VCH, Weinheim, Germany, 2003 • Capillary Electrophoresis of Carbohydrates, ed. P. Thibault and S. Honda, Humana Press, Totowa, NJ 2003 • Chemistry and Biology of Hyaluron, ed. H.C. Garg and C.A. Hales, Elsevier, Amsterdam, Netherlands, 2004 • Tomasik, P. and Schilling, C.H., Chemical modification of starch, Adv.Carbohydr.Chem.Biochem. 59, 1760-403, 2004 • de Lederkremer, R.M. and Gallo-Rodriguez, C., Naturally occurring monosaccharides: properties and synthesis, Adv. Carbohydr.Chem.Biochem. 59, 9-67, 2004 • Handbook of Carbohydrate Engineering, ed. K.J. Yarema, CRC/Taylor & Francis, Boca Raton, FL, USA, 2005 • Chemistry of Polysaccharides, ed. G.E. Zaikov, VSP Brill, Leiden, Netherlands, 2005 • Polysaccharides for Drug Delivery and Pharmaceutical Applications, ed. R.H. Marchessault, F. Ravenelle, and X.X. Zho, American Chemical Society, Washington, DC, USA, 2006 • Dictionary of Carbohydrates, 2nd edn., ed. P.M. Collins, Chapman and Hall/CRC, Boca Raton, FL, 2006
Recent references for selected monosaccharides Structures for these carbohydrates are available on p. 390
Aldaric acids
• Fonseca, A., Utilization of tartaric acid and related compounds by yeasts: taxonomic implications, Can.J.Microbiol. 38, 1242-1251, 1992 • Talemaka. M., Yan, X., Ono, H., et al., Caffeic acid derivatives in the roots of yacon (Smallanthus sonchifolius), J.Agric.Food Chem. 51, 793-796, 2003 • Lakatos, A., Bertani, R., Kiss, T., et al., Al(III) ion complexes of saccharic acid and mucic acid: a solution and solid-state study, Chemistry 10, 1281-1290, 2004 • Dornyei, A., Garribba, E., Jakusch, T., et al., Vanadium (IV,V) complexes of D-saccharic and mucic acids in aqueous solution, Dalton Trans.June 21(12), 1882-1892, 2004 • Gonzalez, J.C., Daier, V., Garcia, S., et al., Redox and complexation chemistry of the Cr(VI)/Cr(V)-D-galacturonic acid system, Dalton Trans. Augtust 7(15), 2288-2296, 2004
Aldonic acids
• Limberg, G., Klaffke, W., and Thiem, J., Conversion of aldonic acids to their corresponding 2-keto-3-deoxy analogs by the non-carbohydrate enzyme dihydroxy acid dehydratase (DHAD), Bioorg.Med.Chem. 3, 487, 494, 1995 • Yang, B.Y. and Montgomery, R., Oxidation of lactose with bromine, Carbohydr.Res. 340, 2698-2705, 2005
Allose altrose allulose
• Kemp, M.B. and Quayle, J.R., Microbial growth on C1 compounds. Incorporation of C1 units into allulose phosphate
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Introduction to Carbohydrates by extracts of Pseudomonas methanica, Biochem.J. 99, 41-48, 1966 • Hough, L. and Stacey, B.E., Biosynthesis of allitol and D-allulose in Itea plants–incorporation of 14CO2, Phytochemistry 5, 215-222, 1966 • Cree, G.M. and Perlin, A.S., O-Isopropylidene derivatives of D-allulose (D-psicose) and D-erythro-hexopyranos-2,3diulose, Canad.J.Biochem. 46, 765-770, 1968 • Poulson, T.S., Chang, Y.Y., and Hove-Jensen, B., D-Allose catabolism of Escherichia coli: Involvement of alsI and regulation of als regulon expression by allose and ribose, J.Bacteriol. 181, 7126-7130, 1999
Arabinose
• Quiocho, F.A., Molecular features and basic understanding of protein-carbohydrate interactions: the arabinose-binding protein-sugar complex, Curr.Top.Microbiol.Immunol. 139, 135-148, 1988 • Sultana, I., Mizanur, R.M., Takeshita, K., et al., Direct production of D-arabinose from D-xylose by a coupling reaction using D-xylose isomerase, D-tagatose 3-epimerase and D-arabinose isomerase, J.Biosci.Bioeng. 95, 342-347, 2003 • Kim, P., Current studies on biological tagatose production using L-arabinose isomerase: a review and future perspective, Appl.Microbiol.Biotechnol. 65, 243-249, 2004 • Wang, H. and Ng, T.B., First report of an arabinose-specific fungal lectin, Biochem.Biophys.Res.Commun. 337, 621-625, 2005 • Pramod, S.N. and Venkatesh, Y.P., Utility of pentose colorimetric assay for the purification of potato lectin, an arabinoserich glycoprotein, Glycoconj.J. 23, 481-488, 2006 • Bercier, A., Plantier-Royon, R., and Portella, C., Convenient conversion of wheat hemicelluloses pentoses (d-xylose and l-arabinose) into a common intermediate, Carbohydr.Res. 342, 2450-2455, 2007
Arabitol (arabinitol)
• de Repentigny, L. and Reiss, E., Current trends in immunodiagnosis of candidiasis and aspergillosis, Rev.Infect.Dis. 6, 301-312, 1984 • Christensson, B., Sigmundsdottir, G., and Larsson, L., D-Arabinitol–a marker for invasive candidiasis, Med. Mycol. 37, 391-396, 1999 • Mohan, S. and Pinto, B.M., Zwitterionic glycosidase inhibitors: salacinol and related analogues, Carbohydr.Res. 342, 1551-1580, 2007
Arabonic acid
• Jahn, M., Baynes, J.W., and Spiteller, G., The reation of hyaluronic acid and its monomers, glucuronic acid and N-acetylglucosamine, with reactive oxygen species, Carbohydr.Res. 321, 228-234, 1999 • Soroka, N.V., Kulminskaya, A.A., Eneyskaya, E.V., et al., Synthesis of arabinitol 1-phosphate and its use for characterization of arabinitol-phosphate dehydrogenase, Carbohydr.Res. 340, 539-546, 2005 • Nunes, F.M., Reis, A., Domingues, M.R., and Coimbra, M.A., Characterization of galactomannan derivatives in roasted coffee beverages, J.Agric.Food Chem. 54, 34283439, 2006
Butanetetrol
• Sakamoto, I., Ichimura, K., and Ohrui, H., Synthesis of 2-C-methyl-D-erythritol and 2-C-methyl-L-threitol; deter-
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385 mination of the absolute configuration of 2-C-methyl1,2,3,4-butanetetrol isolated, Biosci.Biotechnol.Biochem. 64, 1915-1922, 2000 • Romero, C.M., Lozano, J.M., Sancho, J., and Giraldo, G.I., Thermal stability of b-lactoglobulin in the presence of aqueous solution of alcohols and polyols, Int,J.Biol. Macromol. 40, 423-428, 2007
Conduritol
• Quaronine, A., Gershon, E., and Semenza, G., Affinity labeling of the active sites in the sucrase-isomaltase complex from small intestine, J.Biol.Chem. 249, 6424-6433, 1974 • Kwon, Y.U. and Chung, S.K., Facile synthetic routes to all possible enantiomeric pairs of conduritol stereoisomers via efficient enzymatic resolution of conduritol B and C derivatives, Org.Lett. 3, 3013-3016, 2001 • Freeman, S., Hudlicky, T., New oligomers of conduritol-F and mucoinositol. Synthesis and biological evaluation as glycosidase inhibitors, Bioorg.Med.Chem.Lett. 14, 12091212, 2004 • Cere, V., Minzoni, M., Pollicino, S., et al., A general procedure of the synthesis of stereochemically pure conduritol derivatives practical also for solid-phase chemistry, J.Comb.Chem. 8, 74-78, 2006
Dialdoloses
• Thealander, O., Acids and other oxidation products, in The Carbohydrates Chemistry and Biology. 2nd edn., ed. W.Pigman and D. Horton, Academic Press, New York, NY, USA Chapter 23, pps. 1013-1100, 1980 • Green, J.W., Oxidative reactions and degradations, in The Carbohydrates Chemistry and Biology. 2nd edn., ed. W.Pigman and D. Horton, Academic Press, New York, NY, USA Chapter 24, pps. 1101-1166, 1980 • Avigad, G., Amorel, D., Asensio, C., and Horecker, B.L., The D-galactose oxidase of Polyporus circinatus, J.Biol.Chem. 237, 2736-2743, 1962
Fructose
• Benkovic, S.J. and deMaine, M.M., Mechanism of action of fructose 1,6-bisphosphatase, Adv.Enzymol.Relat.Areas Mol.Biol. 53, 45-82, 1982 • Hers, H.G., The discovery and the biological role of fructose 2,6-bisphosphate, Biochem.Soc.Trans. 12, 729-735, 1984 • Hanover, L.M. and White, J.S., Manufacturing, composition, and applications of fructose, Am.J.Clin.Nutr. 58 (5 suppl), 724S-732S, 1993 • Schalkwijk, C.G., Stehouwer, C.D., and van Hinsbergh, V.W., Fructose-mediated non-enzymatic glycation: sweet coupling or bad modification, Diabetes Metab.Res.Rev. 20, 369-382, 2004 • Reddy, M.R. and Erion, M.D., Computer-aided drug design strategies used in the discovery of fructose 1,6-bisphosphatase inhibitors, Curr.Pharm.Des. 11, 283-294, 2005
Galactose
• Mann, B.J., Mirelman, D., and Petri, W.A., Jr., The D-galactose-inhibitable lectin of Entamoeba histolytica, Carbohydr.Res. 213, 331-338, 1991 • McPherson, M.J., Stevens, C., Baron, A.J., et al., Galactose oxidase: molecular analysis and mutagenesis studies, Biochem.Soc.Trans. 21, 752-756, 1993
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386 • Halcrow, M., Phillips, S., and Knowles, P., Amine oxidases and galactose oxidase, Subcell.Biochem. 35, 183-231, 2000 • Whittaker, J.W., Galactose oxidase, Adv.Protein Chem. 60, 1-49, 2002 • Whittakcr, J.W., The radical chemistry of galactose oxidase, Arch.Biochem.Biophys. 433, 227-239, 2005 • Cho, C.S., Seo, S.J., Park, I.K., et al., Galactose-carrying polymers as extracellular matrices for liver tissue engineering, Biomaterials 27, 576-585, 2006
Glucitol (sorbitol)
• Jeffery, J. and Jornvall, H., Sorbitol dehydrogenase, Adv. Enzymol.Relat.Areas Mol.Biol. 61, 47-106, 1988 • Suarez, G., Nonezymatic browning of proteins and the sorbitol pathway, Prog.Clin.Biol.Res. 304, 14-162, 1989 • Silveira, M.M. and Jonas, R., The biotechnological production of sorbitol, Appl.Microbiol.Biotechnol. 59, 400-408, 2002 • Dworacka, M., Winiarska, H., Szymanska, M., et al., 1,5-anhydro-D-glucitol: a novel marker of glucose excursions, Int.J.Clin.Pract.Suppl.I July(129), 40-44, 2002 • El-Kabbani, O., Darmanin, C., and Chung, R.P., Sorbitol dehydrogenase: structure, function and ligand design, Curr.Med.Chem. 11, 365-476, 2004 • Jonas, R. and Silveira, M.M., Sorbitol can be produced not only chemically but also biotechnologically, Appl.Biochem. Biotechnol. 1991, 321-336, 2004 • Obrosova, I.G., Increased sorbitol pathway activity generates oxidative stress in tissue sites for diabetic complications, Antioxid.Redox.Signal. 7, 1543-1552, 2005 • Yardimci, H. and Leheny, R.L., Aging of the JohariGoldstein relaxation in the glass-forming liquids sorbitol and xylitol, J.Chem.Phys. 124, 214503, 2006 • Pedruzzi, I., Malvessi, E., Mata, V.G., et al., Quantification of lactobionic acid and sorbitol from enzymatic reaction of fructose and lactose by high-performance liquid chromatography, J.Chromatog. A 1145, 128-132, 2007 • Shah, P.P. and Roberts, C.J., Molecular solvation in watermethanol and water-sorbitol mixtures: the roles of preferential hydration, hydrophobicity, and the equation of state, J.Phys.Chem.B. 111, 4467-4476, 2007 • Jungo, C., Schenk, J., Pasquier, M., et al., A quantitative analysis of the benefits of mixed feeds of sorbitol and methanol for the production of recombinant avidin with Pichia pastoris, J.Biotechnol. 131, 57-66, 2007
Glucose
• Fraser, C.G., Analytical goals for glucose analyses, Ann. Clin.Biochem. 23, 379-389, 1986 • Carruthers, A., Facilitated diffusion of glucose, Physiol.Rev. 70, 1135-1176, 1990 • James, D.E. Piper, R.C., and Slot, J.W., Targeting of mammalian glucose transporters, J.Cell.Sci. 104, 607-612, 1993 • Mueckler, M., Facilitative glucose transporters, Eur. J. Biochem. 219, 713-725, 1994 • Bhosale, S.H., Rao, M.B., and Deshpande, V.V., Molecular and industrial aspects of glucose isomerase, Microbiol.Rev. 60, 280-300, 1996 • Schmidt, K.C., Lucignani, G., and Sokoloff, L., Fluorine18-fluorodeoxyglucose PET to determine regional cerebral glucose utilization: a re-examination, J.Nucl.Med. 37, 394399, 1996
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Handbook of Biochemistry and Molecular Biology • Pischetsrieder, M., Chemistry of glucose and biochemical pathways for biological interest, Perit.Dial.Int. 20(suppl 2), S26-S30, 2000 • Bonnefont-Ruousselot, D., Glucose and reactive oxygen species, Curr.Opin.Clin.Nutr.Metab.Care 5, 510568, 2002 • Anthony, C., The quinoprotein dehydrogenases for methanol and glucose, Arch.Biochem.Biophys. 428, 2-9, 2004 • Fang, H., Kaur, G., and Wang, B., Progress in boronic acidbased fluorescent glucose sensors, J.Fluoresc. 14, 481-489, 2004
Glucose oxidation
• Merbouh, N., Francois Thaburet, J., Ibert, M., et al., Facile nitroxide-mediated oxidations of D-glucose to D-glucaric acid, Carbohydr.Res. 336, 75-78, 2001 • Ibert, M., Marsais, F., Merbouh, N., and Bruckner, C., Determination of the side-products formed during the nitroxide-mediated bleach oxidation of glucose to glucaric acid, Carbohydr.Res. 337, 1059-1063, 2002 • Fischer, K. and Bipp, H.P., Generation of organic acids and monosaccharides by hydrolytic and oxidative transformation of food processing residues, Biosour.Technol. 96, 831842, 2005
Glucuronic acid
• Glucuronic Acid Free and Combined, Chemistry, Biochemistry, Pharmacology, and Medicine, ed. G.J. Dutton, Academic Press, New York, NY, USA, 1966 • Clarke, D.J.. and Buschell, B., The uridine diphosphate glucuronosyltransferase multigene family: Function and regulation, in Conjugation-Deconjugation Reactions in Drug Metabolism and Toxicity, ed. F.C. Kauffman, SpingerVerlag, Berlin, Germany, 1994 • Bedford, C.G., Glucuronic acid conjugates, J.Chromatogr. B.Biomed.Sci.Appl. 717 313-326, 1998 • Durham, T.B. and Miller, M.J., Conversion of glucuronic acid glycosides to novel bicyclic beta-lactams, Org.Lett. 4, 135-138, 2002 • Pitt, N., Duane, R.M., O’Brien, A., et a., Synthesis of a glucuronic acid and glucose conjugate library and evaluation of effects on endothelial cell growth, Carbohydr.Res. 339, 1873-1887, 2004 • Engstrom, K.M., Daanen, J.F., Wagaw, S., and Stewart, A.O., Gram scale synthesis of the glucuronide metabolite of ABT-724, J.Org.Chem. 71, 8278-8383, 2006 • Sorich, M.J., McKinnon, R.A., Miners, J.O., and Smith, P.A., The importance of local chemical structure for chemical metabolism by human uridine-5’-diphosphate-glucuronosyltransferase, J.Chem.Inf.Model. 46, 2692-2697, 2006 • Jantti, S.E., Kiriazis, A., Reinila, R.R., et al., Enzymeassistance and characterization of glucuronide conjugates of neuroactive steroids, Steroids 72, 287-296, 2007
Glycodiuloses
• Avigad, G., and Englard, S., 5-Ketofructose I. Chemical characterization and analytical determination of the dicarbonylhexose produced by Gluconobacter cerinus, J.Biol. Chem. 240, 2290-2296, 1965 • Yamada, Y., Aida, K., Uemura, T., Enzymatic studies on oxidation of sugar and sugar alcohols. 2. Purification and properties of NADPH-linked 5-ketofructose reductase, J.Biochem. 61, 803-811, 1967
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Introduction to Carbohydrates
387
• Schrimsher, J.L., Wingfield, P.T., Bernard, A., et al., Purification and characterization of 5-ketofructose reductase from Erwinia citreus, Biochem.J. 253, 511-516, 1988 • White, R.H., and Xu, H.M., Methylglyoxal is an intermediate in the biosynthesis of 6-deoxy-5-ketofructose-1-phosphate: A precursor for aromatic amino acid biosynthesis in Methanocaldococcus jannaschii, Biochemstry 45, 1235512379, 2006
• Johnson, L.N., Acharya, K.R., Jordon, M.D., and McLaughlin, P.J., Refined crystal structures of the phosphorylase-heptulose-2-phosphate-oligosaccharide-AMPcomplex, J.Mol.Biol. 211, 645-661, 1990 • Gomez, R.V., Kolender, A.A., and Valero, O., Synthesis of polyhydroxyl amino acids based on D- an L-alanine from D-glycero-D-gulo-heptone-1,4 lactone, Carbohydr.Res.341, 1498-1504, 2006
Lactone formation
Glycosonic Acids
• Truesdell, S.J., Sims, J.C., Boerman, P.A., et al., Pathways for metabolism of ketoaldonic acids in an Erwinia sp., J.Bacteriol. 173, 6651-6666, 1991
Glycosuloses
• Fry, S.C., Dumville, J.C., and Miller, J.G., Fingerprinting of polysaccharides attacked by hydroxyl radicals in vitro and in the cell wall of ripening pear fruit, Biochem.J. 357, 727737, 2001 • Baker N, Egyud LG. 3-Deoxy-D-glucosulose in fed and fasted mouse livers. Biochim. Biophys. Acta, 1968 Sep 3;165(2):293-6
Heptoses and heptose derivatives
• Adams, G.A., Quadling, C., and Perry, M.B., D-glyceroD-manno-hepose as a component of lipopolysaccharides from gram negative bacteria, Canad.J.Microbiol. 13, 16051613, 1967 • Leshem, B., Sharoni, Y., and Dimant, E., The hyperglycemic effect of 1-deoxy-D-manno-heptulose. Inhibition of hexokinase, glucokinase, and insulin release in vitro, Canad.J.Biochem. 52, 1078-1081, 1974 • Shaw, P.E., Wilson, C.W., III, and Knight, R.J., Jr., Highperformance liquid chromatographic analysis of D-mannoheptulose, perseitol, glucose, and fructose in avocado cultivers, J.Agric.Food Chem. 28, 379-382, 1980 • Penner, J.L., and Aspinall, G.O., Diversity of lipopolysaccharide saccharides from gram-negative bacteria, J.Infect. Dis. 176(Suppl 2), S135-S138, 1997 • Müller-Loennies, S., Brade, L., and Brade, H., Neutralizing and cross-reactive antibodies against enterobacterial lipopolysaccharides, Int.J.Med.Microbiol. 297, 321-340, 2007
• de Lederkremer, R.M. and Marino, C., Acids and other products of the oxidation of sugars, Adv.Carbohydr.Chem. Biochem. 58, 199-306, 2003
Octuloses
• Weiser, W., Lehmann, J., Chiba, S., et al., Steric course of the hydration of D-gluco-octenitol catalyzed by a-glucosidase and by trehalose, Biochemistry 27, 2294-2300, 1988 • Williams, J.F., Clark, M.G., and Arora, K.K., 14C labelling of octulose bisphosphates by L-type pentose pathway reactions in liver in situ and in vitro, Biochem.Int. 11, 97-106, 1985 • Williams, J.F. and MacLeod, J.K., The metabolic significance of octulose phosphates in the photosynthetic carbon reduction cycle in spinach, Photosynth.Res. 90, 125-148, 2006
Shikimic acid
• Davis, B.D., Aromatic biosynthesis. I. The role of shikimic acid, J.Biol.Chem. 191, 315-325, 1951 • Yoshida, S., and Hasegawa, M., A micro-colorimetric method for the determination of shikimic acid, Arch. Biochem.Biophys. 70, 377-381, 1957 • Cox, G.B. and Gibson, F., The role of shikimic acid in the biosynthesis of vitamin K2, Biochem.J. 100, 1-6, 1966 • Stavric, B. and Stoltz, D.R., Shikimic acid, Food Cosmet. Toxicol. 14, 141-145, 1976 • Kramer, M., Bongaerts, J., Bovenberg, R., et al., Metabolic engineering for microbial production of shikimic acid, Metab.Eng. 5, 277-283, 2003
Validatol
• Horri, S., Iwasa, T., Mizuta, E., and Kameda, Y., Studies on validamycins, new antibiotics. VI. Valdiamine, hydroxyvalidamine and validatol, new cyclitols, J.Antibiot. 24, 59-63, 1971
SOME TRIVIAL NAMES FOR MONOSACCHARIDESa Allose (All) Altrose (Alt) Erythrose Erythrulose Fructose (Fru) Galactosamine (GalN) Galactose (Gal) Glucose (Glu) Glyceraldehyde Glycerol (Gro) Gulose (Gul) Idose (Ido) Lyxose (Lyx) a
9168_Book.indb 387
allo-hexose altro-hexose erythro-tetrose glycero-tetrulose arabino-hex-2-ulose 2-amino-2-deoxygalactose galacto-hexose gluco-hexose 2,3-dihydroxy-propanal 1,2,3-propanetriol gulo-hexose ido-hexose lyxo-hexose
Mannosamine (ManN) Mannose (Man) Psicose (Psi) Quinovose (Qui) Rhamnosamine (RhaN) Rhamnose (Rha) Ribose (Rib) Ribulose (Rul) Sorbose (Sor) Tagatose (Tag) Talose (Tal) Tartaric acid Xylose (Xyl)
2-amino-2-deoxy-mannose manno-hexose ribo-hex-2-ulose 6-deoxyglucose 2-amino-2,6-dideoxymannose 6-deoxymannose ribo-pentose erythro-pent-2-ulose xylo-hex-2-ulose lyxo-hex-2-ulose talo-hexose Erythraric/Threaric Acid xylo-pentose
Adapted from McNaught, A.D., Nomenclature of carbohydrates, IUPAC Pure & Applied Chemistry 68, 1919-2008, 1996
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Selected references for disaccharides Structures for these carbohydrates are available on p.390.
Cellobiose
• Camevascini, G., A cellulose assay coupled to cellobiose dehydrogenase, Anal.Biochem. 147, 419-427, 1985 • Ichikawa, Y., Ichikawa, R., and Kuzuhara, H., Synthesis, from cellobiose, of a trisaccharide closely related to the GlNAc - - -GlcA - - - - -GlcN segment of the antithrombin-binding sequence of heparin, Carbohydr.Res. 141, 273-282, 1985 • Ali, S.A., Eary, J.F., Warren, S.D., et al., Synthesis and radioiodination of tyramine cellobiose for labeling monoclonal antibodies, Int.J.Rad.Appl.Instrum.B. 15, 557-561, 1988 • Tewari, Y.B., and Goldberg, R.N., Thermodynamics of hydrolysis of disaccharides, cellobiose, gentiobiose, isomaltose, and maltose, J.Biol.Chem. 264, 3966-3971, 1989 • Reizer, J., Reizer, A., and Saier, M.H., Jr,. The cellobiose permease of Escherichia coli consists of three proteins and is homologous to the lactose permease of Staphylococcus aureus, Res.Microbiol. 141, 1061-1067, 1990 • Kremer, S.M. and Wood, P.M., Production of Fenton’s reagent by cellobiose oxidase from cellulolytic cultures of Phanerochaete chrysoporium, Eur.J.Biochem. 208, 807-814, 1992 • Henriksson, G., Johansson, G., and Pettersson, G., A critical review of cellobiose dehydrogenases, J.Biotechnol. 78, 91-113, 2000 • Maeda, A., Kataoka, H., Adachi, S., and Matsuno, R., Transformation of cellubiose to 3-ketocellobiose by the EDTA-treated Agrobacterium tumefaciens cells, J.Biosci. Bioeng. 95, 608-611, 2003 • Mason, M.G., Nicholis, P., and Wilson, M.T., Rotting by radicals - the role of cellobiose oxidoreductase?, Biochem. Soc.Trans. 31, 1335-1336, 2003 • Koto, S., Hirooka, M., Tashiro, T., et al., Simple preparations of alkyl and cycloalkyl a-glycosidases of maltose, cellobiose, and lactose, Carbohydr.Res. 339, 2415-2424, 2004 • Stoica, L., Ludwig, R., Haltrich, D., and Gorton, L., Thirdgeneration biosensor for lactose based on newly discovered cellobiose dehydrogenase, Anal.Chem. 78, 393-398, 2006 • Zamocky, M., Ludwig, R., Peterbauer, C., et al., Cellobiose dehydrogenase - a flavocytochrome from wood-degrading, phytopathogenic and saprotropic fungi, Curr.Protein Pept. Sci. 7. 255-280, 2006
Isomaltose
• Dowd, M.K., Reilly, P.J., and French, A.D., Relaxed-residue conformational mapping of the three linkage bonds of isomaltose and gentiobiose with MM3 (92), Biopolymers 34, 625-638, 1994 • Brand, D.A., Lium H.S., and Zhu, Z.S., The dependence of glucan conformation dynamics on linkage position and stereochemistry, Carbohydr.Res. 278, 11-26, 1995 • Vetere, A., Gamini, A., Campa, C., and Paoletti, S., Regiospecific transglycolytic synthesis and structural characterization of 6-O-glucopyranosyl-glucopyranose (isomaltose), Biochem.Biophys.Res.Commun. 274, 99-104, 2000 • Hendrix, D.L. and Salvucci, M.E., Isobemisiose: an unusual trisaccharide abundant in the silverleaf whitefly, Bemisia argentifolii, J.Insect Physiol. 47, 423-432, 2001 • Streigel, A.M., Anomeric configuration, glycosidic linkage, and the solution conformational entropy of O-linked disaccharides, J.Am.Chem.Soc. 125, 4146-4148, 2003
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• Kanou, M., Nakanishi, K., Hashimoto, A., and Kameoka, T., Influence of monosaccharides and its glycosidic linkage on infrared spectral characteristics of disaccharides in aqueous solutions, Appl.Spectrosc. 59, 885-892, 2005 • Cai, Y., Liu, J., Shi, Y., et al., Determination of several sugars in serum by high-performance anion-exchange chromatography with pulsed amperometric detection, J.Chromatog.A. 1085, 98-103, 2005 • Pereira, C.S., Kony, D., Baron, R., et al., Conformational and dynamical properties of disaccharides in water: a molecular dynamics study, Biophys.J. 90, 4337-4344, 2006 • Mikami, B., Iwamoto, N., Malle, D., et al., Crystal structure of pullulanase: evidence for parallel binding of oligosaccharides in the active site, J.Mol.Biol. 359, 690-707, 2006 • Maruta, K., Watanabe, H., Nishimoto, T., et al., Acceptor specificity of trehalose phosphorylase from Thermoanaerobacter brockii: production of novel nonreducing trisaccharide, 6-O-a-galactopyranosyl trehalose, J.Biosci.Bioeng. 101, 385-390, 2006
Isomaltulose
• Bucke, C., Carbohydrate transformation by immobilized cells, Biochem.Soc.Symp. 48, 25-38, 1983 • Takazoe, I., Frostell, G., Ohta, K., et al., Palatinose - a sucrose substitute. Pilot studies, Swed.Dent.J. 9, 81-87, 1985 • Thompson, J., Robrish, S.A., Pikis, A., et al., Phosphorylation and metabolism of sucrose and its five linkage-isomeric a-D-glucosyl-D-fructoses by Klebsiella pneumoniae, Carbohydr.Res. 331, 149-161, 2001 • Lina, B.A., Jonker, D., and Kozianowski, G., Isomaltulose (Palatinose): a review of biological and toxicological studies, Food Chem.Toxicol. 40, 1375-1381, 2002 • Zhang, D. Li, N., Swaminathan, K., and Zhang, L.H., A motif rich in charged residues determines product specificity in isomaltulose synthase, FEBS Lett. 534, 151-155, 2003 • Ahn, S.J., Yoo, J.H., Lee, M.C., et al., Enhanced conversion of sucrose to isomaltulose by a mutant of Erwinia rhapontici, Biotechnol.Lett. 25, 1179-1183, 2003 • Kawaguti, H.Y., Buzzato, M.F., and Sato, H.H., Isomaltulose production using free cells: optimization of a culture medium containing agricultural wastes and conversion in repeatedbatch processes, J.Ind.Microbiol.Biotechnol. 34, 261-269, 2007 • Park, S.E., Cho, M.B., Lim, J.K., et al., A new colorimetric method for determining the isomerization activity of sucrose isomerase, Biosci.Biotechnol.Biochem. 71, 583-586, 2007 • Achten, J., Jentjens, R.L., Brouns, F., and Jeukendrup, A.E., Exogenous oxidation of isomaltulose is lower than that of sucrose during exercise in men, J.Nutr. 137, 1143-1148, 2007
Lactose
• Yang, S.T. and Silva, E.M., Novel products and new technologies for use of a familiar carbohydrate, milk lactose, J.Dairy Sci. 78, 2541-2562, 1995 • Sahin-Toth, M., Dunten, R.L., and Kaback, H.R., The lactose permease of Escherichia coli: a paradigm for membrane transport proteins, Soc.Gen.Physiol.Ser. 48, 1-9, 1993 • Ramakrishnan, B., Boeggeman, E., and Qasba, P.K., b-1,4Galactosyltransferase and lactose synthase: molecular mechanical devices, Biochem.Biophys.Res.Commun. 291, 1113-1118, 2002 • Adam, A.C., Rubio-Texeira, M., and Poaina, J., Lactose: the milk sugar from a biotechnological perspective, Crit.Rev. Food Sci.Nutr. 44, 553-557, 2004
4/16/10 1:20 PM
Introduction to Carbohydrates • Guan, L. and Kaback, H.R., Lessons from lactose permease, Annu.Rev.Biophys.Biomol.Struct. 35, 67-91, 2006 • Pedruzzi, I., Malvessi, E., Mata, V.G., et al., Quantification of lactobionic acid and sorbitol from enzymatic reaction of fructose and lactose by high-performance liquid chromatography, J.Chromatog.A. 1145, 128-132, 2007 • Jurs, S. and Thiem, J., From lactose towards a novel galactosylated cyclooctenone, Carbohydr.Res. 342, 1238-1243, 2007 • Meltretter, J., Seeber, S., Humeny, A., et al., Site-specific formation of Maillard, oxidation, and condensation products from whey proteins during reaction with lactose, J.Agric.Food Chem. 55, 6096-6103, 2007 • Higl, B., Kurtmann, L., Carlsen, C.U., et al., Impact of water activity, temperature, and physical state on the storage stability of Lactobacillus paracasei ssp. paracasei freeze-dried in a lactose matrix, Biotechnol.Prog. 23, 794-800, 2007
Maltose
• 1H NMR studies of maltose, maltoheptaose, a-,b-, and g-cyclodextrins, and complexes in aqueous solutions with hydroxyl protons as structural probes, J.Org.Chem. 68, 1671-1678, 2003 • Hernandez-Luis, F., Amado-Gonzalez, E., and Esteso, M.A., Activity coefficients of NaCl in trehalose-water and maltosewater at 298.15 K, Carbohydr.Res. 388, 1415-1424, 2003 • Kawakami, K. and Ida, Y., Direct observation of the enthalpy relaxation and the recovery processes of maltosebased amorphous formulation by isothermal microcalorimetry, Pharm.Res. 20, 1430-1436, 2003 • Lourdin, D., Colonna, P., and Ring, S.G., Volumetric behavior of maltose-water, maltose-glycerol and starch-sorbitolwater systems mixtures in relation to structural relaxation, Carbohydr.Res. 338, 2883-2887, 2003 • Haghight, K.S., Imura, Y., Oomori, T., et al,. Decomposition kinetics of maltose in subcritical water, Biosci.Biotechnol. Biochem. 68, 91-95, 2004 • Mundt, S. and Wedzicha, B.L., Comparative study of the composition of melanodins, from glucose and maltose, J.Agric.Food Chem. 52, 4256-4260, 2004 • Weise, S.E., Kim, K.S., Stewart, R.P., and Sharkey, T.D., b-Maltose is the metabolically active anomer of maltose during transitory starch degradation, Plant Physiol. 137, 756-761, 2005 • Mundt, S. and Wedzicha, B.L., Role of glucose in the Maillard browning of maltose and glycine: a radiochemical approach, J.Agric.Food Chem. 53, 6798-6803, 2005 • Rodriguez, S., Lona, L.M., and Franco, T.T., The effect of maltose on dextran yield and molecular weight distribution,. Bioprocess Biosyst.Eng. 28, 9-14, 2005 • Shirke, S. and Ludescher, R.D., Dynamic site heterogeneity in amorphous maltose and mannitol from spectral heterogeneity in erythrosine B phosphorescence, Carbohydr.Res. 340, 2661-2669, 2005 • Noel, T.R., Parker, R., Brownsely ,G.J., et al., Physical aging of starch, maltodexin, and maltose, J.Agric.Food Chem. 53, 8580-8585, 2005 • Sanz, M.L., Cote, G.L., Gibson, G.R., and Rastall, R.A., Influence of glycosidic linkages and molecular weight on the fermentation of maltose-based oligosaccharides by human gut bacteria, J.Agric.Food Chem. 54, 9779-9784, 2006 • Jarusiewicz, J.A., Sherma, J., and Fried, B., Thin layer chromatographic analysis of glucose and maltose in estivated Biomphalaria glabrata snails and those infected with Schistomsoma mansoni, Comp.Biochem.Physiol.B.Biochem. Mol.Biol. 145, 346-349, 2006
9168_Book.indb 389
389 Sucrose
• Kitts, D.D., Wu, C.H., Kopec, A. and Nagasawa, T. Chemistry and genotoxicity of caramelized sucrose, Mol. Nutr.Food Res. 50, 1180-1190, 2006 • Dangaran, K.L. and Krochta, J.M., Kinetics of sucrose crystallization in whey protein films, J.Agric.Food Chem. 54, 7152-7158, 2006 • Blanshard, J.M., Muhr, A.H., and Gough, A., Crystallization from concentrated sucrose solutions, Adv.Exp.Med.Biol. 302, 639-655, 1991 • Edye, L.A. and Clarke, M.A., Sucrose loss and color formation in sugar manufacture, Adv.Exp.Med.Biol. 434, 123-133, 1998 • Starzak, M., Peacock, S.D., and Mathiouthi, M., Hydration number and water activity models for the sucrose-water system: a critical review, Crit.Rev.Food Sci.Nutr. 40, 327367, 2000 • Salerno, G.L. and Curatti, L., Origin of sucrose metabolism in higher plants: when, how and why?, Trends Plant Sci. 8, 63-69. 2003 • Queneau, Y., Jarosz, S., Lewandowski, B., and Fitremann, J., Sucrose chemistry and applications of sucrochemicals, Adv.Carbohydr.Chem.Biochem. 61, 217-292, 2007 • Jaradat, D.M., Mebs, S., Checinska, L., and Luger, P., Experimental charge density of sucrose at 20K: bond topological, atomic, and intermediate quantitative properties, Carbohydr.Res. 342, 1480-1489, 2007 • Bhugra, C., Ramphatla, S., Bakri, A., et al., Prediction of the onset of crystallization of amorphous sucrose below the calorimetric glass transition temperature from correlations with mobility, J.Pharm.Sci. 96, 1258-1269, 2007 • Quintas, M., Brandao, T.R., Silva, C.L., and Cunha, R.L., Modeling viscosity temperature dependence of supercooled sucrose solutions - the random-walk approach, J.Phys.Chem.B. 111, 3192-3196, 2007 • Tombari, E., Salvetti, G., Ferrari, C., and Johari, G.P., Kinetics and thermodynamics of sucrose hydrolysis from real-time enthalpy and heat capacity measurements, J.Phys. Chem.B. 111, 496-501, 2007
Trehalose
• Lillford, P.J. and Holt, C.B., In vitro uses of biological cryoprotectants, Philo.Trans.B.Soc.Lond.B.Biol.Sci. 357, 945951, 2002 • Kawai, K., Hagiwara, T., Takai, R., and Suzuki, T., Comparative investigation by two analytical approaches of enthalpy relaxation for glassy glucose, sucrose, maltose, and trehalose, Pharm.Res. 22, 490-495, 2005 • Cordone, L., Cottone, G., Giuffrida, S., et al., Internal dynamics and protein-matrix coupling in trehalose-coated proteins, Biochim.Biophys.Acta. 1749, 252-281, 2005 • Lerbret, A., Bordat, P. Affouard, F., et al., How homogeneous are the trehalose, maltose, and sucrose water solutions? An insight from molecular dynamics simulations, J.Phys.Chem.B. 109, 11046-11057, 2005 • Pereira, C.S. and Hunenberger, P.M., Interaction of the sugars trehalose, maltose and glucose with a phospholipid bilayer: a comparative molecular dynamics study, J.Phys. Chem.B 110, 11572-11581, 2006 • Empadinhas, N. and da Costa, M.S., Diversity and biosynthesis of compatible solutes in hyper/thermophiles, Int. Microbiol. 9, 199-206, 2006 • Lerbret, A., Bordat, P., Affouard, F., et al., How do trehalose, maltose, and sucrose influence some structural and
4/16/10 1:20 PM
Handbook of Biochemistry and Molecular Biology
390 dynamical properties of lysozyme? Insight from molecular dynamics simulations, J.Phys.Chem.B. 111, 9410-9420, 2007 • Crowe, J.H., Trehalose as a “chemical chaperone”: fact and fantasy, Adv.Exp.Med.Biol. 594, 143-158, 2007
• O-Glycosides are composed of saccharides where the hydroxyl group of a hemiacetal is replaced by a alkyloxy or aryloxy group. A thioglycoside is a glycoside where the hydroxyl group of a hemiacetal is replaced by a alkylthio or arylthio group. The replacement of the hydroxyl group of a hemiacetal with an alkylamine or arylamine function is called a glycosylamine. Replacement of the hydroxyl group with an alkyl or aryl group results in a C-glycoside. A C-glycoside is also referred to as an anhydroalditol. A C-glycoside is not cleaved by hydrolysis. The carbohydrate portion may be a five-membered ring (furanoside), a six-membered ring (pyranoside), or rarely a seven-membered ring (septanoside).
Trehalulose
• Ooshima, T., Izumitani, A., Minami, T., et al., Trehalulose does not induce dental caries in rats infected with mutans streptococci, Caries Res. 25, 277-282, 1991 • Salvucci, M.E., Distinct sucrose isomerases catalyze trehalulose synthesis in whiteflies, Bemisia argentifolii, and Erwinia rhaptici, Comp.Biochem.Physiol.B.Biochem.Mol. Biol. 135, 385-395, 2003 • Ravaud, S., Watzlawick, H., Haser, R., et al., Expression, purification, crystallization and preliminary x-ray crystallographic studies of the trehalulose synthase MutB from Pseudomonas mesoacidophila MX-45, Acta Crystallogr. Sect.F. Struct.Biol.Cryst.Commun. 61, 100-103, 2005 • Ravaud, S., Robert, X. Watzlawick, H., et al., Trehalulose synthase native and carbohydrate complexed structures provide insights into sucrose isomerization, J.Biol.Chem. 282, 28126-28136, 2007
General reference for glycosides
• Waller, G.R. and Yamasaki, K., Saponins used in traditional and modern medicine, Plenum Press, New York, NY, USA, 1996 • Lehman, J., Carbohydrate Structure and Biology, Thieme, Stuttgart, Germany, 1998 • Naturally Occurring Glycosides, ed. R. Khan, John Wiley & Sons, Ltd., Chichester, United Kingdom, 1999 • Saponins in Food, Feedstuffs and Medicinal Plants, ed .W. Olezek and A. Mersten, Kluwer Academic, Dordrecht, Netherlands, 2000 • Biologically Active Natural Products: Pharmaceuticals, ed. S.J. Cutler and H.G. Cutler, CRC Press, Boca Raton, FL, 2000 • Garegg, P.J., Synthesis and reactions of glycosides, Adv. Carbohydr.Chem.Biochem. 59, 69-134, 2004 • Levy, D.E. and Fügedi, P., The Organic Chemistry of Sugars, Taylor & Francis, Boca Raton, FL, USA, 2006
Glycosides
• The term glycoside refers to a compound composed of a carbohydrate and a non-carbohydrate moiety. The noncarbohydrate moiety is referred to as the aglycone. There are O-glycosides and C-glycosides. The carbohydrate portions generally contain between one and three monosaccharide units connected by a glycosidic bond.
COOH Aldaric Acids
HO
COOH
O
O
C
C
H
OH
OH
(R)-Hydroxybutanedioic acid MW 134.01
COOH
H COOH L-Tartaric acid (2R,3R) MW 150.09 COOH
COOH OH
HO
OH
HO
CH2COOH
Oxalic Acid MW 90.03
H
H
H
OH
HO
H H
H
HO
H
HO
H
OH
HO
H
H
OH
H
OH
H
OH
H
OH
COOH D-Glucaric acid Saccharic acid MW 210.14
COOH D-Mannaric acid Mannosaccharic acid MW 210.14
COOH D-Galactaric acid Mucic acid MW 210.14
COOH HO
H
HO
H
HO H
H OH
COOH H HO
COOH OH
H
OH
H
H
OH
OH
H
OH
O
O
OH H
OH
OH
COOH COOH COOH D-Talaric acid Ribonic acid Xylonic acid Talomucic acid Xylosaccharic acid Altraric acid MW 180.11 MW 210.14 Aldaric acids are a group of carbohydrate dicarboxylic acids characterized by the general formula HOOC-(CHOH)n-COOH
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Introduction to Carbohydrates
391
Aldonic Acids
COOH OH
HO
H O Glycollic acid (hydroxyacetic acid) MW 76.05
OH
CH2OH Glyceric acid 2,3-Dihydroxypropanoic acid MS 106.08
COOH
Allose and Altrose CH2OH
CH2OH O
O
COOH
OH H
OH
HO
H
OH
CH3
CH3 D-Lactic acid (2R)-Hydroxypropanoic acid
L-Lactic acid (2S)-Hydroxypropanoic acid
Lactic acid MW 90.08
OH
H
OH
HO
H H
HO
H
HO
H
OH
HO
H
H
OH
H
OH
H
OH
H
OH
CH2OH Gluconic acid MW 196.16
CH2OH
CH2OH Galactonic acid MW 196.16
Mannonic acid MW 196.16
Aldonic acids are carboxylic acids formed by the oxidation of aldehyde functions
9168_Book.indb 391
H OH
H OH
H
H
HO
OH
OH O HO
HO COOH
OH
OH
OH
H O
COOH
COOH H
OH
OH
H
H
H
OH
H
H
OH
H
OH
OH
CHO
OH CHO
H
OH
HO
H
OH
H
OH
H
OH
H
OH
H
OH
H
OH
CH2OH Allose MW 181.16
H
CH2OH Altrose MW 189.16
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Handbook of Biochemistry and Molecular Biology
392 Arabinose and Derivatives
CHO
O
COOH
HO
OH OH
OH
H
HO
H
H
OH
H
OH
H
OH
H
OH
CH2OH
OH Arabinose (alpha-D-pyranose form)
Arabinose MW 15013
CH2OH HO
CH2OH Arabonic acid (Arabinonic acid MW 166.13
CH2OH H
H
OH
H
OH
HO
H
H
OH
HO
H
CH2OH
CH2OH
D-Arabitol
MW 152.15
L-Arabitol
Conduritol OH
OH
OH
OH OH OH
OH
Conduritol A (5-cyclohexene-1,2,3,4-tetrol) MW 146.4
Butanetetrol CH2OH
OH Conduritol B MW 146.14
OH
CH2OH
H
C
OH
HO
C
H
H
C
OH
H
C
OH
OH OH OH
CH2OH (R*,S*)-1,2,3,4-Butanetetrol Erythritol MW 122.12
9168_Book.indb 392
CH2OH (R*,R*)-1,2,3,4-Butanetetrol Threitol MW 122.12
O Conduritol B epoxide
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Introduction to Carbohydrates
393 Dialdoses CHO
CHO Oxidation
(CHOH)n
(CHOH)n
CH2OH
CHO
Aldose
Dialdose CHO
CHO H
H
OH
HO
H
HO
H
H
Oxidation
OH
HO
H
HO
H
H
OH
OH CHO
CH2OH D-Galactose
D-galacto-Hexosedialdose
Fructose CH2OH O HO
O
H
H
OH
H
OH CH2OH
O
CH2OH
OH
OH
OH OH
OH
CH2OH
OH
OH
OH
alpha-D-pyranose
beta-D-pyranose
HO CH2
OH
O OH
CH2OH OH beta-D-furanose D-Fructose MW 180.16
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Handbook of Biochemistry and Molecular Biology
394 Galactose and Galactose Derivatives CH2OH H
CHO
CHO OH
H
H
OH
OH
HO
H
HO
H
HO
H
HO
H
HO
H
HO
H
H
OH
H
H
OH
CH2OH
CH2OH
D-Galactitol MW 182.17
D-Galactose MW 180.16
OH COOH
D-Galacturonic acid MW 196/14
CHO
COOH
CH2OH H
NH2
H
OH
O
OH HO
H
HO
H
HO
H
HO
H
H
H
OH
OH
OH OH
CH2OH
CH2OH
D-Galactosamine (beta-D-pyranose) 2-amino-2-deoxy-D-galactose MW 179.17
D-Galactonic Acid MW 196.14n
OH OH
OH OH H
H
O
O
H
H HO
H H
H
HO
H
OH OH
D-galactose, alpha-pyranose form
9168_Book.indb 394
NH2
H
OH OH H
D-galactose, beta-pyranose form
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Introduction to Carbohydrates
395
Glucitol (sorbitol)
Different Presentations of Glucose
CHO H
OH
H
1
CH2OH
CH2OH
CHO C
H
O
OH
2
HO
HO
H
H
OH
H
OH
HO
H
HO
H
OH
H
H
OH
H
CH2OH
CH2OH
C
OH
C C
6
4
OH
5
H
H OH
H
4
H
HO
CH2OH
H
6
4
5
HO OH
OH
HO 3
HO
COOH
H
HO
H
H
OH
HO
H
H
OH
H
OH
H
OH
H
OH
H
OH
H
OH
CH2OH
CH2OH
D-Glucose MW 180.16
D-Glucitol (D-sorbitol) MW 182.17 CHO H
D-Gluconic Acid MW 196.16
CHO OH
HO
CH2OH
H
H
NH2
HO
OH
H
OH
H
OH
H
OH
COOH
9168_Book.indb 395
CH2OH Glucosamine 2-Amino-2-deoxyglucose MW 179.17
OH
H
OH
H
Oxidation
COOH
HO
OH H
H
OH
H
OH
H
OH
H
OH CH2OH
COOH D-Glucuronic Acid
D-Gluconic Acid
oxidation
oxidation COOH H HO
Glucuronic Acid 194.14
H
H
H
H
H
OH
HO
H
CH2OH D-Glucose
CHO H
OH
OH
HO
OH
1
OH
CHO
Oxidation
H
H
H
H
OH
O
D-Glucose, beta-pyranose form
Glucose Oxidation
H
H 2
OH alpha-D-glucose. Haworth representation
CHO
OH
H OH
O OH
CH2OH
OH
D-Glucose, alpha-pyranose form
CH2OH
Glucose and Glucose Derivatives
H
1
H
3
D-Glucose Fischer projection
Sorbose (D-gluco-2-ulose) 180.16
O 2
HO
OH
H
5
6
OH CH2OH
Sorbitol (D-glucitol) MW 182.17
D-Glucose MW 180.16
H
OH
3
OH H
H
OH
H
OH
COOH D-Glucaric Acid
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Handbook of Biochemistry and Molecular Biology
396 Glucuronic Acid
CHO H
OH
HO
H
H
OH
H
OH COOH
D-Glucuronic Acid
COOH O
C
H +
OH O
OH
O
Uridine Diphosphate
HO
CH3
NH
Acetaminophen
OH
Uridine diphosphate glucuronosyltransferase
glucuronidation O
COOH
C O
O
CH3
NH
OH H
OH OH
9168_Book.indb 396
Conjugate product, glucuronidate
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Introduction to Carbohydrates
397 O
Glycodiuloses CH2OH O HO
H
H
OH
H
OH
O 5-ketofructose reductase
HO
H
H
OH
HO H
CH3
O H
OH
H
OH CH2OH
1-deoxy-2,3-hexodiulose
H
OH
OH
CH3
CH3 O
H
O
OH
6-deoxy-5-ketofructose 1-phosphate
CH3 O
P
O
CH2OH D-threo-2,5-hexodiulose 5-keto-D-fructose
O
H
O
CH2OH D-Fructose
9168_Book.indb 397
H2C
CH2OH
H
OH
OH
O
O
O
OH CH2OH
1-deoxy-2,4-hexodiulose
H
OH CH2OH
1-deoxy-3,4-hexodiulose
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Handbook of Biochemistry and Molecular Biology
398 Glycosulose
Glycosuloses are obtained by the oxidation of a secondary hydroxyl groups in an aldose yielding a ketone aldo-2-uloses CHO
CHO
CHO H
OH
HO
O
O
H
oxidation
HO
H
H
H
H
OH
H
OH
H
OH
H
OH
H
OH
H
OH CH2OH
CH2OH
CH2OH D-Glucose
D-arabino-2-ulose D-glucosone
3-Deoxy-D-erythro-hexose-2-ulose
Glycosonic Acids (ketoaldonic acids) are obtained by the oxidation of secondary hydroxyl group in an aldonic acid yielding a ketone COOH O HO
H
H
OH
H
OH CH2OH
D-arabino-2-hexosonic acid
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Introduction to Carbohydrates
399
Heptose Derivatives
OH
HO
H
HO
H
HO
H
HO
H
H
OH
H
OH
H
OH
H
OH
H
OH
H
OH
H
OH
H
OH
H
CH2OH
HO
CH2OH
D-glycero-D-manno-heptitol Volemitol MW 212.20
CH2OH
H O
CH2OH
D-glycero-D-galacto-heptitol Perseitol MW 212.20
9168_Book.indb 399
CH2OH
CH2OH
CH2OH
altro-3-Heptulose Coriose MW 210.18
CHO
CHO
HO
H
HO
H
HO
H
HO
H
O HO
H
H
OH
H
OH
H
OH
H
OH
H
OH
H
OH
H
OH
H
OH
H
OH
CH2OH
CH2OH
D-glycero-D-manno-heptose
D-glycero-D-manno-heptose
CH2OH D-altro-2-heptulose Sedoheptulose
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Handbook of Biochemistry and Molecular Biology
400 Heptoses and heptonic acids CHO
HO
H
HO
H
CHO
HO
OH
H
Lactone Formation
CH2OH
H O
OH
H
HO
OH
H
OH
H
OH
OH
OH
OH
H
HO
H
H
OH
H
H
O
HO HO
H
H
OH
OH H
OH
CH2OH
CH2OH
D-glucose
D-glycero-D-galacto-heptose MW 210.18 alpha-D-pyranose form
D-glucose
CHO H
OH
H
OH
CH2OH
O
OH HO
H
H
OH
H
OH
COOH
HO H
H
OH
HO
H
HO
H
H
OH H
OH OH
OH
OH OH
H
CH2OH D-glycero-D-gulo-heptose MW 210.18 alpha-D-pyranose form
HO
H
OH
CH2OH
H
D-gluconic acid
D-glucono-1,5-lactone
O
O
HO
COOH
O
HO
COOH H
OH
HO
HO H HO
O
OH H
H
OH
H
OH
O
OH
H
O gamma-hydroxybutyric acid
gamma-butyrolactone
OH OH
OH
CH2OH D-glycero-D-gulo-heptonic acid MW 220.18
COOH
HO
OH
OH Shikimic acid 3,4,5-Trihydroxy-1-cyclohexene-1-carboxylic acid (3R,4S,5R) MW 174.15
9168_Book.indb 400
4/16/10 1:21 PM
Introduction to Carbohydrates
401
OH
OH
OH CH2OH Validatol 4-Hydroxymethyl-1,2,3-hexanetriol, (1S,2R,3R,4S) MW 162.19
Cellobiose; Lactose: Maltose
OH
CH2OH O
O HO
OH O
OH CH2OH
OH Cellobiose; MW 342.30 4-O-beta-D-glucopyranosyl-D-glucose
OH
CH2OH O
OH
OH
OH
O HO
OH H
H
O CH2OH
OH Lactose; MW 342,3 4-O-beta-D-galactopyranosyl-D-glucose CH2OH
CH2OH O
O
OH
OH
OH O
OH OH
OH
Maltose, MW 342.30 4-O-alpha-D-glucopyranosyl-D-glucose
9168_Book.indb 401
4/16/10 1:21 PM
Handbook of Biochemistry and Molecular Biology
402 Melibiose Trehalose Isomaltose
OH
CH2OH
OH
O
OH
OH OH O
O
OH
OH Melibiose MW 342.30 6-O-alpha-D-galactopyranosyl-D-glucose
OH
CH2OH O HO
CH2OH
OH O
OH
OH
O
OH Trehalose MW 342.30 alpha-D-glucopyranosyl-alpha-D-glucopyranoside OH
CH2OH
OH
O OH OH O
OH OH
O OH
Isomaltose MW 342.30 Brachiose 6-O-alpha-D-glucopyranosyl-D-glucose
9168_Book.indb 402
4/16/10 1:21 PM
Introduction to Carbohydrates
403
Sucrose Trehalulose Isomaltulose CH2OH O
CH2OH
O OH
HO O
OH
CH2OH OH
OH Sucrose MW 342.30 alpha-D-glucopyranosyl-beta-D-fructofuranoside CH2OH O
O
HO
OH
O
OH
OH
OH Trehalulose alpha-D-glucopyranosyl-1-1-D-fructofuranose
CH2OH OH
CH2OH O
CH2OH
O OH
O HO
OH
OH OH OH Isomaltulose' Palatinose alpha-D-glucopyranosyl-1,6-D-fructofuranose
9168_Book.indb 403
4/16/10 1:21 PM
404
Chemical modification of carbohydrates General Modification of Carbohydrates
• Hoover, R. and Sosulski, F.W., Composition, structure, functionality, and chemical modification of legume starches: a review, Can.J.Physiol.Pharmacol. 69, 79-92, 1991 • Stevens, C.V., Meriggi, A., and Booten, K., Chemical modification of inulin, a valuable renewable resource, and its industrial applications, Biomacromolecules 2, 1-16, 2001 • Lin, C.H. and Lin, C.C., Enzymatic and chemical approaches for the synthesis of sialyl glycoconjugates, Adv. Exp.Med.Biol. 491, 215-230, 2001 • Kellam, B., De Bank, P.A., and Shakesheff, K.M., Chemical modification of mammalian cell surfaces, Chem.Soc.Rev. 32, 327-337k, 2003 • Tomasik, P. and Schilling, C.H., Chemical modification of starch, Adv.Carbohydr.Chem.Biochem. 59, 175-403, 2004 • Jayakumuar, R., Nwe, N., Tokura, S., et al,. Sulfated chitin and chitosan as novel biomaterials, Int.J.Biol.Macromol. 40, 175-181, 2007
Glycol cleavage
• Salomies, H., Luukkanen, L., and Knuutila, R., Oxidation of beta-blocking agents - - VII. Periodate oxidation of labetalol, J.Pharm.Biomed.Anal. 7, 1447-1451, 1989 • Chai, W., Stoll, M.S., Cashmore, G.C., and Lawson, A.M., Specificity of mild periodate oxidation of oligosaccharidealditols: relevance to the analysis of the core-branching pattern of O-linked glycoprotein oligosaccharides, Carbohydr. Res. 239, 107-115, 1993 • Brevnov, M.G., Gritsenko, O.M., Mikhailov, S.N., et al., DNA duplexes with reactive dialdehyde groups as novel reagents for cross-linking to restriction-modification enzymes, Nucleic Acids Res. 25, 3302-3309, 1997 • Zhong, Y.L. and Shing, T.K., Efficient and facile glycol cleavage oxidation using improved silica gel-supported sodium metaperiodate, J.Org.Chem. 62, 2622-2624, 1997 • Bhavandandan, V.P.. Ringler, N.J., and Gowda, D.C., Identification of the glycosidically bound sialic acid in mucin glycoproteins that reacts like “free sialic acid” in the Warren assay, Glycobiology 8, 1077-1086, 1998 • Balakrishnan, B., Lesieur, S., Labarre, D., and Jayakrishnan, A., Periodate oxidation of sodium alginate in water and in
9168_Book.indb 404
Handbook of Biochemistry and Molecular Biology ethanol-water mixture: A comparative study, Carbohydr. Res. 340, 1425-1429, 2005 • Liu, B., Burdine, L., and Kodadek, T., Chemistry of periodatemediated cross-linking of 3,4-dihydroxyphenylalaninecontaining molecules to proteins, J.Am.Chem.Soc. 128, 15228-15235, 2006 • Perlin, A.S., Glycol-cleavage oxidation, Adv.Carbohydr. Chem.Biochem. 60, 184-210, 2006
Enzymatic oxidation of carbohydrates
• De Lederkremer, R.M. and Marino, C., Acids and other products of the oxidation of sugars, Adv.Carbohydr.Chem. Biochem. 58, 199-306, 2003 • Varela, O., Oxidative reactions and degradations of sugars and polysaccharides, Adv.Carbohydr.Chem.Biochem. 58, 308-369, 2003 • van Hellemond, E.W., Leferink, N.G.,H, Heuts, D.P.H.M., et al., Occurrence and biocatalytic potential of carbohydrate oxidases, Adv.Appd.Microbiol. 60, 17-54, 2006 • Perlin, A.S., Glycol-cleavage oxidation, Adv.Carb.Chem. Biochem. 60, 184-250, 2006
Glucose oxidase
• Leskovac, V., Trivic, S., Wohlfahrt, G., et al., Glucose oxidase from Aspergillus niger: the mechanism of action of oxygen, quinines, and one-electron acceptors, Int.J.Biochem.Cell. Biol. 37, 731-750, 2005 • Willner, I., Baron, R., and Willner,B., Integrated nanoparticlebiomolecule systems for biosensing and bioelectronics, Biosens.Bioelectron. 22, 1841-1852, 2007
Galactose oxidase
• Whittacker, J.W., The radical chemistry of galactose oxidase, Arch.Biochem.Biophys. 433, 227-239, 2005 • Kondakova, L., Yanishpolskii, V., Tertykh, V., and Bulglova, T., Galactose oxidase immobilized on silica in an analytical determination of galactose-containing carbohydrates, Analyt.Sci. 23, 97-101, 2007 • Alberton, D., de Olivera, L.S., Peralta, R.M. et al., Production, purification, and characterization of a novel galactose oxidase from Fusarium acumindatum, J.Basic Microbiol. 47, 203-212, 2007
4/16/10 1:21 PM
Introduction to Carbohydrates
405
Glycol Cleavage
R
HIO4
R
R
O
R
OH
R
IO4H2
O
R
IO4H2 R
O
R
R
OH
R
OH
R 2
O
R
R
R
O
O
R
OH
R
Pb(OAc)2 R
Pb(OAc)2
O R
R
R
R H
C
OH
H
C +
O
H
C
OH
H
C
O
R
+ H2O
R
R
Pb(OAc)2
9168_Book.indb 405
R
+ H2O
R
4/16/10 1:21 PM
9168_Book.indb 407
Table A: Natural Alditols, Inositols, Inososes, and Amino Alditols and Inosamines TABLE A: NATURAL ALDITOLS, INOSITOLS, INOSOSES, AND AMINO ALDITOLS AND INOSAMINES (Continued)
Substancea (Synonym) Derivative (A)
Chemical Formula (B)
Chromatography, R Value, and Referenced Melting Point °C (C)
Specific Rotationb [a]D Alditols
(D)
Referencec (E)
ELC (F)
PPC
TLC
4.69 (3) 3.60 (6) 8xyll (7) 5.94 (8)
32f (4)
62f (5)
(G)
(H)
(I)
Glycerol (glycerine) Triacetate Trimethyl ether Trimethylsilyl ether Tris(o-nitrobenzoate)
C3H8O3 C9H14O6 C6H14O3
20 4 Oil, bp 148
None (meso) None None
1 1 1
C24H17N3O12
190–192
None
9egp
1-Deoxyglycerole (1,2-propanediol, propylene glycol) Bis(p-nitrobenzoate)
C3H8O3 C17H14N2O8
Oil, bp 188–189 125–126
Racemic
1,10 9egp
5rib (2)
3.00 (11)
35f (12)
80f (5)
Erythritol Tetraacetate Tetramethyl ether Trimethylsilyl ether Tetrakis(p-nitrobenzoate)
C4H10O4 C12H18O8 C8H18O4
118–120 85
None (meso) None None
13 13
53rib (2)
23f (4)
51f (5)
C32H22N4O16
251–252
None
9egp
14.1 (3) 13.2 (6) 26xyll (7) 28.5 (14)
1,4-Dideoxyerythritol (2,3-butyleneglycol) Dibenzoate
C4H10O2 C18H18O4
25,34 77
None (meso)
15 16
1,4-Dideoxy-d-threitol Diacetate
C4H10O2 C8H14O4
19 Oil, bp 192–194 (745 mm) 142–144
−13 +1.4
15 17
4 07
Bis(p-nitrobenzoate)
C18H16N2O8
−52.7±0.5 (c 4, CHCl3)
18
l-Threitol Tetraacetate
C4H10O6 C12H18O10
−4.5 −32 (C2H5OH)
19,22p
Trimethylsilyl ether Di-O-benzylidene ether Tetrakis(p-nitrobenzoate)
88–89 Oil, bp 145 (0.05 mm)
C18H18O4 C32H22N4O16
218–220 219–221f
+87.2 (c 0.4, acetone)
22p 23
1,4-Dideoxy-l-threitol Bis(p-nitrobenzoate)
C4H10O2 C18H16N2O8
Oil, bp ca 170 141–143
+12.4 +52 (CHCl3)
18 18
1,4-Dideoxy-dl-threitol Diacetate
C4H10O2 C8H14O4
7.6 41–41.5
Racemic Racemic
24 24
d-Arabinitol (d-arabitol) Pentaacetate
C5H12O5 C15H22O10
103 76
+7.8 (c 8, borax solution) +37.2 (CHCl3)
25 27
l-Arabinitol
C5H12O5
101–102
Pentaacetate Pentamethyl ether Trimethylsilyl ether
C15H22O10 C10H22O5
72–73
−7.2 (c 9, borax solution) −32 (c 0.4, 5% molybdate)
28p, 30cp 29 30
Ribitol (adonitol) Pentaacetate Pentamethyl ether Trimethylsilyl ether
C5H12O5 C15H22O10 C10H22O5
102 51
None (meso) None None None
33 34
24rib (2)
GLC
67f (35t)
6rib (2)
55f (12)
96rib (2) 45xyllf (21)
144glcf (20) 27.5f (14)
124rib (2)
38.1 (3)
14f (4)
38.1 (3)
32f (26) 70f (31)
44.4 (6) 105xyll (7) 40glcl (32) 76rib (2)
39.9 (3) 40.0 (6) 60xyll (7) 32.8, 38.5 (35g)
14f (4)
37f (26) 92f (35t)
4/16/10 1:21 PM
9168_Book.indb 408
Substancea (Synonym) Derivative (A)
Chemical Formula (B)
408
TABLE A: NATURAL ALDITOLS, INOSITOLS, INOSOSES, AND AMINO ALDITOLS AND INOSAMINES (Continued) Chromatography, R Value, and Referenced Melting Point °C (C)
Specific Rotationb [a]D (D)
Referencec (E)
ELC (F)
GLC (G)
PPC (H)
TLC (I)
Alditols (Continued) Xylitol Pentaacetate Pentamethyl ether Trimethylsilyl ether
C5H12O5 C15H22O10 C10H22O5
61,92.5–93.5 62.5–63
None (meso) None None None
28,36 36
155rib (2)
Galactitol (dulcitol) Hexaacetate Hexamethyl ether Trimethylsilyl ether
C6H14O6 C18H26O12 C12H26O6 C24H62O6Si6
186–187 167.5–168.5
37e 37g
145rib (2)
78
None (meso) None None None
d-Glucitol (sorbitol) Hexaacetate Hexamethyl ether Trimethylsilyl ether
C6H14O6 C18H26O12 C12H26O6
112 99
−1.8 (15°) +12.5 (c 0.8, CHCl3)
39 40,41
1,5-Anhydro-d-glucitol (polygalitol) Tetraacetate
C6H12O5 C14H20O9 C6H14O6
140–141 73–74 73.5
+42.4 +38.9 (CHCl2) −3.5 (c 10)
42 42 43
C18H26O12
121.5
−25.7 (CHCl3)
43,45
d-Mannitol
C6H14O6
166
−0.21
46
Hexaacetate Hexamethyl ether Trimethylsilyl ether
C18H26O12 C12H26O6
126
+16 (5% molybdate) +18.8 (acetic acid)
29 47,48
1,5-Anhydro-d-mannitol (styrachitol) Tetraacetate
C6H12O5 C14H20O9
157 66–67
−49.9 −20.9 (C2H5OH)
49 51
d-Mannitol 1-acetate
C8H16O7
124–125
+4
52cp
d-glycero-d-galacto- Heptitol (l-glycero-d-mannoheptitol, perseitol)
C7H16O7
183–185,188
−1.1
53,54
Heptaacetate
C21H30O14
119–120.5
+24.5 (5% molybdate) −14 (CHCl3)
29 53
d-glycero-d-gluco-Heptitol (l-glycero-d-talo-heptitol, b-sedoheptitol) Tri-O-methylene-b-sedoheptitol
C7H16O7
131–132
+46 (5% molybdate)
55c
C10H16O7
Sublimes 130, 276–278d
−23.3 (c 0.4, CHCl3)
56
d-glycero-d-ido-Heptitol
C7H16O7
0.0
57c
Heptaacetate Heptabenzoate
C21H30O14 C56H44O14
180–181
+24 (CHCl3)
57
l-glycero-d-ido-Heptitol Heptaacetate d-glycero-d-manno-
C7H16O7 C21H30O14 C7H16O7
111–112 175–176 153
None (meso) None +2.6
57c 57 58
Heptitol (d-glycero-d-talo-heptitol, volemitol) Heptaacetate
C21H30O14
62
+55 (5% molybdate) +36.1 (CHCl3)
29 58, 59
l-Iditol Hexaacetate
84 161rib (2)
30.3 (3) 52.8 (6) 100xyll (7) 46myol (84) 144.4 (6) 388xyll (7) 12.47 (38) 144.4 (6) 246xyll (7) 27 (32)
173rib (2)
14f (4)
26f (26)
7f (4)
24f (5)
8f (4)
22f (5)
92sorl (44) 153xyllf (21)
130rib (2)
38.1 (3)
8f (4)
83f (50a)
140rib (2)
100perl (55a)
171rib (2)
>120suc (55b)
168rib (2)
78–82 gal (57a)
621aral (57b)
719aral (57b) 140rib (2)
74gal (57a)
Handbook of Biochemistry and Molecular Biology
127.2 (6) 284xyll (7) 9.46 (8)
27f (5)
4/16/10 1:21 PM
Substancea (Synonym) Derivative (A)
Chemical Formula (B)
Chromatography, R Value, and Referenced Melting Point °C
Specific Rotationb [a]D
(C)
(D)
Referencec (E)
ELC (F)
GLC
PPC
(G)
(H)
TLC (I)
Alditols (Continued) d-erythro-d-galacto- Octitol Octaacetate
C8H18O8 · H2O
169–170
−11 (5% molybdate)
55c
C24H34O16
99–100
+2 (CHCl3)
55
83perl (55a)
Inositols C7H14O6
Betitol (a dideoxyinositol)
C6H12O4
224
d-Bornesitol (d-myo-inositol monomethyl ether) Pentaacetate Trimethylsilyl ether
C7H14O6 C17H24O11
201–202 138–139
+31.4 +11.8 (c 0.8, acetone)
62 62
20rib (2)
l-Bornesitol (1-O-methyl l-myo-inositol) Pentaacetate
C7H14O6 C17H24O11
205–206 142–143, 157
−32.1 −11.2 (CHCl3)
63, 65cp 64
15glc (82)
Conduritol (a 2,3-dehydro-2,3-dideoxy-inositol) Dihydroconduritol Tetraacetate
C6H10O4 C6H12O4 C14H18O4
142–143 204 bp 165 (0.6 mm)
None (meso) None None
67 68 68
dambonitol (1,3-di-O-methyl-myo-inositol) Tetraacetate
C8H16O6 C16H24O10
206, 210 202
None (meso) None
69, 70 71c
0glc (82)
20f (70)
d-Inositol [d-inositol, chiro- (+)-inositol,(+)-inositol, d-chiro-inositol]
C6H12O6
246–247d
+60 +65
72c, 73
83glc (82)
17f 12f (72, 74)
Hexaacetate Hexabenzoate
C18H24O12 C48H36O12
230–235 215–220 252–253
+68
74 74 74,75
l-Inositol [l inositol, levo-inositol, chiro-(−)-inositol] Hexaacetate Trimethylsilyl ether
C6H12O6 C18H24O12
247 96
−64.1
1, 76p 1
23rib (2)
49pinl (132)
d, l-Inositol Hexaacetate Hexabenzoate Trimethylsilyl ether
C6H12O6 C18H24O12 C48H36O12
253 111 213
Racemic None None
77 77 78cp
1-O-Methyl-(+)-inositol Pentaacetate
C7H14O6 C17H14O11
207–208 110.5–111.5
+60.7 +29.1 (CHCl3)
75 75
32glcf (82)
1-O-Methyl-muco-inositol Pentaacetate Pentabenzoate myo-Inositol (meso-inositol) Hexaacetate
C7H14O6 C17H14O6 C42H34O11 C6H12O6 C18H24O12
Gum bp ca 200 (vac) Amorphous 95–100 225–227 206–208 221–213 118–119
80cp,81 80 80 72p 83cp 85 84g
30glc (80)
85,86 86cpt
43rib (2)
Trimethylsilyl ether
C24H60O6Si6
neo-Inositol Hexaacetate
C6H12O6 C18H24O12
Sublimes, melts 164
314 251–253
+157 (c 0.01)
60
32f (60)
61
+64.5
None (meso) None
86myol (84) 8.27 (93)
19glc (66)
82myol (84) 9.62 (93) 13.3 (79)
16rib (2)
109pinl (81)
13.42 (93) 10.3, 24.3 (8, 79) 9.17 (93)
2f(4)
27f (50b) 79f (50a)
409
4/16/10 1:21 PM
Asteritol (an inositol monomethyl ether)
Table A: Natural Alditols, Inositols, Inososes, and Amino Alditols and Inosamines
9168_Book.indb 409
TABLE A: NATURAL ALDITOLS, INOSITOLS, INOSOSES, AND AMINO ALDITOLS AND INOSAMINES (Continued)
9168_Book.indb 410
Substancea (Synonym) Derivative (A)
Chemical Formula (B)
410
TABLE A: NATURAL ALDITOLS, INOSITOLS, INOSOSES, AND AMINO ALDITOLS AND INOSAMINES (Continued) Chromatography, R Value, and Referenced Melting Point °C (C)
Specific Rotationb [a]D (D)
Referencec (E)
ELC (F)
GLC (G)
PPC (H)
TLC (I)
Inositols (Continued) Laminitol (6-C-methyl-myo inositol) Hexaacetate
C7H14O6 C19H26O12
226–269 153
−3 −19.6±1 (CHCl3)
87c, 88cp 88
Leucanthemitol (a dehydro dideoxy inositol)
C6H10O4
Dihydroleucanthemitol
C6H12O4
Liniodendritol (1,4-di-O-methyl-myo-inositol) Tetraacetate
131–132
+101.5
89,90
161
−40
90
C8H16O6 C16H24O10
224 139
−25 −24
66,91 91
Mytilitol (a C-methyl-scyllo-inositol) Hexaacetate
C7H14O6 C19H26O12
259 180–181
None (meso) None
92 92
290 (205s) >280d
None (meso) None
143 143
342–345d
None
143
221–223 283–286d
None (meso) None
144 145
N,N′-Diacetyl triacetate
C6H14N2O3 C6H14N2O3 · 2HBr C16H24N2O8
340–350
None
144
Deoxy-N-methylstreptamine (hyosamine)
C7H16N2O3
183–186 160–162 130–133d
+39.8 −31.1 −17.8
146 146 147i
204 238–240d
Racemic
144 147
None (meso)
143
None None
143 148
N,N′-Diacetyl tetraacetate Bluensidine (3-O-carbamoyl-1-deoxy-1-guanidinoscyllo-inositol) Hydrochloride N,N′-Diacetyl tetraacetate neo-Inosamine-2 (2-amino-2-deoxy-neo-inositol) Hydrochloride N-Acetyl pentaacetate scyllo-Inosamine
Streptamine (1,3-diamino-1,3-dideoxy-scyllo-inositol) Dihydroiodide N,N′-Diacetyl tetraacetate 2-Deoxystreptamine Dihydrobromide
N,N′-Diacetyl triacetate Dipicrate Streptidine (1,3-dideoxy-1,3-diguanidinoscyllo-inositol) N,N′-Diacetyl tetraacetate Dipicrate
C7H16N2O3 · H2O C17H26N2O8 C8H18N6O4 C20H30N6O10 C20H24N12O18
342–345 284–285d
1
3.14 (8)
47glcn (149)
Handbook of Biochemistry and Molecular Biology
N-Acetyl pentaacetate
None None
4/16/10 1:21 PM
Substancea (Synonym) Derivative (A)
Chemical Formula (B)
Chromatography, R Value, and Referenced Melting Point °C (C)
Specific Rotationb [a]D (D)
Referencec (E)
ELC (F)
GLC (G)
PPC (H)
TLC (I)
Inositols (Continued) Validamine [1S-(1,2,4/3,5)-5-hydroxymethyl-2,3,4trihydroxycyclohexylamine] Hydrochloride
C7H15NO4
+60.6
150
C7H15NO4 · HCl
229–232d
+57.4 (N HCl)
123
Hydroxyvalidamine epi-Validamine
C7H15NO5 C7H15NO4
164–165 210
+80.7 +5.8
123 150
Valienamine (1,6-dehydro-validamine) Hydrochloride
C7H13NO4 C7H13NO4 · HCl C17H23NO9
No constants known
+68.6 (N HCl)
150 150
+30.2 (CHCl3)
150
N-Acetyl tetraacetate
95
Compiled by George G. Maher. a b c d
e f g h i
In order of increasing carbon chain length in the parent compounds grouped in the classes – alditols, inositols, inososes, amino alditols, and inosamines. [a]D for 1–5 g solute, c, per 100 ml aqueous solution at 20–25°C unless otherwise given. References for melting point and specific rotation data. Letter indicates the reference also has chromatographic data according to: c = column, e = electrophoresis, g = gas, p = paper, and t = thin-layer. R value times 100, given relative to that of the compound indicated by abbreviation: f = s olvent front, gal = galactose, glc = glucose, glcl = glucitol, glcn = glucosamine, myol = myo-inositol, perl = perseitol, pinl = pinitol, rib = ribose, sorl = sorbitol, suc = sucrose, xyll = xylitol, (as the pentaacetate or the pentamethyl ether, as pertains), and aral = arabinitol (as the pentaacetate). Under gas chromatography (Column GLC or G) numbers without code indication signify retention time in minutes. The conditions of the chromatography are correlated with the reference given in parentheses and are found in Table 5. Said to exist as a phosphate ester also.10 Data given are for the enanthiomorphic isomer. The author names as 3-dehydroquinic acid, but it is actually 5-dehydroquinic. The early given name, l-quercitol, of this compound does not make it the enanthiomorph of d-quercitol; other isomeric relations are involved. This compound is isomeric with the previous one in regard to the N-methyl group position.
Table A: Natural Alditols, Inositols, Inososes, and Amino Alditols and Inosamines
9168_Book.indb 413
TABLE A: NATURAL ALDITOLS, INOSITOLS, INOSOSES, AND AMINO ALDITOLS AND INOSAMINES (Continued)
413
4/16/10 1:21 PM
Handbook of Biochemistry and Molecular Biology
414
References 1. Pollock and Stevens, Dictionary of Organic Compounds, Oxford University Press, New York, 1965. 2. Frahn and Mills, Aust. J. Chem., 12, 65 (1959). 3. Dooms, Declerck, and Verachtert, J. Chromatogr., 42, 349 (1969). 4. Bourne, Lees, and Weigel, J. Chromatogr., 11, 253 (1963). 5. de Simone and Vicedomini, J. Chromatogr., 37, 538 (1968). 6. Sawardeker, Sloneker, and Jeanes, Anal. Chem., 37, 1602 (1965). 7. Whyte, J. Chromatogr., 87, 163 (1973). 8. Roberts, Johnston, and Fuhr, Anal. Biochem., 10, 282 (1965). 9. Dutton and Unrau, Can. J. Chem., 43, 924, 1738 (1965). 10. Lindberg, Ark. Kemi. Mineral. Geol., 23, A 2 (1946–1947). 11. Weatherall, J. Chromatogr., 26, 251 (1967). 12. Borecký and Gasparič, Collect. Czech. Chem. Commun., 25, 1287 (1960). 13. Bamberger and Landsiedl, Monatsh. Chem., 21, 571 (1900). 14. Dutton, Gibney, Jensen, and Reid, J. Chromatogr., 36, 152 (1968). 15. Ward, Pettijohn, Lockwood, and Coghill, J. Am. Chem. Soc., 66, 541 (1944). 16. Ciamician and Silber, Ber. Dtsch. Chem. Ges., 44, 1280 (1911). 17. Morell and Auernheimer, J. Am. Chem. Soc., 66, 792 (1944). 18. Rubin, Lardy, and Fischer, J. Am. Chem. Soc., 74, 425 (1952). 19. Bertrand, C. R. Acad. Sci., 130, 1472 (1900). 20. Batt, Dickens, and Williamson, Biochem. J., 77, 272 (1960). 21. Oades, J. Chromatogr., 28, 246 (1967). 22. Hu, McComb, and Rendig, Arch. Biochem. Biophys., 110, 350 (1965). 23. Dutton and Unrau, J. Chromatogr., 20, 78 (1965). 24. Wilson and Lucas, J. Am. Chem. Soc., 58, 2396 (1936). 25. Asahina and Yanagita, Ber. Dtsch. Chem. Ges., 67, 799 (1934). 26. Němec, Kefurt, and Jarý, J. Chromatogr., 26, 116 (1967). 27. Frèrejacque, C. R. Acad. Sci., 208, 1123 (1939). 28. Onishi and Suzuki, Agric. Biol. Chem., 30, 1139 (1966). 29. Richtmyer and Hudson, J. Am. Chem. Soc., 73, 2249 (1951). 30. Touster and Harwell, J. Biol. Chem., 230, 1031 (1958). 31. Grasshof, J. Chromatogr., 14, 513 (1964). 32. Dutton, Reid, Rowe, and Rowe, J. Chromatogr., 47, 195 (1970). 33. Wessely and Wang, Monatsh. Chem., 72, 168 (1938). 34. Binkley and Wolform, J. Am. Chem. Soc., 70, 2809 (1948). 35. Gregory, J. Chromatogr., 36, 342 (1968). 36. Wolfrom and Kohn, J. Am. Chem. Soc., 64, 1739 (1942). 37. Wells, Pittman, and Egan, J. Biol. Chem., 239, 3192 (1964). 38. Horowitz and Delman, J. Chromatogr., 21, 302 (1966). 39. Von Lippmann, Ber. Dtsch. Chem. Ges., 60, 161 (1927). 40. Haas and Hill, Biochem. J., 26, 987 (1932). 41. Jeger, Norymberski, Szpilfogel, and Prelog, Helv. Chim. Acta, 29, 684 (1946). 42. Richtmyer, Carr, and Hudson, J. Am. Chem. Soc., 65, 1477 (1943). 43. Bertrand, Bull. Soc. Chim. Fr. Ser. 3, 33, 166 (1905). 44. Britton, Biochem. J., 85, 402 (1962). 45. Perlin, Mazurek, Jaques, and Kavanagh, Carbohydr. Res., 7, 369 (1968). 46. Braham, J. Am. Chem. Soc., 41, 1707 (1919). 47. Patterson and Todd, J. Chem. Soc. (Lond.), p. 2876 (1929). 48. Iwate, Chem. Zentralbl., 2, 177 (1929). 49. Zervas, Ber. Dtsch. Chem. Ges., 63, 1689 (1930). 50. Hay, Lewis, and Smith, J. Chromatogr., 11, 479 (1963). 51. Asahina, Ber. Dtsch. Chem. Ges., 45, 2363 (1912). 52. Lindberg, Acta Chem. Scand., 7, 1119, 1123 (1953). 53. Jones and Wall, Nature, 189, 746 (1961). 54. Maquenne, Ann. Chim. Phys. Ser. 6, 19, 5 (1890). 55. Charlson and Richtmyer, J. Am. Chem. Soc., 82, 3428 (1960). 56. Buck, Foster, Richtmyer, and Zissis, J. Chem. Soc. (Lond.), p. 3633 (1961). 57. Onishi and Perry, Can. J. Microbiol., 11, 929 (1965). 58. Bougault and Aliard, C.R. Acad. Sci., 135, 796 (1902). 59. Maclay, Hann, and Hudson, J. Org. Chem., 9, 293 (1944). 60. Ackerman, Hoppe-Seyler’s Z. Physiol. Chem., 336, 1 (1964). 61. Von Lippmann, Ber. Dtsch. Chem. Ges., 34, 1159 (1901). 62. King and Jurd, J. Chem. Soc. (Lond.), p. 1192 (1953). 63. Bien and Ginsburg, J. Chem. Soc. (Lond.), p. 3189 (1958).
9168_Book.indb 414
64. 65. 66. 67. 68. 69. 70. 71. 72. 73. 74. 75. 76. 77. 78. 79. 80. 81. 82. 83. 84. 85. 86. 87. 88. 89. 90. 91. 92. 93. 94. 95. 96. 97. 98. 99. 100. 101. 102. 103. 104. 105. 106. 107. 108. 109. 10. 1 111. 112. 113. 114. 115. 116. 117. 118. 119. 120. 21. 1 122. 123. 124. 125. 126.
Pilouvier, C.R. Acad. Sci., 241, 983 (1955). Post and Anderson, J. Am. Chem. Soc., 84, 478 (1962). Angyal and Bender, J. Chem. Soc. (Lond.), p. 4718 (1961). Kübler, Arch. Pharm., 246, 620 (1908). Dangschat and Fischer, Naturwissenschaften, 27, 756 (1939). DeJong, Recl. Trav. Chim. Pays-Bas, 27, 257 (1908). Kiang and Loke, J. Chem. Soc. (Lond.), p. 480 (1956). Angyal, Gilham, and MacDonald, J. Chem. Soc. (Lond.), p. 1417 (1957). Ballou and Anderson, J. Am. Chem. Soc., 75, 648 (1953). Umezawa, Okami, Hashimoto, Suhara, Hamada, and Takeuchi, J. Antibiot. Ser. A, 18, 101 (1965). Dzhumyrko and Shinkaxenko, Chem. Nat. Compd. (USSR), 7, 638 (1971). Foxall and Morgan, J. Chem. Soc. (Lond.), p. 5573 (1963). Smith, Biochem. J., 57, 140 (1954). Tanret, C. R. Acad. Sci., 145, 1196 (1907). Cosgrove, Nature, 194, 1265 (1962). Lee and Ballou, J. Chromatogr., 18, 147 (1965). Adhikari, Bell, and Harvey, J. Chem. Soc. (Lond.), p. 2829 (1962). Utkin, Chem. Nat. Compd. (USSR), 4, 234 (1968). Angyal and McHugh, J. Chem. Soc. (Lond.), p. 1423 (1957). Lindberg, Acta Chem. Scand., 9, 1093 (1955). Loewus, Carbohydr. Res., 3, 130 (1966). Allen, J. Am. Chem. Soc., 84, 3128 (1962). Cosgrove and Tate, Nature, 200, 568 (1963). Lindberg and Wickberg, Ark. Kemi, 13, 447 (1959). Posternak and Falbriard, Helv. Chim. Acta, 44, 2080 (1961). Kindl, Kremlicka, and Hoffman-Ostenhof, Monatsh. Chem., 97, 1783 (1966). Plouvier, C. R. Acad. Sci., 255, 360 (1962). Plouvier, C. R. Acad. Sci., 241, 765 (1955). Ackermann, Ber. Dtsch. Chem. Ges., 54, 1938 (1921). Krzeminski and Angyal, J. Chem. Soc. (Lond.), p. 3251 (1962). Bourne, Hutson, and Weigel, J. Chem. Soc. (Lond.), p. 4252 (1960). Maquenne, Ann. Chim. Phys. Ser. 6, 22, 264 (1891). Anderson, Fischer, and MacDonald, J. Am. Chem. Soc., 74, 1479 (1952). Pease, Reider, and Elderfield, J. Org. Chem., 5, 198 (1940). Plouvier, C. R. Acad. Sci., 243, 1913 (1956). Anderson, Takeda, Angyal, and McHugh, Arch. Biochem. Biophys., 78, 518 (1958). DeJong, Recl. Trav. Chim. Pays-Bas, 25, 48 (1906). Adams, Pease, and Clark, J. Am. Chem. Soc., 62, 2194 (1940). Haustveit and Wold, Carbohydr. Res., 29, 325 (1973). Bourne, Percival, and Smestad, Carbohydr. Res., 22, 75 (1972). Prunier, Ann. Chim. Phys. Ser. 5, 15, 5 (1878). McCasland, Naumann, and Durham, Carbohydr.Res., 4, 516 (1967). Bauer and Moll, Arch. Pharm., 280, 37 (1942). Plouvier, C.R. Acad. Sci., 253, 3047 (1961). Gorter, Annchem, 359, 221 (1908). Haslam, Turner, Sargent, and Thompson, J. Chem. Soc. (Lond.), p. 1493 (1971). Ervig and Koenigs, Ber. Dtsch. Chem. Ges., 22, 1457 (1889). Weiss, Davis, and Mingioli, J. Am. Chem. Soc., 75, 5572 (1953). Adlersberg and Sprinson, Biochemistry, 3, 1855 (1964). Shyluk, Youngs, and Gamborg, J. Chromatogr., 26, 268 (1967). Muller, J. Chem. Soc. (Lond.), p. 1767 (1907). Posternak, Helv. Chim. Acta, 25, 746 (1942). Ueno, Hasegawa, and Tsuchiya, Carbohydr. Res., 29, 520 (1973). Sherrard and Kurth, J. Am. Chem. Soc., 51, 3139 (1929). Eijkman, Ber. Dtsch. Chem. Ges., 24, 1278 (1891). Eijkman, Recl. Trav. Chim. Pays-Bas, 4, 32 (1885). McCrindle, Overton, and Raphael, J. Chem. Soc. (Lond.), p. 1560 (1960). Grewe, Buttner, and Burmeister, Angew. Chem., 69, 61 (1957). Salamon and Davis, J. Am. Chem. Soc., 75, 5567 (1953). Horii, Iwasa, Mizuta, and Kameda, J. Antibiot. Ser. A, 24, 59 (1971). Power and Tutin, J. Chem. Soc. (Lond.), p. 624 (1904). Angyal, Gorin, and Pitman, J. Chem. Soc. (Lond.), p. 1807 (1965). Posternak and Schopfer, Helv. Chim. Acta, 33, 343 (1950).
4/16/10 1:21 PM
Table A: Natural Alditols, Inositols, Inososes, and Amino Alditols and Inosamines 27. 1 128. 129. 130. 131. 132. 133. 134. 135. 136. 137.
Nakajima and Kurihara, Ber. Dtsch. Chem. Ges., 94, 515 (1961). Angyal, Gorin, and Pitman, J. Chem. Soc. (Lond.), p. 1807 (1965). Stanacev and Kates, J. Org. Chem., 26, 912 (1961). Posternak, Helv. Chim. Acta, 19, 1333 (1936). Magasanik and Chargaff, J. Biol. Chem., 175, 929 (1948). Post and Anderson, J. Am. Chem. Soc., 84, 471 (1962). Magasanik and Chargaff, J. Biol. Chem., 174, 173 (1948). Berman and Magasanik, J. Biol. Chem., 241, 800 (1966). Stevens, Gillis, French, and Haskell, J. Am. Chem. Soc., 80, 6088 (1958). Johnson, Gourlay, Tarbell, and Autrey, J. Org. Chem., 28, 300 (1963). Nakajima, Kurihara, Hasegawa, and Kurokawa, Justus Liebigs Ann. Chem., 689, 243 (1965). 138. Bannister and Argoudelis, J. Am. Chem. Soc., 85, 119 (1963). 39. Allen, J. Am. Chem. Soc., 78, 5691 (1956). 1 140. Patrick, Williams, Waller, and Hutchings, J. Am. Chem. Soc., 78, 2652 (1956).
9168_Book.indb 415
415
41. Walker and Walker, Biochim. Biophys. Acta, 170, 219 (1968). 1 142. Carter, Clark, Lytle, and McCasland, J. Biol. Chem., 175, 683 (1948). 143. Peck, Hoffhine, Peel, Graber, Holly, Mozingo, and Folkers, J. Am. Chem. Soc., 68, 776 (1946). 144. Nakajima, Hasegawa, and Kurihara, Justus Liebigs Ann. Chem., 689, 235 (1965). 145. Maeda, Murase, Mawatari, and Umezawa, J. Antibiot. Ser. A, 11, 73 (1958). 146. Neuss, Koch, Malloy, Day, Huckstep, Dorman, and Roberts, Helv. Chim. Acta, 53, 2314 (1970). 147. Kondo, Sezaki, Koika, and Akita, J. Antibiot. Ser. A, 18, 192 (1965). 148. Peck, Graber, Walti, Peel, Hoffhine, and Folkers, J. Am. Chem. Soc., 68, 29 (1946). 149. Nakajima, Kurihara, and Hasegawa, Ber. Dtsch. Chem. Ges., 95, 141 (1962). 150. Horii and Kameda, J. chem. Soc. D Chem. Commun., 746, 747 (1972).
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9168_Book.indb 417
Table B: Natural Acids of Carbohydrate Derivation TABLE B: NATURAL ACIDS OF CARBOHYDRATE DERIVATION (Continued)
Substancea (Synonym) Derivative
(A)
Chemical Formula (B)
Melting Point °C (C)
Specific Rotationb [a]D (D)
Chromatography, R Value, and Referenced Referencec (E)
ELC (F)
GLC (G)
PPC (H)
TLC (I)
Aldonic Acids Glycollic acid (hydroxyacetic acid) Acetate Ammonium salt Trimethylsilyl ester ether
C2H4O3 C4H6O4 C4H7NO3
80 66–68 102
None None None
1,5c 1 1
d-Glyceric acid Amide Calcium salt Methyl ester methyl ether
C3H6O4 C3H7NO3 CaC6H10O8 C6H12O4
Gum 99.5–100
dextro −63.1 (CH3OH) +10.9
6 7 8p
l-Glyceric acid Calcium salt Trimethylsilyl ester ether d-Lactic acid (2-hydroxypropionic acid, 3-deoxy-d-glyceric acid) Acetate Amide
C3H6O4 CaC6H10O8
Gum 134–135
levo −12 (30°)
1 9
C3H6O3 C5H8O4 C3H7NO2
26–27 Oil, bp 171–172 49–51
−2.3 +54.3 [a]18Hg +22.2
1,5c 1 1
l-Lactic acid Methyl ester methyl ether
C3H6O3 C5H10O3
25–26 Oil, bp 45 (22 mm)
+3.8 (15°) −95.5
1 1
Trimethylsilyl ester ether
48Cl (2) 10.45MU (3) 85f (8)
55f (4)
20glc (15)
16f (16)
16f (28)
35f (32)
73f (28)
40fg (16) 75fg (23)
24xyll (11)
42Cl (2)
16glc (10)
10.65MU (3)
d,l-Lactic acid Acetate Amide
C3H6O3 C5H8O4 C3H7NO2
18 57–60 75.5
Racemic Racemic Racemic
1 1 1
3-Hydroxypropionic acid (2-deoxyglyceric acid) Methyl ester methyl ether
C3H6O3 C5H10O3
Syrup Oil, bp 142–143
None None
1 1
Hydroxypyruvic acid (2-keto-glyceric acid, 2-triulosonic acid) p-Nitrophenylhydrazone
C3H4O4 C9H9N3O5
260
None None
12 1
Pyruvic acid (2-keto-3 deoxyglyceric acid) p-Nitrophenylhydrazone Methyl ester Trimethylsilyl ester
C3H4O3 C9H9N3O4 C3H6O3
13.6 220 Oil, bp 134–137
None None None
1 1 1
50Cl (2)
d-Arabinonic acid (arabonic acid) Phenylhydrazide Tetraacetate Trimethylsilyl ester ether
C5H10O6 C11H16N2O5 C13H18O10
114–116 208–209 135–136
+10.5 (c6) −13 +32.5
5c,13 17 18
8.6e (14)
l-Arabinonic acid Amide 1,4-Lactone (g-lactone)
C5H10O6 C5H11NO5 C5H8O5
118–119 135–136 95–98
−9.6→−41.7f +37.2 −71.6
19 20 21,22cp
417
Lactone trimethylsilyl ether
75f (4)
10.91MU (3)
55glc (10)
97glc (10)
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418
9168_Book.indb 418
TABLE B: NATURAL ACIDS OF CARBOHYDRATE DERIVATION (Continued) Substancea (Synonym) Derivative
(A)
Chemical Formula (B)
Melting Point °C (C)
Specific Rotationb [a]D (D)
Chromatography, R Value, and Referenced Referencec (E)
ELC (F)
GLC (G)
PPC (H)
ALDONIC ACIDS (Continued) C10H20O6 215 C11H16H2O5
24
Pentulosonic acid, 3-deoxy- d-glycero-2-(2-keto-3-deoxyd-arabonic acid) 2,4-Dinitrophenylhydrazone
C5H8O5
29
61f (29)
29
14f (29)
Pentulosonic acid, d-threo-4- (4-keto-d-arabonic acid) Brucine salt
C5H8O6 C28H34N2O10
Pentulosonic acid, 3-deoxy- l-glycero-2- (2-keto-3-deoxyl-arabonic acid) 2,4-Dinitrophenylhydrazone
C5H8O5
Methyl ester methyl ether Phenylhydrazide
C11H12N4O8
C11H10N4O7h
No constants known 163
No constants known −10.3 −29.4
27 27
No constants known 220–223d
No constants known −22.7 (c 0.3, dioxane)
28
154–155
62f (32)
29f (28)
C5H6O4 C5H10O6
114g
+82.7g
1,4-Lactone Phenylhydrazide
C5H8O5 C11H16N2O5
108–110g 163
+70g +13.7
5c,25,36g 45g 46p 26cp
82f (28)
d-Ribonic acid Amide 1,4-Lactone Methyl ester methyl ether
C5H10O6 C5H11NO5 C5H8O5 C10H20O6
112–113 136–137 77
−17f +17 +17→+8 (13 days)
5c,31 33 34,35p
d-Xylonic acid Amide Brucine salt 1,4-Lactone Lactone trimethylsilyl ether Methyl ester methyl ether Trimethylsilyl ester ether
C5H10O6 C5H11NO5 C28H36N2O10 C5H8O5
Syrup 81–82 170–172 98–101
−2.9→+20.1 +44.5→+23.8 −37.4 +91.8→+86.7
5c,36 37 38 21
l-Xylonic acid
C5H10O6 C28H36N2O10 C5H8O5 C13H18O10
No constants known +24.3 −82.2 −4.5 (c 2, C2H5OH)
26
Brucine salt 1,4-Lactone Tetraacetate
No constants known 177–178 97 86–88
d-Altronic acid 1,4-Lactone Phenylhydrazide
C6H12O7 C6H10O6 C12H18N2O6
110g
+8 +35 +18.4g
5c,8c,47g 8p 42,47g
d-Fuconic acid
C6H12O6 C6H10O5 C11H22O6
No constants known
43p
1,4-Lactone Methyl ester methyl ether
No constants known 104g
C10H20O6
38f (32) 73f (23) 61f (32)
142xyll (11) 9.1 (14) e
79f (23)
126glc (10) 269xyll (11) 48glc (10)
26 39 40
48
132galn (41)
438xyll (11)
Handbook of Biochemistry and Molecular Biology
1,4-Lactone
150–152
(I)
30
l-Lyxonic acid
f
295xyll (11)
TLC
4/16/10 1:21 PM
Substancea (Synonym) Derivative
(A)
Chemical Formula (B)
Melting Point °C (C)
Specific Rotationb [a]D (D)
Chromatography, R Value, and Referenced Referencec (E)
ELC (F)
GLC (G)
PPC (H)
TLC (I)
Aldonic Acids (Continued) Hexulosonic acid, 3,6-dideoxy- d-threo-2- (2-keto-3-deoxy-dfuconic acid)
C6H10O5
No constants known
No constants known
44
d-Galactonic acid
C6H12O7
122 148 175 110–112, 132–133
−11.2→−57.6f −13.6→−17 +31.5 −73→−63.7
49,50 21 51 8,21,52
131–132 203
+12 (CHCl3) +10.4
53 24
No constants known −30 +77
5c,8c
−5 −22.5 (50% C2H5OH)
58 58
+15
59cp,60
−270 (c 0.5, C5H5N) +13.9 (C5H5N)
60
Amide 1,4-Lactone Methyl ester methyl ether Pentaacetate Phenylhydrazide
C6H13NO6 C6H10O6 C12H24O7 C16H22O12 C12H18N2O6
C15H22O12
No constants known 175 110, 134–135 Oil 132–133
Hexulosonic acid, d-lyxo-2- (2-keto-d-galactonic acid) Brucine salt
C6H10O7 C29H36N2O11
169 172
Hexulosonic acid, 3-deoxy- d-threo-2 (2-keto-3-deoxy-dgalactonic acid) Lactone phenylhydrazone
C6H10O6
l-Galactonic acid Amide 1,4-Lactone Lactone trimethylsilyl ether Pentaacetate
Phenylhydrazone phenylhydrazide Potassium salt Hexulosonic acid, d-arabino-5- (5-keto- l-galactonic or d-tagaturonic acid) Brucine salt Calcium salt · 5H2O d-Gluconic acid Amide 1,4-Lactone Lactone trimethylsilyl ether 1,5-Lactone Methyl ester methyl ether Pentaacetate Trimethylsilyl ester ether
C6H13NO6 C6H10O6
−14 (28°, CHCl3)
213–214
C18H22N4O4 C6H9KO6
204–205 159–163d
C6H10O7
108–109
C29H36N2O11
148–149, 189–190dg
−17g
62,64g
−14
63
C6H12O7 C6H13NO6 C6H10O6
120–121 143–144 133–135
−6.9→+7.3 +31.2 +68→+17.7
5c,21 20,65 8c,21
C6H10O6 C12H24O7 C16H22O12
150–152
+66→+8.8
21
110–111
+11.5 (CHCl3)
40
C10H19NO8
No constants known 92–95d 69–73
No constants known +20.9 (CH3OH) +32.3 (CH3OH)
66
C10H20N2O7 · HCl C10H17NO7 · HCl
38f (32)
1185xyll (11)
54 55,56 57 54
C12H14N2O4
CaC12H18O14 · 5H2O
8.4e (14)
66myo (57)
100galn (41) 78f (23)
68f (71)
57f (61)
60 61 62p
66 66
62galn (41)
25Cl (2) 198glc (10)
10glc (15)
40f (32)
210glc (15)
75f (23)
708xyll (11) l00glc (10)
419
d-Gluconic acid, 6-O-(N,N-dimethylglycyl)- (pangamic acid, vitamin B15) Amide hydrochloride Lactone hydrochloride
C6H12O7
78f (44)
Table B: Natural Acids of Carbohydrate Derivation
9168_Book.indb 419
TABLE B: NATURAL ACIDS OF CARBOHYDRATE DERIVATION (Continued)
4/16/10 1:21 PM
420
9168_Book.indb 420
TABLE B: NATURAL ACIDS OF CARBOHYDRATE DERIVATION (Continued) Substancea (Synonym) Derivative
(A)
Chemical Formula (B)
Melting Point °C (C)
Specific Rotationb [a]D (D)
Chromatography, R Value, and Referenced Referencec (E)
ELC (F)
GLC (G)
PPC (H)
TLC (I)
Aldonic Acids (Continued) Hexonic acid, 2-deoxy-d-arabino- (2-deoxy-d-gluconic acid) 1,4-Lactone Methyl ester methyl ether Phenylhydrazide
C6H12O6 C6H10O5 C11H22O6 C12H18N2O5
Hexulosonic acid, d-arabino-2- (2-keto-d-gluconic acid) Brucine salt Calcium salt · 3H2O Methyl ester methyl ether Phenylhydrazone phenylhydrazide
C6H10O7 C29H36N2O11 CaC12H18O11 · 3H2O C11H20O7 C18H24N4O6
Hexulosonic acid, 3-deoxy-d-erythro-2- (2-keto-3-deoxyd-gluconic acid) Calcium salt · 1/2H2O Phenylhydrazone Hexulosonic acid, d-xylo-5- (5-keto-d-gluconic acid) Brucine salt Calcium salt · 3H2O Methyl ester methyl ether
C6H10O7 C29H36N2O11 CaC12H18O14 · 3H2O C11H20O7
Hex-2,5-diulosonic acid, d-threo- (2,5-diketo-d-gluconic acid)
C6H8O7
+2 +68
156
67,68cpt 68,69
61f (70) 353xyll (11)
69 −99.6 (dil HCl) −59.4 (c 0.4) −70.8
5c,72 74p 63,76p
121
−36.1 (H2O, C5H5N)
72
C6H10O6
119.5–120
−45.2
60,75
CaC12H18O12 · 1/2H2O C12H16N2O5
229d
−29.2 (c 6) +168 (C5H5N)
77p 60
−14.5 −24 −11.7 (dil HCl)
78,79 64 79
9.3e (14)
No constants known −51 ± 5 +57.2 (C5H5N)
82p
100kgu (88)
59glc (83)
82 82 5c,21,g8 84 85 85,86
25Clg (2)
10glcg (15)
40fg (32)
30f (89a) 84galn (41)
31fg (32) 53fg (32)
CaC12H14O14 · 3H2O C18H16N4O13
179–182 153d
174–175d
No constants known 156–157d
10.7e (14)
116galn (62)
120–121g 181–182 203–204
–6.9→+7.3g −6.4 (c 8) −25.4 −11.7
l-Gulonic acid 1,4-Lactone Lactone trimethylsilyl ether Methyl ester methyl ether
C6H12O7 C6H10O6
Syrup 183–185
0 +55
5c,87 56
47kgu (88)
Hexulosonic acid, l-xylo-2- (2-keto- l-gulonic acid) Brucine salt · H2O Sodium salt Trimethylsilyl ester ether
C6H10O7 C29H36N2O11 · H2O C6H9NaO7
170–171 114 145
−48.8
100kgu (88)
−24.4
63 90 90
Hexulosono-1,4-lactone, l-xylo-2- (2-keto- l-gulono-g-lactone, l-ascorbic acid)i Bis(phenylhydrazone) Trimethylsilyl ester ether
C6H8O6
190d
+24.7
96c
25Cl (2)
C18H18N4O4
187
Hexulosono-1,4-lactone 2-sulfate, l-xylo-2 (ascorbic acid sulfate) Ammonium salt
C6H8O9S C6H10NO9S
70myo (57) 632xyll (11)
C12H24O7
96c 96
24f (80eg) 79f (81) 538xyll (11)
C6H12O7 BaC12H22O14 C29H38N2O11 C12H18N2O6
+98.5 (pH 8.6) +202.8
18glc (73)
348xyll (11)
l-Gluconic acid Barium salt Brucine salt Phenylhydrazide
1
10glc (15)
35glc (73) 164glc (91) 90f (89b)
57f (32)
40f (96)
39f (97p)
183glc
Handbook of Biochemistry and Molecular Biology
Calcium salt · 3H2O Bis(2,4-dinitrophenylhydrazone)
142–144 93–95
4/16/10 1:21 PM
Substancea (Synonym) Derivative
(A)
Chemical Formula (B)
Melting Point °C (C)
Specific Rotationb [a]D (D)
Chromatography, R Value, and Referenced Referencec (E)
ELC (F)
GLC (G)
PPC (H)
TLC (I)
Aldonic Acids (Continued) Hexulosonic acid, l-xylo-3- (3-keto-l-gulonic acid)
C6H10O7
No constants known
No constants known
92
Hexulosonic acid, d -lyxo-5- (5-keto-l-gulonic acid or d-fructuronic acid) Brucine salt Potassium salt
C6H10O7
No constants known 195–197 160–165
No constants known −15.5 +11
62,71
Hex-2,3-diulosonic acid, l-threo- (2,3-diketo-l-gulonic acid)
C6H8O7
No constants known 281
No constants known
94,95
No constants known 113d 67–68 No constants known 190–192 Syrup 115–117 179
No constants known +5.5 No constants known −17 +50,+4.5 +12.5 +8
203 151–152 158–160
−15.6 −27.8 +51.5 +114→+30.3
5c,103 105 8,21 21
68–70 212–214
+23 (CHCl3) +15.8
106 42,107
No constants known
No constants known
108
17k (108)
−17.9
109c
82f (110c)
+4→−32.7 +27.5 −39.2,−51 −100→−35.1
5c, 36, 86 111 8,21 21
No constants known +42.4 −5.8 +33
112c
23f (113)
114 113 115
39f (113)
Bis(2,4-dinitrophenylhydrazone) Hexulosonic acid, 4-deoxy-d-threo-5 (4-deoxy-5-keto-d-idonic acid)j Phenylosazone Sodium salt l-Idonic acid Brucine salt 1,4-Lactone Phenylhydrazide Sodium salt
C29H36N2O11 C6H9KO7
C18H16N4O13 C6H10O6 C18H22N4O4 C6H9NaO6 C6H12O7 C29H38N2O11 C6H10O6 C12H18N2O6 C6H11NaO7
d-Mannonic acid Brucine salt 1,4-Lactone 1,5-Lactone Methyl ester methyl ether Pentaacetate Phenylhydrazide
C6H12O7 C29H38N2O11 C6H10O6 C6H10O6 C12H24O7 C16H22O12 C12H18N2O6
Hex-2,5-diulosonic acid, 3-deoxy-d-glycero- (3-deoxy-2,5-diketo-dmannonic acid)
C6H8O6
Hexulosonic acid, 2,3,6-trideoxy-d-glycero-4-(4-keto-2,3,6trideoxy-d-mannonic acid, 5-hydroxy-4-ketohexanoic acid)
C6H10O4
l-Mannonic acid, 6-deoxy-(l-rhamnonic acid) Amide 1,4-Lactone 1,5-Lactone Methyl ester methyl ether
C6H12O6 C6H13NO5 C6H10O5 C6H10O5 C11H20O5
Heptulosonic acid, 3-deoxy-d-arabino-2-
C7H12O7 C7H15NO7 C7H10O6 C7H10O6
No constants known 96 167
62f (41)
62,64 93
78f (81)
95 98,99 98 98 5c,100
40f (98)
47kgu (88)
8 8,101 101 102
111f (41)
47kgu (88)
4f (104) 112f (41)
62fg (23)
779xyll (11)
69f (23) 361xyll (11)
421
Ammonium salt 1,4-Lactone 1,5-Lactone
141–142 149–151 172–182
52kgu (88)
Table B: Natural Acids of Carbohydrate Derivation
9168_Book.indb 421
TABLE B: NATURAL ACIDS OF CARBOHYDRATE DERIVATION (Continued)
4/16/10 1:21 PM
422
9168_Book.indb 422
TABLE B: NATURAL ACIDS OF CARBOHYDRATE DERIVATION (Continued) Substancea (Synonym) Derivative
(A)
Methyl ester glycoside 7-Phosphate Octulosonic acid, 3-deoxy- d-manno-2-
Chemical Formula (B)
C9H14O6 C7H13O10P
Melting Point °C (C)
Specific Rotationb [a]D (D)
Aldonic Acids (Continued) 148 +78.2 (CH3OH)
C8H14O8
No constants known 125–126 192–194 98–103
No constants known +41.3 +31.8
Chromatography, R Value, and Referenced Referencec (E)
ELC (F)
GLC (G)
115 116c
Ammonium salt · H2O 1,4-Lactone Pentaacetate 8-Phosphate
C8H17NO8 · H2O C8H12O7 C18H24O13 C8H15O11P
Glyoxylic acid Methyl ester Phenylhydrazone Trimethylsilyl ester Trimethylsilyl ester Malonic semi-aldehyde (2-deoxy-glyceruronic acid, formylacetic acid) Semicarbazone
C2H2O3 C3H4O3 C8H8N2O2
98 53 144,137d
None None None
1,5c 1 1
C3H4O3
No constants known 116d
No constants known
8
PPC (H)
TLC (I)
49f (112) 100kdo (117)
52kdh (116)
118c 118c 118c
25ara (116) 51f (119)
63f (118)
Uronic Acids
C4H7N3O3
No constants known
No constants known
8
d-Lyxuronic acid
C5H8O6
No constants known
No constants known −23→−53 −37.7→−23g
120
a- d-Galacturonic acid · H2O
120 121 122
p-Bromophenylhydrazone salt
Br2C18H24N4O6
159–160 (110–115s) 145–146
2,3,4-Tri-O-methyl ether
C12H22O7
160
+9 ± 2 (c 0.7, CH3OH) +27→
C6H10O7
160
+27→+55.6
123
p-Bromophenylhydrazone Brucine salt Methyl ester β-methyl pyranoside
Br2C12H15BrN2O6 C29H36N2O11 C8H14O7
150–151 180 194–196
+11.5±2 (CH3OH) −7.7 −49 (CH3OH)
124 123 125pt
β- d-Glucuronic acid Brucine salt · H2O Phenylhydrazone phenylhydrazide phenylhydrazide 2,3,4-Tri-O-methyl ether
C6H10O7 C29H36N2O11 · H2O C18H22N4O5
156 156–157 182
+11.7→ + 36.3 −15.1
126,127 128 129
β- d-Galacturonic acid
Trimethylsilyl ether
C6H10O7 · H2O
140g 164d
C12H22O7
12.65MU (3)
+97.9→+50.9
123
7.9e (14)
85f (41)
124 123
67tmg (138a) 18glc (153.141c)
9.3e (14)
32f (32)
46f (41)
372tmg (156) 7 (154)
84tmg (138a)
13f (4)
Handbook of Biochemistry and Molecular Biology
C3H4O4
CaC10H14O12 · 2H2O C6H10O6 C23H28N6O4
30f (16)
1
Glyceruronic acid (tartonic semialdehyde)
Calcium salt · 2H2O Methyl ester Phenylosazone salt
57Cl (2)
4/16/10 1:21 PM
Substancea (Synonym) Derivative
(A)
Chemical Formula (B)
Melting Point °C (C)
Specific Rotationb [a]D (D)
Chromatography, R Value, and Referenced Referencec (E)
ELC (F)
GLC (G)
PPC
TLC
125f (41)
58f (32)
(H)
(I)
Uronic Acids (Continued) d-Glucurono-3,6-lactone
C6H8O6
163–165, 180
+18.6
130,131
d-Glucuronic acid, 3-O-methylp-Bromophenylosazone salt Methyl ester a-methyl pyranoside
C7H12O7 Br3C25H39N6O5 C9H16O7
Syrup 157 88.5–89
+6 −104→−14 +150 (CH3OH)
132,133 134 135ct
d-Glucuronic acid, 4-O-methyl-
C7H12O7
Syrup
+48,+82
136p,137p
C8H15O6 C8H14O7 C9H16O7
232 123–124 203–216
+143 (c0.7) +41 +145.5 (CH3OH, 15°)
139
l-Glucuronic acid 3,6-Lactone
C6H10O7 C6H8O6
174–176
Lactone 1,2,5-triacetate l-Guluronic acid
C12H14O9 C6H10O7
Amide a-methyl-pyranoside Methyl ester Methyl ester a-methyl pyranoside
9.2e (14)
145galu (138b)
140
−33.3 (c 0.5) −19
141c 142pt
195–196 Syrup
−85.4 (CHCl3)
142 143,147cp
C6H8O6
141–142
+81.7
146ct
Hexulosuronic acid, 4-deoxy- l-erythro-5- (4-deoxy-5-ketod-mannuronic acid)
C6H8O6
No constants known
No constants known
148c
26l (148)
Hexulosuronic acid, 4-deoxy- l-threo-5- (4-deoxy-5-ketol-iduronic acid) Methyl ester β-methyl glucoside Methyl ester a-methyl glycoside
C6H8O6
No constants known
No constants known +192.5 (CH3OH) −67 (CH3OH)
108c
26l (1480)
125 125
76f (149)
l-Iduronic acid
C6H10O7
131–133
+37→+33
150,151
3,6-Lactone
C6H8O6
Syrup
+30 (18°)
131
Sodium salt
C6H9NaO7
C6H10O7 · H2O
120–130 (110s)
+16→−6.1 (c 6.8)
155
C6H8O6
140–141
+89.3
155
C6H10O7 Br2C18H24N4O6
165–167 143–144d
155 −47.9→−23.9 +48.5 ± 1(CH3OH) 124
3,6-Lactone
C8H12O6 C6H12O6
40asc (141a) 310glcu (141 b) 81manu (144)
79glcu (152)
Trimethylsilyl ether a- d-Mannuronic acid · H2O 3,6-Lactone β- d-Mannuronic acid p-Bromophenylhydrazone salt
28glc (153,141c)
5 (154) 100manu (144)
28glc (153,141c) 377glcu (141b)
Table B: Natural Acids of Carbohydrate Derivation
9168_Book.indb 423
TABLE B: NATURAL ACIDS OF CARBOHYDRATE DERIVATION (Continued)
53glc (145) 150glc (145)
30f (125)
65glc (131,141c) 85glc (131,141c) 264glcu (141c) 22f (80)
36f (32)
52f (153)
53f (32)
Aldaric Acids C2H2O4 C2H2O4 · 2H2O C2H4N2O2
189–190 102, 150–160s 320d
None None None
1 1 1
70Cl (2)
88f (4)
423
4/16/10 1:21 PM
Oxalic acid Dihydrate Diamide
424
9168_Book.indb 424
TABLE B: NATURAL ACIDS OF CARBOHYDRATE DERIVATION (Continued) Substancea (Synonym) Derivative
(A)
Dimethyl ester Trimethylsilyl ester
Chemical Formula (B)
C4H6O4
Melting Point °C (C)
Specific Rotationb [a]D (D)
Aldaric Acids (Continued) 54 None
Chromatography, R Value, and Referenced Referencec (E)
ELC (F)
1
C3H4O4 C3H6N2O2 C5H8O4
135–136 170 Bp 181
None None None
1 1 1
63Cl (2)
C3H4O5 C3H6N2O3 C5H8O5
141–142 198 44–45
None (meso) None None
1 1 1
57Cl (2)
d-Threaric acid (l-tartaric) Trimethylsilyl ester ether
C4H6O6
170
−15
157
59Cl (2)
l-Threaric acid (d-tartaric) Diamide Dimethyl ester
C4H6O6 C4H8N2O4 C6H10O6
170 195 48, 61.5
+15 (15°) +106.5 +2.7
158,159 160 161–163
l-Malic acid (hydroxysuccinic acid, deoxytartaric acid) Acetate Diamide Dimethyl ester Trimethylsilyl ester ether
C4H6O5 C6H8O6 C4H8N2O3 C6H10O5
100 132 156–157
−2.3 (c 8.4)
164,165 167 168 1
Oxaloacetic acid (hydroxymaleic acid, ketosuccinic acid)
C4H4O5 C4H6N2O3 C6H8O5
No constants known 124d 77
Allaric acid (allo-mucic acid)
C6H10O5
188–192d
d-Galactaric acid (mucic acid)
C6H10O8
215d
None (meso)
170
C8H14O8 C18H22N4O6
184–186 242
None None
171 170
d-Glucaric acid (saccharic acid) Diamide 1,4-Lactone · H2O 6,3-Lactone Bis(phenylhydrazide)
C6H10O8 C6H12N2O6 C6H8O7 · H2O C6H8O7 C18H22N4O6
125–126 172–173 90–95 143–145 209–210
+6.9→+20.6 +13.3
172 20 173 173 173
d-Mannaric acid (d-manno-saccharic acid) Diamide Bis(phenylhydrazide)
C6H10O8 C6H12N2O6 C18H22N4O6
128.5 188–189.5 214–216d
+3.5–+48.7 −24.4
174 20 175
Dimethyl ester Bis(phenylhydrazide)
PPC (H)
TLC (I)
12.MU (3) 73f (4) 28glc (10) 58f (4)
16.8MU (3)
57Cl (2)
56f (166a)
60f (166b)
15MU (3) 48Cl (2)
1 1
15.65MU (3)
169 200pa (171cp)
90mal (170)
10.8e (14)
2f (89b)
59f (4)
23f (173) 30f (173)
43f (32) 85f (32)
Handbook of Biochemistry and Molecular Biology
Diamide Dimethyl ester Trimethylsilyl ester ether
No constants known
(G)
11.14MU (3)
Malonic acid (deoxytartonic acid) Diamide Dimethyl ester Trimethylsilyl ester Tartronic acid Diamide Dimethyl ester Trimethylsilyl ester ether
−37.9 +6
GLC
4/16/10 1:21 PM
Substancea (Synonym) Derivative
(A)
Chemical Formula (B)
Specific Rotationb [a]D
Melting Point °C (C)
(D)
Chromatography, R Value, and Referenced Referencec (E)
ELC (F)
GLC (G)
PPC (H)
TLC (I)
Amino Sugar Acids Glycine (aminoacetic acid, amino deoxyglycollic acid) Amide Hydrochloride Methyl ester N,N-Trimethylsilyl amine ester
C2H5NO2 C2H6N2O C2H5NO2 · HCl C3H7NO2
233–236d 65–67 185 Bp 54 (50 mm)
None None None None
176 1 1 1
Sarcosine (N-methylaminoacetic acid) Hydrochloride N-Trimethylsilyl amine ester
C3H7NO2 C3H7NO2 · HCl
212–213d 168–170
None None
1 1
l-Serine (2-amino-2-deoxy-l-glyceric acid, 2-amino-3hydroxypropionic acid) Methyl ester hydrochloride N-Trimethylsilyl amine ester ether
C3H7NO3
228dg
−7.3 (c 5.5, 26°)
1,180p
C4H9NO3 · HCl
167
l-Alanine (2-amino-2,3-di-deoxy-l-glyceric acid, deoxy-l-serine, 2-aminopropionic acid) Amide Hydrochloride N-Trimethylsilyl amine ester
C3H7NO2
297d
+2.7
1
C3H8N2O C3H7NO2 · HCl
72 204
+10.4
1 1
Acetoacetic acid, 2-amino-
C4H7NO3
No constants known +2.8
181 181
C6H11NO3 · HCl
l-Xylonic acid, 2-amino-2-deoxy (polyoxamic acid) N-Acetyl 1,4-lactone
C5H11NO5 C7H11NO5
171–173d 150–152
l-Xylonic acid, 2-amino-2-deoxy-5-O-carbamoyl-
C6H12N2O6
226–232d
+1.3
181
l-Xylonic acid, 2-amino-2,3-dideoxy-
C5H11NO4
Syrup
+11
181
l-Xylonic acid, 2-aminocarbamoylN-Acetyl 1,4-lactone
C6H12N2O5 C8H12N2O5
215–216d 181–191
+5.8
181 181
d-Galactonic acid, 2-amino-2-deoxy- (d-galactosaminic acid, chondrosaminic acid) N-Acetyl acid · H2O
C6H13NO6
198–203d
−5 (c 0.6)
60
C8H15NO7 · H2O
102–103
−33.8→−16 (3 days)
182pt
22f (179)
28f (178)
47f (179)
49f (178)
26f (179)
11.43MU (3) 215ala (177) 13.8MU (3) 100ala (177)
11.05MU (3)
1 1
C8H13NO6
165
d-Alluronic acid, 3-acetamido-3-deoxy-
C8H13NO7
182–183
3-Amino-3-deoxy- d-allose·hydrochloride
C6H13NO5 · HCl
157–160d
+25 (c 0.7)
199
d-Alluronic acid, 5-amino-5-deoxy-
C6H11NO6
No constants known
No constants known
181
d-Galacturonic acid, 2-amino-2-deoxy- (d-galactosaminuronic acid)
C6H11NO6
160d
+84 (pH 2, HCl)
186p
a- d-Galactosamine · HCl
C6H13NO5 · HCl
185
+121→+80
187
N-Acetyl 1, 4-lactone
32f (178)
13.18MU (3)
1
No constants known 95d
Ethyl ester hydrochloride
150ala (177)
Table B: Natural Acids of Carbohydrate Derivation
9168_Book.indb 425
TABLE B: NATURAL ACIDS OF CARBOHYDRATE DERIVATION (Continued)
183 184
68gNUA (192a)
46f (185)
425
69F (194)
4/16/10 1:21 PM
426
9168_Book.indb 426
TABLE B: NATURAL ACIDS OF CARBOHYDRATE DERIVATION (Continued) Substancea (Synonym) Derivative
(A)
Chemical Formula (B)
Melting Point °C (C)
Specific Rotationb [a]D (D)
Chromatography, R Value, and Referenced Referencec (E)
ELC (F)
GLC (G)
PPC (H)
TLC (I)
Amino Sugar Acids (Continued) β- d-Galactosamine · HCl
C6H13NO5 · HCl
187
+44→
187
d-Glucuronic acid, 2-amino-2-deoxy- (d-glucosaminuronic acid)
C6H11NO6
120–172d
+55
188,189
7.2m (188cp)
57F (185)
a- d-Glucosamine
C6H13NO5
88
+100→+47
190
9m (188cp)
76f (194)
β- d-Glucosamine Methyl a-glycoside N-Acetyl furanurono-1,4-lactone
C6H13NO5 C7H13NO6 C8H11NO6
110–111 203–207 (196s) 177–178
+28→+47 +126.3 +43.6
190 189 191
d-Guluronic acid, 2-amino-2-deoxy- (d-gulosaminuronic acid)
C6H11NO6
No constants known
No constants known
193c
d-Gulosamine · HCl
C6H13NO5 · HCl
150–170d
+34→−19
200,201
Hexuronic acid, 2-amino-2-deoxy-
C6H11NO6
+11.5
192c
N-Trimethylsilyl amine ester ether
71gNUA (193a)
43glcN (193b) 77f (194)
40gNUA (192a)
128eic (192c)
27glcN (192b)
No constants known +30.5 (c 0.3) +87 (CHCl3)
196 196
d-Mannuronic acid, 2-amino-2-deoxy- (d-mannosaminuronic acid)
C6H11NO6
92–94
−9.9 (c 0.6)
197,198
d-Mannosamine hydrochloride
C6H13NO5 · HCl
178–180d
−3
202
Destomic acid
C7H15NO7
207–209d
211 211
C8H17NO7 · HCl
150–151d
+1.9 −12.1→−30.6 (2 N HCl)
Muramic acid (2-amino-3-O-(d-1-carboxyethyl)-2-deoxy-d-glucose)
C9H17NO7
155
+165→+123 (3 h)
203cp,204cp 13glcN (210a)
76f (194)
N-Acetylmuramic acid
C11H19NO8
122–124
+59→+39 (6 h)
205
100glcN (205)
Methyl a-pyranoside N-Acetyl methyl a-pyranoside methyl ester
Methyl ester · HCl
195
C13H21NO9 · 1/2 H2O
176
+56 (c 0.6)
49glcN (193b) 80f (194)
211
N-Acetyl trimethylsilyl ether glycoside N-Acetyl muramic acid 6-acetate · 1/2 H2O
115gNUA (193a)
194gNAc (210b)
206,207
N-Acetyl 6-acetate trimethylsilyl ether glycoside
152myot (206a) 175myot (206a)
N-Glycolyl muramic acid · H2O
C11H19NO9 · H2O
+56 (50% C2H5OH)
208
manno-Muramic acid [2-amino-3-O- (d-1-carboxyethyl)-2-deoxy-d-mannose]
C9H17NO7
+21 (c 0.6, 79% C2H5OH)
209c
13glcN (210a)
245gNAc (206b)
130gNac (208a)
62gNAc (208b)
180glcN (209a)
25f (209b)
Handbook of Biochemistry and Molecular Biology
C7H11NO4 C10H15NO5
No constants known >270d 145–146
Hex-2-enuronic acid, 4-amino-2,3,4-trideoxy- d-erythro
C6H9NO4
90glcN (189) 38f (191)
4/16/10 1:21 PM
Substancea (Synonym) Derivative
(A)
Chemical Formula (B)
Melting Point °C (C)
Specific Rotationb [a]D (D)
Chromatography, R Value, and Referenced Referencec (E)
Amino Sugar Acids (Continued)
(F)
(G)
PPC (H)
No constants known
183–186d
−31.7
212,213
C17H23N3O7
204–205
−100 (c 0.3, 1:1 H2O, CH3SOCH3)
215
N-Acetyl neuraminic acid 4-acetate
C13H21NO10
200d
−62±1
217
N-Acetyl neuraminic acid 7-acetate · H2O
C13H21NO10 · H2O
138–140d
+6.2±2
217
233nana (217)
N-Acetyl neuraminic acid 7,8(9)-diacetate · CH3OH
C15H23NO11 · CH3OH 130–131d
+9.2±2
217
466nana (217)
N-Glycoly lneuraminic acid
C11H19NO10
189–191d
−33.6
218c, 220ep
30f (218)
N-Acetoglycolyl-4-C methyl-4,9-dideoxyneuraminic acid
C14H23NO9
No constants known
No constants known
N-Glycolyl sialic acid 4-acetate
C13H21NO11
No constants known
No constants known
123nana (221ct)
N-Glycolyl-8-O-methyl-neuraminic acid
C12H21NO10 Unknown
No constants known No constants known
25–35f (222)
Hf-Neuraminic acid
No constants known No constants known
N-Acetyl trimethylsilyl ether glycoside Nonulosonic acid, 5-acetamino-3,5-dideoxy- d-glycero- d-galacto(N-acetylneuraminic acid, gynaminic acid, lactaminic acid, sialic acid) Quinoxaline deriv.
C11H19NO9
n
Methyl glycoside tetratrifluoroacetate
Methyl glycoside tetratrifluoroacetate
189gNAc (210b)
GLC
No constants known
N-Acetyl manno-muramic acid
C11H19NO8
ELC
19.6 (209c)
TLC (I)
160gNAC (209a) 14f (212)
17f (214)
80–82 (216)
86–88 (216)
47nana (219c)
Table B: Natural Acids of Carbohydrate Derivation
9168_Book.indb 427
TABLE B: NATURAL ACIDS OF CARBOHYDRATE DERIVATION (Continued)
144nana (219c)
31f (223cegt)
Compiled by George G. Maher. In order of increasing carbon chain length in the parent compounds grouped in the classes-aldonic, uronic, aldaric, and amino sugar acids. [a]D for 1–5 g solute, c, per 100 ml aqueous solution at 20–25°C, unless otherwise given. c References for melting point and specific rotation data. Letter indicates the reference also has chromatographic data according to: c = column, e = electrophoresis, g = gas, p = paper, and t = thin-layer. d R value times 100, given relative to that of the compound indicated by abbreviation: f = solvent front, ala = alanine, ara = arabinose, asa = ascorbic acid, Cl = chloride ion, eic = eicosane, galn = galactono-1,4-lactone, galU = galacturonic acid, glc = glucose, glcN = glucosamine, glcU = glucuronic acid, gNAc = N-acetyl-glucosamine, gNUA = glucosaminuronic acid, kdh = 3-deoxy-erythro-hexulosonic acid, kdo = 3-deoxy-manno-octulosonic acid, kgu = 2-keto-gulonic acid, mal = malonic acid, manU = mannuronic acid, myo = myo-inositol, myot = myo-inositol trimethylsilyl ether, MU = methylene standard hydrocarbon units, nana = N-acetyl-neuraminic acid, pa = picric acid, rha = rhamnose, tmg = 2,3,4,6-tetra-O-methyl glucose, and xyll = xylitol pentamethylether. Under gas chromatography (column Glc or G) numbers without code indications signify retention time in minutes. The conditions of the chromatography are correlated with the reference given in parentheses and are found in Table E. e Value is in cm/h. f Equilibrates with the lactone. g Data given are for the enanthiomorphic isomer. h The analytical elemental analysis indicates the compound is an anhydride. i The enol form is l-ascorbic acid. j Reference 99 terms this compound 2-deoxy-5-keto-d-gluconic acid; neither name nor structure seem definite. k Value is in cm/9 h. l Value is in cm/24 h. m Value is in cm/1.5 h. n Some workers relate the formula C H NO whose elemental analysis is little different from that of C H NO . 12 21 10 11 19 9 a
b
427
4/16/10 1:21 PM
Handbook of Biochemistry and Molecular Biology
428
Rererences 1. Pollock and Stevens, Dictionary of Organic Compounds, Oxford University Press, New York, 1965. 2. Gross, Chem. Ind., p. 1219 (1959). 3. Butts, Anal. Biochem., 46, 187 (1972). 4. Baraldi, J. Chromatogr., 42, 125 (1969). 5. Carlsson and Samuelson, Anal. Chim. Acta, 49, 248 (1970). 6. Frankland and McGregor, J. Chem. Soc. (Lond.), p.513 (1893). 7. Frankland, Wharton, and Aston, J. Chem. Soc. (Lond.), p. 269 (1901). 8. Isherwood, Chen, and Mapson, Biochem. J., 56, 1–15 (1954). 9. Wolfrom and DeWalt, J. Am. Chem. Soc., 70, 3148 (1948). 10. Verhaar and de Wilt, J. Chromatogr., 41, 168 (1969). 11. Whyte, J. Chromatogr., 87, 163 (1973). 12. Hough and Jones, J. Chem. Soc. (Lond.), p. 4052 (1952). 13. Robbins and Upson, J. Am. Chem. Soc., 62, 1074 (1940). 14. Theander, Sven. Kem. Tidskr., 70, 393 (1958). 15. Bourne, Hutson, and Weigel, J. Chem. Soc. (Lond.), p. 5153 (1960). 16. Wolfrom, Patin, and Lederkremer, J. Chromatogr., 17, 488 (1965). 17. Hardegger, Kreiss, and El Khadem, Helv. Chim. Acta, 35, 618 (1952). 18. Robbins and Upson, J. Am. Chem. Soc., 62, 1074 (1940). 19. Rehorst, Ber. Dtsch. Chem. Ges., 63, 2280 (1930). 20. Hudson and Komatsu, J. Am. Chem. Soc., 41, 1141 (1919). 21. Isbell and Frush, J. Res. Nat. Bur. Stand., 11, 649 (1933). 22. Assarson, Lindberg, and Vorbrueygen, Acta Chem. Scand., 13, 1395 (1959). 23. Němec, Kefurt, and Jarý, J. Chromatogr., 26, 116 (1967). 24. Bates, Polarimetry, Saccharimetry and the Sugars, Nat. Bur. Stand. Circ. C440, U.S. Govt. Print. Off., Washington, D.C., 1942, 790. 25. Gardner and Wenis, J. Am. Chem. Soc., 73, 1855 (1951). 26. Kanfer, Ashwell, and Burns, J. Biol. Chem., 235, 2518 (1960). 27. Liebster, Kulhanek, and Tadra, Chem. Listy, 47, 1075 (1953). 28. Weimberg, J. Biol. Chem., 234, 727 (1959). 29. Palleroni and Doudoroff, J. Biol. Chem., 223, 499 (1956). 30. Kurata and Sakurai, Agric. Biol. Chem., 32, 1250 (1968). 31. Ladenberg, Tishler, Wellmann, and Babson, J. Am. Chem. Soc., 66, 1217 (1944). 32. Hay, Lewis, and Smith, J. Chromatogr., 11, 479 (1963). 33. Wolfrom, Bennett, and Crum, J. Am. Chem. Soc., 80, 944 (1958). 34. Steiger, Helv. Chim. Acta, 19, 189 (1936). 35. Hough, Jones, and Mitchell, Can. J. Chem., 36, 1720 (1958). 36. Rehorst, Justus Liebigs Ann. Chem., 503, 143, 154 (1933). 37. Weerman, Recl. Trav. Chim. Pays-Bas, 37, 15, 40 (1917). 38. Menzinsky, Ber. Dtsch. Chem. Ges., 68, 822 (1935). 39. Heyns and Stein, Justus Liebigs Ann. Chem., 558, 194 (1947). 40. Major and Cook, J. Am. Chem. Soc., 58, 2474, 2477 (1936). 41. Hickman and Ashwell, J. Biol. Chem., 241, 1424 (1966). 42. Hickman and Ashwell, J. Biol. Chem., 235, 1566 (1960). 43. Dahms and Anderson, J. Biol. Chem., 247, 2222, 2228 (1972). 44. Dahms and Anderson, J. Biol. Chem., 247, 2233 (1972). 45. Isbell, J. Res. Nat. Bur. Stand., 29, 227 (1942). 46. Gorin and Perlin, Can. J. Chem., 34, 693 (1956). 47. Humoller, McManus, and Austin, J. Am. Chem. Soc., 58, 2479 (1936). 48. Mortensson-Egnund, Schöyen, Howe, Lee, and Harboe, J. Bacteriol., 98, 924 (1969). 49. Kiliani, Ber. Dtsch. Chem. Ges., 55, 75 (1922). 50. Pryde, J. Chem. Soc. (Lond.), p. 1808 (1923). 51. Glattfeld and MacMillan, J. Am. Chem. Soc., 56, 2481 (1934). 52. Levene and Meyer, J. Biol. Chem., 46, 307 (1921). 53. Hurd and Sowden, J. Am. Chem. Soc., 60, 235 (1938). 54. Wolfrom, Berkebile, and Thompson, J. Am. Chem. Soc., 71, 2360 (1949). 55. Fukunaga and Kubata, Bull. Chem. Soc. Jap., 13, 272 (1938). 56. Wolfrom and Anno, J. Am. Chem. Soc., 74, 5583 (1952). 57. Loewus, Carbohydr. Res., 3, 130 (1966). 58. Ettel, Liebster, and Tadra, Chem. Listy, 46, 45 (1952). 59. Claus, Biochem. Biophys. Res. Commun., 20, 745 (1965). 60. Kuhn, Weiser, and Fischer, Justus Liebigs Ann. Chem., 628, 207 (1959). 61. Ley and Doudoroff, J. Biol. Chem., 227, 745 (1957). 62. Ashwell, Wahba, and Hickman, J. Biol. Chem., 235, 1559 (1960). 63. Regna and Caldwell, J. Am. Chem. Soc., 66, 243, 244, 246 (1944).
9168_Book.indb 428
64. Hart and Everett, J. Am. Chem. Soc., 61, 1822 (1939). 65. Wolfrom, Thompson, and Evans, J. Am. Chem. Soc., 67, 1793 (1945). 66. Yurkevich, Vereikina, Dolgikh, and Preobrazheuskii, J. Gen. Chem. USSR, 37, 1201 (1967). 67. Hughes, Overend, and Stacey, J. Chem. Soc. (Lond.), p. 2846 (1949). 68. Bauer and Biely, Collect. Czech. Chem. Commun., 33, 1165 (1968). 69. Fischer and Dangschat, Helv. Chim. Acta, 20, 705 (1937). 70. Williams and Egan, J. Bacteriol., 77, 167 (1959). 71. Kilgore and Starr, Biochim. Biophys. Acta, 30, 652 (1958). 72. Ohle and Berend, Ber. Dtsch. Chem. Ges., 60, 1159 (1927). 73. Waldi, J. Chromatogr., 18, 417 (1965). 74. Cirelli and de Lederkremer, Chem. Ind., 1139 (1971). 75. Paerels, Recl. Trav. Chim. Pays-Bas, 80, 985 (1961). 76. Henderson, J. Am. Chem. Soc., 79, 5304 (1957). 77. Merrick and Roseman, J. Biol. Chem., 235, 1274 (1960). 78. Boutroux, Ann. Chim. Phys. Ser. 6, 21, 565 (1890). 79. Barch, J. Am. Chem. Soc., 55, 3656 (1933). 80. Strobel, J. Biol. Chem., 245, 32 (1970). 81. Rosenthal, Spaner, and Brown, J. Chromatogr., 13, 152 (1964). 82. Wakisaka, Agric. Biol. Chem., 28, 819 (1964). 83. Katznelson, Tanenbaum, and Tatum, J. Biol. Chem., 204, 43 (1953). 84. Hudson, J. Am. Chem. Soc., 73, 4498 (1951). 85. Upson, Sands, and Whitnah, J. Am. Chem. Soc., 50, 519 (1928). 86. Barber and Hassid, Bull. Res. Counc. Isr. Sect. A., 11, 249 (1963). 87. Burns, J. Am. Chem. Soc., 79, 1257 (1957). 88. Okazaki, Kanzaki, Sasajima, and Terada, Agric. Biol. Chem., 33, 207 (1969). 89. Puhakainen and Hanninen, Acta Chem. Scand., 26, 3599 (1972). 90. Heyns, Justus Liebigs Ann. Chem., 558, 177 (1947). 91. deWilt, J. Chromatogr., 63, 379 (1971). 92. Grollman and Lehninger, Arch. Biochem. Biophys., 69, 458 (1957). 93. Okazaki, Kanzaki, Doi, Nara, and Motizuki, Agric. Biol. Chem., 32, 1250 (1968). 94. Smiley and Ashwell, J. Biol. Chem., 236, 357 (1961). 95. Penney and Zilva, Biochem. J., 37, 403 (1943). 96. Mead and Finamore, Biochemistry, 8, 2652 (1969). 97. Mumma and Verlangieri, Biochim. Biophys. Acta, 273, 249 (1972). 98. Berman and Magasanik, J. Biol. Chem., 241, 807 (1966). 99. Anderson and Magasanik, J. Biol. Chem., 246, 5653, 5662 (1971). 100. Takagi, Agric. Biol. Chem., 26, 717 (1962). 101. Hamilton and Smith, J. Am. Chem. Soc., 76, 3543 (1954). 102. Kanzaki and Okazaki, Agric. Biol. Chem., 34, 432 (1970). 103. Levene, J. Biol. Chem., 59, 123 (1924). 104. Phillips and Criddle, J. Chem. Soc. (Lond.), p. 3404 (1960). 105. Pervozvanski, Microbiology (USSR), 8, 915 (1939). 106. Wolfrom, Konigsberg, and Weisblat, J. Am. Chem. Soc., 61, 576 (1939). 107. Gakhokidge and Gvelukashvili, J. Gen. Chem. USSR, 22, 143 (1952). 108. Preiss and Ashwell, J. Biol. Chem., 238, 1571, 1577 (1963). 109. Bloom and Westerfeld, Biochemistry, 5, 3204 (1966). 110. Hirabayashi and Harada, Agric. Biol. Chem., 33, 276 (1969). 111. Kuhn, Bister, and Dafeldecker, Justus Liebigs Ann. Chem., 617, 115 (1958). 112. Srinivasan and Sprinson, J. Biol. Chem., 234, 716 (1959). 113. Paerels and Geluk, Recl. Trav. Chim. Pays-Bas, 89, 813 (1970). 114. Charon and Szabo, J. Chem. Soc. (Lond.), Perk I, 1175 (1973). 115. Adlersberg and Sprinson, Biochemistry, 3, 1855 (1964). 116. Ghalambor, Levine, and Heath, J. Biol. Chem., 241, 3207 (1966). 117. Dröge, Lehmann, Lüderitz, and Westphal, Eur. J. Biochem., 14, 175 (1970). 118. Hershberger, Davis, and Binkley, J. Biol. Chem., 243, 1578, 1585 (1968). 119. Levin and Racker, J. Biol. Chem., 234, 2532 (1959). 120. Ameyama and Kondo, Bull. Agric. Chem. Soc. Jap., 22, 271, 380 (1958). 121. Hulyalkar and Perry, Can. J. Chem., 43, 3241 (1965). 122. Bergmann, Ber. Dtsch. Chem. Ges., 54, 1362 (1921). 123. Ehrlich and Schubert, Ber. Dtsch. Chem. Ges., 62, 1987, 2022 (1929). 124. Niemann, Schoeffal, and Link, J. Biol. Chem., 101, 337 (1933). 125. Kováč, Hirsch, and Kováčik, Carbohydr. Res., 32, 360 (1974). 126. Winmann, Ber. Dtsch. Chem. Ges., 62, 1637 (1929). 127. Ehrlich and Rehorst, Ber. Dtsch. Chem. Ges., 58, 1989 (1925). 128. Ehrlich and Rehorst, Ber. Dtsch. Chem. Ges., 62, 628 (1929).
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Table B: Natural Acids of Carbohydrate Derivation 29. Bergmann and Wolff, Ber. Dtsch. Chem. Ges., 56, 1060 (1923). 1 130. Goebel and Babers, J. Biol. Chem., 100, 573, 743 (1933). 131. Fischer and Schmidt, Ber. Dtsch. Chem. Ges., 92, 2184 (1954). 132. Das Gupta and Sarkar, Text. Res. J., 24, 705, 1071 (1954). 133. Marsh, J. Chem. Soc. (Lond.), p. 1578 (1952). 134. Levene and Meyer, J. Biol. Chem., 60, 173 (1924). 135. Kovác, Carbohydr. Res., 31, 323 (1973). 136. Currie and Timell, Can. J. Chem., 37, 922 (1959). 137. Jones and Painter, J. Chem. Soc. (Lond.), p. 669 (1957). 138. Tyler, J. Chem. Soc. (Lond.), p. 5288, 5300 (1965). 139. Jones and Nunn, J. Chem. Soc. (Lond.), p. 3001 (1955). 140. Wacek, Leitinger, and Hochbahn, Monatsh. Chem., 90, 555, 562 (1959). 141. Charalampous and Lyras, J. Biol Chem., 228, 1 (1957). 142. Sowa, Can. J. Chem., 47, 3931 (1969). 143. Sutter and Reichstein, Helv. Chim. Acta, 21, 1210 (1938). 144. Haug and Larsen, Acta Chem. Scand., 15, 1395 (1961). 145. Gunther and Schweiger, J. Chromatogr., 34, 498 (1968). 146. Fischer and Dörfel, Hoppe-Seyler’s Z. Physiol. Chem., 302, 186 (1955). 147. Whistler and Schweiger, J. Am. Chem. Soc., 80, 5701 (1958). 148. Preiss and Ashwell, J. Biol. Chem., 237, 309, 317 (1962). 149. Heim and Neukom, Helv. Chim. Acta, 45, 1737 (1962). 150. Cifonelli, Ludowieg and Dorfman, J. Biol. Chem., 233, 541 (1958). 151. Shafizadeh and Wolfrom, J. Am. Chem. Soc., 77, 2568 (1955). 152. St. Cyr, J. Chromatogr., 47, 284 (1970). 153. Fischer and Dörfel, Hoppe-Seyler’s Z. Physiol. Chem., 301, 224 (1955). 154. Lehtonen, Kärkkäinen, and Haahti, Anal. Biochem., 16, 526 (1966). 155. Schoeffel, Link, J. Biol. Chem., 100, 397 (1933). 156. Stephen, Kaplan, Taylor, and Leisegang, Tetrahedron Suppl., 7, 233 (1966). 157. Pasteur, Ann. Chim. Phys. Ser., 3, 28, 71 (1850). 158. Walden, Ber. Dtsch. Chem. Ges., 29, 1701 (1896). 159. Fandolt, Ber. Dtsch. Chem. Ges., 6, 1075 (1873). 160. Frankland, and Slater, J. Chem. Soc. (Lond.), 83, 1354 (1903). 161. Anschütz, and Pictet, Ber. Dtsch. Chem. Ges., 13, 1176 (1880). 162. Patterson, J. Chem. Soc. (Lond.), 85, 765 (1904). 163. Frankland and Wharton, J. Chem. Soc. (Lond.), 69, 1310 (1896). 164. Pasteur, Justus Liebigs Ann. Chem., 82, 331 (1852). 165. Schneider, Ber. Dtsch. Chem. Ges., 13, 620 (1880). 166. Walczyk and Burczyk, Chem. Anal. (Warsaw), 17, 404 (1972). 167. Anschütz and Bennert, Justus Liebigs Ann. Chem., 254, 165 (1889). 168. Lutz, Dissertation, University of Rostock (1899); Chem. Zentralbl., II, 1013 (1900). 169. Bond, D., J. Chem. Soc. D, p. 338 (1969). 170. Anet and Reynolds, Nature, 174, 930 (1954). 171. Kessler, Neufeld, Feingold, and Hassid, J. Biol. Chem., 236, 308 (1961). 172. Rehorst, Ber. Dtsch. Chem. Ges., 61, 163 (1928). 173. Marsh, Biochem. J., 86, 77 1963; 87, 82 (1963); 89, 108 (1963). 174. Rehorst, Ber. Dtsch. Chem. Ges., 65, 1476 (1932). 175. Matsui, Okada, and Ishidata, J. Biochem. (Tokyo), 57, 715 (1965). 176. Tobie and Ayres, J. Am. Chem. Soc., 64, 725 (1942). 177. Katz and Lewis, Anal. Biochem., 17, 306 (1966).
9168_Book.indb 429
429 178. Wright, Jr.Burton, and Berry, Jr. Arch Biochem. Biophys., 86, 94 (1960). 179. Frei, Fukui, Lieu , T., and Frodyma, Chemia (Aarau), 20, 24 (1966). 180. Fusari, Haskell, Frohardt, and Bartz, J. Am. Chem. Soc., 76, 2881 (1954). 181. Isono, Asahi, and Suzuki, J. Am. Chem. Soc., 91, 7490 (1969). 182. Zissis, Diehl, and Fletcher, Jr. Carbohydr. Res., 28, 327 (1973). 183. Karrer and Mayer, Helv. Chim. Ada, 20, 407 (1937). 184. Iwasaki, Yakugaku Zasshi, 82, 1380 (1962); Chem. Abstr., 59, 758 (1963). 185. Heyns, Kiessling, Lindenberg, and Paulsen, Ber. Dtsch. Chem. Ges., 92, 2435 (1959). 186. Heyns and Beck, Ber. Dtsch. Chem. Ges., 90, 2443 (1957). 187. Levene, J. Biol. Chem., 57, 337 (1923). 188. Williamson and Zamenhof, J. Biol. Chem., 238, 2255 (1963). 189. Heyns, Paulsen, Ber. Dtsch. Chem. Ges., 88, 188 (1955). 190. Westphal and Holzmann, Ber. Dtsch. Chem. Ges., 75, 1274 (1942). 191. Weidmann, Fauland, Helbig, and Zimmerman, Justus Liebigs Ann. Chem., 694, 183 (1966). 192. Romanowska and Reinhold, Eur. J. Biochem., 36, 160 (1973). 193. Torii, Sakakibara, Kuroda, Eur. J. Biochem., 37, 401 (1973). 194. Crumpton, Biochem. J., 72, 479 (1959). 195. Ōtake, Takeuchi, Endō, and Yonehara, Tetrahedron Lett., 1405 (1965). 196. Watanabe, Goody, Fox, Tetrahedron, 26, 3883 (1970). 197. Kundu, Crawford, Prajsnar, Reed, and Rosenthal, Carbohydr. Res., 12, 225 (1970). 198. Perkins, Biochem. J., 86, 475 (1963); 89, 104P (1963). 199. Koto, Kawakatsu, and Zen, Bull. Chem. Soc. Jap., 46, 876 (1973). 200. Tarasiejska and Jeanloz, J. Am. Chem. Soc., 79, 2660 (1957). 201. Kuhn and Bister, Justus Liebigs Ann. Chem., 617, 92 (1958). 202. Lemieux and Nagabhushan, Can. J. Chem., 46, 401 (1968). 203. Strange and Kent, Biochem. J., 71, 333 (1959). 204. Lambert and Zilliken, Ber. Dtsch. Chem. Ges, 93, 2915 (1960). 205. Flowers and Jeanloz, J. Org. Chem., 28, 1564, 2983 (1963). 206. Osawa, Sinay, Halford, and Jeanloz, Biochemistry, 8, 3369 (1969). 207. Ghuysen and Strominger, Biochemistry, 2, 1119 (1963). 208. Sinay, Carbohydr. Res., 16, 113 (1971). 209. Sinay, Halford, Choudhary, Gross, and Jeanloz, J. Biol. Chem., 247, 391 (1972). 210. Hoshino, Zehavi, Sinay, and Jeanloz, J. Biol. Chem., 247, 381 (1972). 211. Kondo, Akita, and Sezaki, J. Antibiot. (Tokyo) Ser. A, 19, 137 (1966). 212. Faillard, Hoppe-Seyler’s Z. Physiol. Chem., 307, 62 (1957). 213. Zilliken and McGlick, Naturwissenschaften, 43, 536 (1956). 214. Khorlin and Privalova, Chem. Nat. Compd. (U S S R), 3, 159 (1967). 215. Kuhn and Baschang, Justus Liebigs Ann. Chem., 659, 156 (1962). 216. Zanetta, Breckenridge, and Vincendon, J. Chromatogr., 69, 291 (1972). 217. Blix and Lindberg, Acta Chem. Scand., 14, 1809 (1960). 218. Faillard and Blohm, Hoppe-Seyler’s Z. Physiol. Chem., 341, 167 (1965). 219. Hotta, Kurokawa, and Isaka, J. Biol. Chem., 245, 6307 (1970). 220. Brunetti, Jourdian, and Roseman, J. Biol. Chem., 237, 2447 (1962). 221. Hakomori and Saito, Biochemistry, 8, 5082 (1969). 222. Warren, Biochim. Biophys. Acta, 83, 129 (1964). 223. Isemura, Zahn, and Schmid, Biochem. J., 131, 509 (1973).
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9168_Book.indb 431
Table C: Natural Aldoses
TABLE C: NATURAL ALDOSES (Continued) Substancea (Synonym) Derivative (A)
Chemical Formula
Chromatography, R Value, and Referenced Melting Point °C
(B)
Specific Rotationb [α]D
(C)
Referencec
(D)
ELC
(E)
GLC
(F)
PPC
(G)
TLC
(H)
(I)
Acetaldehyde (deoxyglycolaldehyde) 2,4-Dinitrophenylhydrazone
C2H4O C8H8N4O4
Liquid, bp 21 146, 163.5–164.5
None None
1 1
0.6 (2)
52f (3)
d-Glyceraldehyde (glycerose) Dimethone 2,4-Dinitrophenylhydrazone Trimethylsilyl ether
C3H6O3 C19H28O6 C9H10N4O6 C5H10O5
Syrup 199–201 155–156 138–139
+13.5±0.5 +197.5 (c 0.7, C2H5OH) −(29 (CH3OH) +5.6 (c 10) (15°)
4 8 8 10 11,12
dek is hydrodynamically mobile and electro-kinetically active, and a particle (if spherical) behaves hydrodynamically as if it had a radius a + dek. The space charge for x < dek is hydrodynamically immobile, but can still be electrically conducting. The potential at the plane where slip with respect to bulk solution is postulated to occur is identified as the electrokinetic or zeta-potential, z. The diffuse charge at the solution side of the slip plane equals the negative of the electrokinetic (particle) charge, σek. General experience indicates that the plane of shear is located very close to the OHP. Both planes are abstractions of reality. The OHP is interpreted as a sharp boundary between the diffuse and the non diffuse parts of the EDL, but it is very difficult to locate it exactly. Likewise, the slip plane is interpreted as a sharp boundary between the hydrodynamically mobile and immobile fluid. In reality, none of these transitions is sharp. However, liquid motion may be hindered in the region where ions experience strong interactions with the surface. Therefore, it is feasible that the immobilization of the fluid extends further out of the surface than the beginning of the diffuse part of the EDL. This means that, in practice, the z-potential is equal to or lower in magnitude than the diffuse-layer potential, ψd. In the latter case, the difference between yd and z is a function of the ionic strength: at low ionic strength, the decay of the potential as a function of distance is small and z = ψd; at high ionic strength, the decay is steeper and |z | ≤ | ψd|. A similar reasoning applies to the electrokinetic charge, as compared to the diffuse charge.
1.5 Basic problem: Evaluation of ζ-potentials
The notion of slip plane is generally accepted in spite of the fact that there is no unambiguous way of locating it. It is also accepted that z is fully defined by the nature of the surface, its charge (often determined by pH), the electrolyte concentration in the solution, and the nature of the electrolyte and of the solvent. It can be said that for any interface with all these parameters fixed, z is a welldefined property. Experience demonstrates that different researchers often find different z-potentials for supposedly identical interfaces. Sometimes, the surfaces are not in fact identical: the high specific surface area and surface reactivity of colloidal systems make z very sensitive to even minor amounts of impurities in solution. This can partly explain variations in electrokinetic determinations from one laboratory to another. Alternatively, since z is not a directly measurable property, it may be that an inappropriate model has been used to convert the electrokinetic signal into a z-potential. The level of sophistication required (for the model) depends on the situation and on the particular phenomena investigated. The choice of measuring technique and of the theory used depends to a large extent on the purpose of the electrokinetic investigation. There are instances in which the use of simple models can be justified, even if they do not yield the correct z-potential. For example, if electrokinetic measurements are used as a sort of quality-control tool, one is interested in rapidly (online) detecting modifications in the electrical state of the interface rather than
4/16/10 1:25 PM
Measurement and Interpretation of Electrokinetic Phenomena in obtaining accurate z-potentials. On the other hand, when the purpose is to compare the calculated values of z of a system under given conditions using different electrokinetic techniques, it may be essential to find a true z-potential. The same applies to those cases in which z will be used to perform calculations of other physical quantities, such as the Gibbs interaction energy between particles. Furthermore, there may be situations in which the use of simple theories may be misleading even for simple quality control. For example, there are ranges of z-potential and double-layer thickness for which the electrophoretic mobility does not depend linearly on z, as assumed in the simple models. Two samples might have the same true z-potential and quite different mobilities because of their different sizes. The simple theory would lead us to believe that their electrical surface characteristics are different when they are not. An important complicating factor in the reliable estimation of z is the possibility that charges behind the plane of shear may contribute to the excess conductivity of the double layer (stagnant-layer or inner-layer conductivity.) If it is assumed that charges located between the surface and the plane of shear are electrokinetically inactive, then the z-potential will be the only interfacial quantity explaining the observed electrokinetic signal. Otherwise, a correct quantitative explanation of EKP will require the additional estimation of the stagnant-layer conductivity (SLC). This requires more elaborate treatments [2, 3, 13–17] than standard or classical theories, in which only conduction at the solution side of the plane of shear is considered. It should be noted that there are a number of situations where electrokinetic measurements, without further interpretation, provide extremely useful and unequivocal information, of great value for technological purposes. The most important of these situations are • Identification of the isoelectric point (or point of zero z-potential) in titrations with a potential determining ion (e.g., pH titration). • Identification of the isoelectric point in titrations with other ionic reagents such as surfactants or polyelectrolytes. • Identification of a plateau in the adsorption of an ionic species indicating optimum dosage for a dispersing agent. In these cases, the complications and digressions, which are discussed below, are essentially irrelevant. The electrokinetic property (or the estimated z-potential) is then zero or constant and that fact alone is of value.
1.6 Purpose of the document
The present document is intended to deal mainly with the following issues, related to the role of the different EKP as tools for surface chemistry research. Specifically, its aims are: • Describe and codify the main and related EKP and the quantities involved in their definitions. • Give a general overview of the main experimental techniques that are available for electrokinetic characterization. • Discuss the models for the conversion of the experimental signal into z-potential and, where appropriate, other double-layer characteristics. • Identify the validity range of such models, and the way in which they should be applied to any particular experimental situation. The report first discusses the most widely used EKP and techniques, such as electrophoresis, streaming-potential, streaming
9168_Book.indb 649
649
current, or electro-osmosis. Attention is also paid to the rapidly growing techniques based on dielectric dispersion and electro-acoustics.
2. Elementary theory of electrokinetic phenomena All electrokinetic effects originate from two generic phenomena, namely, the electro-osmotic flow and the convective electric surface current within the EDL. For nonconducting solids, Smoluchowski [18] derived equations for these generic phenomena, which allowed an extension of the theory to all other specific EKP. Smoluchowski’s theory is valid for any shape of a particle or pores inside a solid, provided the (local) curvature radius a largely exceeds the Debye length k−1: κ a >> 1
(2)
where k is defined as 1/ 2
∑ N e 2 z 2n κ = i =1 i i ε rsε 0 kT
(3)
with e the elementary charge, zi, ni the charge number and number concentration of ion i (the solution contains N ionic species), e rs the relative permittivity of the electrolyte solution, e 0 the electric permittivity of vacuum, k the Boltzmann constant, and T the thermodynamic temperature. Note that under condition (2), a curved surface can be considered as flat for any small section of the double layer. This condition is traditionally called the “thin double-layer approximation”, but we do not recommend this language, and we rather refer to this as the “large ka limit”. Many aqueous dispersions satisfy this condition, but not those for very small particles in low ionic strength media. Electro-osmotic flow is the liquid flow along any section of the double layer under the action of the tangential component Et of an external field E. In Smoluchowski’s theory, this field is considered to be independent of the presence of the double layer, i.e., the distortion of the latter is ignored*. Also, because the EDL is assumed to be very thin compared to the particle radius, the hydrodynamic and electric field lines are parallel for large ka. Under these conditions, it can be shown [3] that at a large distance from the surface the liquid velocity (electro-osmotic velocity), neo, is given by
veo = −
ε rsε 0ζ E η
(4)
where h is the dynamic viscosity of the liquid. This is the Smoluchowski equation for the electro-osmotic slip velocity. From this, the electro-osmotic flow rate of liquid per current, Qeo,I (m3 s−1 A−1), can be derived
Qeo,I =
ε εζ Qeo = − rs 0 ηK L I
(5)
KL being the bulk liquid conductivity (S m−1) and I the electric current (A). It is impossible to quantify the distribution of the electric field and the velocity in pores with unknown or complex geometry. * The approximation that the structure of the double layer is not affected by the applied field is one of the most restrictive assumptions of the elementary theory of EKP.
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Handbook of Biochemistry and Molecular Biology
650 However, this fundamental difficulty is avoided for kα >> 1, when eqs. 4 and 5 are valid [3]. Electrophoresis is the counterpart of electro-osmosis. In the latter, the liquid moves with respect to a solid body when an electric field is applied, whereas during electrophoresis the liquid as a whole is at rest, while the particle moves with respect to the liquid under the influence of the electric field. In both phenomena, such influence on the double layer controls the relative motions of the liquid and the solid body. Hence, the results obtained in considering electroosmosis can be readily applied for obtaining the corresponding formula for electrophoresis. The expression for the electrophoretic velocity, that is, the velocity of the particle with respect to a medium at rest, becomes, after changing the sign in eq. 4
ve =
ε rsε 0ζ E η
(6)
and the electrophoretic mobility, ue
ue =
ε rsε 0ζ η
(7)
This equation is known as the Helmholtz–Smoluchowski (HS) equation for electrophoresis. Let us consider a capillary with circular cross-section of radius a and length L with charged walls. A pressure difference between the two ends of the capillary, Dp, is produced externally to drive the liquid through the capillary. Since the fluid near the interface carries an excess of charge equal to σek, its motion will produce an electric current known as streaming current, Istr:
I str = −
ε rsε 0 a 2 ∆p ζ η L
(8)
The observation of this current is only possible if the extremes of the capillary are connected through a low-resistance external circuit (short-circuit conditions). If this resistance is high (open circuit), transport of ions by this current leads to the accumulation of charges of opposite signs between the two ends of the capillary and, consequently, to the appearance of a potential difference across the length of the capillary, the streaming-potential, Ustr. This gives rise to a conduction current, Ic:
Ic = K L a2
U str L
(9)
The value of the streaming-potential is obtained by the condition of equality of the conduction and streaming currents (the net current vanishes)
U str ε rsε 0ζ = ∆p ηK L
(10)
For large ka, eq. 10 is also valid for porous bodies. As described, the theory is incomplete in mainly three aspects: (i) it does not include the treatment of strongly curved surfaces (i.e., surfaces for which the condition kα >> 1 does not apply); (ii) it neglects the effect of surface conduction both in the diffuse and the inner part of the EDL; and (iii) it neglects EDL polarization. Concerning the first point, the theoretical analysis described above is based on the assumption that the interface is flat or that its radius of curvature at any point is much larger than the
9168_Book.indb 650
double-layer thickness. When this condition is not fulfilled, the Smoluchowski theory ceases to be valid, no matter the existence or not of surface conduction of any kind. However, theoretical treatments have been devised to deal with these surface curvature effects. Roughly, in order to check if such corrections are needed, one should simply calculate the product ka, where a is a characteristic radius of curvature (e.g., particle radius, pore or capillary radius). When describing the methods below, we will give details about analytical or numerical procedures that can be used to account for this effect. With respect to surface conductivity, a detailed account is given in Section 3 and mention will be made to it where necessary in the description of the methods. Here, it may suffice to say that it may be important when the z-potential is moderately large (>50 mV, say.) Finally, the polarization of the double layer implies accumulation of excess charge on one side of the colloidal particle and depletion on the other. The resulting induced dipole is the source of an electric field distribution that is superimposed on the applied field and affects the relative solid/liquid motion. The extent of polarization depends on surface conductivity, and its role in electrokinetics will be discussed together with the methodologies.
3. Surface conductivity and electrokinetic phenomena Surface conduction is the name given to the excess electric conduction that takes place in dispersed systems owing to the presence of the electric double layers. Excess charges in them may move under the influence of electric fields applied tangentially to the surface. The phenomenon is quantified in terms of the surface conductivity, Ks , which is the surface equivalent to the bulk conductivity, KL . Ks is a surface excess quantity just as the surface concentration Γi of a certain species i. Whatever the charge distribution, Ks can always be defined through the two-dimensional analog of Ohm’s law jσ = K σ E
(11)
where js is the (excess) surface current density (A m−1). A measure of the relative importance of surface conductivity is given by the dimensionless Dukhin number, Du, relating surface (Ks) and bulk (KL) conductivities Du =
Kσ K La
(12)
where a is the local curvature radius of the surface. For a colloidal system, the total conductivity, K, can be expressed as the sum of a solution contribution and a surface contribution. For instance, for a cylindrical capillary, the following expression results:
(
)
K = K L + 2 K σ /a = K L (1 + 2 Du)
(13)
The factor 2 in eq. 13 applies for cylindrical geometry. For other geometries, its value may be different. As mentioned, HS theory does not consider surface conduction, and only the solution conductivity, KL, is taken into account to derive the tangential electric field within the double layer. Thus, in addition to eq. 2, the applicability of the HS theory requires
Du > 1, and z is small. Substitution of this expression for Dud in eq. 16 yields
Du =
zeζ 2 3m K σi − 1 1 + σ d 1 + 2 cosh κa 2kT K z
(20)
9168_Book.indb 651
(21)
4.1.2 How and under which conditions the electrophoretic mobility can be converted into ζ-potential
As discussed above, it is not always possible to rigorously obtain the z-potential from measurements of electrophoretic mobility only. We give here some guidelines to check whether the system under study can be described with the standard electrokinetic models:
a. Calculate ka for the suspension. b. If ka >> 1 (ka > 20, say), we are in the large ka regime, and simple analytical models are available. b.1 Obtain the mobility ue for a range of indifferent electrolyte concentrations. If ue decreases with increasing electrolyte concentration, use the HS formula, eq. 7, to obtain z. b.1.1 If the z value obtained is low (z ≤ 50 mV, say), concentration polarization is negligible, and one can trust the value of z. b.1.2 If z is rather high (z > 50 mV, say), then HS theory is not applicable. One has to use more elaborate models. The possibilities are: (i) the numerical calculations of O’Brien and White [22]; (ii) the equation derived by Dukhin and Semenikhin [5] for symmetrical z-z electrolytes 3 ηe 3 y ek −6 ue = 2 ε rsε 0 kT 2
y ek (1 + 3m/z 2 )sinh 2 ( zy ek /4 ) + [2 z −1 sinh( zy ek /2) − 3my ek ]lncosh( zy ek /4 ) × κ a + 8(1 + 3m/z 2 )sinh 2 ( zy ek /4 ) − (24 m/z 2 )lncosh( zy ek /4 )
This equation shows that, in general, Du is dependent on the z-potential, the ion mobility in bulk solution, and Ksi/Ksd. Now, the condition Du > 1, rather low values of z, and Ksi /Ksd < 1.
where ue is the quantity of interest, the electrophoretic mobility.
(19)
ve = ue E
2
2 kT ε rsε 0 m± = 3 e η D±
651
(22)
where m was defined in eq. 18 and y ek =
eζ kT
(23)
is the dimensionless z-potential. For aqueous solutions, m is about 0.15. O’Brien [4] found
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Handbook of Biochemistry and Molecular Biology
652 that eq. 22 can be simplified by neglecting terms of order (kα)−1 as follows: 3 ηe ue 2 ε rsε 0 kT y ek ln 2 1 − exp(− zy ek ) − 6 2 z 3 = y ek − zy ek 2 κa exp − 2+ 1 + 3m / z 2 2
{
conductivity, K P. With that aim, it can be modified to read [2]
}
ε rsε 0 Du kT 2 ln 2 − 1 ζ 1+ η 1 + Du e ζ z
(25)
with Du including both the stagnant- and diffuselayer conductivities. This requires additional information about the value of Ksi (see Section 3).
c. If ka is low, the O’Brien and White [22] numerical calculations remain valid, but there are also several analytical approximations. For ka < 1, the Hückel–Onsager (HO) equation applies [4]:
ue =
2 ε rsε 0 ζ 3 η
(26)
d. For the transition range between low and high ka, Henry’s formula can be applied if z is presumed to be low (> 1 (a is the capillary radius), the HS formula can be used I str ε ε ζ Ac = − rs 0 ∆p η L
(33)
where Ac is the capillary cross-section, and L its length. If instead of a single capillary, the experimental system is a porous plug or a membrane, eq. 33 remains approximately valid, provided that ka >> 1 everywhere in the pore walls. In the case of porous plugs, attention has to be paid to the fact that a plug is not a system of straight parallel capillaries, but a random distribution of particles with a resulting porosity and tortuosity, for which an equivalent capillary length and cross-section is just a simplified model. In addition, the use of eq. 33 requires that the conduction current in the system is determined solely by the bulk conductivity of the supporting solution. It often happens that surface conductivity is important, and, besides that, the ions in the plug behave with a lower mobility than in solution. A c/L can be estimated experimentally as follows [40, 41]. Measure the resistance, R∞ , of the plug or capillary wetted by a highly concentrated (above 10 −2 mol/L, say) electrolyte solution, with conductivity K∞L . Since for such a high ionic strength the double-layer contribution to the overall conductivity is negligible, we may write
Ac 1 = ∞ L K L R∞
(34)
In addition, theoretical or semi-empirical models exist that relate the apparent values of Ac and L (external dimensions of the plug) to the volume fraction, f, of solids in the plug. For instance, according to [42]
Ac Acap = ap exp( Bφ ) L L
(35)
where B is an empirical constant that can be experimentally determined by measuring the electro-osmotic volume flow for different plug porosities. In eq. 35, L ap and Acap are the apparent (externally measured) length and cross-sectional area of the plug, respectively. An alternative expression was proposed in [43]:
Ac Acap -5/2 = ap φL L L
(36)
where f L is the volume fraction of liquid in the plug (or void volume fraction). Other estimates of Ac /L can be found in [44–46]. For the case of a close packing of spheres, theoretical treatments are available involving the calculation of streaming current using cell models. No simple expressions can be given in this case; see [3, 47–52] for details.
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656
1.0
(Istr)/(Istr-Smol.)
0.8
b. The most frequent case (except for high ionic strengths, or high KL) is that surface conductance, Ks , is significant. Then the following equation should be used:
Slit channel
0.6
U str ε rsε 0ζ 1 = ∆p η K L (1 + 2 Du)
Cylindrical channel
0.4
where Du is given by eqs. 12 and 20. An empirical way of taking into account the existence of surface conductivity is to measure the resistance R∞ of the plug or capillary in a highly concentrated electrolyte solution of conductivity K∞l. As for such a solution, Du is negligible, one can write
0.2 0
0.1
1
κa
10
100
Figure 3: Streaming current divided by the applied pressure difference, eqs. 37 and 39, relative to the Smoluchowski formula, eq. 33, plotted as a function of the product ka (a: capillary radius, or slit half-width) for slit- and cylindrical-shaped capillaries.
b. If ka is intermediate (ka ≈ 1–10, say), the HS equation is not valid. For low z, curvature effects can be corrected by means of the Burgen and Nakache theory [4, 49]:
I str ε rsε 0ζ Ac [1 − G(κ a)] = ∆p η L
(37)
tanh(κ a) κa
2 I1(κ a) κ aI 0 (κ a)
U str ε rsε 0ζ RS = ∆p η K L∞R∞
U str ε rsε 0ζ RS = ∆p η K L∞R∞
4.2.1.2 Streaming potential
The streaming potential difference (briefly, streaming potential) Ustr can be measured between two electrodes, upstream and downstream in the liquid flow, connected via a high-input impedance voltmeter. The quantity of interest is, in this case, the ratio between the streaming potential and the pressure drop, Ustr/Dp (V Pa−1). The conversion into z-potentials can be realized in a number of cases.
a. If kα >> 1 and surface conduction can be neglected, the HS formula can be used:
9168_Book.indb 656
U str ε rsε 0ζ 1 = ∆p η KL
(40)
(ε
(44)
ε κζ
rs 0
η
)
2
RS K L∞R∞
(45)
Figure 4 illustrates some results that can be obtained by using eq. 44. 1.0 (Ustr)/(Ustr-Smol.)
c. If the z-potential is not low and ka is small, no simple expression for Istr can be given, and only numerical procedures are available [52].
2 I1(κ a) κ aI 0 (κ a) 2 I (κ a) I12 (κ a) 1 − β 1 − 1 − 2 κ aI 0 (κ a) I 0 (κ a) 1−
where
where I0 and I1 are the zeroth- and first-order modified Bessel functions of the first kind, respectively. Fig. 3 illustrates the importance of this curvature correction.
(43)
c. If ka is intermediate (ka ~ 1…10) and the z-potential is low, Rice and Whitehead’s corrections are needed [50]. For a cylindrical capillary, the result is
β= (39)
(42)
where Rs is the resistance of the plug in the solution under study, of conductivity KL . Now, eq. 41 can be approximated by
(38)
for slit-shaped capillaries (2a corresponds in this case to the separation of the parallel solid walls). In the case of cylindrical capillaries of radius a, the calculattion was first carried out by Rice and Whitehead [50]. They found that the function G(ka) in eq. 37 reads G(κ a) =
2K σ K L∞R∞ = K L + Rs a
where G(κ a) =
(41)
0.8
ζ = 25 mV ζ = 50 mV
0.6 0.4 0.2 0.0 0.1
1
κa
10
100
Figure 4: Streaming potential divided by the applied pressure difference, eq. 44, relative to its Smoluchowski value, eq. 40, as a function of the product ka (a: capillary radius), for the ζ-potentials indicated. Surface conductance is neglected.
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Measurement and Interpretation of Electrokinetic Phenomena
d. As in the case of streaming current, for high z-potentials, only numerical methods are available (see, e.g., [53] for details).
In practice, instead of potential or current measurements for just one driving pressure, the streaming potential and streaming current are mostly measured at various pressure differences applied in both directions across the capillary system, and the slopes of the functions Ustr = Ustr(Dp) and Istr = Istr(Dp) are used to calculate the z-potential. This makes it possible to detect electrode asymmetry effects and correct for them. It is also advisable to verify that the Dp dependencies are linear and pass through the origin.
4.2.2 Samples that can be studied
Streaming potential/current measurements can be applied to study macroscopic interfaces of materials of different shape. Single capillaries made of flat sample surfaces (rectangular capillaries) and cylindrical capillaries can be used to produce microchannels for streaming potential/current measurements. Further, parallel capillaries and irregular capillary systems such as fiber bundles, membranes, and particle plugs can also be studied. Recall, however, the precautions already mentioned in connection with the interpretation of results in the case of plugs of particles. Other effects, including temperature gradients, Donnan potentials, or membrane potential can contribute to the observed streaming potential or electro-osmotic flow. An additional condition is the constancy of the capillary geometry during the course of the experiment. Reversibility of the signal upon variations in the sign and magnitude of Dp is a criterion for such constancy. Most of the materials studied so far by streaming potential/ current measurements, including synthetic polymers and inorganic non-metals, are insulating. Either bulk materials or thin films on top of carriers can be characterized. In addition, in some cases, semiconductors [54] and even bulk metals [55] have been studied, proving the general feasibility of the experiment. Note that streaming potential/current measurements on samples of different geometries (flat plates, particle plugs, fiber bundles, cylindrical capillaries,…) each require their own set-up.
4.2.3 Sample preparation
The samples to be studied by streaming potential/current measurements have to be mechanically and chemically stable in the aqueous solutions used for the experiment. First, the geometry of the plug must be consolidated in the measuring cell. This can be checked by rinsing with the equilibrium liquid through repeatedly applying Dp in both directions until finding a constant signal. Another issue to consider is the necessity that the solid has reached chemical equilibrium with the permeating liquid; this may require making the plug from a suspension of the correct composition, followed by rinsing. Checking that the experimental signal does not change during the course of measurement may be a good practice. The presence or formation of air bubbles in the capillary system has to be avoided.
require effective electrical insulation of the set-up in order to prevent short-circuiting. However, such low ionic strength values can only be attained if the solid sample is extremely pure and insoluble. The upper value of electrolyte concentration depends on the sensitivity of the electrometer and on the applied pressure difference; usually, solutions above 10 −1 mol/L of 1-1 charge-type electrolyte are difficult to measure by the present techniques.
4.3 Electro-osmosis 4.3.1 Operational definitions; recommended symbols and terminology; conversion of the measured quantities into ζ-potential
In electro-osmosis, a flow of liquid is produced when an electric field E is applied to a charged capillary or porous plug immersed in an electrolyte solution. If kα >> 1 everywhere at the solid/liquid interface, far from that interface the liquid will attain a constant (i.e., independent of the position in the channel) velocity (the electro-osmotic velocity) veo, given by eq. 4. If such a velocity cannot be measured, the convenient physical quantity becomes the eoelectro-osmotic flow rate, Qeo (m3 s−1), given by Qeo =
eo
⋅ dS
(46)
where dS is the elementary surface vector at the location in the channel where the fluid velocity is veo. The counterparts of Qeo are Qeo, E (flow rate divided by electric field) and Qeo, I (flow rate divided by current). These are the quantities that can be related to the z-potential. As before, several cases can be distinguished:
a. If ka >> 1 and there is no surface conduction:
Qeo,E ≡
Qeo,I ≡
Qeo ε εζ = − rs 0 Ac E η
ε εζ 1 Qeo = − rs 0 η KL I
(47)
b. With surface conduction, the expression for Qeo,E is as in eq. 47, and that for Qeo,I is
Qeo,I = −
ε rsε 0ζ 1 η K L (1 + 2 Du)
(48)
In eq. 48, the empirical approach for the estimation of Du can be followed:
No standard samples have been developed specifically so far for streaming potential/current measurements, although several materials have been frequently analyzed and may, therefore, serve as potential reference samples [56, 57].
4.2.3.2 Range of electrolyte concentrations
9168_Book.indb 657
∫∫ v Ac
4.2.3.1 Standard samples
From the operational standpoint, there is no lower limit to the ionic strength of the systems to be investigated by these methods, although in the case of narrow channels, very low ionic strengths
657
Qeo,I = −
(49)
c. Low z-potential, finite surface conduction, and arbitrary capillary radius [46]:
Qeo,E = −
ε rsε 0ζ Ac[1 − G(κ a)] η
Qeo,I = −
ε rsε 0ζ [1 − G(κ a)] η K L (1 + 2 Du)
≅−
ε rsε 0ζ RS η K L∞R∞
(50)
ε rsε 0ζ RS[1 − G(κ a)] η K L∞R∞
where the function G(ka) is given by eq. 38.
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658
d. When z is high and the condition ka >> 1 is not fulfilled, no simple expression can be given for Qeo.
As in the case of streaming potential and current, the procedures described can be also applied to either plugs or membranes. If the electric field E is the independent variable (see eq. 47), then Ac must be estimated. In that situation, the recommendations suggested in Section 4.2.1 can be used, since eq. 47 can be written as
Qeo,E ε ε ζ Ac = − rs 0 ∆Vext η L
(51)
where DVext is the applied potential difference.
4.3.2 Samples that can be studied
The same samples as with streaming current/potential, see Section 4.2.2.
conductivity of disperse systems. When dealing with such heterogeneous systems as colloidal dispersions, these quantities are defined as the electric permittivity and conductivity of a sample of homogeneous material, that when placed between the electrodes of the measuring cell, would have the same resistance and capacitance as the suspension. The dielectric investigation of dispersed systems involves determinations of their complex permittivity, e*(w) (F m−1) and complex conductivity K*(w) (S m−1) as a function of the frequency w (rad s−1) of the applied ac field. These quantities are related to the volume, surface, and geometrical characteristics of the dispersed particles, the nature of the dispersion medium, and also to the concentration of particles, expressed either in terms of volume fraction, f (dimensionless) or number concentration N (m−3). It is common to use the relative permittivity e *r (w) (dimensionless), instead of the permittivity
ε * (ω ) = ε r* (ω )ε 0
(52)
4.3.3 Sample preparation and standard samples
e 0 being the permittivity of vacuum. K*(w) and e*(w) are not independent quantities:
4.4 Experimental determination of surface conductivity
See Section 4.2.3 referring to streaming potential/current determination.
Surface conductivities are excess quantities and cannot be directly measured. There are, in principle, three methods to estimate them. i. In the case of plugs, measure the plug conductivity Kplug as a function of KL . The latter can be changed by adjusting the electrolyte concentration. The plot of Kplug vs. KL has a large linear range which can be extrapolated to KL = 0 where the intercept represents Ks . This method requires a plug and seems relatively straightforward. ii. For capillaries, deduce Ks from the radius dependence of the streaming potential, using eq. 41 and the definition of Du (eq. 12). This method is rather direct, but requires a range of capillaries with different radii, but identical surface properties [58, 59]. iii. Utilize the observation that, when surface conductivity is not properly accounted for, different electrokinetic techniques may give different values for the z-potential of the same material under the same solution conditions. Correct the theories by inclusion of the appropriate surface conductivity, and find in this way the value of Ks that harmonizes the z-potential. This method requires insight into the theoretical backgrounds [60, 61], and it works best if the two electrokinetic techniques have a rather different sensitivity for surface conduction (such as electrophoresis and LFDD).
In many cases, it is found that the surface conductivity obtained in one of these ways exceeds Ksd, sometimes by orders of magnitude. This means that Ksi is substantial. The procedure for obtaining Ksi consists of subtracting Ksd from Ks . For Ksd, Bikerman’s equation (eq. 17) can be used. The method is not direct because this evaluation requires the z-potential, which is one of the unknowns; hence, iteration is required.
4.5 Dielectric dispersion 4.5.1 Operational definitions; recommended symbols and terminology; conversion of the measured quantities into ζ-potential
The phenomenon of dielectric dispersion in colloidal suspensions involves the study of the dependence on the frequency of the applied electric field of the electric permittivity and/or the electric
9168_Book.indb 658
K * (ω ) = K DC − iωε * (ω ) = K DC − iωε 0ε r* (ω )
(53)
or, equivalently,
Re[ K (ω )] = K DC + ωε 0 Im[ε r* (ω )]
Im[ K * (ω )] = −ωε 0 Re[ε r* (ω )]
(54)
where KDC is the direct-current (zero frequency) conductivity of the system. The complex conductivity K* of the suspension can be expressed as K * (ω ) = K L + δ K * (ω )
(55)
where dK*(w) is usually called conductivity increment of the suspension. Similarly, the complex dielectric constant of the suspension can be written in terms of a relative permittivity increment or, briefly, dielectric increment de *r (w):
ε r* (ω ) = ε rs + δε r* (ω )
(56)
As in homogeneous materials, the electric permittivity is the macroscopic manifestation of the electrical polarizability of the suspension components. Mostly, more than one relaxation frequency is observed, each associated with one of the various mechanisms contributing to the system’s polarization. Hence, the investigation of the frequency dependence of the electric permittivity or conductivity allows us to obtain information about the characteristics of the disperse system that are responsible for the polarization of the particles. The frequency range over which the dielectric dispersion of suspensions in electrolyte solutions is usually measured extends between 0.1 kHz and several hundred MHz. In order to define in this frame the low-frequency and high-frequency ranges, it is convenient to introduce an important concept dealing with a point in the frequency scale. This frequency corresponds to the reciprocal of the Maxwell–Wagner–O’Konski relaxation time t MWO
ω MWO ≡
1
τ MWO
≡
(1 − φ )K p + (2 + φ )K L ε 0 (1 − φ )ε rp + (2 + φ )ε rs ε rs
(57)
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Measurement and Interpretation of Electrokinetic Phenomena
Maxwell–Wagner model was generalized by O’Konski [61], who first took the surface conductivity Ks explicitly into account. In his treatment, the conductivity of the particle is modified to include the contributions of both the solid material and the excess surface conductivity. This effective conductivity will be called Kpef:
and it is called the Maxwell–Wagner–O’Konski relaxation frequency. In eq. 57, e rp is the relative permittivity of the dispersed particles. For low volume fractions and low permittivity of the particles (e rp ω MWO
(60)
ε rp ε rs ≠ K p KL
(61)
the conditions of continuity of the normal components of the current density and the electrostatic induction on both sides of the surface are inconsistent with each other. This results in the formation of free ionic charges near the surface. The finite time needed for the formation of such a free charge is in fact responsible for the Maxwell–Wagner dielectric dispersion. In the Maxwell–Wagner model, no specific properties are assumed for the surface, which is simply considered as a geometrical boundary between homogeneous phases. The
9168_Book.indb 659
ε rs* = ε rs − i
KL ωε 0
(63)
K pef ωε 0
(64)
and for the particle
ε rp* = ε rp − i
In terms of these quantities, the Maxwell–Wagner– O’Konski theory gives the following expression for the complex dielectric constant of the suspension:
4.6 Dielectric dispersion and ζ-potential: Models
a. Middle-frequency range: Maxwell–Wagner–O’Konski relaxation There are various mechanisms for the polarization of a heterogeneous material, each of which is always associated with some property that differs between the solid, the liquid, and their interface. The most widely known mechanism of dielectric dispersion, the Maxwell–Wagner dispersion, occurs when the two contacting phases have different conductivities and electric permittivities. If the ratio erp/Kp is different from that of the dispersion medium, i.e., if
2K σ (62) a Both the conductivity and the dielectric constant can be considered as parts of a complex electric permittivity of any of the system’s components. Thus, for the dispersion medium K pef = K p +
the characteristic value of the displacement current density exceeds that of conduction currents, and the space distribution of the local electric fields is determined by polarization of the molecular dipoles, rather than by the distribution of ions.
659
ε r* = ε rs*
ε rp* + 2ε rs* + 2φ (ε rp* − ε rs* ) ε rp* + 2ε rs* − φ (ε rp* − ε rs* )
(65)
b. Low-frequency range: dilute suspensions of nonconducting spherical particles with ka >> 1, and negligible Ksi At moderate or high z-potentials, mobile counterions are more abundant than coions in the EDL. Therefore, the contribution of the counterions and the coions to surface currents in the EDL differs from their contribution to currents in the bulk solution. Such difference gives rise to the existence of a field-induced perturbation of the electrolyte concentration, dc(r), in the vicinity of the polarized particle. The ionic diffusion caused by dc(r) provokes a lowfrequency dependence of the particle’s dipole coefficient, C*0 (see below). This is the origin of the LFDD (α-dispersion) displayed by colloidal suspensions. Recall that the dipole coefficient relates the dipole moment d* to the applied field E. For the case of a spherical particle of radius a, the dipole coefficient is defined through the relation
d * = 4πε rsε 0 a3C 0* E
(66)
The calculation of this quantity proves to be essential for evaluation of the dielectric dispersion of the suspension [63–65]. A model for the calculation of the low-frequency conductivity increment dK*(w) and relative permittivity increment de *r(w) from the dipole coefficient C*0 when ka >> 1 is described in Appendix I. There it is shown that, in the absence of SLC, the only parameter of the solid/ liquid interface that is needed to account for LFDD is the z-potential. The overall behavior is illustrated in Fig. 5 for a dilute dispersion of spherical nonconducting particles (a = 100 nm, e rp = 2) in a 10 −3 mol/L KCl solution (e rs = 78.5), and with negligible ionic conduction in the stagnant layer. In this figure, we plot the variation of the real and imaginary parts
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660
Figure 5: Real and imaginary parts of the dielectric increment de *r (divided by the volume fraction f) for dilute suspensions of 100-nm particles in KCl solution, as a function of the frequency of the applied field for ka=10 and z = 100 mV. The arrows indicate the approximate location of the α (low frequency) and Maxwell– Wagner–O’ Konski relaxations. of de *r with frequency. Note the very significant effect of the double-layer polarization on the low-frequency dielectric constant of a suspension. The variation with frequency is also noticeable, and both the a- (around 105 s−1) and Maxwell–Wagner (~2 × 107 s−1) relaxations are observed, the amplitude of the latter being much smaller than that of the former for the conditions chosen. No other electrokinetic technique can provide such a clear account of the double-layer relaxation processes. The effect of z-potential on the frequency dependence of the dielectric constant is plotted in Fig. 6: the dielectric increment always increases with z, as a consequence of the larger concentration of counterions in the EDL: all processes responsible for LFDD are amplified for this reason. The procedure for obtaining z from LFDD measurements is somewhat involved. It is advisable to determine experimentally the dielectric constant (or, equivalently, the conductivity) of the suspension over a wide frequency
Figure 6: Real part of the dielectric increment de *r (per volume fraction) as a function of the frequency of the applied field for dilute suspensions of 100-nm particles in KCl solution. The ζ-potentials are indicated, and in all cases, ka = 10.
9168_Book.indb 660
range, and use eqs. I.2 and I.3 (see Appendix I) to estimate the LFDD curve that best fits the data. A simpler routine is to measure only the low-frequency values, and deduce z using the same equations, but substituting w = 0. However, the main experimental problems occur at low frequencies (see Section 4.5.3). c. Dilute suspensions of nonconducting spherical particles with arbitrary ka, and negligible Ksi In this situation, there are no analytical expressions relating LFDD measurements to z. Instead, numerical calculations based on DeLacey and White’s treatment [66] are recommended. As before, the computing routine should be constructed to perform the calculations a number of times with different z-potentials as inputs, until agreement between theory and experiment is obtained over a wide frequency range (or, at least, at low frequencies). d. Dilute suspensions of nonconducting spherical particles with ka >> 1 and SLC The problem of generalizing the theory by taking into account surface conduction caused by ions situated in the hydrodynamically stagnant layer has been dealt with in [60, 61, 64]. In theoretical treatments, SLC is equated to conduction within the Stern layer. According to these models, the dielectric dispersion is determined by both z and Ksi. This means that, as discussed before, additional information on Ksi (see the methods described in Section 4.4) must accompany the dielectric dispersion measurements. Using dielectric dispersion data alone can only yield information about the total surface conductivity [66]. e. Dilute suspensions of nonconducting spherical particles with arbitrary ka and SLC. Only numerical methods are available if this is the physical nature of the system under study. The reader is referred to [13–16, 61, 62, 67–69]. Figure 7 illustrates how important the effect of SLC onRe [de*r(w)] can be for the same conditions as in Fig. 5. Roughly, the possibility of increased ionic mobilities in the stagnant layer brings about a systematically larger dielectric increment of the suspension: surface currents are larger for a conducting stagnant layer, and hence the electrolyte concentration gradients, ultimately responsible for the dielectric dispersion, will also be increased.
Figure 7: Real part of the dielectric increment (per volume fraction) of dilute suspensions as in Fig. 5. The curves correspond to increasing importance of SLC; the ratios between the diffusion coefficients of counterions in the stagnant layer and in the bulk electrolyte are indicated (the lower curve corresponds to zero SLC).
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Measurement and Interpretation of Electrokinetic Phenomena
f. Nondilute suspensions of nonconducting spherical particles with large ka and negligible Ksi Considering that many suspensions of technological interest are rather concentrated, the possibilities of LFDD in the characterization of moderately concentrated suspensions have also received attention. This requires establishing a theoretical basis relating the dielectric or conductivity increments of such systems to the concentration of particles [70–72]. Here, we focus on a simplified model [73] that allows the calculation of the volume-fraction dependence of both the low-frequency value of the real part of de*r, and of the characteristic time τα of the α-relaxation. The starting point is the assumption that LD, the length scale over which ionic diffusion takes place around the particle, can be averaged in the following way between the values of a very dilute (LD ≈ a) and a very concentrated (LD ≈ b – a; b is half the average distance between the centers of neighboring particles) dispersion:
1 1 LD = 2 + a (b − a)2
−1/ 2
(67)
or, in terms of the particle volume fraction
1 LD = a 1 + −1/3 (φ − 1)2
−1/ 2
(68)
From these expressions, the simplified model allows us to obtain the dielectric increment at low frequency as follows. Let us call
∆ε (0 ) ≡
Re[δε r* (0 )] φ
(69)
the specific (i.e., per unit volume fraction) dielectric increment (for w → 0). In the case of dilute suspensions, this quantity is a constant (independent of f), that we denote Ded(0):
∆ε (0 ) ≡
Re[δε r* (0 )] φ φ →0
(70)
The model allows us to relate eq. 69 with eq. 70 through the volume-fraction dependence of LD:
1 ∆ε (0 ) = ∆ε d (0 ) 1 + −1/3 2 (φ − 1)
−3/ 2
(71)
A similar relationship can be established between dilute and concentrated suspensions in the case of the relaxation frequency wa =1/ta:
1 ω α = ω α d 1 + −1/3 (φ − 1)2
(72)
where w ad is the reciprocal of the relaxation time for a dilute suspension. Using this model, the dielectric increment and characteristic frequency of a concentrated suspension can be related to those corresponding to the dilute case which, in turn, can be related, as discussed above, to the z-potential and other double-layer parameters. A general treatment of the problem, valid for arbitrary values of ka and z can be found in [74].
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661
Summing up, we can say that the dielectric dispersion of suspensions is an interesting physical phenomenon, extremely sensitive to the characteristics of the particles, the solution, and their interface. It can provide invaluable information on the dynamics of the EDL and the processes through which it is altered by the application of an external field. Because of the experimental difficulties involved in its determination, it is unlikely that dielectric dispersion measurements alone can be useful as a tool to obtain the z-potential of the dispersed particles.
4.6.1 Experimental techniques available
One of the most usual techniques for measuring the dielectric permittivity and/or the conductivity of suspensions as a function of the frequency of the applied field, is based on the use of a conductivity cell connected to an impedance analyzer. This technique has been widely employed since it was first proposed by Fricke and Curtis [75]. In most modern setups, the distance between electrodes can be changed (see, e.g., [69, 76–79]). The need for variable electrode separation stems from the problem of electrode polarization at low frequencies, since at sufficiently low frequencies the electrode impedance dominates over that of the sample. The method makes use of the assumption that electrode polarization does not depend on their distance. A so-called quadrupole method has been recently introduced [80] in which the correction for electrode polarization is optimally carried out by proper calibration. Furthermore, the method based on the evaluation of the logarithmic derivative of the imaginary part of raw e*(w) data also seems to be promising [81]. These are not, however, the only possible procedures. A fourelectrode method has also been employed with success [60, 61, 68] in this case, since the sensing and current-supplying electrodes are different, polarization is not the main problem, but the electronics of the experimental set-up is rather complicated.
4.6.2 Samples for LFDD measurements
There are no particular restrictions to the kind of colloidal particles that can be studied with the LFDD technique. The obvious precautions involve avoiding sedimentation of the particles during measurement, and control of the stability of the suspensions. LFDD quantities are most sensitive to particle size, particle concentration, and temperature. Hence, the constancy of the latter is essential. Another important concern deals with the effect of electrode polarization. Particularly at low frequencies, electrode polarization can be substantial and completely invalidate the data. This fact imposes severe limitations on the electrolyte concentrations that can be studied; it is very hard to obtain data for ionic strengths in excess of 1 to 5 mmol L−1.
4.7 Electroacoustics 4.7.1 Operational definitions; recommended symbols and terminology; experimentally available quantities Terminology
The term “electroacoustics” refers to two kinds of closely related phenomena: • Colloid vibration current (ICV) and colloid vibration potential (UCV) are two phenomena in which a sound wave is passed through a colloidal dispersion and, as a result, electrical currents and fields arise in the suspension. When the wave travels through a dispersion of particles whose
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662 density differs from that of the surrounding medium, inertial forces induced by the vibration of the suspension give rise to a motion of the charged particles relative to the liquid, causing an alternating electromotive force. The manifestations of this electromotive force may be measured in a way depending on the relation between the impedance of the suspension and the properties of the measuring instrument, either as ICV (for small impedance of the meter) or as UCV (for large one). • The reciprocal effect of the above two phenomena is the electrokinetic sonic amplitude (ESA), in which an alternating electric field is applied to a suspension and a sound wave arises as a result of the motion of the particles caused by their ac electrophoresis. Colloid vibration potential/current may be considered as the ac analog of sedimentation potential/current. Similarly, ESA may be considered as the ac analog of classical electrophoresis. The relationships between electroacoustics and classical EKP may be used for testing modern electroacoustic theories, which should at least provide the correct limiting transitions to the well-known and well-established results of the classical electrokinetic theory. A very important advantage of the electroacoustic techniques is the possibility they offer to be applied to concentrated dispersions.
4.7.1.1 Experimentally available quantities
4.7.1.1.1 Colloid vibration potential (UCV) If a standing sound wave is established in a suspension, a voltage difference can be measured between two different points in the standing wave. If measured at zero current flow, it is referred to as colloid vibration potential. The measured voltage is due to the motion of the particles: it alternates at the frequency of the sound wave and is proportional to another measured value, DP, which is the pressure difference between the two points of the wave. The kinetic coefficient, equal to the ratio
)
(
U CV U CV ω , φ , ∆ρ /ρ , K σ i ,ζ ,η, a = ∆p ∆p
(73)
characterizes the suspension. In eq. 73, Dr is the difference in density between the particles and the suspending fluid, of density ρ. 4.7.1.1.2 Colloid vibration current (ICV) If the measurements of the electric signal caused by the sound wave in the suspension, are carried out under zero UCV conditions (short-circuited), an ac ICV can be measured. Its value, measured between two different points in the standing wave, is also proportional to the pressure difference between those two points, and the kinetic coefficient ICV/Dp characterizes the suspension and is closely related to UCV/Dp:
(
)
I CV I CV U ω , φ , ∆ρ / ρ , K σ i ,ζ ,η, a = K * CV = ∆p ∆p ∆p
AESA AESA = (ω , φ , ∆ρ / ρ , K σ i ,ζ ) E E
(75)
Measurement of ICV or AESA rather than UCV has the operational advantage that it enables measurement of the kinetic characteristics, ICV/Dp and AESA/E, which are independent of the complex conductivity K* of the suspension, and thus knowledge of K* is not a prerequisite for the extraction of the z-potential from the interpretation of the electroacoustic measurements. Note, however, that if SLC is significant, as with the other EKP, additional measurements will be needed, as both z and Ksi are required to fully characterize the interface.
4.7.2 Estimation of the ζ-potential from UCV, ICV, or A ESA
There are two recent methods for the theoretical interpretation of the data of electroacoustic measurements and extracting from them a value for the z-potential. One is based on the symmetry relation proposed in [82, 83] to express both kinds of electroacoustic phenomena (colloid vibration potential/current and ESA) in terms of the same quantity, namely the dynamic electrophoretic mobility, ud* , which is the complex, frequency-dependent analog of the normal direct current (dc) electrophoretic mobility. The second method is based on the direct evaluation of ICV without using the symmetry relations, and hence it is not necessarily based on the concept of dynamic electrophoretic mobility. Both methods for z-potential determination from electroacoustic measurements are briefly described below. Using the dynamic mobility method has some advantages: (i) the zero frequency limiting value of ud* is the normal electrophoretic mobility, and (ii) the frequency dependence of ud* can be used to estimate not only the z-potential, but also (for particle radius > −40 nm) the particle size distribution. Since the calculation of the z-potential in the general case requires a knowledge of ka it is helpful to have available the most appropriate estimate of the average particle size (most colloidal dispersions are polydisperse, and there are many possible “average” sizes which might be chosen). Although the calculation of the z-potential from the experimental measurements would be a rather laborious procedure, the necessary software for effecting the conversion is provided as an integral part of the available measuring systems, for both dilute and moderately concentrated sols. The effects of SLC can also be eliminated in some cases, without access to alternative measuring devices, simply by undertaking a titration with an indifferent electrolyte. In the case of methods based on the direct evaluation of ICV, the use of different frequencies, if available, or of acoustic attenuation measurements, allows the determination of particle size distributions.
(74)
4.7.1.1.3 Electrokinetic sonic amplitude (ESA) This refers to the measurement of the sound wave amplitude, which is caused by the application of an alternating electric field to a suspension of particles of which the density is different from that of the suspending medium. The ESA signal (i.e., the amplitude AESA of the sound pressure wave generated by the applied electric field) is proportional to the field strength E, and the kinetic coefficient
9168_Book.indb 662
AESA/E can be expressed as a function of the characteristics of the suspension
4.8 Method based on the concept of dynamic electrophoretic mobility
The symmetry relations lead to the following expressions, relating the different electroacoustic phenomena to the dynamic electrophoretic mobility, ud* [82, 83]:
∆ρ ud* U CV ∝φ ∆p ρ K*
(76)
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Measurement and Interpretation of Electrokinetic Phenomena
∆ρ * I CV ∝φ ud ∆p ρ
(77)
AESA ∆ρ * ∝φ ud E ρ
(78)
Although the kinetic coefficients on the right-hand side of these relations (both magnitude and phase) can readily be measured at any particle concentration, there is some difficulty (see below) in the conversion of ud* to a z-potential, except for the simplest case of spherical particles in fairly dilute suspensions (up to a few vol %). In this respect, the situation is similar to that for the more conventional electrophoretic procedures. There are, though, some offsetting advantages. In particular, the ability to operate on concentrated systems obviates the problems of contamination which beset some other procedures. It also makes possible meaningful measurements on real systems without the need for extensive dilution, which can compromise the estimation of z-potential, especially in emulsions systems.
4.9 Dilute systems (up to ~4 vol %)
1. For a dilute suspension of spherical particles with ka >> 1 and arbitrary z-potential, the following equation can be used [84], which relates the dynamic mobility with the z-potential and other particle properties:
2ε ε ζ ud* = rs 0 (1 + f )G(α ) 3η
(79)
The restriction concerning double-layer thickness requires in practice that ka > ~20 for reliable results, although the error is usually tolerable down to about ka = 13. The function f is a measure of the tangential electric field around the particle surface. It is a complex quantity, given by
f=
1 + iω ′ − [2 Du + iω ′(ε rp/ε rs )] 2(1 + iω ′ ) + [2 Du + iω ′(ε rp/ε rs )]
(80)
where w¢ ≡ werse0/KL is the ratio of the measurement frequency, w, to the Maxwell–Wagner relaxation frequency of the electrolyte. If it can be assumed that the tangential current around the particle is carried essentially by ions outside the shear surface, then Du is given by the Dud (eqs. 16–19); see also [85]*. The function G (a) is also complex and given by
G(α ) =
1 + (1 + i ) α/2 α 1 + (1 + i ) α / 2 + i (3 + 2∆ρ/ρ ) 9
(81)
It is a direct measure of the inertia effect. The dimensionless parameter α is defined as
ω a2 ρ α= η
(82)
* If this assumption breaks down, Du must be estimated by reference to a suitable surface conductance model or, preferably, by direct measurement of the conductivity over the frequency range involved in the measurement (normally from about 1 to 40 MHz) [86]. Another procedure involved the analysis of the results of a salt titration (see previous subsection).
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so G is strongly dependent on the particle size. G varies monotonically from a value of unity, with zero phase angle, when a is small (less than 0.1 mm typically) to a minimum of zero and a phase lag of π/4 when a is large (say, a > 10 mm). Equations 80–82 are, as mentioned above, applicable to systems of arbitrary z-potential, even when conduction occurs in the stagnant layer, in which case Du in eq. 80 must be properly evaluated. They have been amply confirmed by measurements on model suspensions [86], and have proved to be of particular value in the estimation of high z-potentials [87]. The maximum that appears in the plot of dc mobility against z-potential ([22], see also Fig. 2) gives rise to an ambiguity in the assignment of the z-potential, which does not occur when the full dynamic mobility spectrum is available. 2. For double layers with ka < ~20, there are approximate analytical expressions [88, 89] for dilute suspensions of spheres, but they are valid only for low z-potentials. They have been checked against the numerical solutions for general ka and z [90, 91], and those numerical calculations have been subjected to an experimental check in [92]. 3. The effect of particle shape has been studied in [93,94], again in the limit of dilute systems with thin double layers. This analysis has been extended in [95] to derive formulae for cylindrical particles with zero permittivity and low z-potentials, but for arbitrary ka. The results are consistent with those of [93, 94].
4.10 Concentrated systems
The problem of considering the effect on the electroacoustic signal of hydrodynamic or electrostatic interactions between particles in concentrated suspensions was first theoretically tackled [96] by using the cell model of Levine and Neale [97, 98] to provide a solution that was claimed to be valid for UCV measurements on concentrated systems (see [87] for a discussion on the validity of such approach in the high-frequency range). It is possible to deal with concentrated systems at high frequency without using cell models in the case of near neutrally buoyant systems (where the relative density compared to water is in the range 0.9–1.5) using a procedure developed by O’Brien [99, 100]. In that case, the interparticle interactions can be treated as pairwise additive and only nearest-neighbor interactions need to be taken into account. An alternative approach is to estimate the effects of particle concentration considering in detail the behavior of a pair of particles in all possible orientations [101, 102]. Empirical relations have been developed that appear to represent the interaction effects for more concentrated suspensions up to volume fractions of 30% at least. In [103], an example can be found where the dynamic mobility was analyzed assuming the system to be dilute. The resulting value of the z-potential (zapp) was then corrected for concentration using the semi-empirical relation:
ζ corr = ζ app exp{2φ[1 + s(φ )]}, with s(φ ) =
1 1 + (0.1 / φ )4
(83)
Finally, O’Brien et al. [104] have recently developed an effective analytical solution to the concentration problem for the dynamic mobility.
4.11 Method based on the direct evaluation of the ICV
This approach [105] applies a “coupled phase model” [106–109] for describing the speed of the particle relative to the liquid. The
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664 Kuwabara cell model [110] yields the required hydrodynamic parameters, such as the drag coefficient, whereas the Shilov– Zharkikh cell model [111] was used for the generalization of the Kuwabara cell model to the electrokinetic part of the problem. The method allows the study of polydisperse systems without using any superposition assumption. It is important in concentrated systems, where superposition does not work because of the interactions between particles. An independent exact expression for ICV in the quasi-stationary case of low frequency, using Onsager’s relationship and the HS equation, and neglecting the surface conductivity effect (Du 40). For e rs > 40, dissociation of most dissolved electrolytes is complete, and all equations concerning EDL or EKP remain unmodified, except that a lower value for e rs has to be used. For moderately polar solvents, the electrolyte dissociation is incomplete, which means that the concentration of charged species (i.e., the species that play a role in EDL or EKP) may be lower than the corresponding electrolyte concentration. Moreover, the charge number of the charge carriers can become lower. For instance, in solutions of Ba(NO3)2, we may find not (only) Ba2+ and NO -3 ions, but (also) Ba(NO3)+ complexes. The category of weakly polar solvents is a transition to the class of nonpolar liquids, for which the relative permittivity (at infinite frequency) equals the square of the index of refraction. Such liquids exhibit no self-dissociation, and the notion of electrolytes almost completely dissociated loses its meaning. However, even in such media, special types of molecules may dissociate to some extent and give rise to the formation of EDL and EKP. It is this group that we shall now emphasize. Unlike in aqueous media, dissociation in these solvents occurs only for potential electrolytes that contain ions of widely different sizes. Once dissociation occurs, the tendency for re-dissociation is relatively small because the charge on the larger ion is distributed over a large area. However, the concentration of ions (c+, c−) is very small. An indication of the magnitude of c+ and c− can be obtained from conductivity measurements. Particles embedded in such a liquid can become charged when one type of ion can be preferentially adsorbed. Typically, the resulting surface charges, s 0, are orders of magnitude lower than in aqueous systems. However, because of the very low EDL capacitance, the resulting surface potentials, y0, are of the same order of magnitude as in aqueous systems. The very slow decay of the potential with distance has two consequences. First, as the decay between surface and slip plane is negligible, y0≈z This simplifies the analysis. Second, the slow decay implies that colloid particles “feel” each other’s presence at long range. So, even dilute sols may behave physically as “concentrated” and formation of small clusters of coagulated particles is typical. Interestingly, electrophoresis can help in making a distinction between concentrated and dilute systems by studying the dependence of the electrophoretic mobility on the concentration of dispersed particles. If there is no dependence, the behavior is that of a dilute system. In this case, equations devised for dilute systems can be used. Otherwise, the behavior is effectively that of a concentrated system, and ESA or UCV measurements are more appropriate [86].
5.2 Experimental requirements of electrokinetic techniques
The methods described are also applicable to electrokinetic measurements in nonaqueous systems, but some precautions need to be taken. Microelectrophoresis cells are often designed for aqueous and water-like media whose conductivity is high relative to that of the material from which the cell is made. A homogeneous electric field between the electrodes of such cells filled with well- or moderately conducting liquids is readily achieved. However, when the cell is filled with a low-conductivity liquid, the homogeneity of the electric field can be disturbed by the
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more conducting cell walls and/or by the surface conduction due to adsorption of traces of water dissolved from the solvents of low polarity on the more hydrophilic cell walls. Special precautions (coating the walls with hydrophobic layers) are necessary to improve the field characteristics. The electric field in cells of regular geometrical shape is calculated from the measured current density and the conductivity of the nonaqueous liquid. Because of the low ionic strength of the latter, electrode polarization may occur and it can sometimes be observed as bubble formation. Hence, an additional pair of electrodes is often used for the measurements of the voltage gradient across the cell. For the correct measurement of the streaming potential in nonaqueous systems, attention must be paid to ensure that the resistance of the capillary, plug, or diaphragm filled with liquid is at least 100 times less than the input impedance of the electrical measuring device. The usual practice is to use millivoltmeter-electrometers with input resistance higher than 1014 Ω and platinum gauze electrodes placed in contact with the ends of the capillary or plug for both resistance and streaming potential measurements. The resistance is usually measured with an ac bridge in the case of polar solvents (typical frequencies used are around a few kHz) and by dc methods in the case of nonpolar or weakly polar liquids. The use of data recording on the electrometer output is common practice to check the rate of attainment of equilibrium and possible polarization effects.
5.3 Conversion of electrokinetic data into ζ-potentials
The first step in interpreting the electrokinetic behavior of nonaqueous systems must be to check whether the system behaves as a dilute or as a concentrated system (see Section 5.2). In the dilute regime, all theories described for aqueous systems can be used, provided one can find the right values of the essential parameters ka, Kp, and Ks . The calculation of k requires knowledge of the ionic strength of the solution; this, in turn, can be estimated from the measurement of the dialysate conductivity, KL, and knowledge of the mobilities and valences of the ionic species. The effective conductivity of the solid particle, Kpef, including its surface conductivity* can be calculated from the experimental values of conductivities of the liquid KL, and of a dilute suspension of particles, K, with volume fraction f using either Street’s equation
K pef K
L
= 1+
2 3
K KL
−1
φ
(85)
or Dukhin’s equation
K pef
K
L
=2
(1 − φ ) − (1 − φ )
K KL
K KL
(1 + φ / 2)
(
− 1 + 2φ
)
(86)
which accounts for interfacial polarization. *
The low conductivity of nonaqueous media with respect to aqueous solutions is the main reason why often the finite bulk conductivity of the dielectric solids cannot be neglected. In contrast, if one can assume that no water is adsorbed at the interface, any SLC effect can be neglected. Furthermore, the joint adsorption of the ionic species of both signs or (and) the adsorption of such polar species as water at the solid/liquid interface can produce an abnormally high surface conduction. This is not due to the excess of free charges in the EDL, commonly taken into account in the parameter Ks of the generalized theories of EKP. Rather, it is conditioned by the presence of thin, highly conducting adsorption layers. Therefore, the ratio Kpef /KL is an important parameter that has to be estimated for nonaqueous systems.
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666 For the estimation of the z-potential in the case of electrophoresis, considering that low ka values are not rare, it is suggested to use Henry’s theory, after substitution of Kpef estimated as described above, for the particle conductivity. In formulas, it is suggested to employ eqs. 29 and 30. Concerning streaming potential/current or electro-osmotic flow measurements, all the above-mentioned features of nonaqueous systems must be taken into account to obtain correct values of z-potential from this sort of data in either single capillaries, porous plugs, or diaphragms. In view of the low ka values normally attained by nonaqueous systems, the Rice and Whitehead curvature corrections are recommended [50]; see eqs. 37–39, 44, and 50. Finally, electroacoustic characterization of z-potential in nonaqueous suspensions requires subtraction of the background arising from the equilibrium dispersion medium. This is imperative because the electroacoustic signal generated by particles in low- or nonpolar media is very weak. It is recommended to make measurements at several volume fractions in order to ensure that the signal comes, in fact, from the particles. Note that there is one more issue that complicates the formulation of a rigorous electrokinetic theory in concentrated nonaqueous systems, namely, the conductivity of a dispersion of particles in such media becomes position-dependent, as the double layers of the particles may occupy most of the volume of the suspension. This is a significant theoretical obstacle in the elaboration of the theory. In the case of EKP based on the application of nonstationary fields (dielectric dispersion, electroacoustics), this problem can be overcome. This is possible because one of the peculiarities of low- and nonpolar liquids compared to aqueous systems is the very low value of the Maxwell–Wagner–O’Konski frequency, eqs. 57 and 58. This means that in the modern electroacoustic methods based on the application of electric or ultrasound fields in the MHz frequency region, all effects related to conductivity can be neglected, because that frequency range is well above the Maxwell–Wagner characteristic frequency of the liquid. This makes electroacoustic techniques most suitable for the electrokinetic characterization of suspensions in nonaqueous media.
6. Remarks on non-ideal surfaces 6.1 General comments
The general theory of EKP described so far strictly applies to ideal, nonporous, and rigid surfaces or particles. By ideal, we mean smooth (down to the scale of molecular diameters) and chemically homogeneous, and we use rigid to describe those particles and surfaces that do not deform under shear. We will briefly indicate interfaces that are nonporous and rigid as hard interfaces. For hard particles and surfaces, there is a sharp change of density in the transition between the particle or surface and the surrounding medium. Hard particles effectively lead to stacking of water (solvent, in general) molecules; that is, only for hard surfaces the notion of a stagnant layer close to the real surface is conceptually simple. Not many surfaces fulfill these conditions. For instance, polystyrenesulfonate latex colloid has, in fact, a heterogeneous interface: the hydrophilic sulfate end-groups of its polymer chains occupy some 10 % of the total surface of the particles, the remainder being hydrophobic styrene. Moreover, the sulfate groups will protrude somewhat into the solution. Generally speaking, many interfaces are far from molecularly smooth, as they may contain pores or can be somewhat deformable. Such interfaces can briefly be indicated as soft interfaces. For soft interfaces, such as, for instance, rigid particles with “soft” or
9168_Book.indb 666
“hairy” polymer layers, gel-type penetrable particles and water– air or water–oil interfaces, the molecular densities vary gradually across the phase boundary. A main problem in such cases is the description of the hydrodynamics, and in some cases it is even questionable if a discrete slip plane can be defined operationally. The difficulties encountered when interpreting experimental results obtained for non-ideal interfaces depend on the type and magnitude of the non-idealities and on the aim of the measurements. In practice, one can always measure some quantity (like ue), apply some equation (like HS) to compute what we can call an “effective” z-potential, but the physical interpretation of such a z-potential is ambiguous. We must keep in mind that the obtained value has only a relative meaning and should not be confused with an actual electrostatic potential close to the surface. Nevertheless, such an “effective z” can help us in practice, because it may give some feeling for the sign and magnitude of the part of the double-layer charge that controls the interaction between particles. When the purpose of the measurement is to obtain a realistic value of the z-potential, there is no general recipe. It may be appropriate to use more than one electrokinetic method and to take into account the specific details of the non-ideality as well as possible in each model for the calculation of the z-potential. If the z-potentials resulting from both methods are similar, physical meaning can be assigned to this value. Below, we will discuss different forms of non-ideality in somewhat more detail. We will mainly point out what the difficulties are, how these can be dealt with and where relevant literature can be found.
6.2 Hard surfaces
Some typical examples of non-ideal particles that still can be considered as hard are discussed below. Attention is paid to size and shape effects, surface roughness, and surface heterogeneity. For hard non-ideal surfaces, both the stagnant-layer concept and the z-potential remain locally defined and experimental data provide some average electrokinetic quantity that will lead to an average z-potential. The kind of averaging may depend on the electrokinetic method used, therefore, different methods may give different average z-potentials.
6.2.1 Size effects
For rigid particles that are spherical with a homogeneous charge density, but differ in size, a rigorous value of the z-potential can be found if the electrokinetic quantity measured is independent of the particle radius, a. In general, this will be the case for ka >> 1 (HS limit). In the case of electrophoresis, the mobility is also independent of a for ka > 1 (HS equation valid) or if ka 1 (HS limit) or kR > 1, where a is a characteristic dimension of the system (curvature radius of the solid particle, capillary radius, equivalent pore radius of the plug,…), and furthermore, the surface conductance of any kind must be low, i.e., Du(z,Ksi) « 1. Thus, in the absence of independent information about Ksi, additional electrokinetic determinations can only be avoided for sufficiently large particles and high electrolyte concentrations. Another caveat can be given, even if the previously mentioned conditions on dimensions and Dukhin number are met. For concentrated systems, the possibility of the overlap of the double layers of neighboring particles cannot be neglected if the concentration of the dispersed solids in a suspension or a plug is high. In such cases, the validity of the HS equation is also doubtful and cell models for either electrophoresis, streaming potential, or electroosmosis are required. Use of the latter two kinds of experiments or of electroacoustic or LFDD measurements is recommended. In all cases, a proper model accounting for interparticle interaction must be available.
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669
8. Appendix I. Calculation of the low-frequency dielectric dispersion of suspensions Neglecting SLC, the complex conductivity increment is related to the dipole coefficient as follows [62–64]:
δ K * (ω ) =
3φ K LC 0* = δ K * ω →0 a3
+ 9φ K L
( R + − R − )H iωτ α 2 AS 1 + S iωτ α + iωτ α
(I.1)
Its low-frequency value is a real quantity:
δ K*
2 Du(ζ ) − 1 3( R + − R − )H = 3φ K L − ω →0 2B 2 Du(ζ ) + 2
(I.2)
The dielectric increment of the suspension can be calculated from δ K *
ω →0
as follows:
δε r* (ω ) = −
(
1 δ K * −δ K * ω =0 ωε 0
)
9 τ ( R + − R − )H 1 = φε rs′ α 2 AS τ el 1 + S iωτ α + iωτ α
(I.3)
Here:
( ) − 1 + 6m exp( ∓ ) − 1 ± z zy ek
R =4 ±
exp ∓ zy2
zy ek 2
±
κa
κa
(I.4) κ a ek
and
τα =
a2 1 2 Def S
(I.5)
is the value of the relaxation time of the low-frequency dispersion. It is assumed that the dispersion medium is an aqueous solution of an electrolyte of z-z charge type. The definitions of the other quantities appearing in eqs. I.1–5 and the z-potential are as follows:
Def =
A = 4 Du(ζ ) + 4
(I.6)
(I.7)
B = ( R + + 2)( R − + 2) − U + − U − − (U + R − + U − R + )/2 (I.8) S=
2D+ D− D+ + D−
H=
B A
(I.9)
( R + − R − )(1 − z 2 ∆ 2 ) − U + + U − + z ∆(U + + U − ) (I.10) A
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670
U± =
zyζ 48m ± ln cosh 4 κa
∆=
D− − D+
(
z D− + D+
)
(I.11)
(I.12)
The factor 1/S is of the order of unity, and comparison of eqs. 58 and I.5 leads to the important conclusion that
τα ≈ (κ a)2 τ MWO
(I.13)
This means that for the case of ka >> 1, the characteristic frequency for the α-dispersion is (ka)2 times lower than that of the Maxwell–Wagner dispersion.
9. Acknowledgments Financial assistance from IUPAC is gratefully acknowledged. The Coordinators of the MIEP working party wish to thank all members for their effort in preparing their contributions, suggestions, and remarks.
10. References 1. S. S. Dukhin. Adv. Colloid Interface Sci. 61, 17 (1995). 2. R. J. Hunter. Foundations of Colloid Science, Chap. 8, Oxford University Press, Oxford (2001). 3. J. Lyklema. Fundamentals of Interfaces and Colloid Science, Vol. II, Chaps. 3, 4, Academic Press, New York (1995). 4. R. J. Hunter. Zeta Potential in Colloid Science, Academic Press, New York (1981). 5. S. S. Dukhin and B. V. Derjaguin. “Electrokinetic phenomena”, in Surface and Colloid Science, Vol. 7, Chap. 2, E. Matijević (Ed.), John Wiley, New York (1974). 6. A. V. Delgado (Ed.). Interfacial Electrokinetics and Electrophoresis, Marcel Dekker, New York (2001). 7. Manual of symbols and terminology for physicochemical uantities and units, Appendix II: Definitions, terminology and symbols in colloid and surface chemistry. Part I. Pure Appl. Chem. 31, 577 (1972). 8. I. Mills, T. Cvitaš, K. Homann, N. Kallay, K. Kuchitsu. Quantities, Units and Symbols in Physical Chemistry, 2nd ed., Sect. 2.14, International Union of Pure and Applied Chemistry, Blackwell Science, Oxford (1993). 9. A. D. McNaught and A. Wilkinson. Compendium of Chemical Terminology. IUPAC Recommendations, 2nd ed., International Union of Pure and Applied Chemistry, Blackwell Science, Oxford (1997). 10. J. Lyklema. Pure Appl. Chem. 63, 885 (1995). 11. J. Lyklema, S. Rovillard, J. de Coninck. Langmuir 14, 5659 (1998). 12. J. W. Lorimer. J. Membr. Sci. 14, 275 (1983). 13. C. F. Zukoski and D. A. Saville. J. Colloid Interface Sci. 114, 32 (1986). 14. C. S. Mangelsdorf and L. R. White. J. Chem. Soc., Faraday Trans. 86, 2859 (1990). 15. C. S. Mangelsdorf and L. R. White. J. Chem. Soc., Faraday Trans. 94, 2441 (1998). 16. C. S. Mangelsdorf and L. R. White. J. Chem. Soc., Faraday Trans. 94, 2583 (1998). 17. J. Lyklema and M. Minor. Colloids Surf., A 140, 33 (1998). 18. M. von Smoluchowski. In Handbuch der Electrizität und des Magnetismus (Graetz), Vol. II, p. 366, Barth, Leipzig (1921). 19. J. J. Bikerman. Z. Physik. Chem. A163, 378 (1933). 20. J. J. Bikerman. Kolloid Z. 72, 100 (1935). 21. J. J. Bikerman. Trans. Faraday Soc. 36, 154 (1940). 22. R. W. O’Brien and L. R. White. J. Chem. Soc., Faraday Trans. II 74, 1607 (1978).
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113. R. J. Hunter and R. W. O’Brien. In Encyclopedia of Colloid and Surface Science, A. Hubbard (Ed.), p. 1722, Marcel Dekker, New York (2002). 114. H. Ohshima. In Electrical Phenomena at Interfaces. Fundamentals, Measurements and Applications, H. Ohshima and K. Furusawa (Eds.), Chap. 2, Marcel Dekker, New York (2002). 115. F. Carrique, F. J. Arroyo, A. V. Delgado. J. Colloid Interface Sci. 206, 206 (1998). 116. S. S. Dukhin. Adv. Colloid Interface Sci. 44, 1 (1993). 117. R. W. O’Brien and D. N. Ward. J. Colloid Interface Sci. 121, 402 (1988). 118. M. C. Fair and J. L. Anderson. J. Colloid Interface Sci. 127, 388 (1989). 119. D. Velegol, J. L. Anderson, Y. Solomentsev. In Interfacial Electrokinetics and Electrophoresis, A. V. Delgado (Ed.), Chap. 6, Marcel Dekker, New York (2002). 120. J. Y. Kim and B. J. Yoon. In Interfacial Electrokinetics and Electrophoresis, A. V. Delgado (Ed.), Chap. 7, Marcel Dekker, New York (2002). 121. C. Grosse and V. N. Shilov. J. Colloid Interface Sci. 193, 178 (1997). 122. C. Grosse, S. Pedrosa, V. N. Shilov. J. Colloid Interface Sci. 220, 31 (1999). 123. S. S. Dukhin, R. Zimmermann, C. Werner. Colloids Surf., A 195, 103 (2001). 124. J. Y. Walz. Adv. Colloid Interface Sci. 74, 119 (1998). 125. S. A. Allison. Macromolecules 29, 7391 (1996). 126. M. Teubner. J. Chem. Phys. 76, 5564 (1982). 127. M. C. Fair and J. L. Anderson. Langmuir 8, 2850 (1992). 128. D. Velegol, J. L. Anderson, S. Garoff. Langmuir 12, 675 (1996). 129. J. L. Anderson, D. Velegol, S. Garoff. Langmuir 16, 3372 (2000). 130. J. L. Anderson. J. Colloid Interface Sci. 105, 45 (1985). 131. H. van Olphen. An Introduction to Clay Colloid Chemistry, John Wiley, New York (1977). 132. S. Akari, W. Schrepp, D. Horn. Langmuir 12, 857 (1996). 133. K. E. Bremmell, G. J. Jameson, S. Biggs. Colloids Surf., A 139, 199 (1998). 134. M. A. Cohen-Stuart, F. H. W. H. Waajen, S. S. Dukhin. Colloid Polym. Sci. 262, 423 (1984). 135. L. K. Koopal, V. Hlady, J. Lyklema. J. Colloid Interface Sci. 121, 49 (1988). 136. L. Kong, J. K. Beattie, R. J. Hunter. Phys. Chem. Chem. Phys. 3, 87 (2001). 137. H. Ohshima. J. Colloid Interface Sci. 163, 474 (1995). 138. H. Ohshima and K. Makino. Colloids Surf., A 109, 71 (1996). 139. H. N. Stein. In Interfacial Electrokinetics and Electrophoresis, A. V. Delgado (Ed.), Chap. 21, Marcel Dekker, New York (2002). 140. R. Hidalgo-Álvarez, A. Martín, A. Fernández, D. Bastos, F. Martínez, F. J. de las Nieves. Adv. Colloid Interface Sci. 67, 1 (1996). 141. H. Ohshima. In Interfacial Electrokinetics and Electrophoresis, A. V. Delgado, (Ed.), Chap. 5, Marcel Dekker, New York (2002). 142. H. J. Keh. In Interfacial Electrokinetics and Electrophoresis, A. V. Delgado (Ed.), Chap. 15, Marcel Dekker, New York (2002). 143. C. Tanford and J. A. Reynolds. Biochim. Biophys. Acta 457, 133 (1976). 144. D. Stigter. Cell Biophys. 11, 139 (1987). 145. D. Stigter. J. Phys. Chem. B 104, 3402 (2000). 146. R. J. Hill, D. A. Saville, W. B. Russel. J. Colloid Interface Sci. 258, 56 (2003). 147. A. Martín-Rodríguez, J. L. Ortega-Vinuesa, R. Hidalgo-Álvarez. In Interfacial Electrokinetics and Electrophoresis, A. V. Delgado (Ed.), Chap. 22, Marcel Dekker, New York (2002). 148. E. Lee, F. Y. Yen, J. P. Hsu. In Interfacial Electrokinetics and Electrophoresis, A. V. Delgado (Ed.), Chap. 23, Marcel Dekker, New York (2002). 149. R. Barchini, H. P. van Leewen, J. Lyklema. Langmuir 16, 8238 (2000). 150. A. Van der Wal, M. Minor, W. Norde, A. J. B. Zehnder, J. Lyklema. Langmuir 13, 165 (1997). 151. S. A. Nespolo, M. A. Bevan, D. Y. C. Chan, F. Grieser, G. W. Stevens. Langmuir 17, 7210 (2001). 152. C. Yang, T. Dabros, D. Li, J. Czarnecki, J. H. Masliyah. J. Colloid Interface Sci. 243,128 (2001).
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11. List of symbols Note: SI base (or derived) units are given in parentheses for all quantities, except dimensionless ones. a (m) particle radius, local curvature radius, capillary radius capillary cross-section Ac (m2) Acap (m2) apparent (externally measured) capillary cross-section A ESA (Pa) electrokinetic sonic amplitude b (m) half distance between neighboring particles c (mol m−3) electrolyte concentration c+(c−) (mol m−3) concentration of cations (anions) C*0 dipole coefficient of particles d (m) distance between the surface and the outer Helmholtz plane d* (C m) complex dipole moment dek (m) distance between the surface and the slip plane D (m2 s−1) diffusion coefficient of counterions (or average diffusion coefficient of ions) D+ (D−) (m2 s−1) diffusion coefficient of cations (anions) Du Dukhin number Dud Dukhin number associated with diffuselayer conductivity Dui Dukhin number associated with stagnant-layer conductivity e (C) elementary charge E (V m−1) applied electric field Esed(V m−1) sedimentation field Et (V m−1) tangential component of external field F (C mol−1) Faraday constant f1(ka), F1(ka, Kp) Henry’s functions I (A) electric current intensity I0 I1 zeroth- (first-) order modified Bessel functions of the first kind Ic (A) conduction current ICV (A) colloid vibration current Istr (A) streaming current js (Am−1) surface current density jstr(A m−2) streaming current density k (J K−1) Boltzmann constant K (S m−1) total conductivity of a colloidal system KDC (S m−1) direct current conductivity of a suspension KL (S m−1) conductivity of dispersion medium K∞l (S m−1) conductivity of a highly concentrated ionic solution Kp (S m−1) conductivity of particles Kplug (S m−1) conductivity of a plug of particles Kpef (S m−1) effective conductivity of particles Krel ratio between particle and liquid conductivities K* (S m−1) complex conductivity of a suspension Ks (S) surface conductivity Ksd (S) diffuse-layer surface conductivity Ksi (S) stagnant-layer surface conductivity L (m) capillary length, characteristic dimension L ap (m) apparent (externally measured) capillary cross-section
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LD (m) ionic diffusion length m dimensionless ionic mobility of counterions m+ (m−) dimensionless ionic mobility of cations (anions) n (m−3) number concentration of particles NA (mol−1) Avogadro constant number concentration of type i ions ni (m−3) Qeo (m3 s−1) electro-osmotic flow rate Qeo,E (m4 s−1 V−1) electro-osmotic flow rate per electric field Qeo,I (m3 C−1) electro-osmotic flow rate per current r (m) spherical or cylindrical radial coordinate R (m) roughness of a surface Rs (W) electrical resistance of a capillary or porous plug in an arbitrary solution R∞ (W) electrical resistance of a capillary or porous plug in a concentrated ionic solution T (K) thermodynamic temperature Ud* (m2 s−1 V−1) dynamic electrophoretic mobility UCV (V) colloid vibration potential ne (m2 s−1 V−1) electrophoretic mobility Used (V) sedimentation potential Ustr (V) streaming potential ne (m s−1) electrophoretic velocity neo (m s−1) electro-osmotic velocity n L (m s−1) liquid velocity in electrophoresis cell yek dimensionless z-potential z common charge number of ions in a symmetrical electrolyte zi charge number of type i ions a relaxation of double-layer polarization, degree of electrolyte dissociation, dimensionless parameter used in electroacoustics b (m) distance between the solid surface and the inner Hemholtz plane (see also eq. 45 for another use of this symbol) Gi (m−2) surface concentration of type i ions d c (mol m−3) field-induced perturbation of electrolyte amount concentration dK* (S m−1) conductivity increment of a suspension d er relative dielectric increment of a suspension Dp (Pa) applied pressure difference Dpeo (Pa) electro-osmotic counter-pressure DVext (V) applied potential difference De (0) low-frequency dielectric increment per volume fraction Ded(0) value of Δe(0) for suspensions with low volume fractions Δρ (kg m−3) density difference between particles and dispersion medium e* (F m−1) complex electric permittivity of a suspension er* complex relative permittivity of a suspension e rp relative permittivity of the particle * complex relative permittivity of a erp particle e rs relative permittivity of the dispersion medium
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Measurement and Interpretation of Electrokinetic Phenomena ers* complex relative permittivity of the dispersion medium e 0 (F m−1) electric permittivity of vacuum z (V) electrokinetic or z-potential zapp (V) electrokinetic or z-potential not corrected for the effect of particle concentration h (Pa s) dynamic viscosity k (m−1) reciprocal Debye length density of dispersion medium r (kg m−3) r p (kg m−3) density of particles r s (kg m−3) density of a suspension s d (C m−2) diffuse charge density s ek (C m−2) electrokinetic charge density s i (C m−2) surface charge density at the inner Helmholtz plane s 0 (C m−2) titratable surface charge density
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673
t MWO (s) characteristic time of the Maxwell– Wagner–O’Konski relaxation t a (s) relaxation time of the low-frequency dispersion f volume fraction of solids f L volume fraction of liquid in a plug yd (V) diffuse-layer potential yi (V) inner Helmholtz plane potential y0 (V) surface potential w (s−1) angular frequency of an ac electric field w MWO (s−1) Maxwell–Wagner–O’Konski characteristic frequency w a (s−1) characteristic frequency of the a-relaxation w ad (s−1) characteristic frequency of the a-relaxation for a dilute suspension
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Measurement of pH Definition, standards, and procedures (IUPAC Recommendations 2002) Working Party on pH
R. P. Buck (Chairman)1, S. Rondinini (Secretary)2,‡, A. K. Covington (Editor)3, F. G. K. Baucke4, C. M. A. Brett5, M. F. Camões6, M. J. T. Milton7, T. Mussini8, R. Naumann9, K. W. Pratt10, P. Spitzer11, and G. S. Wilson12 101 Creekview Circle, Carrboro, NC 27510, USA; 2 Dipartimento di Chimica Fisica ed Elettrochimica, Università di Milano, Via Golgi 19, I-20133 Milano, Italy; 3 Department of Chemistry, The University, Bedson Building, Newcastle Upon Tyne, NE1 7RU, UK; 4 Schott Glasswerke, P.O. Box 2480, D-55014 Mainz, Germany; 5Departamento de Química, Universidade de Coimbra, P-3004-535 Coimbra, Portugal; 6 Departamento de Química e Bioquimica, University of Lisbon (SPQ/DQBFCUL), Faculdade de Ciencias, Edificio CI-5 Piso, P-1700 Lisboa, Portugal; 7National Physical Laboratory, Centre for Optical and Environmental Metrology, Queen’s Road, Teddington, Middlesex TW11 0LW, UK; 8 Dipartimento di Chimica Fisica ed Elettrochimica, Università di Milano, Via Golgi 19, I-20133 Milano, Italy; 9MPI for Polymer Research, Ackermannweg 10, D-55128 Mainz, Germany; 10 Chemistry B324, Stop 8393, National Institute of Standards and Technology, 100 Bureau Drive, ACSL, Room A349, Gaithersburg, MD 20899-8393, USA; 11 Physikalisch-Technische Bundesanstalt (PTB), Postfach 33 45, D-38023 Braunschweig, Germany; 12 Department of Chemistry, University of Kansas, Lawrence, KS 66045, USA 1
‡
Corresponding author
Abstract: The definition of a “primary method of measurement” [1] has permitted a full consideration of the definition of primary standards for pH, determined by a primary method (cell without transference, Harned cell), of the definition of secondary standards by secondary methods, and of the question whether pH, as a conventional quantity, can be incorporated within the internationally accepted system of measurement, the International System of Units (SI, Système International d’Unités). This approach has enabled resolution of the previous compromise IUPAC 1985 Recommendations [2]. Furthermore, incorporation of the uncertainties for the primary method, and for all subsequent measurements, permits the uncertainties for all procedures to be linked to the primary standards by an unbroken chain of comparisons. Thus, a rational choice can be made by the analyst of the appropriate procedure to achieve the target uncertainty of sample pH. Accordingly, this document explains IUPAC recommended definitions, procedures, and terminology relating to pH measurements in dilute aqueous solutions in the temperature range 5–50°C. Details are given of the primary and secondary methods for measuring pH and the rationale for the assignment of pH values with appropriate uncertainties to selected primary and secondary substances.
Contents 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.
Introduction and scope . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 676 Activity and the definition of pH . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 676 Traceability and primary methods of measurement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 676 Harned cell as a primary method for the absolute measurement of pH . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 677 Sources of uncertainty in the use of the harned cell . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 678 Primary buffer solutions and their required properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 679 Consistency of primary buffer solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 679 Secondary standards and secondary methods of measurement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 682 Consistency of secondary standard buffer solutions established with respect to primary standards . . . . . . . . . . . . . . . . . . . . . . . 684 Target uncertainties for the measurement of secondary buffer solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 685 Calibration of pH meter-electrode assemblies and target uncertainties for unknowns . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 685 Glossary [2,15,44] . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 686 Annex: measurement uncertainty . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 687 Summary of recommendations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 691 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 691
Reproduced from: Pure Appl. Chem., Vol. 74, No. 11, pp. 2169–2200, 2002. © 2002 IUPAC
675
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676
BIPM CRMs EUROMET NBS NIST NMIs PS LJP RLJP SS
Abbreviations used
Bureau International des Poids et Mesures, France certified reference materials European Collaboration in Metrology (Measurement Standards) National Bureau of Standards, USA, now NIST National Institute of Science and Technology, USA national metrological institutes primary standard liquid junction potential residual liquid junction potential secondary standard
1. Introduction and scope 1.1 pH, a single ion quantity
The concept of pH is unique among the commonly encountered physicochemical quantities listed in the IUPAC Green Book [3] in that, in terms of its definition [4],
pH = −lg aH
it involves a single ion quantity, the activity of the hydrogen ion, which is immeasurable by any thermodynamically valid method and requires a convention for its evaluation.
1.2 Cells without transference, Harned cells
As will be shown in Section 4, primary pH standard values can be determined from electrochemical data from the cell without transference using the hydrogen gas electrode, known as the Harned cell. These primary standards have good reproducibility and low uncertainty. Cells involving glass electrodes and liquid junctions have considerably higher uncertainties, as will be discussed later (Sections 5.1, 10.1). Using evaluated uncertainties, it is possible to rank reference materials as primary or secondary in terms of the methods used for assigning pH values to them. This ranking of primary (PS) or secondary (SS) standards is consistent with the metrological requirement that measurements are traceable with stated uncertainties to national, or international, standards by an unbroken chain of comparisons each with its own stated uncertainty. The accepted definition of traceability is given in Section 12.4. If the uncertainty of such measurements is calculated to include the hydrogen ion activity convention (Section 4.6), then the result can also be traceable to the internationally accepted SI system of units.
1.3 Primary pH standards
In Section 4 of this document, the procedure used to assign primary standard [pH(PS)] values to primary standards is described. The only method that meets the stringent criteria of a primary method of measurement for measuring pH is based on the Harned cell (Cell I). This method, extensively developed by R. G. Bates [5] and collaborators at NBS (later NIST), is now adopted in national metrological institutes (NMIs) worldwide, and the procedure is approved in this document with slight modifications (Section 3.2) to comply with the requirements of a primary method.
1.4 Secondary standards derived from measurements on the Harned cell (Cell I)
Values assigned by Harned cell measurements to substances that do not entirely fulfill the criteria for primary standard status are secondary standards (SS), with pH(SS) values, and are discussed in Section 8.1.
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1.5 Secondary standards derived from primary standards by measuring differences in pH
Methods that can be used to obtain the difference in pH between buffer solutions are discussed in Sections 8.2–8.5 of these Recommendations. These methods involve cells that are practically more convenient than the Harned cell, but have greater uncertainties associated with the results. They enable the pH of other buffers to be compared with primary standard buffers that have been measured with a Harned cell. It is recommended that these are secondary methods, and buffers measured in this way are secondary standards (SS), with pH(SS) values.
1.6 Traceability
This hierarchical approach to primary and secondary measurements facilitates the availability of traceable buffers for laboratory calibrations. Recommended procedures for carrying out these calibrations to achieve specified uncertainties are given in Section 11.
1.7 Scope
The recommendations in this Report relate to analytical laboratory determinations of pH of dilute aqueous solutions (≤0.1 mol kg–1). Systems including partially aqueous mixed solvents, biological measurements, heavy water solvent, natural waters, and hightemperature measurements are excluded from this Report.
1.8 Uncertainty estimates
The Annex (Section 13) includes typical uncertainty estimates for the use of the cells and measurements described.
2. Activity and the definition of pH 2.1 Hydrogen ion activity
pH was originally defined by Sørensen in 1909 [6] in terms of the concentration of hydrogen ions (in modern nomenclature) as pH = −lg(cH/c°) where cH is the hydrogen ion concentration in mol dm–3, and c° = 1 mol dm–3 is the standard amount concentration. Subsequently [4], it has been accepted that it is more satisfactory to define pH in terms of the relative activity of hydrogen ions in solution
pH = −lg aH = −lg(mHg H/m°)
(1)
where aH is the relative (molality basis) activity and g H is the molal activity coefficient of the hydrogen ion H+ at the molality mH, and m° is the standard molality. The quantity pH is intended to be a measure of the activity of hydrogen ions in solution. However, since it is defined in terms of a quantity that cannot be measured by a thermodynamically valid method, eq. 1 can be only a notional definition of pH.
3. Traceability and primary methods of measurement 3.1 Relation to SI
Since pH, a single ion quantity, is not determinable in terms of a fundamental (or base) unit of any measurement system, there was some difficulty previously in providing a proper basis for the traceability of pH measurements. A satisfactory approach is now available in that pH determinations can be incorporated into the SI if they can be traced to measurements made using a method that fulfills the definition of a “Primary method of measurement” [1].
3.2 Primary method of measurement
The accepted definition of a primary method of measurement is given in Section 12.1. The essential feature of such a method is that it
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Measurement of pH Definition, Standards, and Procedures must operate according to a well-defined measurement equation in which all of the variables can be determined experimentally in terms of SI units. Any limitation in the determination of the experimental variables, or in the theory, must be included within the estimated uncertainty of the method if traceability to the SI is to be established. If a convention is used without an estimate of its uncertainty, true traceability to the SI would not be established. In the following section, it is shown that the Harned cell fulfills the definition of a primary method for the measurement of the acidity function, p(aHgCl), and subsequently of the pH of buffer solutions.
The cell without transference defined by Cell I
known as the Harned cell [7], and containing standard buffer, S, and chloride ions, in the form of potassium or sodium chloride, which are added in order to use the silver–silver chloride electrode. The application of the Nernst equation to the spontaneous cell reaction:
1/2
EI = E°− [(RT/F)ln 10] lg[(mHg H/m°)(mClg Cl/m°)]
p(aHg Cl) = −lg(aHg Cl) = (EI – E°)/[(RT/F)ln 10] + lg(mCl/m°) (2′) where E° is the standard potential difference of the cell, and hence of the silver–silver chloride electrode, and g Cl is the activity coefficient of the chloride ion. Note 1: The sign of the standard electrode potential of an electrochemical reaction is that displayed on a high-impedance voltmeter when the lead attached to standard hydrogen electrode is connected to the minus pole of the voltmeter. The steps in the use of the cell are summarized in Fig. 1 and described in the following paragraphs. The standard potential difference of the silver–silver chloride electrode, E°, is determined from a Harned cell in which only HCl is present at a fixed molality (e.g., m = 0.01 mol kg–1). The application of the Nernst equation to the HCl cell
gives
Pt | H2 | HCl(m) | AgCl | Ag
EIa = E° – [(2RT/F)ln 10] lg[(mHCl/m°)(g ±HCl)]
Cell Ia
(3)
where EIa has been corrected to 1 atmosphere partial pressure of hydrogen gas (101.325 kPa) and g±HCl is the mean ionic activity coefficient of HCl.
4.2 Activity coefficient of HCl
The values of the activity coefficient (g±HCl) at molality 0.01 mol kg–1 and various temperatures are given by Bates and Robinson [8]. The standard potential difference depends in some not entirely
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Determine p(aHγCl)o by extrapolation
Linear extrapolation
Calculate pH
Bates–Guggenheim convention for the calculation of γClo based on Debye–Hückel theory
(2)
which can be rearranged, since a H = mHg H/m°, to give the acidity function
Fill Harned Cell with HCl, at, e.g., mHCl = 0.01 mol kg–1
Either, Literature value for γ+ HCl, – at e.g., mHCl = 0.01 mol kg–1 or, by extrapolation based on Debye—Hückel theory
H2 + AgCl → Ag(s) + H+ + Cl −
yields the potential difference EI of the cell [corrected to 1 atm (101.325 kPa), the partial pressure of hydrogen gas used in electrochemistry in preference to 100 kPa] as
Notional definition
Fill Harned Cell with buffer at the known ionic strength Measure p(aH γCl) for at least 3 molalities of added chloride
4.1 Harned cell
Pt | H2| buffer S, Cl–| AgCl |Ag
pH = –lg aH
Measure E°Cl|AgCl|Ag
4. Harned cell as a primary method for the absolute measurement of pH
677
Figure 1 Operation of the Harned cell as a primary method for the measurement of absolute pH. understood way on the method of preparation of the electrodes, but individual determinations of the activity coefficient of HCl at 0.01 mol kg–1 are more uniform than values of E°. Hence, the practical determination of the potential difference of the cell with HCl at 0.01 mol kg–1 is recommended at 298.15 K at which the mean ionic activity coefficient is 0.904. Dickson [9] concluded that it is not necessary to repeat the measurement of E° at other temperatures, but that it is satisfactory to correct published smoothed values by the observed difference in E° at 298.15 K.
4.3 Acidity function
In NMIs, measurements of Cells I and Ia are often done simultaneously in a thermostat bath. Subtracting eq. 3 from eq. 2 gives
∆E = EI − EIa = −[(RT/F)ln 10]{lg[(mHg H/m°)(mClg Cl/m°)] − lg[(mHCl/m°)2g 2 ±HCl]}
(4)
which is independent of the standard potential difference. Therefore, the subsequently calculated pH does not depend on the standard potential difference and hence does not depend on the assumption that the standard potential of the hydrogen electrode, E°(H+|H2) = 0 at all temperatures. Therefore, the Harned cell can give an exact comparison between hydrogen ion activities at two different temperatures (in contrast to statements found elsewhere, see, for example, ref. [5]). The quantity p(aHg Cl) = −lg(aHg Cl), on the left-hand side of eq. 2′, is called the acidity function [5]. To obtain the quantity pH (according to eq. 1), from the acidity function, it is necessary to evaluate lg g Cl by independent means. This is done in two steps:
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678 (i) the value of lg(aHg Cl) at zero chloride molality, lg(aHg Cl)°, is evaluated and (ii) a value for the activity of the chloride ion g °Cl , at zero chloride molality (sometimes referred to as the limiting or “trace” activity coefficient [9]) is calculated using the Bates– Guggenheim convention [10]. These two steps are described in the following paragraphs.
4.4 Extrapolation of acidity function to zero chloride molality
The value of lg(aHg Cl)° corresponding to zero chloride molality is determined by linear extrapolation of measurements using Harned cells with at least three added molalities of sodium or potassium chloride (I < 0.1 mol kg–1, see Sections 4.5 and 12.6)
−lg(aHg Cl) = −lg(aHg Cl)° + SmCl
(5)
where S is an empirical, temperature-dependent constant. The extrapolation is linear, which is expected from Brønsted’s observations [11] that specific ion interactions between oppositely charged ions are dominant in mixed strong electrolyte systems at constant molality or ionic strength. However, these acidity function measurements are made on mixtures of weak and strong electrolytes at constant buffer molality, but not constant total molality. It can be shown [12] that provided the change in ionic strength on addition of chloride is less than 20 %, the extrapolation will be linear without detectable curvature. If the latter, less-convenient method of preparation of constant total molality solutions is used, Bates [5] has reported that, for equimolal phosphate buffer, the two methods extrapolate to the same intercept. In an alternative procedure, often useful for partially aqueous mixed solvents where the above extrapolation appears to be curved, multiple application of the Bates–Guggenheim convention to each solution composition gives identical results within the estimated uncertainty of the two intercepts.
4.5 Bates–Guggenheim convention
The activity coefficient of chloride (like the activity coefficient of the hydrogen ion) is an immeasurable quantity. However, in solutions of low ionic strength (I < 0.1 mol kg–1), it is possible to calculate the activity coefficient of chloride ion using the Debye– Hückel theory. This is done by adopting the Bates–Guggenheim convention, which assumes the trace activity coefficient of the chloride ion g °Cl is given by the expression [10]
lg g °Cl = −AI /(1 + Ba I ) 1/2
1/2
(6)
where A is the Debye–Hückel temperature-dependent constant (limiting slope), a is the mean distance of closest approach of the ions (ion size parameter), Ba is set equal to 1.5 (mol kg–1)–1/2 at all temperatures in the range 5–50 °C, and I is the ionic strength of the buffer (which, for its evaluation requires knowledge of appropriate acid dissociation constants). Values of A as a function of temperature can be found in Table A.9 and of B, which is effectively unaffected by revision of dielectric constant data, in Bates [5]. When the numerical value of Ba = 1.5 (i.e., without units) is introduced into eq. 6 it should be written as
1/2
1/2
lg g °Cl = −AI /[1 + 1.5 (I/m°) ]
(6′)
The various stages in the assignment of primary standard pH values are combined in eq. 7, which is derived from eqs. 2′, 5, 6′,
pH(PS) = lim mCl → 0 {(EI – E°)/[(RT/F)ln 10] + lg(mCl/m°)} − AI1/2/[1 + 1.5 (I/m°)1/2], (7)
and the steps are summarized schematically in Fig. 1.
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5. Sources of uncertainty in the use of the harned cell 5.1 Potential primary method and uncertainty evaluation
The presentation of the procedure in Section 4 highlights the fact that assumptions based on electrolyte theories [7] are used at three points in the method:
i. The Debye–Hückel theory is the basis of the extrapolation procedure to calculate the value for the standard potential of the silver–silver chloride electrode, even though it is a published value of g±HCl at, e.g., m = 0.01 mol kg–1, that is recommended (Section 4.2) to facilitate E° determination. ii. Specific ion interaction theory is the basis for using a linear extrapolation to zero chloride (but the change in ionic strength produced by addition of chloride should be restricted to no more than 20 %). iii. The Debye–Hückel theory is the basis for the Bates– Guggenheim convention used for the calculation of the trace activity coefficient, g °Cl.
In the first two cases, the inadequacies of electrolyte theories are sources of uncertainty that limit the extent to which the measured pH is a true representation of lg aH. In the third case, the use of eq. 6 or 7 is a convention, since the value for Ba is not directly determinable experimentally. Previous recommendations have not included the uncertainty in Ba explicitly within the calculation of the uncertainty of the measurement. Since eq. 2 is derived from the Nernst equation applied to the thermodynamically well-behaved platinum–hydrogen and silver–silver chloride electrodes, it is recommended that, when used to measure –lg(aHg Cl) in aqueous solutions, the Harned cell potentially meets the agreed definition of a primary method for the measurement. The word “potentially” has been included to emphasize that the method can only achieve primary status if it is operated with the highest metrological qualities (see Sections 6.1–6.2). Additionally, if the Bates–Guggenheim convention is used for the calculation of lg g °Cl , the Harned cell potentially meets the agreed definition of a primary method for the measurement of pH, subject to this convention if a realistic estimate of its uncertainty is included. The uncertainty budget for the primary method of measurement by the Harned cell (Cell I) is given in the Annex, Section 13. Note 2: The experimental uncertainty for a typical primary pH(PS) measurement is of the order of 0.004 (see Table 4).
5.2 Evaluation of uncertainty of the Bates–Guggenheim convention
In order for a measurement of pH made with a Harned cell to be traceable to the SI system, an estimate of the uncertainty of each step must be included in the result. Hence, it is recommended that an estimate of the uncertainty of 0.01 (95% confidence interval) in pH associated with the Bates–Guggenheim convention is used. The extent to which the Bates–Guggenheim convention represents the “true” (but immeasurable) activity coefficient of the chloride ion can be calculated by varying the coefficient Ba between 1.0 and 2.0 (mol kg–1)1/2. This corresponds to varying the ion-size parameter between 0.3 and 0.6 nm, yielding a range of ±0.012 (at I = 0.1 mol kg–1) and ±0.007 (at I = 0.05 mol kg–1) for g °Cl calculated using equation [7]. Hence, an uncertainty of 0.01 should cover the full extent of variation. This must be included in the uncertainty of pH values that are to be regarded as traceable
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Measurement of pH Definition, Standards, and Procedures to the SI. pH values stated without this contribution to their uncertainty cannot be considered to be traceable to the SI.
5.3 Hydrogen ion concentration
It is rarely required to calculate hydrogen ion concentration from measured pH. Should such a calculation be required, the only consistent, logical way of doing it is to assume g H = g Cl and set the latter to the appropriate Bates–Guggenheim conventional value. The uncertainties are then those derived from the Bates– Guggenheim convention.
5.4 Possible future approaches
Any model of electrolyte solutions that takes into account both electrostatic and specific interactions for individual solutions would be an improvement over use of the Bates–Guggenheim convention. It is hardly reasonable that a fixed value of the ionsize parameter should be appropriate for a diversity of selected buffer solutions. It is hoped that the Pitzer model of electrolytes [13], which uses a virial equation approach, will provide such an improvement, but data in the literature are insufficiently extensive to make these calculations at the present time. From limited work at 25 °C done on phosphate and carbonate buffers, it seems that changes to Bates–Guggenheim recommended values will be small [14]. It is possible that some anomalies attributed to liquid junction potentials (LJPs) may be resolved.
6. Primary buffer solutions and their required properties 6.1 Requisites for highest metrological quality
In the previous sections, it has been shown that the Harned cell provides a primary method for the determination of pH. In order for a particular buffer solution to be considered a primary buffer solution, it must be of the “highest metrological” quality [15] in accordance with the definition of a primary standard. It is recommended that it have the following attributes [5: p. 95;16,17]: • High buffer value in the range 0.016–0.07 (mol OH–)/pH • Small dilution value at half concentration (change in pH with change in buffer concentration) in the range 0.01–0.20 • Small dependence of pH on temperature less than ±0.01 K–1 • Low residual LJP 5 years) is a requirement not met by borax [16]. There are also doubts about the extent of polyborate formation in 0.05 mol kg–1 borax solutions, and hence this solution is not accorded primary status.
6.2 Primary standard buffers
Since there can be significant variations in the purity of samples of a buffer of the same nominal chemical composition, it is essential that the primary buffer material used has been certified with values that have been measured with Cell I. The Harned cell has been used by many NMIs for accurate measurements of pH of
9168_Book.indb 679
679
buffer solutions. Comparisons of such measurements have been carried out under EUROMET collaboration [18], which have demonstrated the high comparability of measurements (0.005 in pH) in different laboratories of samples from the same batch of buffer material. Typical values of the pH(PS) of the seven solutions from the six accepted primary standard reference buffers, which meet the conditions stated in Section 6.1, are listed in Table 2. These listed pH(PS) values have been derived from certificates issued by NBS/NIST over the past 35 years. Batch-to-batch variations in purity can result in changes in the pH value of samples of at most 0.003. The typical values in Table 2 should not be used in place of the certified value (from a Harned cell measurement) for a specific batch of buffer material. The required attributes listed in Section 6.1 effectively limit the range of primary buffers available to between pH 3 and 10 (at 25 °C). Calcium hydroxide and potassium tetroxalate have been excluded because the contribution of hydroxide or hydrogen ions to the ionic strength is significant. Also excluded are the nitrogen bases of the type BH+ [such as tris(hydroxymethyl)aminomethane and piperazine phosphate] and the zwitterionic buffers (e.g., HEPES and MOPS [19]). These do not comply because either the Bates–Guggenheim convention is not applicable, or the LJPs are high. This means the choice of primary standards is restricted to buffers derived from oxy-carbon, -phosphorus, -boron, and mono, di-, and tri-protic carboxylic acids. In the future, other buffer systems may fulfill the requirements listed in Section 6.1.
7. Consistency of primary buffer solutions 7.1 Consistency and the liquid junction potential
Primary methods of measurement are made with cells without transference as described in Sections 1–6. Less-complex, secondary methods use cells with transference, which contain liquid junctions. A single LJP is immeasurable, but differences in LJP can be estimated. LJPs vary with the composition of the solutions forming the junction and the geometry of the junction. Equation 7 for Cell I applied successively to two primary standard buffers, PS1, PS2, gives ∆pHI = pHI(PS2) − pHI(PS1) = lim mCl→0{EI(PS2)/k − EI(PS1)/k} 1/2 – A{I(2)1/2/[1 + 1.5 (I(2)/m°) ] − I(1)1/2/[1 + 1.5 (I(1)/m°)1/2]} (8) where k = (RT/F)ln 10 and the last term is the ratio of trace chloride activity coefficients lg[g °Cl(2)/g °Cl(1)], conventionally evaluated via B-G eq. 6′. Note 4: Since the convention may unevenly affect the g °Cl(2) and g °Cl(1) estimations, ∆pHI differs from the true value by the unknown contribution: lg[g °Cl(2)/g °Cl(1)] – A{I(1) 1/2/[1 + 1.5(I(1)/m°)1/2] – I(2) 1/2/[1 + 1.5(I(2)/m°) 1/2]}. A second method of comparison is by measurement of Cell II in which there is a salt bridge with two free-diffusion liquid junctions
Pt | H2 | PS2 ¦ KCl (≥3.5 mol dm–3) ¦ PS1 | H2 | Pt
Cell II
for which the spontaneous cell reaction is a dilution,
H+(PS1) → H+(PS2)
which gives the pH difference from Cell II as
∆pHII = pHII(PS2) − pHII(PS1) = EII/k – [(Ej2 – Ej1)/k]
(9)
where the subscript II is used to indicate that the pH difference between the same two buffer solutions is now obtained from Cell II. ∆pHII differs from ∆pHI (and both differ from the true value ∆pHI) since it depends on unknown quantity, the residual LJP,
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Table 1 Summary of Useful Properties of Some Primary and Secondary Standard Buffer Substances and Solutions [5]
Salt or Solid Substance Potassium tetroxalate dihydrate Potassium tetroxalate dihydrate Potassium hydrogen tartrate (sat. at 25 °C) Potassium dihydrogen citrate Potassium hydrogen phthalate Disodium hydrogen orthophosphate + potassium dihydrogen
orthophosphate + potassium dihydrogen
orthophosphate Disodium tetraborate decahydrate Disodium tetraborate decahydrate Sodium hydrogen carbonate + sodium carbonate Calcium hydroxide (sat. at 25°C)
Molality/ mol kg-1
Molar Mass/g mol-1
Density/ g dm-3
Amount Conc. at 20 çC/ mol dm-3
Mass/g to Make 1 dm3
Dilution Value DpH1/2
Buffer Value (β)/ mol OH- dm-3
pH Temperature Coefficient/ K-1
KH3C4O8 · 2H2O
0.1
254.191
1.0091
0.09875
25.101
KH3C4O8 · 2H2O
0.05
254.191
1.0032
0.04965
12.620
0.186
0.070
0.001
KHC4H4O6
0.0341
188.18
1.0036
0.034
6.4
0.049
0.027
-0.0014
KH2C6H5O7
0.05
230.22
1.0029
0.04958
11.41
0.024
0.034
-0.022
KHC8H4O4
0.05
204.44
1.0017
0.04958
10.12
0.052
0.016
0.00012
Na2HPO4
0.025
141.958
1.0038
0.02492
0.080
0.029
-0.0028
KH2PO4
0.025
136.085
Na2HPO4
0.03043
141.959
0.07
0.016
-0.0028
KH2PO4
0.00869
136.085
Na2B4O7 · 10H2O
0.05
381.367
Na2B4O7 · 10H2O
0.01
NaHCO3 Na2CO3 Ca(OH)2
3.5379 3.3912
1.0020
0.08665
4.302
0.03032
1.179
1.0075
0.04985
19.012
381.367
1.0001
0.00998
3.806
0.01
0.020
-0.0082
0.025
84.01
1.0013
0.02492
2.092
0.079
0.029
-0.0096
0.025 0.0203
105.99 74.09
0.9991
0.02025
2.640 1.5
-0.28
0.09
-0.033
Handbook of Biochemistry and Molecular Biology
orthophosphate Disodium hydrogen
Molecular Formula
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Table 2 Typical Values of pH(PS) for Primary Standards at 0–50 çC (see Section 6.2) Primary Standards (PS)
0
5
10
15
20
Sat. potassium hydrogen tartrate (at 25 °C)
Temp./çC 25
30
35
37
40
50
3.557
3.552
3.549
3.548
3.547
3.549
0.05 mol kg-1 potassium dihydrogen citrate
3.863
3.840
3.820
3.802
3.788
3.776
3.766
3.759
3.756
3.754
3.749
0.05 mol kg-1 potassium hydrogen phthalate
4.000
3.998
3.997
3.998
4.000
4.005
4.011
4.018
4.022
4.027
4.050
0.025 mol kg-1 disodium hydrogen phosphate + 0.025 mol kg–1 potassium dihydrogen phosphate
6.984
6.951
6.923
6.900
6.881
6.865
6.853
6.844
6.841
6.838
6.833
0.03043 mol kg–1 disodium hydrogen phosphate + 0.008695 mol kg–1 potassium dihydrogen phosphate
7.534
7.500
7.472
7.448
7.429
7.413
7.400
7.389
7.386
7.380
7.367
0.01 mol kg–1 disodium tetraborate
9.464
9.395
9.332
9.276
9.225
9.180
9.139
9.102
9.088
9.068
9.011
0.025 mol kg–1 sodium hydrogen carbonate + 0.025 mol kg–1 sodium carbonate
10.317
10.245
10.179
10.118
10.062
10.012
9.966
9.926
9.910
9.889
9.828
RLJP = (Ej2 − Ej1), whose exact value could be determined if the true ∆pH were known. Note 5: The subject of liquid junction effects in ion-selective electrode potentiometry has been comprehensively reviewed [20]. Harper [21] and Bagg [22] have made computer calculations of LJPs for simple three-ion junctions (such as HCl + KCl), the only ones for which mobility and activity coefficient data are available. Breer, Ratkje, and Olsen [23] have thoroughly examined the possible errors arising from the commonly made approximations in calculating LJPs for three-ion junctions. They concluded that the assumption of linear concentration profiles has less-severe consequences (~0.1–1.0 mV) than the other two assumptions of the Henderson treatment, namely constant mobilities and neglect of activity coefficients, which can lead to errors in the order of 10 mV. Breer et al. concluded that their calculations supported an earlier statement [24] that in ion-selective electrode potentiometry, the theoretical Nernst slope, even for dilute sample solutions, could never be attained because of liquid junction effects. Note 6: According to IUPAC recommendations on nomenclature and symbols [3], a single vertical bar ( | ) is used to represent a phase boundary, a dashed vertical bar ( ¦ ) represents a liquid–liquid junction between two electrolyte solutions (across which a potential difference will occur), and a double dashed vertical bar ( ¦¦ ) represents a similar liquid junction, in which the LJP is assumed to be effectively zero (~1 % of cell potential difference). Hence, terms such as that in square brackets on the right-hand side of eq. 9 are usually ignored, and the liquid junction is represented by ¦¦. However, in the Annex, the symbol ¦ is used because the error associated with the liquid junction is included in the analysis. For ease of comparison, numbers of related equations in the main text and in the Annex are indicated.
9168_Book.indb 681
Note 7: The polarity of Cell II will be negative on the left, i.e., − | +, when pH(PS2) > pH(PS1). The LJP Ej of a single liquid junction is defined as the difference in (Galvani) potential contributions to the total cell potential difference arising at the interface from the buffer solution less that from the KCl solution. For instance, in Cell II, Ej1 = E(S1) – E(KCl) and E j2 = E(S2) – E(KCl). It is negative when the buffer solution of interest is acidic and positive when it is alkaline, provided that Ej is principally caused by the hydrogen, or hydroxide, ion content of the solution of interest (and only to a smaller degree by its alkali ions or anions). The residual liquid junction potential (RLJP), the difference Ej(right) – Ej(left), depends on the relative magnitudes of the individual Ej values and has the opposite polarity to the potential difference E of the cell. Hence, in Cell II the RLJP, Ej1(PS1) – Ej2(PS2), has a polarity + | − when pH(S2) > pH(S1). Notwithstanding the foregoing, comparison of pHII values from the Cell II with two liquid junctions (eq. 9) with the assigned pHI(PS) values for the same two primary buffers measured with Cell I (eq. 8) makes an estimation of RLJPs possible [5]:
[pHI(PS2) − pHII(PS2)] − [pHI(PS1) − pHII(PS1)] = (Ej2 − Ej1)/k = RLJP
(10)
With the value of RLJP set equal to zero for equimolal phosphate buffer (taken as PS1) then [pHI(PS2) − pHII(PS2)] is plotted against pH(PS). Results for free-diffusion liquid junctions formed in a capillary tube with cylindrical symmetry at 25 °C are shown in Fig. 2 [25, and refs. cited therein]. Note 8: For 0.05 mol kg–1 tetroxalate, the published values [26] for Cell II with free-diffusion junctions are wrong [27,28].
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8. Secondary standards and secondary methods of measurement
RLJP in Terms of pH
0.02 0.015 0.01
Tartrate
0.005
Tetroxalate
0 –0.005 –0.01
Acetate
0.025 phosphate 1:3.5 phosphate
Phthalate
0
2
4
6
8
Carbonate
Calcium hydroxide
8.1 Secondary standards derived from Harned cell measurements
0.01 borax
10
12
14
pH (S)
FigUre 2 Some values of residual LJPs in terms of pH with reference to the value for 0.025 mol kg–1 Na2HPO4 + 0.025 mol kg–1 KH2PO4 (0.025 phosphate buffer) taken as zero [25]. Values such as those shown in Fig. 2 give an indication of the extent of possible systematic uncertainties for primary standard buffers arising from three sources:
i. Experimental uncertainties, including any variations in the chemical purity of primary buffer materials (or variations in the preparation of the solutions) if measurements of Cells I and II were not made in the same laboratory at the same occasion. ii. Variation in RLJPs between primary buffers. iii. Inconsistencies resulting from the application of the Bates–Guggenheim convention to chemically different buffer solutions of ionic strengths less than 0.1 mol kg–1. It may be concluded from examination of the results in Fig. 2, that a consistency no better than 0.01 can be ascribed to the primary pH standard solutions of Table 2 in the pH range 3–10. This value will be greater for less reproducibly formed liquid junctions than the free-diffusion type with cylindrical symmetry. Note 9: Considering the conventional nature of eq. 10, and that the irreproducibility of formation of geometrydependent devices exceeds possible bias between carefully formed junctions of known geometry, the RLJP contribution, which is included in the difference between measured potential differences of cells with transference, is treated as a statistical, and not a systematic error. Note 10: Values of RLJP depend on the Bates–Guggenheim convention through the last term in eq. 8 and would be different if another convention were chosen. This interdependence of the single ion activity coefficient and the LJP may be emphasized by noting that it would be possible arbitrarily to reduce RLJP values to zero for each buffer by adjusting the ion-size parameter in eq. 6.
7.2 Computational approach to consistency
The consistency between conventionally assigned pH values can also be assessed by a computational approach. The pH values of standard buffer solutions have been calculated from literature values of acid dissociation constants by an iterative process. The arbitrary extension of the Bates–Guggenheim convention for chloride ion, to all ions, leads to the calculation of ionic activity coefficients of all ionic species, ionic strength, buffer capacity, and calculated pH values. The consistency of these values with primary pH values obtained using Cell I was 0.01 or lower between 10 and 40 °C [29,30].
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Substances that do not fulfill all the criteria for primary standards but to which pH values can be assigned using Cell I are considered to be secondary standards. Reasons for their exclusion as primary standards include, inter alia:
i. Difficulties in achieving consistent, suitable chemical quality (e.g., acetic acid is a liquid). ii. High LJP, or inappropriateness of the Bates–Guggenheim convention (e.g., other charge-type buffers).
Therefore, they do not comply with the stringent criterion for a primary measurement of being of the highest metrological quality. Nevertheless, their pH(SS) values can be determined. Their consistency with the primary standards should be checked with the method described in Section 7. The primary and secondary standard materials should be accompanied by certificates from NMIs in order for them to be described as certified reference materials (CRMs). Some illustrative pH(SS) values for secondary standard materials [5,17,25,31,32] are given in Table 3.
8.2 Secondary standards derived from primary standards
In most applications, the use of a high-accuracy primary standard for pH measurements is not justified, if a traceable secondary standard of sufficient accuracy is available. Several designs of cells are available for comparing the pH values of two buffer solutions. However, there is no primary method for measuring the difference in pH between two buffer solutions for reasons given in Section 8.6. Such measurements could involve either using a cell successively with two buffers, or a single measurement with a cell containing two buffer solutions separated by one or two liquid junctions.
8.3 Secondary standards derived from primary standards of the same nominal composition using cells without salt bridge
The most direct way of comparing pH(PS) and pH(SS) is by means of the single-junction Cell III [33].
Pt | H2 | buffer S2 ¦ ¦ buffer S1 | H2 | Pt
Cell III
The cell reaction for the spontaneous dilution reaction is the same as for Cell II, and the pH difference is given, see Note 6, by
pH(S2) − pH(S1) = EIII/k
(11) cf.(A-7)
The buffer solutions containing identical Pt | H2 electrodes with an identical hydrogen pressure are in direct contact via a vertical sintered glass disk of a suitable porosity (40 µm). The LJP formed between the two standards of nominally the same composition will be particularly small and is estimated to be in the µV range. It will, therefore, be less than 10 % of the potential difference measured if the pH(S) values of the standard solutions are in the range 3 ≤ pH(S) ≤ 11 and the difference in their pH(S) values is not larger than 0.02. Under these conditions, the LJP is not dominated by the hydrogen and hydroxyl ions but by the other ions (anions, alkali metal ions). The proper functioning of the cell can
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Table 3 Values of pH(SS) of Some Secondary Standards from Harned Cell I Measurements Temp./°C Secondary Standards 0.05 mol kg potassium tetroxalatea [5,17] 0.05 mol kg-1 sodium hydrogen diglycolateb [31] 0.1 mol dm-3 acetic acid + 0.1 mol dm-3 sodium acetate [25] 0.1 mol dm-3 acetic acid + 0.1 mol dm-3 sodium acetate [25] 0.02 mol kg-1 piperazine phosphatec [32] 0.05 mol kg-1 tris hydrochloride + 0.01667 mol kg-1 trisc [5] 0.05 mol kg-1 disodium tetraborate Saturated (at 25°C) calcium hydroxide [5]
0
5
10
15
20
25
30
37
40
50
1.67
1.67
1.67
1.68
1.68
1.68
1.69
1.69
1.71
3.47
3.47
3.48
3.48
3.49
3.50
3.52
3.53
3.56
4.68
4.67
4.67
4.66
4.66
4.65
4.65
4.66
4.66
4.68
4.74
4.73
4.73
4.72
4.72
4.72
4.72
4.73
4.73
4.75
6.58
6.51
6.45
6.39
6.34
6.29
6.24
6.16
6.14
6.06
8.47
8.30
8.14
7.99
7.84
7.70
7.56
7.38
7.31
7.07
9.51 13.42
9.43 13.21
9.36 13.00
9.30 12.81
9.25 12.63
9.19 12.45
9.15 12.29
9.09 12.07
9.07 11.98
9.01 11.71
-1
a
potassium trihydrogen dioxalate (KH3C4O8) sodium hydrogen 2,2¢-oxydiacetate c 2-amino-2-(hydroxymethyl)-1,3 propanediol or tris(hydroxymethyl)aminomethane b
be checked by measuring the potential difference when both sides of the cell contain the same solution.
8.4 Secondary standards derived from primary standards using cells with salt bridge
The cell that includes a hydrogen electrode [corrected to 1 atm (101.325 kPa) partial pressure of hydrogen] and a reference electrode, the filling solution of which is a saturated or high concentration of the almost equitransferent electrolyte, potassium chloride, hence minimizing the LJP, is, see Note 6: Ag|AgC|KCl (≥3.5 mol dm−3) ¦¦ buffer S | H2 | Pt
Note 12: Cell IV is written in the direction: reference| indicator
i. for conformity of treatment of all hydrogen ion-responsive electrodes and ion-selective electrodes with various choices of reference electrode, and partly, ii for the practical reason that pH meters usually have one low impedance socket for the reference electrode, assumed negative, and a high-impedance terminal with a different plug, usually for a glass electrode.
With this convention, whatever the form of hydrogen ionresponsive electrode used (e.g., glass or quinhydrone), or whatever the reference electrode, the potential of the hydrogen-ion responsive electrode always decreases (becomes more negative) with increasing pH (see Fig. 3). This convention was used in the 1985 document [2] and is also consistent with the treatment of ion-selective electrodes [35]. In effect, it focuses attention on the indicator electrode, for which the potential is then given by the Nernst equation for the singleelectrode potential, written as a reduction process, in accord with the Stockholm convention [36]:
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For Ox + ne−→ Red, E = E° − (k/n) lg(ared/aox)
H+ + e−→ 1/2H2 E = E° + klg aH+ = E° − kpH
The equation for Cell IV is, therefore: pH(S) = –[EIV(S) – EIV°′ ]/k
Cell IV
Note 11: Other electrolytes, e.g., rubidium or cesium chloride, are more equitransferent [34].
(where a is activity), or, for the hydrogen gas electrode at 1 atm partial pressure of hydrogen gas:
(12)
Eo –k = –(RT/F) ln 10
+
E(H+|H2, Pt)
Ej
Ej
0
pH
12
FigURE 3 Schematic plot of the variation of potential dif– + + ference ( ____ ) for the cell Ag|AgCl|KCl H (buffer)|H 2|Pt with pH and illustrating the choice of sign convention. The effect of LJP is indicated ( ____ ) with its variation of pH as given by the Henderson equation (see, e.g., ref. [5]). The approximate linearity (----) in the middle pH region should be noted. Both lines have been grossly exaggerated in their deviation from the Nernst line since otherwise they would be indistinguishable from each other and the Nernst line. For the calomel electrode Hg|Hg 2 Cl 2|KCl and the thallium amalgam|thallium(I) chloride electrode Hg|Τl(Hg)|TlCl|KCl, or any other constant potential reference electrode, the diagram is the same.
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684 in which EIV°′ is the standard potential, which includes the term lg aCl/m°, and Ej is the LJP. Note 13: Mercury–mercury(I) chloride (calomel) and thallium amalgam–thallium (I) chloride reference electrodes are alternative choices to the silver–silver chloride electrode in Cell IV. The consecutive use of two such cells containing buffers S1 and S2 gives the pH difference of the solutions pH(S2) − pH(S1) = −[EIV(S2) − EIV(S1)]/k
transference, or the irreversible inter-diffusion of ions, and hence an LJP contribution to the measured potential difference. The value of this potential difference depends on the ionic constituents, their concentrations and the geometry of the liquid junction between the solutions. Hence, the measurement equations contain terms that, although small, are not quantifiable, and the methods are secondary not primary.
9. Consistency of secondary standard buffer solutions established with respect to primary standards
(13) cf. (A-8)
Note 14: Experimentally, a three-limb electrode vessel allowing simultaneous measurement of two Cell IIs may be used [25] with the advantage that the stability with time of the electrodes and of the liquid junctions can be checked. The measurement of cells of type II, which has a salt bridge with two liquid junctions, has been discussed in Section 7. Cells II and IV may also be used to measure the value of secondary buffer standards that are not compatible with the silver– silver chloride electrode used in Cell I. Since the LJPs in Cells II and IV are minimized by the use of an equitransferent salt, these cells are suitable for use with secondary buffers that have a different concentration and/or an ionic strength greater than the limit (I ≤ 0.1 mol kg−1) imposed by the Bates–Guggenheim convention. They may, however, also be used for comparing solutions of the same nominal composition.
8.5 Secondary standards from glass electrode cells
Measurements cannot be made with a hydrogen electrode in Cell IV, for example, if the buffer is reduced by hydrogen gas at the platinum (or palladium-coated platinum) electrode. Cell V involving a glass electrode and silver–silver chloride reference electrode may be used instead in consecutive measurements, with two buffers S1, S2 (see Section 11 for details).
8.6 Secondary methods
The equations given for Cells II to V show that these cannot be considered primary (ratio) methods for measuring pH difference [1], (see also Section 12.1) because the cell reactions involve
9.1 Summary of procedures for establishing secondary standards
The following procedures may be distinguished for establishing secondary standards (SS) with respect to primary standards:
i. For SS of the same nominal composition as PS, use Cells III or II. ii. For SS of different composition, use Cells IV or II. iii. For SS not compatible with platinum hydrogen electrode, use Cell V (see Section 11.1). Although any of Cells II to V could be used for certification of secondary standards with stated uncertainty, employing different procedures would lead to inconsistencies. It would be difficult to define specific terminology to distinguish each of these procedures or to define any rigorous hierarchy for them. Hence, the methods should include estimates of the typical uncertainty for each. The choice between methods should be made according to the uncertainty required for the application (see Section 10 and Table 4).
9.2 Secondary standard evaluation from primary standards of the same composition
It is strongly recommended that the preferred method for assigning secondary standards should be a procedure in which measurements are made with respect to the primary buffer of nominally the same chemical composition. All secondary standards should be accompanied by a certificate relating to that
Table 4 Summary of Recommended Target Uncertainties U(pH) (For coverage factor 2) PRIMARY STANDARDS Uncertainty of PS measured (by an NMI) with Harned Cell I 0.004 Repeatability of PS measured (by an NMI) with Harned Cell I 0.0015 Reproducibility of measurements in comparisons with Harned Cell I 0.003 Typical variations between batches of PS buffers 0.003 SECONDARY STANDARDS Value of SS compared with same PS material with Cell III 0.004 Value of SS measured in Harned Cell I Value of SS labeled against different PS with Cell II or IV Value of SS (not compatible with Pt | H2) measured with Cell V Multipoint (5-point) calibration Calibration (2-point) by bracketing Calibration (1-point), DpH = 3 and assumed slope
0.01 0.015 0.02
Comments
EUROMET comparisons
increase in uncertainty is negligible relative to PS used e.g., biological buffers
example based on phthalate
ELECTRODE CALIBRATION 0.01–0.03 0.02–0.03 0.3
Note: None of the above includes the uncertainty associated with the Bates–Guggenheim convention so the results cannot be considered to be traceable to SI (see Section 5.2).
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685
particular batch of reference material as significant batch-tobatch variations are likely to occur. Some secondary standards are disseminated in solution form. The uncertainty of the pH values of such solutions may be larger than those for material disseminated in solid form.
The potential difference of Cell V is made up of contributions arising from the potentials of the glass and reference electrodes and the liquid junction (see Section 7.1). Various random and systematic effects must be noted when using these cells for pH measurements:
9.3 Secondary standard evaluation when there is no primary standard of the same composition
It may sometimes be necessary to set up a secondary standard when there is no primary standard of the same chemical composition available. It will, therefore, be necessary to use either Cells II, IV, or V, and a primary or secondary standard buffer of different chemical composition. Buffers measured in this way will have a different status from those measured with respect to primary standards because they are not directly traceable to a primary standard of the same chemical composition. This different status should be reflected in the, usually larger, uncertainty quoted for such a buffer. Since this situation will only occur for buffers when a primary standard is not available, no special nomenclature is recommended to distinguish the different routes to secondary standards. Secondary buffers of a composition different from those of primary standards can also be derived from measurements on Cell I, provided the buffer is compatible with Cell I. However, the uncertainty of such standards should reflect the limitations of the secondary standard (see Table 4).
10. Target uncertainties for the measurement of secondary buffer solutions
10.1 Uncertainties of secondary standards derived from primary standards
Cells II to IV (and occasionally Cell V) are used to measure secondary standards with respect to primary standards. In each case, the limitations associated with the measurement method will result in a greater uncertainty for the secondary standard than the primary standard from which it was derived. Target uncertainties are listed in Table 4. However, these uncertainties do not take into account the uncertainty contribution arising from the adoption of the Bates–Guggenheim convention to achieve traceability to SI units.
10.2 Uncertainty evaluation [37]
Summaries of typical uncertainty calculations for Cells I–V are given in the Annex (Section 13).
11. Calibration of pH meter-electrode assemblies and target uncertainties for unknowns 11.1 Glass electrode cells
Practical pH measurements are carried out by means of Cell V reference electrode | KCl (c ≥ 3.5 mol dm–3) ¦¦ solution[pH(S) or pH(X)] | glass electrode Cell V and pH(X) is obtained, see Note 6, from eq. 14
pH(X) = pH(S) – [EV(X) – EV(S)]
(14)
This is a one-point calibration (see Section 11.3). These cells often use glass electrodes in the form of single probes or combination electrodes (glass and reference electrodes fashioned into a single probe, a so-called “combination electrode”).
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i. Glass electrodes may exhibit a slope of the E vs. pH function smaller than the theoretical value [k = (RT/F)ln 10], often called a sub-Nernstian response or practical slope k′, which is experimentally determinable. A theoretical explanation for the sub-Nernstian response of pH glass electrodes in terms of the dissociation of functional groups at the glass surface has been given [38]. ii. The response of the glass electrode may vary with time, history of use, and memory effects. It is recommended that the response time and the drift of the electrodes be taken into account [39]. iii. The potential of the glass electrode is strongly temperature-dependent, as to a lesser extent are the other two terms. Calibrations and measurements should, therefore, be carried out under temperature-controlled conditions. iv. The LJP varies with the composition of the solutions forming the junction, e.g., with pH (see Fig. 2). Hence, it will change if one solution [pH(S) or pH(X)] in Cell V is replaced by another. It is also affected by the geometry of the liquid junction device. Hence, it may be different if a free-diffusion type junction, such as that used to measure the RLJP (see Section 7.1), is replaced by another type, such as a sleeve, ceramic diaphragm, fiber, or platinum junction [39,40]. v. Liquid junction devices, particularly some commercial designs, may suffer from memory and clogging effects. vi. The LJP may be subject to hydrodynamic effects, e.g., stirring.
Since these effects introduce errors of unknown magnitude, the measurement of an unknown sam ple requires a suitable calibration procedure. Three procedures are in common use based on calibrations at one point (one-point calibration), two points (two-point calibration or bracketing), and a series of points (multipoint calibration).
11.2 Target uncertainties for unknowns
Uncertainties in pH(X) are obtained, as shown below, by several procedures involving different numbers of experiments. Numerical values of these uncertainties obtained from the different calibration procedures are, therefore, not directly comparable. It is, therefore, not possible at the present time to make a universal recommendation of the best procedure to adopt for all applications. Hence, the target uncertainty for the unknown is given, which the operator of a pH meter electrode assembly may reasonably seek to achieve. Values are given for each of the three techniques (see Table 4), but the uncertainties attainable experimentally are critically dependent on the factors listed in Section 11.1 above, on the quality of the electrodes, and on the experimental technique for changing solutions. In order to obtain the overall uncertainty of the measurement, uncertainties of the respective pH(PS) or pH(SS) values must be taken into account (see Table 4). Target uncertainties given below, and in Table 4, refer to calibrations performed by the use of standard buffer solutions with an uncertainty U[pH(PS)] or U[pH(SS)] d 0.01. The overall uncertainty becomes higher if standards with higher uncertainties are used.
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A single-point calibration is insufficient to determine both slope and one-point parameters. The theoretical value for the slope can be assumed, but the practical slope may be up to 5% lower. Alternatively, a value for the practical slope can be assumed from the manufacturer’s prior calibration. The one-point calibration, therefore, yields only an estimate of pH(X). Since both parameters may change with age of the electrodes, this is not a reliable procedure. Based on a measurement for which ∆pH = |pH(X) − pH(S)| = 5, the expanded uncertainty would be U = 0.5 in pH(X) for k′ = 0.95k, but assumed theoretical, or U = 0.3 in pH(X) for ∆pH = |pH(X) – pH(S)| = 3 (see Table 4). This approach could be satisfactory for certain applications. The uncertainty will decrease with decreasing difference pH(X) – pH(S) and be smaller if k′ is known from prior calibration.
11.4 Two-point calibration {target uncertainty, U[pH(X)] = 0.02–0.03 at 25 çC}
In the majority of practical applications, glass electrodes cells (Cell V) are calibrated by two-point calibration, or bracketing, procedure using two standard buffer solutions, with pH values pH(S1) and pH(S2), bracketing the unknown pH(X). Bracketing is often taken to mean that the pH(S1) and pH(S2) buffers selected should be those that are immediately above and below pH(X). This may not be appropriate in all situations and choice of a wider range may be better. If the respective potential differences measured are EV(S1), EV(S2), and EV(X), the pH value of the unknown, pH(X), is obtained from eq. 15
pH(X) = pH(S1) – [EV(X) – EV(S1)]/k′
(15) cf. (A-10)
where the practical slope factor (k′) is given by
k′ = [EV(S1) – EV(S2)]/[pH(S2) − pH(S1)]
(16)
An example is given in the Annex, Section 13.
11.5 Multipoint calibration {target uncertainty: U[pH(X)] = 0.01–0.03 at 25 çC}
Multipoint calibration is carried out using up to five standard buffers [39,40]. The use of more than five points does not yield any significant improvement in the statistical information obtainable. The calibration function of Cell V is given by eq. 17
EV(S) = EV° – k′pH(S)
(17) cf. (A-11)
where EV(S) is the measured potential difference when the solution of pH(S) in Cell V is a primary or secondary standard buffer. The intercept, or “standard potential”, EV° and k′, the practical slope are determined by linear regression of eq. 17 [39–41]. pH(X) of an unknown solution is then obtained from the potential difference, EV(X), by
pH(X) = [EV° − EV(X)]/k′
(18) cf. (A-12)
Additional quantities obtainable from the regression procedure applied to eq. 17 are the uncertainties u(k′) and u(EV°) [40]. Multipoint calibration is recommended when minimum uncertainty and maximum consistency are required over a wide range of pH(X) values. This applies, however, only to that range of pH values in which the calibration function is truly linear. In nonlinear regions of the calibration function, the two-point method has clear advantages provided that pH(S1) and pH(S2) are selected to be as close to pH(X) as possible. Details of the uncertainty computations for the multipoint calibration have been given [40], and an example is given in
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the Annex. The uncertainties are recommended as a means of checking the performance characteristics of pH meter-electrode assemblies [40]. By careful selection of electrodes for multipoint calibration, uncertainties of the unknown pH(X) can be kept as low as U[pH(X)] = 0.01. In modern microprocessor pH meters, potential differences are often transformed automatically into pH values. Details of the calculations involved in such transformations, including the uncertainties, are available [41].
12. Glossary [2,15,44] 12.1 Primary method of measurement
A primary method of measurement is a method having the highest metrological qualities, whose operation can be completely described and understood, for which a complete uncertainty statement can be written down in terms of SI units. A primary direct method measures the value of an unknown without reference to a standard of the same quantity. A primary ratio method measures the value of a ratio of an unknown to a standard of the same quantity; its operation must be completely described by a measurement equation.
12.2 Primary standard
Standard that is designated or widely acknowledged as having the highest metrological qualities and whose value is accepted without reference to other standards of the same quantity.
12.3 Secondary standard
Standard whose value is assigned by comparison with a primary standard of the same quantity.
12.4 Traceability
Property of the result of a measurement or the value of a standard whereby it can be related to stated references, usually national or international standards, through an unbroken chain of comparisons all having stated uncertainties. The concept is often expressed by the adjective traceable. The unbroken chain of comparisons is called a traceability chain.
12.5 Primary pH standards
Aqueous solutions of selected reference buffer solutions to which pH(PS) values have been assigned over the temperature range 0–50 °C from measurements on cells without transference, called Harned cells, by use of the Bates–Guggenheim convention.
12.6 Bates–Guggenheim convention
A convention based on a form of the Debye–Hückel equation that approximates the logarithm of the single ion activity coefficient of chloride and uses a fixed value of 1.5 for the product Ba in the denominator at all temperatures in the range 0–50 °C (see eqs. 4, 5) and ionic strength of the buffer < 0.1 mol kg–1 .
12.7 Secondary pH standards
Values that may be assigned to secondary standard pH(SS) solutions at each temperature:
i. with reference to [pH(PS)] values of a primary standard of the same nominal composition by Cell III, ii with reference to [pH(PS)] values of a primary standard of different composition by Cells II, IV or V, or
iii by use of Cell I.
Note 15: This is an exception to the usual definition, see Section 12.3.
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Measurement of pH Definition, Standards, and Procedures 12.8 pH glass electrode
Hydrogen-ion responsive electrode usually consisting of a bulb, or other suitable form, of special glass attached to a stem of high-resistance glass complete with internal reference electrode and internal filling solution system. Other geometrical forms may be appropriate for special applications, e.g., capillary electrode for measurement of blood pH.
12.9 Glass electrode error
Deviation of a glass electrode from the hydrogen-ion response function. An example often encountered is the error due to sodium ions at alkaline pH values, which by convention is regarded as positive.
12.10 Hydrogen gas electrode
A thin foil of platinum electrolytically coated with a finely divided deposit of platinum or (in the case of a reducible substance) palladium metal, which catalyzes the electrode reaction: H+ + e → 1/2 H 2 in solutions saturated with hydrogen gas. It is
customary to correct measured values to standard 1 atm (101.325 kPa) partial pressure of hydrogen gas.
12.11 Reference electrode
External electrode system that comprises an inner element, usually silver–silver chloride, mercury–mercury(I) chloride (calomel), or thallium amalgam–thallium(I) chloride, a chamber containing the appropriate filling solution (see 12.14), and a device for forming a liquid junction (e.g., capillary) ceramic plug, frit, or ground glass sleeve.
material measure or a reference material, and the corresponding values realized by standards.
12.17 Uncertainty (of a measurement)
Parameter, associated with the result of a measurement, which characterizes the dispersion of the values that could reasonably be attributed to the measurand.
12.18 Standard uncertainty, u x
Uncertainty of the result of a measurement expressed as a standard deviation.
12.19 Combined standard uncertainty, uc(y)
Standard uncertainty of the result of a measurement when that result is obtained from the values of a number of other quantities, equal to the positive square root of a sum of terms, the terms being the variances, or covariances of these other quantities, weighted according to how the measurement result varies with changes in these quantities.
12.20 Expanded uncertainty, U
Quantity defining an interval about the result of a measurement that may be expected to encompass a large fraction of the distribution of values that could reasonably be attributed to the measurand. Note 16: The fraction may be viewed as the coverage probability or level of confidence of the interval. Note 17: To associate a specific level of confidence with the interval defined by the expanded uncertainty requires explicit or implicit assumptions regarding the probability distribution characterized by the measurement result and its combined standard uncertainty. The level of confidence that may be attributed to this interval can be known only to the extent to which such assumptions may be justified.
12.12 Liquid junction
Any junction between two electrolyte solutions of different composition. Across such a junction there arises a potential difference, called the liquid junction potential. In Cells II, IV, and V, the junction is between the pH standard or unknown solution and the filling solution, or the bridge solution (q.v.), of the reference electrode.
12.13 Residual liquid junction potential error
Error arising from breakdown in the assumption that the LJPs cancel in Cell II when solution X is substituted for solution S in Cell V.
12.14 Filling solution (of a reference electrode)
Solution containing the anion to which the reference electrode of Cells IV and V is reversible, e.g., chloride for silver–silver chloride electrode. In the absence of a bridge solution (q.v.), a high concentration of filling solution comprising almost equitransferent cations and anions is employed as a means of maintaining the LJP small and approximately constant on substitution of unknown solution for standard solution(s).
12.15 Bridge (or salt bridge) solution (of a double junction reference electrode)
Solution of high concentration of inert salt, preferably comprising cations and anions of equal mobility, optionally interposed between the reference electrode filling and both the unknown and standard solution, when the test solution and filling solution are chemically incompatible. This procedure introduces into the cell a second liquid junction formed, usually, in a similar way to the first.
12.16 Calibration
Set of operations that establish, under specified conditions, the relationship between values of quantities indicated by a measuring instrument, or measuring system, or values represented by a
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Note 18: Expanded uncertainty is sometimes termed overall uncertainty.
12.21 Coverage factor
Numerical factor used as a multiplier of the combined standard uncertainty in order to obtain an expanded uncertainty Note 19: A coverage factor is typically in the range 2 to 3. The value 2 is used throughout in the Annex.
13. Annex: measurement uncertainty Examples are given of uncertainty budgets for pH measurements at the primary, secondary, and working level. The calculations are done in accordance with published procedures [15,37]. When a measurement (y) results from the values of a number of other quantities, y = f (x1, x 2, … x i), the combined standard uncertainty of the measurement is obtained from the square root of the expression
uc2 ( y ) =
n
∂f
∑ ∂x ⋅ u (x ) i =1
i
2
i
where ∂∂xf is called the sensitivity coefficient (ci). This equation i holds for uncorrelated quantities. The ∂xi equation for correlated quantities is more complex. The uncertainty stated is the expanded uncertainty, U, obtained by multiplying the standard uncertainty, uc(y), by an appropriate
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coverage factor. When the result has a large number of degrees of freedom, the use of a value of 2 leads to approximately 95% confidence that the true value lies in the range ±U. The value of 2 will be used throughout this Annex. The following sections give illustrative examples of the uncertainty calculations for Cells I–V. After the assessment of uncertainties, there should be a reappraisal of experimental design factors and statistical treatment of the data, with due regard for economic factors before the adoption of more elaborate procedures.
where EIa = Ea - (k/2) lg(p°/pH2), k = (RT/F)ln 10, pH is the 2 partial pressure of hydrogen in Cell Ia, and p° is the standard pressure. 2. Determination of the acidity function, p(aHg Cl), in the buffer-filled cell (Cell I)
Experimental details have been published [42–45].
A-1.1 Measurement equations
The primary method for the determination of pH(PS) values consists of the following steps (Section 4.1): 1. Determination of the standard potential of the Ag | AgCl electrode from the acid-filled cell (Cell Ia)
E° = Ea + 2k lg(mHCl /m°) + 2k lg g HCl - (k/2) lg(p°/pH ) 2 (A-2) cf. (3)
-lg(aHg Cl) = (Eb – E°)/k + lg(mCl /m°) -(1/2) lg(p°/pH2),
(A-3) cf. (2)
-lg(aHg Cl) = -lg(aHg Cl)° + SmCl
(A-4) cf. (5)
where EI = Eb -(k/2) lg(p°/pH2), pH2 is the partial pressure of hydrogen in Cell I, and p° the standard pressure. 3. Extrapolation of the acidity function to zero chloride concentration
A.1 Uncertainty budget for the primary method of measurement using Cell I
4. pH Determination pH(PS) = -lg(aHg Cl)° + lg g °Cl
(A-5)
where lg g °Cl is calculated from the Bates–Guggenheim convention (see eq. 6). Values of the Debye–Hückel limiting law slope for 0 to 50 °C are given in Table A.9 [46].
A-1.2 Uncertainty budget
Example: PS = 0.025 mol kg–1 disodium hydrogen phosphate + 0.025 mol kg–1 potassium dihydrogen phosphate.
Table A.1 Calculation of Standard Uncertainty of the Standard Potential of the Silver–Silver Chloride Electrode (Eç) from Measurements in mHCl = 0.01 mol kg-1 Quantity E/V T/K mHCl/mol kg-1 pH2/kPa DE(Ag/AgCl)/V Bias potential g±
Estimate xi
Standard Uncertainty u(xi)
Sensitivity Coefficient |ci|
Uncertainty Contribution ui(y)
0.464 298.15 0.01 101.000 3.5 × 10-5
2 × 10-5 8 × 10-3 1 × 10-5 0.003 3.5 × 10-5
1 8.1 × 10-4 5.14 1.3 × 10-7 1
2 × 10-5 6.7 × 10-6 5.1 × 10-5 4.2 × 10-7 3.5 × 10-5
0.9042
9.3 × 10-4
0.0568
5.2 × 10-6
uc(E°) = 6.5 × 10-5 V Note 20: The uncertainty of method used for the determination of hydrochloric acid concentration is critical. The uncertainty quoted here is for potentiometric silver chloride titration. The uncertainty for coulometry is about 10 times lower.
Table A.2 Calculation of the Standard Uncertainty of the Acidity Function lg(aHfCl) for mCl = 0.005 mol kg−1 Quantity E/V E°/V T/K mCl/mol kg-1 pH2/kPa
DE(Ag/AgCl)/V
Estimate xi
Standard Uncertainty u(xi)
0.770 0.222 298.15 0.005 101.000
2 × 10-5 6.5 × 10-5 8 × 10-3 2.2 × 10-6 0.003
3.5 × 10-5
3.5 × 10-5
Sensitivity Coefficient |ci| 16.9 16.9 0.031 86.86 2.2 × 10-6 16.9
Uncertainty Contribution ui(y) 3.4 × 10-4 1.1 × 10-3 2.5 × 10-4 1.9 × 10-4 7 × 10-6 5.9 × 10-4
uc[lg(aHgCl)] = 0.0013 Note 21: If, as is usual practice in some NMIs [42–44], acid and buffer cells are measured at the same time, then the pressure measuring instrument uncertainty quoted above (0.003 kPa) cancels, but there remains the possibility of a much smaller bubbler depth variation between cells.
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Table A.3 S1 = Primary Buffer, pH(PS) = 4.005, u(pH) = 0.003; S2 = Secondary Buffer, pH(SS) = 6.86. Free-Diffusion Junctions with Cylindrical Symmetry Formed in Vertical Tubes Were Used [25] Quantity pH(S1) EII/V (Ej2 - Ej1)/V T/K
Standard Uncertainty Sensitivity Coefficient u(xi) |ci|
Estimate xi 4.005 0.2 3.5 × 10-4 298.15
0.003 1 × 10-5 3.5 × 10-4 0.1
Uncertainty Contribution ui(y)
1 16.9 16.9 1.2 × 10-5
0.003 1.7 × 10-4 6 × 10-3 1.2 × 10-6
uc[pH(S2)] = 0.007 Note 22: The error in EII is estimated as the scatter from 3 measurements. The RLJP contribution is estimated from Fig. 2 as 0.006 in pH; it is the principal contribution to the uncertainty.
The standard uncertainty due to the extrapolation to zero added chloride concentration (Section 4.4) depends in detail on the number of data points available and the concentration range. Consequently, it is not discussed in detail here. This calculation may increase the expanded uncertainty (of the acidity function at zero concentration) to U = 0.004. As discussed in Section 5.2, the uncertainty due to the use of the Bates–Guggenheim convention includes two components:
i. The uncertainty of the convention itself, and this is estimated to be approximately 0.01. This contribution to the uncertainty is required if the result is to be traceable to SI, but will not be included in the uncertainty of “conventional” pH values. ii The contribution to the uncertainty from the value of the ionic strength should be calculated for each individual case.
The typical uncertainty for Cell I is between U = 0.003 and U = 0.004.
A.2 Uncertainty budget for secondary pH buffer using Cell II
Pt | H2 | S2 ¦ KCl (≥3.5 mol dm–3) ¦ S1| H2| Pt
Cell II
where S1 and S2 are different buffers.
Therefore, U[pH(S2)] = 0.014.
A.3 Uncertainty budget for secondary pH buffer using Cell III
Pt | H2 | Buffer S2 ¦ Buffer S1 | H2 | Pt
A.3.1 Measurement equations 1. pH(S2) – pH(S1) = (EIII + Ej)/k 2. k = (RT/F)ln 10
Cell III (A-7) cf. (11)
For experimental details, see refs. [16,33,38]. Therefore, U[pH(S2]) = 0.004. The uncertainty is no more than that of the primary standard PS1.
A.4 Uncertainty budget for secondary pH buffer using Cell IV
Ag | AgCl | KCl (≥3.5 mol dm–3) ¦ buffer S1or S2| H2| Pt
Cell IV
A.4.1 Measurement equations
A.2.1 Measurement equations
1. Determination of pH(S2)
1. Determination of pH(S2) pHII(S2)
A.2.2 Uncertainty budget
− pHII(S1) = EII/k − (Ej2 − Ej1)/k
(A-6) cf. (9)
pHIV(S2) − pHIV(S1) = −[EIV(S2) – EIV(S1)]/k − (Ej2 − Ej1)/k (A-8) cf. (13)
2. Theoretical slope, k = (RT/F)ln 10
2. Theoretical slope, k = (RT/F)ln 10
Table A.4 pH (S2) Determination. S1 = Primary Standard (PS) and S2 = Secondary Standard (SS) are of the Same Nominal Composition. Example: 0.025 mol kg-1 Disodium Hydrogen Phosphate + 0.025 mol kg-1 Potassium Dihydrogen Phosphate, PS1 = 6.865, u(pH) = 0.002 Quantity pH(PS1) [E(S2) - E(S1)]/V [Eid(S2) - Eid(S1)]/V Ej/V T/K
Estimate xi
Standard Uncertainty u(xi)
Sensitivity Coefficient |ci|
Uncertainty Contribution ui(y)
6.865 1 × 10-4 1 × 10-6 1 × 10-5 298.15
2 × 10-3 1 × 10-6 1 × 10-6 1 × 10-5 2 × 10-3
1 16.9 16.9 16.9 5 × 10-6
2 × 10-3 16.9 × 10-6 1.7 × 10-5 16.9 × 10-5 1 × 10-8
uc[pH(S2)] = 0.002 Note 23: [Eid(S2) - Eid(S1)] is the difference in cell potential when both compartments are filled with solution made up from the same sample of buffer material. The estimate of Ej comes from the observations made of the result of perturbing the pH of samples by small additions of strong acid or alkali, and supported by Henderson equation considerations, that Ej contributes about 10 % to the total cell potential difference [33].
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Table A.5 Example from the Work of Paabo and Bates [5] Supplemented by Private Communication from Bates to Covington. S1 = 0.05 mol kg-1 Equimolal Phosphate; S2 = 0.05 mol kg-1 Potassium Hydrogen Phthalate. KCl = 3.5 mol dm-3. S1 = Primary Buffer PS1, pH = 6.86, u(pH) = 0.003, S2 = Secondary Buffer SS2, pH = 4.01 Quantity pH(S1) DEIV/V (Ej2 - Ej1)/V T/K
Estimate xi
Standard Uncertainty u(xi)
Sensitivity Coefficient |ci|
Uncertainty Contribution ui(y)
6.86 0.2 3.5 × 10-4 298.15
0.003 2.5 × 10-4 3.5 × 10-4 0.1
1 16.9 16.9 1.78 × 10-3
0.003 4 × 10-3 6 × 10-3 1.78 × 10-4
uc[pH(S2)] = 0.008 Note 24: The estimate of the error in ∆EIV comes from an investigation of several 3.5 mol dm–3 KCl calomel electrodes in phosphate solutions. The RLJP contribution for free-diffusion junctions is estimated from Fig. 2 as 0.006 in pH.
A.5.3 Measurement equations for multipoint calibration
A.4.2 Uncertainty budget Therefore, U[pΗ(S2)] = 0.016.
A.5 Uncertainty budget for unknown pH(X) buffer determination using Cell V
Ag | AgCl | KCl (≥3.5 mol dm–3) ¦ Buffer pH(S) or pH(X) | glass electrode Cell V
A.5.1 Measurement equations: 2-point calibration (bracketing) 1. Determination of the practical slope (k′)
k′ = [(EV(S2) – EV(S1)]/[pH(S2) – pH(S1)] (A-9) cf. (16)
2. Measurement of unknown solution (X)
(X) pH(X) = pH(S1) −[EV(X) − EV(S1)]/k′ – (Ej2 – Ej1)/k′
(A-10) cf. (15)
A.5.2 Uncertainty budget
Example of two-point calibration (bracketing) with a pH combination electrode [47].
EV(S) = EV°– k′pH(S) pH(X) = [EV° − EV(X)]/k′
(A-11) cf. (17) (A-12) cf. (18)
Uncertainty budget: Example: Standard buffers pH(S1) = 3.557, pH(S2) = 4.008, pH(S3) = 6.865, pH(S4) = 7.416, pH(S5) = 9.182; pH(X) was a “readyto-use” buffer solution with a nominal pH of 7. A combination electrode with capillary liquid junction was used. For experimental details, see ref. [41]; and for details of the calculations, see ref. [45]. The uncertainty will be different arising from the RLJPs if an alternative selection of the five standard buffers was used. The uncertainty attained will be dependent on the design and quality of the commercial electrodes selected. Therefore, U[pH(X)] = 0.01.
Table A.6 Primary Buffers PS1, pH = 7.4, u(pH) = 0.003; PS2, pH = 4.01, u(pH) = 0.003. Practical Slope (k¢) Determination Quantity DE/V T/K (Ej2 - Ej1)/V DpH
Estimate xi 0.2 298.15 6 × 10-4 3.39
Standard Uncertainty u(xi) 5 × 10-4 0.1 6 × 10-4 4.24 × 10-3
Sensitivity Coefficient |ci|
Uncertainty Contribution ui(y)
2.95 × 10-1 1.98 × 10-4 2.95 × 10-1 1.75 × 10-2
1.5 × 10-4 1.98 × 10-5 1.8 × 10-4 7.40 × 10-5
uc(k¢) = 2.3 × 10-4
Table A.7 pH(X) Determination Quantity
Estimate xi
Standard Uncertainty u(xi)
Sensitivity Coefficient |ci|
Uncertainty Contribution ui(y)
pH(S1) DE/V (Ej2 - Ej1)/V k¢/V
7.4 0.03 6.00 × 10-4 0.059
0.003 1.40 × 10-5 6.00 × 10-4 2.3 × 10-4
1 16.95 16.95 9.01
0.003 2.37 × 10-4 1.01 × 10-2 2.1 × 10-3
uc[pH(X)] = 1.6 × 10-2 Note 25: The estimated error in DE comes from replicates. The RLJP is estimated as 0.6 mV. Therefore, U[pH(X)] = 0.021.
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Table A.8 Quantity E°/V T/K E(X)/V k¢/V
Estimate xi
Standard Uncertainty u(xi)
Sensitivity Coefficient |ci|
Uncertainty Contribution ui(y)
-0.427 298.15 0.016 0.059
5 × 10-4 0.058 2 × 10-4 0.076 × 10-3
16.96 1.98 × 10-4 16.9 67.6
0.0085 1.15 × 10-5 0.0034 0.0051
uc[pH(X)] = 0.005 Note 26: There is no explicit RLJP error assessment as it is assessed statistically by regression analysis.
Table A.9 Values of the Relative Permittivity of Water [46] and the Debye–Hückel limiting Law Slope for Activity Coefficients as lg f in eq. 6. Values are for 100.000 kPa, but the Difference from 101.325 kPa (1 atm) is Negligible t/çC 0 5 10 15 20 25 30 35 40 45 50
Relative Permittivity
A/mol-½ kg½
87.90 85.90 83.96 82.06 80.20 78.38 76.60 74.86 73.17 71.50 69.88
0.4904 0.4941 0.4978 0.5017 0.5058 0.5100 0.5145 0.5192 0.5241 0.5292 0.5345
14. Summary of recommendations • IUPAC recommended definitions, procedures, and terminology are described relating to pH measurements in dilute aqueous solutions in the temperature range 0–50 °C. • The recent definition of primary method of measurement permits the definition of primary standards for pH, determined by a primary method (cell without transference, called the Harned cell) and of secondary standards for pH. • pH is a conventional quantity and values are based on the Bates–Guggenheim convention. The assigned uncertainty of the Bates–Guggenheim convention is 0.01 in pH. By accepting this value, pH becomes traceable to the internationally accepted SI system of measurement. • The required attributes (listed in Section 6.1) for primary standard materials effectively limit the number of primary substances to six, from which seven primary standards are defined in the pH range 3–10 (at 25 °C). Values of pH(PS) from 0–50 °C are given in Table 2. • Methods that can be used to obtain the difference in pH between buffer solutions are discussed in Section 8. These methods include the use of cells with transference that are practically more convenient to use than the Harned cell, but have greater uncertainties associated with the results. • Incorporation of the uncertainties for the primary method, and for all subsequent measurements, permits the uncertainties for all procedures to be linked to the primary standards by an unbroken chain of comparisons. Despite its conventional basis, the definition of pH, the establishment of pH standards, and the procedures for pH determination
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are self-consistent within the confidence limits determined by the uncertainty budgets. • Comparison of values from the cell with liquid junction with the assigned pH(PS) values of the same primary buffers measured with Cell I makes the estimation of values of the RLJPs possible (Section 7), and the consistency of the seven primary standards can be estimated. • The Annex (Section 13) to this document includes typical uncertainty estimates for the five cells and measurements described, which are summarized in Table 4. • The hierarchical approach to primary and secondary measurements facilitates the availability of recommended procedures for carrying out laboratory calibrations with traceable buffers grouped to achieve specified target uncertainties of unknowns (Section 11). The three calibration procedures in common use, one-point, two-point (bracketing), and multipoint, are described in terms of target uncertainties.
15. References 1. BIPM. Com. Cons. Quantité de Matière 4 (1998). See also: M. J. T. Milton and T. J. Quinn. Metrologia 38, 289 (2001). 2. A. K. Covington, R. G. Bates, R. A. Durst. Pure Appl. Chem. 57, 531 (1985). 3. IUPAC. Quantities, Units and Symbols in Physical Chemistry, 2nd ed., Blackwell Scientific, Oxford (1993). 4. S. P. L. Sørensen and K. L. Linderstrøm-Lang. C. R. Trav. Lab. Carlsberg 15, 6 (1924). 5. R. G. Bates. Determination of pH, Wiley, New York (1973). 6. S. P. L. Sørensen. C. R. Trav. Lab. Carlsberg 8, 1 (1909). 7. H. S. Harned and B. B. Owen. The Physical Chemistry of Electrolytic Solutions, Chap. 14, Reinhold, New York (1958). 8. R. G. Bates and R. A. Robinson. J. Solution Chem. 9, 455 (1980). 9. A. G. Dickson. J. Chem. Thermodyn. 19, 993 (1987). 10. R. G. Bates and E. A. Guggenheim. Pure Appl. Chem. 1, 163 (1960). 11. J. N. Brønsted. J. Am. Chem. Soc. 42, 761 (1920); 44, 877, 938 (1922); 45, 2898 (1923). 12. A. K. Covington. Unpublished. 13. K. S. Pitzer. In K. S. Pitzer (Ed.), Activity Coefficients in Electrolyte Solutions, 2nd ed., p. 91, CRC Press, Boca Raton, FL (1991). 14. A. K. Covington and M. I. A. Ferra. J. Solution Chem. 23, 1 (1994). 15. International Vocabulary of Basic and General Terms in Metrology (VIM), 2nd ed., Beuth Verlag, Berlin (1994). 16. R. Naumann, Ch. Alexander-Weber, F. G. K. Baucke. Fresenius’ J. Anal. Chem. 349, 603 (1994). 17. R. G. Bates. J. Res. Natl. Bur. Stand., Phys. Chem. 66A (2), 179 (1962). 18. P. Spitzer. Metrologia 33, 95 (1996); 34, 375 (1997). 19. N. E. Good, G. D. Wright, W. Winter, T. N. Connolly, S. Isawa, K. M. M. Singh. Biochem. J. 5, 467 (1966). 20. A. K. Covington and M. J. F. Rebelo. Ion-Sel. Electrode Rev. 5, 93 (1983). 21. H. W. Harper. J. Phys. Chem. 89, 1659 (1985). 22. J. Bagg. Electrochim. Acta 35, 361, 367 (1990); 37, 719 (1992). 23. J. Breer, S. K. Ratkje, G.-F. Olsen. Z. Phys. Chem. 174, 179 (1991). 24. A. K. Covington. Anal. Chim. Acta 127, 1 (1981).
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692 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38.
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A. K. Covington and M. J. F. Rebelo. Anal. Chim. Acta 200, 245 (1987). D. J. Alner, J. J. Greczek, A. G. Smeeth. J. Chem. Soc. A 1205 (1967). F. G. K. Baucke. Electrochim. Acta 24, 95 (1979). A. K. Clark and A. K. Covington. Unpublished. M. J. G. H. M. Lito, M. F. G. F. C. Camoes, M. I. A. Ferra, A. K. Covington. Anal. Chim. Acta 239, 129 (1990). M. F. G. F. C. Camoes, M. J. G. H. M. Lito, M. I. A. Ferra, A. K. Covington. Pure Appl. Chem. 69, 1325 (1997). A. K. Covington and J. Cairns. J. Solution Chem. 9, 517 (1980). H. B. Hetzer, R. A. Robinson, R. G. Bates. Anal. Chem. 40, 634 (1968). F. G. K. Baucke. Electroanal. Chem. 368, 67 (1994). P. R. Mussini, A. Galli, S. Rondinini. J. Appl. Electrochem. 20, 651 (1990); C. Buizza, P. R. Mussini, T. Mussini, S. Rondinini. J. Appl. Electrochem. 26, 337 (1996). R. P. Buck and E. Lindner. Pure Appl. Chem. 66, 2527 (1994). J. Christensen. J. Am. Chem. Soc. 82, 5517 (1960). Guide to the Expression of Uncertainty (GUM), BIPM, IEC, IFCC, ISO, IUPAC, IUPAP, OIML (1993). F. G. K. Baucke. Anal. Chem. 66, 4519 (1994).
39. F. G. K. Baucke, R. Naumann, C. Alexander-Weber. Anal. Chem. 65, 3244 (1993). 40. R. Naumann, F. G. K. Baucke, P. Spitzer. In PTB-Report W-68, P. Spitzer (Ed.), pp. 38–51, Physikalisch-Technische Bundesanstalt, Braunschweig (1997). 41. S. Ebel. In PTB-Report W-68, P. Spitzer (Ed.), pp. 57–73, PhysikalischTechnische Bundesanstalt, Braunschweig (1997). 42. H. B. Kristensen, A. Salomon, G. Kokholm. Anal. Chem. 63, 885 (1991). 43. P. Spitzer, R. Eberhadt, I. Schmidt, U. Sudmeier. Fresenius’ J. Anal. Chem. 356, 178 (1996). 44. Y. Ch. Wu, W. F. Koch, R. A. Durst. NBS Special Publication, 260, p. 53, Washington, DC (1988). 45. BSI/ISO 11095 Linear calibration using reference materials (1996). 46. D. A. Archer and P. Wang. J. Phys. Chem. Ref. Data 19, 371 (1990). See also D. J. Bradley and K. S. Pitzer. J. Phys. Chem. 83, 1599 and errata 3799 (1979); D. P. Fernandez, A. R. H. Goodwin, E. W. Lemmon, J. M. H. Levelt Sengers, R. C. Williams. J. Phys. Chem. Ref. Data 26, 1125 (1997). 47. A. K. Covington, R. Kataky, R. A. Lampitt. Unpublished.
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General Comments on Buffers The major factor in biological pH control in eukaryotic cells is the carbon dioxide-bicarbonate-carbonate buffer (Scheme I) system1-4. There other biological buffers such as bulk protein and phosphate anions which can provide some buffering effect, metabolites such as lactic acid which can lower pH and tris(hydroxymethylaminomethyl) methane, (THAM®) has been used to treat acid base disorders5-7. pH control in prokaryotic cells is mediated by membrane transport of various ions including hydrogen, potassium and sodium8-10.
Scheme I CO2 + H2O = H2CO3; H2CO3 + H2O = HCO3–1 + H3O+ × (pKa 6.15); HCO3–1 + H2O = CO3–2– (pKa 10.3) See Jungas, R.L., Best literature values for the pK of carbonic and phosphoric acid under physiological conditions, Anal. Biochem. 349, 1–15, 2006 In the laboratory, the bicarbonate/carbonate buffer system can only be used in the far alkaline range (pH 9-11) and unless “fixed” by a suitable cation such as sodium, can be volatile. A variety of buffers, most notably the “Good” buffers which were developed by Norman Good and colleagues10a, have been developed over the years to provide pH control in in vitro experiments. While effective in controlling pH, the numerous non-buffer effects that buffer salts have on experimental systems are somewhat less appreciated. Some effects, such as observed with phosphate buffers, are based on biologically significant interactions with proteins and, as such, demonstrate specificity. Other effects, such as metal ion chelation, can be considered general. There are some effects where the stability of a reagent is dependent on both pH and buffer species. One example is provided by the stability of phenylmethylsulfonyl fluoride (PMSF)11. PMSF was less stable in Tris buffer than in either HEPES or phosphate buffer; PMSF is less stable in HEPES than in phosphate buffer. Activity was measured by the ability of PMSF to inhibit chymotrypsin; all activity was lost in Tris (10 mM; pH 7.5) after one hour at 25°C while activity was fully retained in phosphate (10 mM, pH 7.5). This is likely a reflection of the nucleophilic property of Tris12,13 which appears to be enhanced in the presence of divalent cations such as zinc14. The loss of activity, presumably the result of the hydrolysis of the fluoride to hydroxyl function, is more marked at more alkaline pH. Tris can also function as phosphoacceptor in assays for alkaline phosphatase but was not as effective as 2-amino-2-methyl-1,3-propanediol15. The various nitrogen-based buffers such as Tris, HEPES, CAP, and BICINE influence colorimetric protein assays 16-18. Other specific examples are presented in Table 1.
References 1. Lubman, R.L. and Crandall, E.D., Regulation of intracellular pH in alveolar epithelial cells, Amer.J.Physiol. 262, L1-L14, 1992 2. Lyall, V. and Biber, T.O.L., Potential-induced changes in intracellular pH, Amer.J.Physiol. 266, F685-F696, 1994 3. Palmer, L.G., Intracellular pH as a regulator of Na+ transport, J.Membrane Biol. 184, 305-311, 2001 4. Vaughn-Jones, R.D. and Spitzer, K.W., Role of bicarbonate in the regulation of intracellular pH in the mammalian ventricular myocyte, Biochem.Cell Biol. 80, 579-596, 2002 5. Henschler, D., Trispuffer(TAHM) als therapeuticum, Deutsch.Med. Wochenschr. 88, 1328-1331, 1963 6. Nahas, G.G., Sutin, K.M., Fermon, C., et al, Guidelines for the treatment of academia with THAM, Drugs 55, 191-224, 1998 7. Rehm, M. and Finsterer, U., Treating intraoperative hypercholoremic acidosis with sodium bicarbonate or tris-hydroxymethyl amino methane, Anesthes.Analg. 96, 1201-1208, 2003 8. Kashket, E.R. and Wong, P.T., The intracellular pH of Escherichia coli, Biochim.Biophys.Acta 193, 212-214, 1969 9. Padan, E. and Schuldiner, S., Intracellular pH regulation in bacterial cells, Methods Enzymol. 125, 327-352, 1986 10. Booth, I.R., The regulation of intracellular pH in bacteria, Novartis Found.Symp. 221, 19-28, 1999 10a. Good, N.E., Winget, G.D., Winter, W., et al., Hydrogen ion buffers for biological research, Biochemistry 5, 467-477, 1966 11. James, G.T., Inactivation of the protease inhibitor phenylmethylsulfonyl fluoride in buffers, Anal.Biochem. 86, 574-579, 1978 12. Acharya, A.S., Roy, R.P., and Dorai, B., Aldimine to ketoamine isomerization (Amadori rearrangement) potential at the individual nonenzymic glycation sites of hemoglobin A: preferential inhibition of glycation by nucleophiles at sites of low isomerization potential, J.Protein Chem. 10, 345-358, 1991 13. Mattson, A., Boutelje, J., Csoregh, I., et al., Enhanced stereoselectivity in pig liver esterase catalyzed diester hydrolysis. The role of a competitive inhibitor, Bioorg.Med.Chem. 2, 501-508, 1994 14. Tomida, H. and Schwartz, M.A., Further studies on the catalysis of hydrolysis and aminolysis of benzylpenicillin by metal chelates, J.Pharm.Sci. 72, 331-335, 1983 15. Stinson, R.A., Kinetic parameters for the cleaved substrate, and enzyme and substrate stability, vary with the phosphoacceptor in alkaline phosphatase catalysis, Clin.Chem. 39, 2293-2297, 1993 16. Kaushal, V. and Barnes, L.D., Effect of zwitterionic buffers on measurement of small masses of protein with bicinchoninic acid, Anal. Biochem. 157, 291-294, 1986 17. Lleu, P.L. and Rebel, G., Interference of Good’s buffers other biological buffers with protein determination, Anal.Biochem. 192, 215-218, 1991 18. Sapan, C.V., Lundblad, R.L., and Price, N.C., Colorimetric protein assay techniques, Biotechnol.Appl.Biochem. 29, 99-108, 1999
693
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Handbook of Biochemistry and Molecular Biology
694 TABLE 1: Effects of Buffers Buffer
Observation
ACES
Competitive inhibitor of g -aminobutyric acid receptor binding1. Competitive inhibitor of g -aminobutyric acid receptor binding1; chelation of calcium ions2. Interacts with DNA yielding distortion of DNA electrophoretograms3. Chelation of calcium ions2; protects liver alcohol dehydrogenase from inactivation by iodoacetic acid4. Anomalous complex formation with nucleic acids5; complex formation with carbohydrates6,7; participant in the modification of arginine residues by 1,2-cyclohexanedione8. Reaction with sulfhydryl compounds9. Enhances rate of reaction of phenylglyoxal with arginine residues in proteins10; modulation of peroxynitrite reactions with proteins11, 12; modulation of Cu2+ oxidation reactions13-15. Chelation of calcium ions2. Free radical generation16,17 and complexation of copper ions18; reported adverse effects in tissue culture19,20. Complexes copper ions21. Adverse effect on smooth muscle contraction22; Oxidation of metal ions22; formation of nitric oxide donors on incubation with peroxynitrite24; slow reaction with hydrogen peroxide25. Catalysis of the racemization of 5-phenylhydantoins26,27. Binding to bile salt-stimulated lipase28; variation in physiological response based on vendor source29; inhibition of a K+-activated phosphatase30. Interaction with extracellular matrices31; inhibition of the interaction of proteoglycans with type 1 collagen32. Chelating agent2; tricine radicals have been reported in the presence of peroxide-forming enzymes33. Nucleophile34,35 and enzyme inhibitor36.
ADA BES BICINE Borate
Cacodylic Acid Carbonate
Citrate HEPES MES MOPS
Phosphate PIPES TES Tricine Tris
References to Table 1 1. Tunnicliff, G. and Smith, J.A., Competitive inhibition of gamma-aminobutyric acid receptor binding by N-hydroxyethylepiperazine-N-2ethanesulfonic acid and related buffers, J.Neurochem. 36, 1122-1126, 1981 2. Durham, A.C., A survey of readily available chelators for buffering calcium ion concentrations in physiological solutions, Cell Calcium 4, 33-46, 1983 3. Stellwagen, N.C., Bossi, A., Gelfi, C. and Righetti, P.G., DNA and buffers: Are there any noninteracting neutral pH buffers?, Anal. Biochem. 287, 167-175, 2000 4. Syvertsen, C. and McKinley-McKee, J.S., Affinity labelling of liver alcohol dehydrogenase. Effect of pH and buffers on affinity labelling with iodoacetic acid and (R,S)-2- bromo-3-(5-imidazolyl)propionic acid, Eur.J.Biochem. 117, 165-170, 1981 5. Biyani, M. and Nishigaki, K., Sequence-specific and nonspecific mobilities of single-stranded oligonucleotides observed by changing the borate buffer concentration, Electrophoresis 24, 628-633, 2003 6. Zittle, Z.A., Reaction of borate with substances of biological interest, Adv.Enzymol.Relat.Sub.Biochem. 12, 493-527, 1951 7. Weitzman, S., Scott, V., and Keegstra, K., Analysis of glycoproteins as borate complexes by polyacrylamide gel electrophoresis, Anal. Biochem. 438-449, 1979 8. Patthy, L. and Smith, E.L., Reversible modification of arginine residues. Application to sequence studies by restriction of tryptic hydrolysis to lysine residues, J.Biol.Chem. 250, 557-564, 1975 9. Jacobson, K.B., Murphey, J.B., and Sarma, B.D., Reaction of cacodylic acid with organic thiols, FEBS Lett. 22, 80-82, 1972 10. Cheung, S.T. and Fonda, M.L., Reaction of phenylglyoxal with arginine. The effect of buffers and pH, Biochem.Biophys.Res.Commun. 90, 940-947, 1979
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11. Uppu, R.M., Squadrito, G.L., and Pryor, W.A., Acceleration of peroxynitrite oxidations by carbon dioxide, Arch.Biochem.Biophys. 327, 335-343, 1996 12. Denicola, A., Freeman, B.A., Trujillo, M., and Radi, R., Peroxynitrite reaction with carbon dioxide/bicarbonate: kinetics and influence on peroxynitrite-mediated oxidations, Arch.Biochem.Biophys. 333, 49-58, 1996 13. Munday, R., Munday, C.M. and Winterbourn, C.C., Inhibition of copper-catalyzed cysteine oxidation by nanomolar concentrations of iron salts, Free Rad.Biol.Med. 36, 757-764, 2004 14. Jansson, P.J., Del Castillo, U., Lindqvist, C., and Nordstrom, T., Effects of iron on vitamin C/copper-induced hydroxyl radical generation in bicarbonate-rich water, Free Rad.Res. 39, 565-570, 2005 15. Ramirez, D.C., Mejiba, S.E. and Mason, R.P., Copper-catalyzed protein oxidation and its modulation by carbon dioxide: enhancement of protein radicals in cells, J.Biol.Chem. 280, 27402-27411, 2005 16. Tadolini, B., Iron autoxidation in Mops and Hepes buffers, Free Radic.Res.Commun. 4, 149-160, 1987 17. Simpson, J.A., Cheeseman, K.H., Smith, S.E., and Dean, R.T., Free-radical generation by copper ions and hydrogen peroxide. Stimulation by Hepes buffer, Biochem.J. 254, 519-523, 1988 18. Sokolowska, M. and Bal, W., Cu(II) complexation by “non-coordinating” N-2-hydroxyethylpiperazine-N’-ethanesulfonic acid (HEPES buffer), J.Inorg.Biochem. 99, 1653-1660, 2005 19. Bowman, C.M., Berger, E.M., Butler, E.N. et al., HEPES may stimulate cultured endothelial-cells to make growth-retarding oxygen metabolites, In Vitro Cell.Devel.Biol. 21, 140-142, 1985 20. Magonet, E., Briffeuil, E., Polimay, Y., and Ronveaux, M.F., Adverseeffects of HEPES on human-endothelial cells in culture, Anticancer Res. 7, 901, 1987 21. Mash, H.E., Chin, Y.P., Sigg, L., et al., Complexation of copper by zwitterionic aminosulfonic (Good) buffers, Anal.Chem. 75, 671-677, 2003 22. Altura, B.M., Carella, A., and Altura, B.T., Adverse effects of Tris, HEPES, and MOPS buffers on contractile responses of arterial and venous smooth muscle induced by prostaglandins, Prostaglandins Med. 5, 123-130, 1980 23. Tadolini, B., and Sechi, A.M., Iron oxidation in Mops and Hepes buffers, Free Radic.Res.Commun. 4, 149-160, 1987 24. Schmidt, K., Pfeiffer, S., and Meyer, B., Reaction of peroxynitrite with HEPES or MOPS results in the formation of nitric oxide donors, Free Radic.Biol.Med. 24, 859-862, 1998 25. Zhao, G. and Chasteen, J.D., Oxidation of Good’s buffers by hydrogen peroxide, Anal.Biochem. 349, 262-267, 2006 26. Dudley, K.H. and Bius, D.L., Buffer catalysis of the racemization reaction of some 5-phenylhydantoins and its relation to in vivo metabolism of ethotoin, Drug.Metab.Dispos. 4, 340-348, 1976 27. Lazarus, R.A., Chemical racemization of 5-benzylhydantoin, J.Org. Chem. 55, 4755-4757, 1990 28. Moore, S.A., Kingston, R.L., Loomes, K.M., et al., The structure of truncated recombinant human bile salt-stimulated lipase reveals bile salt-independent conformational flexibility at the active-site loop and provides insight into heparin binding, J.Mol.Biol. 312, 511-523, 2001 29. Schmidt, J., Mangold, C., and Deitmer, J., Membrane responses evoked by organic buffers in identified leech neurones, J.Exp.Biol. 199, 327-335, 1996 30. Robinson, J.D. and Davis, R.L., Buffer, pH, and ionic strength effects on the (Na+ + K+)-ATPase, Biochim.Biophys.Acta 912, 343-347, 1987 31. Poole, C.A., Reilly, H.C., and Flint, M.H., The adverse effects of HEPES, TES, and BES zwitterionic buffers on the ultrastructure of cultured chick embryo epiphyseal chondrocytes, In Vitro 18, 755-765, 1982 32. Pogány, G., Hernandez, D.J., and Vogel, K.G., The in Vitro interaction of proteoglycans with type I collagen is modulated by phosphate, Archs.Biochem.Biophys. 313, 102-111, 1994 33. Grande, H.J. and Van der Ploeg, K.R., Tricine radicals as formed in the presence of peroxide producing enzymes, FEBS Lett. 95, 352-356, 1978 34. Oliver, R.W. and Viswanatha, T., Reaction of tris(hydroxymethyl) aminomethane with cinnamoyl imidazole and cinnamoyltrypsin, Biochim.Biophys.Acta 156, 422-425, 1968 35. Ray, T., Mills, A., and Dyson, P., Tris-dependent oxidative DNA strand scission during electrophoresis, Electrophoresis 16, 888-894, 1995 36. Qi, Z., Li, X., Sun, D., et al., Effect of Tris on catalytic activity of MP-11, Bioelectrochemistry 68, 40-47, 2006
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List of Buffers Common Name
Chemical Name
ACES
2-[2-amino-2-oxoethyl)amino] ethanesulfonic acid O
M.W 182.20
Properties and Comment One of the several “Good” buffers
H O
N+ H2N
S H
O
OH
Good, N.E., Winget, G.D., Winter, W., et al., Hydrogen ion buffers for biological research, Biochemistry 5, 467-477, 1966; Tunnicliff, G. and Smith, J.A, Competitive inhibition of gamma-aminobutyric acid receptor binding by N-hydroxyethylpiperazine-N’-2-ethanesulfonic acid and related buffers, J.Neurochem. 36, 1122-1126, 1981; Chappel, D.J., N-[(carbamoylmethyl)amino] ethanesulfonic acid improves phenotyping of a-1-antitrypsin by isoelectric focusing on agarose gel, Clin.Chem. 31, 1384-1386, 1985; Liu, Q., Li, X., and Sommer, S.S., pk-Matched running buffers for gel electrophoresis, Anal.Biochem. 270, 112-122, 1999; Taha, M., Buffers for the physiological pH range: acidic dissociation constants of zwitterionic compounds in various hydroorganic media, Ann.Chim. 95, 105-109, 2005. Acetic Acid/Sodium Acetate
acetic acid (usually with sodium hydroxide to provide sodium acetate
O
H3C
60.0/ 82.0
Frequently used in chromatography with cation exchange matrices below pH 6.0; therapeutic use for acid-base disorders; buffer for peritoneal dialysis. It is an organic acid and a natural product (as is the case with citrate and phosphate)
O
OH
H3C
O–
Chohan, I.S., Vermylen, J., Singh, I. et al., Sodium acetate buffer: a diluent of choice in the clot lysis time technique, Thromb.Diath.Haemorrh. 33, 226-229, 1975; Lim, C.K. and Peters, T.J., Ammonium acetate: a general purpose buffer for clinical applications of high-performance liquid chromatography, J.Chromatog. 3126, 397-406, 1984; Kodama, C., Kodama, T., and Yosizawa, Z., Methods for analysis of urinary glycosaminoglycans, J.Chromatog. 429, 293-313, 1988; Stegmann, S., Norgren, R.B., Jr., and Lehman, M.N., Citric acid-ammonium acetate buffer, Biotech.Histochem. 1, 27-28,1991; Cuvelier, A., Bourguignon, J., Muir, J.F., et al., Substitution of carbonate by acetate buffer for IgG coating in sandwich ELISA, J.Immunoassay 17, 371-382, 1996; Urbansky, E.T., Cooper, B.T., and Margerum, D.W., Disproportionation kinetics of hypoiodous acid as catalyzed and suppressed by acetic acid-acetate buffer, Inorg.Chem. 36, 1338-1344, 1997; Watanabe, N., Shirakami, Y., Tomiyoshi, K., et al., Direct labeling of macroaggregated albumin with indium-111-chloride using acetate buffer, J.Nucl.Med. 38, 1590-1592, 1997; Righetti, P.G. and Gelfi, C., Capillary electrophoresis of DNA in the 20-500 bp range: recent developments, J.Biochem.Biophys.Methods 41, 75-90, 1999; Sen Gupta, K.K., Pal, B., and Begum, B.A., Reactivity of some sugars and sugar phosphates toward gold (III) in sodium acetate-acetic acid buffer medium, Carbohydr.Res. 330, 115-123, 2001. Citations for clinical use: Man, N.K., Itakura, Y., Chauveau, P., and Yamauchi, T., Acetate-free biofiltration: state of the art, Contrib. Nephrol. 108, 87-93, 1994; Maiorca, R., Cancarini, G.C., Zubani, R., et al., Differing dialysis treatment strategies and outcome, Nephrol.Dial. Transplant. 11(Suppl 2), 134-139, 1996; Naka, T. and Bellomo, R., Bench-to-bedside review: Treating acid-base abnormalities in the intensive care unit – the role of renal replacement therapy, Crit.Care 8, 108-114, 2004; Khanna, A. and Kurtzman, N.A., Metabolic alkalosis, J.Nephrol. 19(Suppl 9), S86-S96, 2006. ADA
N-(2-amino-2-oxoethyl)-N(carboxymethyl)glycine N-(2-acetamido)iminodiacetic acid
190.2
A “Good” buffer
HOOC O
HOOC
N
C NH2
695
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696
LIST OF BUFFERS (Continued) Common Name
Chemical Name
M.W
Properties and Comment
Good, N.E., Winget, G.D., Winter, W., et al., Hydrogen ion buffers for biological research, Biochemistry 5, 467-477, 1966; Tunnicliff, G. and Smith, J.A., Competitive inhibition of gamma-aminobutyric acid receptor binding by N-2-hydroxyethylpiperazine-N’-2-e-ethanesulfonic acid and related buffers, J.Neurochem. 36, 1122-1126, 1981; Durham, A.C., A survey of readily available chelators for buffering calcium ion concentrations in physiological solutions, Cell Calcium 4, 33-46, 1983; Kaushal, V. and Barnes, L.D., Effect of zwitterionic buffers on measurement of small masses of protein with bicinchoninic acid, Anal.Biochem. 157, 291-294, 1986; Robinson, J.D. and Davis, R.L., Buffer, pH, and ionic strength effects on the (Na+, + K+)-ATPase, Biochim.Biophys.Acta 912, 343-347, 1987; Pietrzkowski, E and Korohoda, W., Extracellular ATP and ADA-buffer enable chick embryo fibroblasts to grow in secondary culture in protein-free, hormone-free, extracellular growth factor-free media, Folia Histochem.Cytobiol. 26, 143-152, 1988; Righetti, P.G., Chiari, M., and Gelfi, C., Immobilized pH gradients: effect of salts, added carrier ampholytes and voltage gradients on protein patterns, Electrophoresis 9, 65-73, 1988; Bers, D.M., Hryshko, L.V., Harrison, S.M., and Dawson, D.D., Citrate decreases contraction and Ca current in cardiac muscle independent of its buffering action, Am.J.Physiol. 260, C900-C909, 1991; Delaney, J.P., Kimm, G.E., and Bonsack, M.E., The influence of luminal pH on the severity of acute radiation enteritis, Int.J.Radiat.Biol. 61, 381-386, 1992; Taha, M., Buffers for the physiological pH range: acidic dissociation constants of zwitterionic compounds in various hydroorganic media, Ann.Chim. 95, 105-109, 2005. BES
N,N-bis(2-hydroxyethyl)2-aminoethanesulfonic acid; N,N-bis(2-hydroxyethyl)taurine O
213.3
A “Good” buffer, not frequently used, similar to MES, HEPES
OH S
HO N
O
OH Good, N.E., Winget, G.D., Winter, W., et al., Hydrogen ion buffers for biological research, Biochemistry 5, 467-477, 1966; Kaushal, V. and Barnes, L.D., Effect of zwitterionic buffers on the measurement of small masses of protein with bicinchoninic acid, Anal.Biochem. 157, 291-294, 1986; MacKerrow, S.D., Merry, J.M., and Hoeprich, P.D., Effects of buffers on testing of Candida species susceptibility to flucytosine, J.Clin.Microbiol. 25, 885-888, 1987; Tuli, R.K. and Holtz, W., The effect of zwitterionic buffers on the feasibility of Boer goat semen, Theriogenology 37, 947-951, 1992; Stellwagen, N.C., Bossi, A., Gelfi, C., and Righetti, P.G., DNA and buffers: are there any noninteracting, neutral pH buffers, Anal.Biochem. 287, 167-175, 2000; Hosse, M. and Wilkinson, K.J., Determination of electrophoretic mobilities and hydrodynamic radii of three humic substances as a function of pH and ionic strength, Environ.Sci.Technol. 35, 4301-4306, 2002; Taha, M., Buffers for the physiological pH range: acidic dissociation contstants of zwitterionic compounds in various hydroorganic media, Ann.Chim. 95, 105-109, 2005. Bicine
N,N-bis-(2-hydroxyethyl)glycine; N,N-bis(2-hydroxyethyl) amino-acetic acid
163.2
HO
N
O C
HO
OH
Kanfer, J.N., Base exchange reactions of the phospholipids in rat brain particles, J.Lipid Res. 13, 468-476, 1972; Williams-Smith, D.L., Bray, R.C., Barber, M.J., et al., Changes in apparent pH on freezing aqueous buffer solutions and their relevance to biochemical electron-paramagnetic-resonance spectroscopy, Biochem.J. 167, 593-600, 1977; Syvertsen, C. and McKinley-McKee, J.S., Affinity labeling of liver alcohol dehydrogenase. Effects of pH and buffers on affinity labeling with iodoacetic acid and (R, S)-2-bromo-3-(5-imidazoyl)propionic acid, Eur.J.Biochem. 117, 165-170, 1981; Ito, S., Takaoka, T., Mori, H., and Teruo, A., A sensitive new method for measurement of guanase with 8-azaguanine in bicine bis-hydroxy ethyl glycine buffer as substrate, Clin.Chim.Acta 115, 135-144, 1981; Nakon, R., Krishnamoorthy, C.R., Free-metal ion depletion by “Good’s” buffers, Science 221, 749-750, 1983; Ito, S., Xu, Y., Keyser, A.J., and Peters, R.L., Histochemical demonstration of guanase in human liver with guanine in bicine buffer as substrate, Histochem.J. 16, 489-499, 1984; Roy, R.N., Gibbons, J.J, Baker, G., and Bates, R.G., Standard electromotive force of the H2-AgCL:Ag cell in 30, 40, and 50 mass% dimethyl sulfoxide/water from -20 to 25o; pK2 and pH values for a standard “Bicine” buffer solution at subzero temperatures, Cryobiology 21, 672-681, 1984; Vaidya, N.R., Gothoskar, B.P., and Banerji, A.P., Column isoelectric focusing in nature pH gradients generated by biological buffers, Electrophoresis 11, 156-161, 1990; Wiltfang, J., Arold, N., and Neuhoff, V., A new multiphasic buffer system for sodium sulfate-polyacrylamide gel electrophoresis of proteins and peptides with molecular masses 100,000-1000, and their detection with picomolar sensitivity, Electrophoresis 12, 352-366, 1991; Rabilloud, T., Vuillard, L., Gilly, C., and Lawrence, J.J., Silver-staining of proteins in polyacrylamide gels: a general overview, Cell.Mol. Biol. 40, 57-75, 1994; Gordon-Weeks, R., Koren’kov, V.D., Steele, S.H., and Leigh, R.A., Tris is a competitive inhibitor of K+ activation of the vacuolar H+-pumping pyrophosphatase, Plant Physiol. 114, 901-905, 1997; Luo, Q., Andrade, J.D., and Caldwell, K.D., Thin-layer ion-exchange
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List of Buffers
697 LIST OF BUFFERS (Continued)
Common Name
Chemical Name
M.W
Properties and Comment
chromatography of proteins, J.Chromatog. A 816, 97-105, 1998; Churchill, T.A. and Kneteman, N.M., Investigation of a primary requirement of organ preservation solutions: supplemental buffering agents improve hepatic energy production during cold storage, Transplanation 65, 551-559, 1998; Taha, M., Thermodynamic study of the second-stage dissociation of N,N-bis-(2-hydroxyethyl)glycine (bicine) in water at different ionic strength and different solvent mixtures, Ann.Chim. 94, 971-978, 2004; Taha, M., Buffers for the physiological pH range: acidic dissociation constants of zwitterionic compounds in various hydroorganic media, Ann.Chim. 95, 105-109, 2005; Williams, T.I., Combs, J.C., Thakur, A.P., et al., A novel Bicine running buffer system for doubled sodium dodecyl sulfate – polyacrylamide gel electrophoresis of membrane proteins, Electrophoresis 27, 2984-2995, 2006. Borate: Sodium Borate (sodium tetraborate/Boric Acid
Na2B4O7/H3BO3 Sodium borate decahydrate is borax)
61.8/ 201.2
Borate buffers have long history of use; borate well-known for interaction with carbohydrates; participates in the reversible modification of arginine residues by 1,2-cyclohexanedione
B(OH)4– + H3O+
B(OH)3 + 2H2O
Adjutantis, G., Electrophoretic separation of filter paper of the soluble liver-cell proteins of the rat using borate buffer, Nature 173, 539-540, 1954; Consden, R. and Powell, M.N., The use of borate buffer in paper electrophoresis of serum, J.Clin.Pathol. 8, 150-152, 1955; Cooper, D.R., Effect of borate buffer on the electrophoresis of serum, Nature 181, 713-714, 1958; Cooper, D.R., Effect of borate buffer on the electrophoresis of serum, Nature 181, 713-714, 1958; Poduslo, J.F., Glycoprotein molecular-weight estimation using sodium dodecyl sulfate-pore gradient electrophoresis: Comparison of Tris-glycine and Tris-borate-EDTA buffer systems, Anal.Biochem. 114, 131-139, 1981; Shukun, S.A. and Zav’yalov, V.P., Peculiar features of application of pH gradients formed in borate buffer with a polyhydroxy compound for separation of proteins in a free-flow electrophoretic apparatus, J.Chromatog. 496, 121-128, 1989; Patton, W.F., Chung-Welch, N., Lopez, M.F., et al., Tris-tricine and Tris-borate buffer systems provide better estimates of huma mesothelial cell intermediate filament protein molecular weights than the standard Tris-glycine system, Anal.Biochem. 197, 25-33, 1991; Roden, L., Yu, H., Jin, J., and Greenshields, J., Separation of N-acetylglucosamine and N-acetylmannosamine by chromatography on Sephadex in borate buffer, Anal.Biochem. 209, 188-191, 1993; Yokota, H., van den Engh, G., Mostert, M., and Trask, B.J., Treatment of cells with alkaline borate buffer extends the capability of interphase FISH mapping, Genomics 25, 485-491, 1995; Biyani, M. and Nishigaki, K., Sequence-specific and nonspecific mobilities of single-stranded oliogonucleotides observed by changing the borate buffer concentration, Electrophoresis 24, 628-633, 2003; Zhao, Y., Yang, X., Jiang, R. et al., Chiral separation of synthetic vicinal diol compounds by capillary zone electrophoresis with borate buffer and b-cyclodextrin as buffer additives, Anal.Sci. 22, 747-751, 2006. Articles focusing on the interaction of borate with carbohydrates and other polyols include: Zittle, C.A., Reaction of borate with substances of biological interest, Adv.Enzymol.Relat.Sub.Biochem. 12, 493-527, 1951; Larsson, U.B. and Samuelson, O. Anion exchange separation of organic acids in borate medium: influence of the temperature, J.Chromatog. 19, 404-411, 1965; Lin, F.M. and Pomeranz, Y., Effect of borate on colorimetric determinations of carbohydrates by the phenol-sulfuric acid method, Anal.Biochem. 24, 128-131, 1968; Haug, A., The influence of borate and calcium on the gel formation of a sulfated polysaccharide from Ulva lactuca, Acta Chem.Scand. B. 30, 562-566, 1976; Weitzman, S., Scott, V., and Keegstra, K., Analysis of glycoproteins as borate complexes by polyacrylamide gel electrophoresis, Anal.Biochem. 97, 438-449, 1979; Honda, S., Takahashi, M., Kakehi, K. and Ganno, S., Rapid, automated analysis of monosaccharides by high-performance anion-exchange chromatograpy of borate complexes with fluorometric detection using 2-cyanoacetamide, Anal.Biochem. 113, 130-138, 1981; Rothman, R.J. and Warren, L., Analysis of IgG glycopeptides by alkaline borate gel filtration chromatography, Biochim.Biophys.Acta 955, 143-153, 1988; Todd, P. and Elsasser, W., Nonamphometric isoelectric focusing: II. Stablity of borate-glycerol pH gradients in recycling isoelectric focusing, Electrophoresis 11, 947-952, 1990. Selected studies on the effect of borate on the modification of arginine with 1,2-cyclohexanedione include: Patthy, L. and Smith, E.L., Reversible modification of arginine residues. Application to sequence studies by restriction of tryptic hydrolysis to lysine residues, J.Biol.Chem. 250, 557-564, 1975; Patthy, L. and Smith, E.L., Identification of functional arginine residues in ribonuclease A and lysozyme, J.Biol.Chem. 250, 565-569,1975; Menegatti, E., Ferroni, R., Benassi, C.A., and Rocchi, R., Arginine modification in Kunitz bovine trypsin inhibitor through 1,2-cyclohexanedione, Int.J.Pept.Protein Res. 10, 146-152, 1977; Kozik, A., Guevara, I. and Zak, Z., 1,2-Cyclohexanedione modification of arginine residues in egg-white riboflavin-binding protein, Int.J.Biochem. 20, 707-711, 1988. Cacodylic Acid
Dimethylarsinic Acid
138.10
Buffer salt in neutral pH range; largely replaced because of toxicity.
O H3C
As
OH
CH3 McAlpine, J.C., Histochemical demonstration of the activation of rat acetylcholinesterase by sodium cacodylate and cacodylic acid using the thioacetic acid method, J.R.Microsc.Soc. 82, 95-106, 1963; Jacobson, K.B., Murphy, J.B., and Das Sarma, B., Reaction of cacodylic acid with organic thiols, FEBS Lett. 22, 80-82, 1972; Travers, F., Douzou, P., Pederson, T., and Gunsalus. I.C., Ternary solvents to investigate proteins at sub-zero temperature, Biochimie 57, 43-48, 1975; Young, C.W., Dessources, C., Hodas, S., and Bittar, E.S., Use of cationic disc electrophoresis near neutral pH in the evaluation of trace proteins in human plasma, Cancer Res. 35, 1991-1995, 1975; Chirpich, T.P., The effect of different buffers on terminal deoxynucleotidyl transferase activity, Biochim.Biophys.Acta 518, 535-538, 1978; Nunes, J.F., Aguas, A.P., and Soares, J.O., Growth of fungi in cacodylate buffer, Stain Technol. 55, 191-192, 1980; Caswell, A.H. and Bruschwig, J.P., Identification and extraction of proteins that compose the triad junction of skeletal muscle, J.Cell Biol. 99, 929-939, 1984; Parks, J.C. and Cohen, G.M., Glutaraldehyde fixatives for preserving the chick’s inner ear, Acta Otolaryngol. 98, 72-80, 1984; Song, A.H. and Asher, S.A., Internal intensity standards for heme protein UV resonance Raman studies: Excitation profiles of cacodylic acid and sodium selenate, Biochemistry 30, 1199-1205, 1991; Henney, P.J., Johnson, E.L., and Cothran, E.G., A new buffer system for acid PAGE typing of equine protease inhibitor, Anim.Genet. 25, 363-364, 1994; Jezewska, M.J., Rajendran, S., and Bujalowski, W., Interactions of the 8-kDa domain of rat DNA polymerase beta with DNA, Biochemistry 40, 3295-3307, 2001; Kenyon, E.M. and Hughes, M.F., A concise review of the toxicity and carcinogenicity of dimethylarsinic acid, Toxicology 160, 227-236, 2001; Cohen, S.M., Arnold, L.L., Eldan, M., et al., Methylated arsenicals: the implications of metabolism and carcinogenicity studies in rodents to human risk management, Crit.Rev.Toxicol. 99-133, 2006.
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698
LIST OF BUFFERS (Continued) Common Name
Chemical Name
CAPS
3-(cyclohexylamino)-1propanesulfonic acid O H N
M.W 221.3
Properties and Comment A zwitterionic buffer similar to a “Good” buffer
OH S O
Lad, P.J. and Leffert, H.L., Rat liver alcohol dehydrogenase. I. Purification and characterization, Anal.Biochem. 133, 350-361, 1983; Kaushal, V. and Barnes, L.D., Effect of zwitterionic buffers on measurement of small masses of protein with bicinchoninic acid, Anal.Biochem. 157, 291-294, 1986; Himmel, H.M. and Heller, W., Studies on the interference of selected substances with two modifications of the Lowry protein determination, J.Clin. Chem.Clin.Biochem. 25, 909-913, 1987; Nguyen, A.L., Luong, J.H., and Masson, C., Determination of nucleotides in fish tissues using capillary electrophoresis, Anal.Chem. 62, 2490-2493, 1990; Jin, Y. and Cerletti, N., Western blotting of transforming growth factor b2. Optimization of the electrophoretic transfer, Appl.Theor.Electrophor. 3, 85-90, 1992; Ng, L.T., Selwyn, M.J., and Choo, H.L., Effect of buffers and osmolality on anion uniport across the mitochondrial inner membrane, Biochim.Biophys.Acta 1143, 29-37, 1993; Venosa, R.A., Kotsias, B.A., and Horowicz, P., Frog striated muscle is permeable to hydroxide and buffer anions, J.Membr.Biol. 139, 57-74, 1994; Righetti, P.G., Bossi, A. and Gelfi, C., Capillary isoelectric focusing and isoelectric buffers: an evolving scenario, J.Capillary Electrophor. 4, 47-59, 1997; Bienvenut, W.V., Deon, C., Sanchez, J.C., and Hochstrasser, D.F., Anal.Biochem. 307, 297-303, 2002; Zaitseva, J., Holland, I.B., and Schmitt, L., The role of CAPS buffer in expanding the crystallization space of the nucleotide-binding domain of the ABC transporter haemolysin B from Escherichia coli, Acta Crystallogr.D.Biol.Crystallogr. 60, 1076-1084, 2004; Kannamkumarath, S.S., Wuilloud, R.G., and Caruso, J.A., Studies of various elements of nutritional and toxicological interest associated with different molecular weight fractions in Brazil nuts, J.Agric.Food Chem. 52, 5773-5780, 2004; Hautala, J.T., Wiedmer, S.K., and Riekkola, M.L., Influence of pH on formation and stability of phosphatidylcholine/phosphatidylserine coatings in fused-silica capillaries, Electrophoresis 26, 176-186, 2005; Taha, M., Buffers for the physiological pH range: acidic dissociation constants of zwitterionic compounds in various hydroorganic media, Ann.Chim. 95, 105-109, 2005; Tu, J., Halsall, H.B., Seliskar, C.J. et al., Estimation of logP(ow) values for neutral and basic compounds by microchip microemulsion electrokinetic chromatography with indirect fluorometric detection (muMEEKC-IFD), J.Pharm.Biomed.Anal. 38, 1-7, 2005. CAPSO
3-(Cyclohexylamino)-2-hydroxy-1propanesulfonic acid OH H N
O
237.3
A zwitterionic buffer similar to a “Good” buffer
OH S O
Delaney, J.P., Kimm, G.E., and Bonsack, M.E., The influence of lumenal pH on the severity of acute radiation enteritis, Int.J.Radiat.Biol. 61, 381-386, 1992; McGregor, D.P., Forster, S., Steven, J. et al., Simultaneous detection of microorganisms in soil suspension based on PCR amplification of bacterial 16S rRNA fragments, BioTechniques 21, 463-466, 1996; Liu, Q. Li, X., and Somer, S.S. pK-Matched running buffers for gel electrophoresis, Anal. Biochem. 270, 112-122, 1999; Quiros, M., Parker, M.C., and Turner, N.J., Tuning lipase enantioselectivity in organic media using solid-state buffers, J.Org.Chem. 66, 5074-5079, 2001; Okuda, M., Iwahori, K., Yamashita, I., and Yoshimura, H., Fabrication of nickel and chromium nanoparticles using the protein cage of apoferritin, Biotechnol.Bioeng. 84, 187-194, 2003; Vespalec, R., Vlckova, M., and Horakova, H., Aggregation and other intermolecular interactions of biological buffers observed by capillary electrophoresis and UV photometry, J.Chromatog.A 1051, 75-84, 2004; Taha, M., Buffers for the physiological pH range: acidic dissociation constants of zwitterionic compounds in various hydroorganic media, Ann.Chim. 95, 105-109, 2005. Carbonate
Sodium bicarbonate Sodium Carbonate Ammonium bicarbonate ammonium carbonate;
The ammonium salt system is a volatile buffer. The carbonate buffer is considered to be a physiological buffer. Bicarbonate buffers are used in renal dialysis.
Nagasawa, K. and Uchiyama, H., Preparation and properties of biologically active fluorescent heparins, Biochim.Biophys.Acta 544, 430-440, 1978; Horejsi, V. and Hilgert, I., Simple polyacrylamide gel electrophoresis in continuous carbonate buffer system suitable for the analysis of ascites fluids of hybridoma bearing mice, J.Immunol.Methods 86, 103-105, 1986; Chang, G.G. and Shiao, S.L., Possible kinetic mechanism of human placental alkaline phosphatase in vivo as implemented in reverse micelles, Eur.J. Biochem. 220, 861-870, 1994; Steinitz, M. and Tamir, S., An improved method to create nitrocellulose particles suitable for the immobilization of antigen and antibody, J.Immunol.Methods 187, 171-177, 1995; Wang, Z., Gurel, O., Baatz, J.E. and Notter, R.H., Acylation of pulmonary surfactant protein-C is required for its optimal surface active interactions with phospholipids, J.Biol.Chem. 271, 19104-19109, 1996; Petersen, A. and Steckhan, E., Continuous indirect electrochemical regeneration of galactose oxidase, Bioorg.Med.Chem. 7, 2203-2208, 1999; Medda, R., Padiglia, A., Messana, T., et al., Separation of diadenosine polyphosphates by capillary electrophoresis, Electrophoresis 21, 2412-2416, 2000; Bartzatt, R., Fluorescent labeling of drugs and simple organic compounds containing amine functional groups, utilizing dansyl chloride in Na2CO3 buffer, J.Pharmacol.Toxicol.Methods 45, 247-253, 2001; Bruno, F., Curini, R., Di Corcia, A., et al., Determination of surfactants and some of their metabolites in untreated and anaerobically digested sewage sludge by subcritical water extraction followed by liquid chromatography-mass spectrometry, Environ.Sci.Technol. 36, 4156-4161, 2002; Chen, X.L., Sun, C.Y., Zhang, Y.Z., and Gao, P.J., Effects of different buffers on the thermostability and autolysis of a cold-adapted proteases MCP-01, J.Protein Chem. 21,523-527, 2002; Duman, M., Saber, R., and Piskin, E., A new approach for immobilization of
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List of Buffers
699 LIST OF BUFFERS (Continued)
Common Name
Chemical Name
M.W
Properties and Comment
oligonucleotides onto piezoelectric quartz crystal for preparation of a nucleic acid sensor following hybridization, Biosens.Bioelectron. 18, 1355-1363, 2003; Talu, G.F. and Diyamandoglu, V., Formate ion decomposition in water under UV irradiation at 253.7 nm, Environ.Sci.Technol. 38, 3984-3993, 2004; Dwight, S.J., Gaylord, B.S., Hong, J.W., and Bazan, G.C., Perturbation of fluorescence by nonspecific interactions between anionic poly(phenylenevinylene)s and proteins. Implications for biosensors, J.Am.Chem.Soc. 126, 16850-16859, 2004; Willems, A.V., Deforce, D.L., Van Peteghem, C.H., and Van Bocxlaer, J.F.,, Development of a quality control method for the characterization of oligonucleotides by capillary zone electrophoresis-electrospray ionization-quadrupole time of flight-mass spectrometry, Electrophoresis 26, 1412-1423, 2005; Asberg, P.,Bjork, P., Hook, F., and Inganas, O., Hydrogels from a water-soluble zwitterionic polythiophene: dynamics under pH change and biomolecular interactions observed using quartz crystal microbalance with dissipation monitoring, Langmuir 21, 7292-7298, 2005; Shah, M., Meija, J., Cabovska, B.,, and Caruso, J.A., Determination of phosphoric acid triesters in human plasma using solid-phase microextraction and gas chromatography coupled to inductively coupled plasma mass spectrometry, J.Chromatog.A. 1103, 329-336, 2006; Di Pasqua, A.J., Goodisman, J., Kerwood, D.J. et al., Activation of carboplatin by carbonate, Chem.Res.Toxicol. 18, 139-149, 2006; Ormond, D.R. and Kral, T.A., Washing methogenic cells with the liquid fraction from a Mars soil stimulant and water mixture, J.Microbiol.Methods 67, 603-605, 2006; Binter, A., Goodisman, J., and Dabrowiak, J.C., Formation of monofunctional cisplatinin-DNA adducts in carbonate buffer, J.Inorg.Biochem. 100, 1219-1224, 2006. Alkaline carbonate buffers have been used as the medium for proteins for application to microplates for immunoassays such as ELISA assays (Rote, N.S., Taylor, N.L. Shigeoka, A.O., et al., Enzyme-linked immunosorbent assay for group B streptococcal antibodies, Infect. Immun. 27, 118-123, 1980; Hubschle, O.J., Lorenz, R.J., and Matheka, H.D., Enzyme-linked immunosorbent assay for detection of bluetongue virus antibodies, Am.J.Vet.Res. 42, 61-65, 1981; Solling, H., and Dinesen, B., The development of a rapid ELISA for IgE utilizing commercially available reagents, Clin.Chim.Acta 130, 71-83, 1983; Mowat, W.P. and Dawson, S., Detection and identification of plant viruses by ELISA using crude sap extracts and unfractionated antisera, J.Virol.Methods 15, 233-247, 1987; Ferris, N.P., Powell, H., and Donaldson, A.I., Use of pre-coated immunoplates and freeze-dried reagents for the diagnosis of foot-and-mouth disease and swine vesicular disease by enzyme-linked immunosorbent assay [ELISA], J.Virol.Methods 19,197-206, 1988; Cutler, S.J. and Wright, D.J., Comparison of immunofluorescence and enzyme linked immunosorbent assays for diagnosing Lyme disease, J.Clin.Pathol. 42, 869-871, 1989; Oshima, M. and Atassi, M.Z., Comparison of peptide-coating conditions in solid phase assays for detection of anti-peptide antibodies, Immunol.Invest. 18, 841-851, 1989; Martin, R.R., Relationships among luteoviruses based on nucleic acid hybridization and serological studies, Intervirology 31,23-30, 1990; Houen, G. and Koch, C., A non-denaturing enzyme linked immunosorbent assay with protein preadsorbed onto aluminum hydroxide, J.Immunol.Methods 200, 99-105, 1997; Shrivastav, T.G., Basu, A., and Kariya, K.P., Substitution of carbonate buffer by water for IgG immobilization in enzyme linked immunosorbent assay, J.Immunoassay Immunochem. 24, 191-203, 2003). Bicarbonate buffers also have an effect on the reaction of phenylglyoxal with proteins (Cheung, S.T. and Fonda, M.L., Reaction of phenylglyoxal with arginine. The effect of buffers and pH, Biochem.Biophys.Res.Commun. 90, 940-947, 1979). Bicarbonate also enhances the binding of iron to transferrin(Matinaho, S., Karhumäki, P., and Parkkinen, J., Bicarbonate inhibits the growth of Staphylococcus epidermidis in platelet concentrates by lowering the level of non-transferrin-bound iron, Transfusion 45, 1768-173, 2005 Cholamine
(2-aminoethyl) trimethylammonium chloride hydrochloride Me
Cl–
Me
N+ H2N
Me
Blasie, C.A. and Berg, J.M., Structure-based thermodynamic analysis of a coupled metal binding-protein folding reaction involving a zinc finger peptide, Biochemistry 41, 15068-15073, 2002; Zwiorek, K., Kloeckner, J., Wagner, E., and Coester, C., Gelatin nanoparticles as a new and simple gene delivery system, J.Pharm.Pharm.Sci. 7, 22-28, 2005. Citric Acid
HO
2-hydroxy-1,2,3propanetricarboxylic acid
H 2C
COOH
C
COOH
H 2C
COOH
192.1
Compounds found in a variety of biological tissues and cells; involved in energy metabolism (citric acid cycle; Krebs cycle; Krebs, H.A., The citric acid cycle and the Szent-Gyorgyi cycle in pigeon breast muscle, Biochem.J. 34, 775-779, 1940). Also used as biological buffer.
Citric acid has three carboxylic acid functions which permits buffering capacity from pH 2.0 to pH 12. Citric acid also chelate divalent cations and is used an anticoagulant for the collection of blood based its ability to chelate calcium ions. Chelation of metal ions is responsible for the observed inhibition of many enzymes. For early observations, see Smith, E.G., Dipeptidases, Methods Enzymol. 2, 93-114, 1955; McDonald, M.R., Deoxyribonucleases, Methods Enzymol. 2, 437-447, 1955; Kornberg, A., Adenosine phosphokinase, Methods Enzymol. 2, 497-500, 1955; Koshland, D.E., Jr., Preparation and properties of acetyl phosphatase, Methods Enzymol. 2, 556-556, 1955. The ability to chelate calcium serves as the basis for use as a decalcification agent. The polyvalent nature of citrate provide some unique characteristics such as the differentiation of muscle fiber types for histochemistry (Matoba, H., and Gollnick, P.D., Influence of ionic composition, buffering agent, and pH on the histochemical demonstration of myofibrillar actomyosin ATPase, Histochemistry 80, 609-614, 1984) and the activation of “prothrombin”( Seegers, W.H., McClaughery, R.I., and Fahey, J.L., Some properties of purified prothrombin and its activation with sodium citrate, Blood 5, 421-433, 1950; Lanchantin, G.F., Friedman, J.A., and Hart, D.W., The conversion of human prothrombin to thrombin by sodium citrate. Analysis of the reaction mixture, J.Biol.Chem. 240, 3276-3282, 1965; Aronson, D.L. and Mustafa, A.J., The activation of human factor X in sodium citrate: the role of factor VII, Thromb.Haemostas. 36, 104-114, 1976). Citrate is a polyvalent anion and like other polyvalent anions such as phosphate and sulfate, citrate can cause a “salting-out” phenomena (Hegardt, F.G. and Pie, A., Sodium citrate salting-out of the human blood serum proteins, Rev.Esp.Fisiol. 24, 161-168, 1968; Carrea, G., Pasta, P., and Vecchio, G., Effect of the lyotropic series of anions on denaturation and renaturation of 20-b-hydroxysteroid dehydrogenase, Biochim.Biophys.Acta 784, 16-23,
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700
LIST OF BUFFERS (Continued) Common Name
Chemical Name
M.W
Properties and Comment
1984; Nakano, T., Yuasa, H., and Kanaya, Y., Suppression of agglomeration in fluidized bed coating. III. Hofmeister series in suppression of particle agglomeration, Pharm.Res. 16, 1616-1620, 1999; Nakano, T. and Yuasa, H., Suppression of agglomeration in fluidized bed coating. IV. Effects of sodium citrate concentration on the suppression of particle agglomeration and the physical properties of HPMC film, Int.J.Pharm. 215, 3-12, 2001; Mani, N. and Jun, H.W., Microencapsulation of a hydrophilic drug into a hydrophobic matrix using a salting-out procedure. I: Development and optimization of the process using factorial design, J.Microencapsulation 21, 125-135, 2004). Citrate also has an effect on partitioning in aqueous two-phase systems (Andrews, B.A., Schmidt, A.S., and Asenjo, J.A., Correlation for the partition behavior of proteins in aqueous two-phase systems: effect of surface hydrophobicity and charge, Biotechnol.Bioeng. 90, 380-390, 2005). Citrate has proved useful in the solubilization of proteins, usually, but not always, from mineralized/calcified matrices (Faludi, E. and Harsanyi, V., The effect of Na3-citrate on the solubility of cryoprecipitate [citrate effect of cryoprecipitate], Haematologia 14, 207-214, 1981; Myllyla, R., Preparation of antibodies to chick-embryo galactosylhydroxylysyl glucosyltransferase and their use for an immunological characterization of the enzyme of collagen synthesis, Biochim.Biophys.Acta 658, 299-307, 1981; Guy, O., RoblesDiaz, G., Adrich, Z., et al., Protein content of precipitates present in pancreatic juice of alcoholic subjects and patients with chronic calcifying pancreatitis, Gastroenterology 84, 102-107, 1983; Collingwood, T.N., Shanmugam, M., Daniel, R.M., and Langdon, A.G., M[III]-facilitated recovery and concentration of enzymes from mesophilic and thermophilic organisms, J.Biochem. Biophys.Methods 19, 281-286, 1989). Citrate is useful for dissociating protein complexes in some situations by binding to specific anion binding sites; the ability of citrate to function as a buffering at low pH is an advantage [Kuo, T.T., Chow, T.Y., Lin, X.T., et al., Specific dissociation of phage Xp12 by sodium citrate, J.Gen.Virol. 10,199-202, 1971(in this case, the dissociation is reflection of metal ion binding; the dissociation is associated with the loss of biological activity – see Lark, K.G. and Adams, M.H., The stability of phage as a function of the ionic environment, Cold Spring Harbor Symposium on Quantitative Biology, 18, 171-183, 1953); Sheffery, M. and Newton, A., Reconstitution and purification of flagellar filaments from Caulobacter crescentus, J.Bacteriol. 132, 1027-1030, 1977; Brooks, S.P. and Nicholls, P., Anion and ionic strength effects upon the oxidation of cytochrome c by cytochrome c oxidase, Biochim.Biophys.Acta 680, 33-43, 1982; Berliner, L.J., Sugawara, Y., and Fenton, J.W., 2nd, Human alpha-thrombin binding to nonpolymerized fibrin-Sepharose: evidence for an anionic binding region, Biochemistry 24, 7005-7009, 1985; Kella, N.K. and Kinsella, J.E., Structural stability of beta-lactoglobulin in the presence of kosmotropic salts. A kinetic and thermodynamic study, Int.J.Pept.Protein Res. 32, 396-405, 1988; Oe, H., Takahashi, N., Doi. E., and Hirose, M., Effects of anion binding on the conformations of the two domains of ovotransferrin, J.Biochem. 106, 858-863, 1989; Polakova, K., Karpatova, M., and Russ, G., Dissociation of b-2-microglobulin is responsible for selective reduction of HLA class I antigenicity following acid treatment of cells, Mol.Immunol. 30, 1223-1230, 1993; Lecker, D.N. and Khan, A., Model for inactivation of a-amylase in the presence of salts: theoretical and experimental studies, Biotechnol.Prog. 14, 621-625, 1998; Rabiller-Baudry, M. and Chaufer, B., Small molecular ion adsorption on proteins and DNAs revealed by separation techniques, J.Chromatog.B.Analyt.Technol.Biomed.Life.Sci. 797, 331-345, 2003; Raibekas, A.A., Bures, E.J., Siska, C.C., et al., Anion binding and controlled aggregation of human interleukin-1 receptor antagonist, Biochemistry 44, 9871-9879, 2005). A special application of citrate dissociation of protein complexes is the isolation and dissociation of antigen-antibody complexes (Woodroffe, A.J. and Wilson, C.B., An evaluation of elution techniques in the study of immune complex glomerulonephritis, J.Immunol. 118, 1788-1794, 1977; Ehrlich, R. and Witz, I.P., The elution of antibodies from viable murine tumor cells, J.Immunol.Methods 26, 345-353, 1979; McIntosh, R.M., Garcia, R., Rubio, L., et al., Evidence of an autologous immune complex pathogenic mechanism in acute poststreptococcal glomerulonephritis, Kidney Int. 14, 501-510, 1978; Theofilopoulos, A.N., Eisenberg, R.A., and Dixon, F.J., Isolation of circulating immune complexes using Raji cells. Separation of antigens from immune complexes and production of antiserum, J.Clin.Invest. 61, 1570-1581, 1978; Tomino, Y., Sakai, H., Endoh, M., et al., Cross-reacivity of eluted antibodies from renal tissues of patients with Henoch-Schonlein purpura nephritis and IgA nephropathy, Am.J.Nephrol. 3, 315-318, 1983 ). A more complex and poorly understood application of citrate buffers is in epitope retrieval (Shi, S.R., Chaiwun, B., Young, L., et al., Antigen retrieval techniques utilizing citrate buffer or urea solution for immunohistochemical demonstration of androgen receptor in formalin-fixed paraffin sections, J.Histochem.Cytochem. 41, 1599-1604, 1993; Langlois, N.E., King, G., Herriot, R., and Thompson, W.D., Non-enzymatic retrieval of antigen permits staining of follicle centre cells by the rabbit polyclonal antibody to protein gene product 9.5, J.Pathol. 173, 249-253, 1994; Leong, A.S., Microwaves in diagnostic immunohistochemistry, Eur.J.Morphol. 34, 381-383, 1996; Lucas, D.R., al-Abbadi, M., Teabaczka, P., et al., c-Kit expression in desmoid fibroblastosis. Comparative immunohistochemical evaluation of two commercial antibodies, Am.J.Clin.Pathol. 119, 339-345, 2003). Additional work has indicated that citrate is useful but not unique for epitope retrieval ( Imam, S.A., Young, L., Chaiwun, B., and Taylor, C.B., Comparison of two microwave based antigen-retrieval solutions in unmasking epitopes in formalin-fixed tissues for immunostaining, Anticancer Res. 15, 1153-1158, 1995; Pileri, S.A., Roncador, G., Ceccarelli, C., et al., Antigen retrieval techniques in immunohistochemistry: comparison of different methods, J.Pathol. 183, 116-123, 1997; Rocken, C. and Roessner, A., An evaluation of antigen retrieval procedures for immunoelectron microscopic classification of amyloid deposits, J.Histochem.Cytochem. 47, 1385-1394, 1999). Citrate buffer has been useful in affinity chromatography (Ishikawa, K. and Iwai, K., Affinity chromatography of cysteine-containing histone, J.Biochem. 77, 391-398, 1975; Chadha, K.C., Grob, P.M., Mikulski, A.J., et al., Copper chelate affinity chromatography of human fibroblast and leucocyte interferons, J.Gen.Virol. 43, 701-706, 1979; Tanaka, H., Sasaki, I., Yamashita, K. et al., Affinity chromatography of porcine pancreas deoxyribonuclease I on DNA-binding Sepharose under non-digestive conditions, using its substrate-binding site, J.Biochem. 88, 797-806, 1980 Smith, R.L. and Griffin, C.A., Separation of plasma fibronectin from associated hemagglutinating activity by elution from gelatin-agarose at pH 5.5, Thromb. Res. 37, 91-101, 1985). Citrate is also used for immunoaffinity chromatography including chromatography on Protein A (Martin, L.N., Separation of guinea pig IgG subclasses by affinity chromatography on protein A-Sepharose, J.Immunol.Methods 52, 205-212, 1982; Compton, B.J., Lewis, M.A., Whigham, F., et al., Analtyical potential of protein A for affinity chromatography of polyclonal and monoclonal antibodies, Anal.Chem. 61, 1314-1317, 1989; Giraudi, G. and Baggiani, C. Strategy for fractionating high-affinity antibodies to steroid hormones by affinity chromatography, Analyst 121, 939-944, 1996; Arakawa, T., Philo, J.S., Tsumoto, K., et al., Elution of antibodies from a Protein-A column by aqueous arginine solutions, Protein Expr. Purif. 36, 244-248, 2004; Ghose, S., McNerney, T., and Hubbard, B., Protein A affinity chromatography for capture and purification of monoclonal antibody and Fc-fusion protein: Practical considerations for process development, in Process Scale Bioseparations for the Biopharmaceutical Industry, ed. A.A. Shukla, M.R. Etzel, and S. Gadam, , ed. A.A. Shukla, M.R. Etzel, and S. Gadam, CRC/Taylor & Francis, Boca Raton, FL., Chapter 16, pps. 462-489, 2007).
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List of Buffers
701 LIST OF BUFFERS (Continued)
Common Name
Chemical Name
HEPES
4-(2-hydroxyethyl)-1piperizineethanesulfonic acid
M.W 238.3
Properties and Comment a “Good” buffer; reagent purity has been an issue; metal ion binding must be considered; there are bufferspecific effects which are poorly understood; component of tissue fixing technique
OH O
S
O
N
N
OH
Good, N.E., Winget, G.D., Winter, W., et al., Hydrogen ion buffers for biological research, Biochemistry 5, 467-477, 1966; Turner, L.V. and Manchester, K.L., Interference of HEPES with the Lowry method, Science 170, 649, 1970; Chirpich, T.P., The effect of different buffers on terminal deoxynucleotidyl transferase activity, Biochim.Biophys.Acta 518, 535-538, 1978; Tadolini, B., Iron autoxidation in Mops and Hepes buffers, Free Radic.Res. Commun. 4, 149-160, 1987; Simpson, J.A., Cheeseman, K.H., Smith, S.E., and Dean, R.T., Free-radical generation by copper ions and hydrogen peroxide. Stimulation by Hepes buffer, Biochem.J. 254, 519-523, 1988; Abas, L. and Guppy M., Acetate: a contaminant in Hepes buffer, Anal.Biochem. 229, 131-140, 1995; Schmidt, K., Pfeiffer, S., and Mayer, B., Reaction of peroxynitrite with HEPES or MOPS results in the formation of nitric oxide donors, Free Radic.Biol.Med. 24, 859-862, 1998; Wiedorn, K.H., Olert, J., Stacy, R.A., et al., HOPE – a new fixing technique enables preservation and extraction of high molecular weight DNA and RNA of >20 kb from paraffin-embedded tissues. Hepes-glutamic acid buffer mediated Organic solvent Protection Effect, Pathol.Res.Pract. 198, 735-740, 2002; Fulop, L., Szigeti, G., Magyar, J., et al., Differences in electrophysiological and contractile properties of mammalian cardiac tissues bathed in bicarbonate – and HEPES-buffered solutions, Acta Physiol.Scand.178, 11-18, 2003; Mash, H.E., Chin, Y.P., Sigg, L., et al., Complexation of copper by zwitterionic aminosulfonic (good) buffers, Anal.Chem. 75, 671-677, 2003 Sokolowska, M., and Bal, W., Cu(II) complexation by “non-coordinating” N-2-hydroxyethylpiperazine-N’-ethanesulfonic acid (HEPES buffer), J.Inorg.Biochem. 99, 1653-1660, 2005; ; Zhao, G. and Chasteen, N.D., Oxidation of Good’s buffers by hydrogen peroxide, Anal.Biochem. 349, 262-267, 2006; Hartman, R.F. and Rose, S.D., Kinetics and mechanism of the addition of nucleophiles to alpha,beta-unsaturated thiol esters, J.Org.Chem. 71, 6342-6350, 2006. MES
1-morpholineethanesulfonic acid; 2-(4-morpholino)ethanesulfonate
198.2
A “Good” buffer
O
O
N S O
OH
Good, N.E., Winget, G.D., Winter, W., et al., Hydrogen ion buffers for biological research, Biochemistry 5, 467-477, 1966; Bugbee, B.G. and Salisbury, F.B., An evaluation of MES (2(N-morpholino)ethanesulfonic acid) and Amberlite 1RC-50 as pH buffers for nutrient growth studies, J.Plant Nutr. 8, 567-583, 1985; Kaushal, V. and Barnes, L.D., Effect of zwitterionic buffers on measurement of small masses of proiten with bicinchoninic acid, Anal. Biochem. 157, 291-294, 1986; Grady, J.K., Chasteen, N.D., and Harris, D.C., Radicals from “Good’s” buffers, Anal.Biochem. 173, 111-115, 1988; Le Hir, M., Impurity in buffer substances mimics the effect of ATP on soluble 5’-nucleotidase, Enzyme 45, 194-199, 1991; Pedrotti, B., Soffientini, A., and Islam, K., Sulphonate buffers affect the recovery of microtubule-associated proteins MAP1 and MAP2: evidence that MAP1A promotes microtubule assembly, Cell Motil.Cytoskeleton 25, 234-242, 1993; Vasseur, M., Frangne, R., and Alvarado, F., Buffer-dependent pH sensitivity of the fluorescent chloride-indicator dye SPQ, Am.J.Physiol. 264, C27-C31, 1993; Frick, J. and Mitchell, C.A., Stabilization of pH in solid-matrix hydroponic systems, HortScience 28, 981-984, 1993; Yu, Q., Kandegedara, A., Xu, Y., and Rorabacher, D.B., Avoiding interferences from Good’s buffers: A continguous series of noncomplexing tertiary amine buffers covering the entire range of pH 3-11, Anal.Biochem. 253, 50-56, 1997; Gelfi, C., Vigano, A., Curcio, M., et al., Single-strand conformation polymorphism analysis by capillary zone electrophoresis in neutral pH buffer, Electrophoresis 21, 785-791, 2000; Walsh, M.K., Wang, X., and Weimer, B.C., Optimizing the immobilization of single-stranded DNA onto glass beads, J.Biochem.Biophys.Methods 47, 221-231, 2001; Hosse, M. and Wilkinson, K.J., Determination of electrophoretic mobilities and hydrodynamic radii of three humic substances as a function of
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702
LIST OF BUFFERS (Continued) Common Name
Chemical Name
M.W
Properties and Comment
pH and ionic strength, Environ.Sci.Technol. 35, 4301-4306, 2001; Mash, H.E., Chin, Y.P., Sigg, L., et al., Complexation of copper by zwitterionic aminosulfonic (good) buffers, Anal.Chem. 75, 671-677, 2003; Ozkara, S., Akgol, S., Canak, Y., and Denizli, A., A novel magnetic adsorbent for immunoglobulin-g purification in a magnetically stabilized fluidized bed, Biotechnol.Prog. 20, 1169-1175, 2004; Hachmann, J.P. and Amshey, J.W., Models of protein modification in Tris-glycine and neutral pH Bis-Tris gels during electrophoresis: effect of pH, Anal.Biochem. 342, 237-345, 2005; Krajewska, B. and Ciurli, S., Jack bean (Canavalia ensiformis) urease. Probing acid-base groups of the active site by pH variation, Plant Physiol. Biochem. 43, 651-658, 2005; Zhao, G. and Chasteen, N.D., Oxidation of Good’s buffers by hydrogen peroxide, Anal.Biochem. 349, 262-267, 2006. MOPS
3-(N-morpholino)propanesulfonic acid; 4-morpholinepropanesulfonic acid
209.3
A “Good” buffer
O O
S
N O
OH
Good, N.E., Winget, G.D., Winter, W., et al., Hydrogen ion buffers for biological research, Biochemistry 5, 467-477, 1966; Altura, B.M., Altura, B.M., Carella, A. and Altura, B.T., Adverse effects of Tris, HEPES and MOPS buffers on contractile responses of arterial and venous smooth muscle induced by prostaglandins, Prostaglandins Med. 5, 123-130, 1980; Tadolini, B., Iron autoxidation in Mops and Hepes buffers, Free Radic.Res.Commun. 4, 149-160, 1987;Tadolini, B. and Sechi, A.M., Iron oxidation in Mops buffer. Effect of phosphorus containing compounds, Free Radic.Res.Commun. 4, 161-172, 1987; Tadolini, B., Iron oxidation in Mops buffer. Effect of EDTA,. hydrogen peroxide and FeCl3, Free Radic.Res.Commun. 4, 172-182, 1987; Ishihara, H. and Welsh, M.J., Block by MOPS reveals a conformation change in the CFTR pore produced by ATP hydrolysis, Am.J.Physiol. 273, C1278-C1289, 1997; Schmidt, K., Pfeiffer, S., and Meyer, B., Reaction of peroxynitrite with HEPES or MOPS results in the formation of nitric oxide donors, Free Radic.Biol.Med. 24, 859-862, 1998; Hodges, G.R. and Ingold, K.U., Superoxide, amine buffers and tetranitromethane: a novel free radical chain reaction, Free Radic.Res. 33, 547-550, 2000; Corona-Izquierdo, F.P. and Membrillo-Hernandez, J., Biofilm formation in Escherichia coli is affected by 3-(N-morpholino)propane sulfonate (MOPS), Res.Microbiol. 153, 181-185, 2002; Mash, H.E., Chin, Y.P., Sigg, L., et al., Complexation of copper by zwitterionic aminosulfonic (good) buffers, Anal.Chem. 75, 671-677, 2003; Denizli, A., Alkan, M., Garipcan, B., et al., Novel metal-chelate affinity adsorbent for purification of immunoglobulin-G from human plasma, J.Chromatog.B.Analyt.Technol.Biomed.Life.Sci. 795, 93-103, 2003; Emir, S., Say, R., Yavuz, H., and Denizli, A., A new metal chelate affinity adsorbent for cytochrome C, Biotechnol.Prog. 20, 223-228, 2004; Cvetkovic, A., Zomerdijk, M., Straathof, A.J., et al., Adsorption of fluorescein by protein crystals, Biotechnol.Bioeng. 87, 658-668, 2004; Zhao, G. and Chasteen, J.D., Oxidation of Good’s buffers by hydrogen peroxide, Anal.Biochem. 349, 2620267, 2006; Vrakas, D., Giaginis, C. and Tsantili-Kakoulidou, A., Different retention behavior of structurally diverse basic and neutral drugs in immobilized artificial membrane and reversed-phase high performance liquid chromatography: comparison with octanol-water partitioning, J.Chromatog.A. 1116, 158-164, 2006; de Carmen Candia-Plata, M., Garcia, J., Guzman, R., et al., Isolation of human serum immunoglobulins with a new salt-promoted adsorbent, J.Chromatog.A. 1118, 211-217, 2006. Phosphate Phosphate buffers are among the most common buffers used for biological studies. It is noted that the use of phosphate solutions in early transfusion medicine lead to the discovery of the importance of calcium ions in blood coagulation (Hutchin, P., History of blood transfusion: A tercentennial look, Surgery 64, 685-700, 1968). Phosphate-buffer saline (PBS; generally 0.01 M sodium phosphate – 0.14 M NaCl, pH 7.2 – Note, an incredible variation in PBS exists so it is necessary to verify composition – the only common factor that this writer finds is 0.01 M (10 mM) phosphate) is extensively used. Sodium phosphate buffers are the most common but there is extensive use of potassium phosphate buffers and mixtures of sodium and potassium. Unfortunately many investigators simply refer to phosphate buffer without respect to counter ion. Also, investigators will prepare a stock solution of sodium phosphate[usually sodium dihydrogen phosphate (sodium phosphate, monobasic) or disodium hydrogen phosphate (sodium phosphate, dibasic) and adjust pH as required with (usually) hydrochloric acid and/or sodium hydrogen. This is not preferable and, if used, must be described in the text to permit other investigators to repeat the experiment. pH changes in phosphate buffers during freezing can be dramatic due to precipitation of phosphate buffer salts (van den Berg, L. and Rose, D., Effect of freezing on the pH and composition of sodium and potassium phosphate solutions: The reciprocal system KH2PO4-Na2PO4-H2O, Arch.Biochem.Biophys. 81, 319-329, 1959; Murase, N. and Franks, F., Salt precipitation during the freeze-concentration of phosphate buffer solutions, Biophys.Chem. 34, 393-300, 1989; Pikal-Cleland, K.A. and Carpenter, J.F., Lyophilization-induced protein denaturation in phosphate buffer systems: monomeric and tetrameric beta-galactosidase, J.Pharm.Sci. 90, 1255-1268, 2001; Gomez, G., Pikal, M., and Rodriguez-Hornedo, N., Effect of initial buffer composition on pH changes during far-from-equilibrium freezing of sodium phosphate buffer solutions, Pharm.Res. 18, 90-97, 2001; Pikal-Cleland, K.A., Cleland, J.L., Anchorodoquy, T.J. and Carpenter, J.F., Effect of glycine on pH changes and protein stability during freeze-thawing in phosphate buffer systems, J.Pharm .Sci. 91, 1969-1979, 2002). Phosphate bind divalent cations in solutions
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List of Buffers
703 LIST OF BUFFERS (Continued)
Common Name
Chemical Name
M.W
Properties and Comment
and can form insoluble salts. Phosphate influences biological reactions by binding cations such as calcium, platinum and iron (Staum, M.M., Incompatibility of phosphate buffer in 99m Tc-sulfur colloid containing aluminum ion, J.Nucl.Med. 13, 386-387, 1972; Frank, G.B., Antagonism by phosphate buffer of the twitch ions in isolated muscle fibers produced by calcium-free solutions, Can.J.Physiol.Pharmacol. 56, 523-526, 1978; Hasegawa, K., Hashi, K., and Okada, R., Physicochemical stability of pharmaceutical phosphate buffer solutions. I. Complexation behavior of Ca(II) with additives in phosphate buffer solutions, J.Parenter.Sci.Technol. 36, 128-133, 1982; Abe, K., Kogure, K., Arai, H., and Nakano, M., Ascorbate induced lipid peroxidation results in loss of receptor binding in tris, but not in phosphate, buffer. Implications for the involvement of metal ions, Biochem.Int. 11, 341-348, 1985; Pedersen, H.B., Josephsen, J., and Keerszan, G., Phosphate buffer and salt medium concentrations affect the inactivation of T4 phage by platinum(II) complexes, Chem.Biol.Interact. 54, 1-8, 1985; Kuzuya, M., Yamada, K., Hayashi, T., et al., Oxidation of low-density lipoprotein by copper and iron in phosphate buffer, Biochim.Biophys.Acta 1084, 198-201, 1991. Also see Wolf, W.J., and Sly, D.A., Effects of buffer cations on chromatography of proteins on hydroxylapatite, J.Chromatog. 15, 247-250, 1964; Taborsky, G., Oxidative modification of proteins in the presence of ferrous ion and air. Effect of ionic constituents of the reaction medium on the nature of the oxidation products, Biochemistry 12, 1341-1348, 1973; Millsap, K.W., Reid, G., van der Mei, H.C., and Busscher, H.J., Adhesion of Lactobacillus species in urine and phosphate buffer to silicone rubber and glass under flow, Biomaterials 18, 87-91, 1997; Gebauer, P. and Bocek, P., New aspects of buffering with multivalent weak acids in capillary zone electrophoresis: pros and cons of the phosphate buffer, Electrophoresis 21, 2809-2813, 2000; Gebauer, P., Pantuikova, P. and Bocek, P., Capillary zone electrophoresis in phosphate buffer – known or unknown?, J.Chromatog.A 894, 89-93, 2000; Buchanan, D.D., Jameson, E.E., Perlette, J., et al., Effect of buffer, electric field, and separation time on detection of aptamers-ligand complexes for affinity probe capillary electrophoresis, Electrophoresis 24, 1375-1382, 2003; Ahmad, I., Fasihullah, Z. and Vaid, F.H., Effect of phosphate buffer on photodegradation reactions of riboflavin in aqueous solution, J.Photochem.Photobiol.B 78, 229-234, 2005. PIPES
piperazine-N,N’-bis(2ethanesulfonic acid) 1,4-piperazinediethane sulfonic acid O
302.4
A “Good” buffer
OH S
N O
O
N S
HO
O
Good, N.E., Winget, G.D., Winter, W., et al., Hydrogen ion buffers for biological research, Biochemistry 5, 467-477, 1966; Olmsted, J.B. and Borisy, G.G., Ionic and nucleotide requirements for microtubule polymerization in vitro, Biochemistry 14, 2996-3005, 1975; Baur, P.S. and Stacey, T.R., The use of PIPES buffer in the fixation of mammalian and marine tissues for electron microscopy, J.Micros. 109, 315-327, 1977; Schiff, R.I. and Gennaro, J.F., Jr., The influence of the buffer on maintenance of tissue liquid in specimens for scanning electron microscopy, Scan.Electron Microsc. (3), 449-458, 1979; Altura, B.M., Altura, B.T., Carella, A., and Turlapty, P.D., Adverse effects of artificial buffers on contractile responses of arterial and venous smooth muscles, Br.J.Pharmacol. 69, 207-214, 1980; Syvertsen, C. and McKinley-McKee, J.S., Affinity labeling of liver alcohol dehydrogenase. Effects of pH and buffers on affinity labelling with iodoacetic acid and (R,S-2-bromo-3-(5-imidazolyl)propionic acid, Eur.J.Biochem. 117, 165-170, 1981; Roy, R.N., Gibbons, J.J., Padron, J.L., et al., Revised values of the paH of monosodium 1,4-piperazinediethanesulfonate (“Pipes”) in water other buffers in isotonic saline at various temperatures, Clin.Chem. 27, 1787-1788, 1981; Waxman, P.G., del Campo, A.A., Lowe, M.C., and Hamel, E., Induction of polymerization of purified tubulin by sulfonate buffers. Marked differences between 4-morpholineethananesulfonate (Mes) and 1,4-piperazineethanes ulfonate(Pipes), Eur.J.Biochem. 129, 129-136, 1981; Yamamoto, K. and Ogawa, K., Effects of NaOH-PIPES buffer used in aldehyde fixative on alkaline phosphatase activity in rat hepatocytes, Histochemistry 77, 339-351, 1983; Haviernick, S., Lalague, E.D., Corvellec, M.R., et al., The use of Hanks’— pipes buffers in the preparation of human, normal leukocytes for TEM observation, J.Microsc. 135, 83-88, 1984; Simpson, J.A., Cheeseman, K.H., Smith, S.E., and Dean, R.T., Free-radical generation by copper ions and hydrogen peroxide. Stimulation by Hepes buffers, Biochem.J. 254, 519-523, 1988; Prutz, W.A. The interaction between hydrogen peroxide and the DNA-Cu(I) complex: effects of pH and buffers, Z.Naturforsch. 45, 1197-1206, 1990; Le Hir, M., Impurity in buffer substances mimics the effects of ATP on soluble 5’-nucleotidase, Enzyme 45, 194-199, 1991; Lee, B.H. and Nowak, T., Influence of pH on the Mn2+ activation of and binding to yeast enolase: a functional study, Biochemistry 31, 2165-2171, 1992; Tedokon, M., Suzuki, K., Kayamori, Y., et al., Enzymatic assay of inorganic phosphate with the use of sucrose phosphorylase and phosphoglucomutase, Clin.Chem. 38, 512-515, 1992; Correla, J.J., Lipscomb, L.D., Dabrowiak, J.C., et al., Cleavage of tubulin by vandate ion, Arch.Biochem.Biophys. 309, 94-104, 1994; Schmidt, J., Mangold, C., and Deitmer, J., Membrane responses evoked by organic buffers in identified leech neurones, J.Exp.Biol. 199,327-335, 1996; Yu, Q., Kandegedara, A., Xu, Y., and Rorabacher, D.B., Avoiding interferences from Good’s buffers: A contiguous series of noncomplexing tertiary amine buffers covering the entire pH range of pH 3-11, Anal.Biochem. 253, 50-56, 1997; Rover Junior, L., Fernandes, J.C., de Oliveira Neto, G., et al., Study of NADH stability using ultraviolet-visible spectrophotometric analysis and factorial design, Anal.Biochem. 260, 50-55, 1998; Moore, S.A., Kingston, R.L., Loomes, K.M., et al., The structure of truncated recombinant human bile salt-stimulated lipase reveals bile salt-independent conformational flexibility at the active-site loop and provides insights into heparin binding, J.Mol.Biol. 3 12, 511-523, 2001; Sani, R.K., Peyton, B.M., and Dohnalkova, A., Toxic effects of uranium on Desulfovibrio desulfuricans G20, Environ.Toxicol.Chem. 25, 1231-1238, 2006.
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704
LIST OF BUFFERS (Continued) Common Name
Chemical Name
TES
N-tris(hydroxymethyl) methyl-2aminoethane-sulfonic acid
M.W 229.3
Properties and Comment A “Good” buffer.
OH
O S
HO
O
NH HO OH Good, N.E., Winget, G.D., Winter, W., et al., Hydrogen ion buffers for biological research, Biochemistry 5, 467-477, 1966; Itagaki, A. and Kimura, G., Tes and HEPES buffers in mammalian cell cultures and viral studies: problem of carbon dioxide requirement, Exp.Cell Res. 83, 351-361, 1974; Bridges, S. and Ward, B., Effect of hydrogen ion buffers on photosynthetic oxygen evolution in the blue-green alga, Agmenellum quadruplicatum, Microbios 15, 49-56, 1976; Bailyes, E.M., Luzio, J.P., and Newby, A.C., The use of a zwitterionic detergent in the solubilization and purification of the intrinsic membrane protein 5’-nucleotidase, Biochem.Soc.Trans. 9, 140-141, 1981; Poole, C.A., Reilly, H.C., and Flint, M.H., The adverse effects of HEPES, TES, and BES zwitterionic buffers on the ultrastructure of cultured chick embryo epiphyseal chondrocytes, In Vitro 18, 755-765, 1982; Nakon, R. and Krishnamoorthy, C.R., Free-metal ion depletion by “Good’s” buffers, Science 221, 749-750, 1983; del Castillo, J., Escalona de Motta, G., Eterovic, V.A., and Ferchmin, P.A., Succinyl derivatives of N-tris (hydroxylmethyl) methyl-2-aminoethane sulphonic acid: their effects on the frog neuromuscular junction, Br.J.Pharmacol. 84, 275-288, 1985; Kaushal, V. and Varnes, L.D., Effect of zwitterionic buffers on measurement of small masses of protein with bicinchoninic acid, Anal.Biochem. 157, 291-294, 1986; Bhattacharyya, A. and Yanagimachi, R., Synthetic organic pH buffers can support fertilization of guinea pig eggs, but not as efficiently as bicarbonate buffer, Gamete Res. 19, 123-129, 1988; Veeck, L.L., TES and Tris (TEST)-yolk buffer systems, sperm function testing, and in vitro fertilization, Fertil.Steril. 58, 484-486, 1992; Kragh-Hansen, U. and Vorum, H., Quantitative analyses of the interaction between calcium ions and human serum albumin, Clin.Chem. 39, 202-208, 1993; Jacobs, B.R., Caulfield, J., and Boldt, J., Analysis of TEST (TES and Tris) yolk buffer effects of human sperm, Fertil.Steril. 63, 1064-1070, 1995; Stellwagne, N.C., Bossi, A., Gelfi, C., and Righetti, P.G., DNA and buffers: are there any noninteracting, neutral pH buffers?, Anal.Biochem. 287, 167-175, 2000; Taylor, J., Hamilton, K.L., and Butt, A.G., HCO3- potentiates the cAMP-dependent secretory response of the human distal colon through a DIDS-sensitive pathway, Pflugers Arch. 442, 256-262, 2001; Taha, M., Buffers for the physiological pH range: acidic dissociation constants of zwitterionic compounds in various hydroorganic media, Ann. Chim. 95, 105-109, 2005. Tricine
N-[tris(hydroxymethyl) methyl] glycine; N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl] glycine
179.2
A “Good” buffer which is also used as a chelating agent, useful for cupric ions. Tricine is also used to complex technetium-99(99mTc) in cancer therapy.
OH
HO
C
NH
O
HO HO Garder, R.S., The use of tricine buffer in animal tissue cultures, J.Cell Biol. 42, 320-321, 1969; Spendlove, R.S., Crosbie, R.B., Hayes, S.F., and Keeler, R.F., TRICINE-buffered tissue culture media for control of mycoplasma contaminants, Proc.Soc.Exptl.Biol.Med. 137, 258-263, 1971; Bates, R.G., Roy, R.N., and Robinson, R.A., Buffer standards of tris(hydroxymethyl)methylglycine (“tricine”) for the physiological range pH 7.2 to 8.5, Anal.Chem. 45, 1663-1666, 1973; Roy, R.N., Robinson, R.A., and Bates, R.G., Thermodynamics of the two dissociation steps of N-tris(hydroxymethyl)methylglycine (“tricine”) in water from 5 to 50 degrees, J.Amer.Chem.Soc. 95, 8231-8235, 1973; Grande, H.J. and van der Ploeg, K.R., Tricine radicals as formed in the presence of peroxide producing enzymes, FEBS Lett. 95, 352-356, 1978; Roy, R.N., Gibbons, J.J., and Baker, G.E., Acid dissociation constants and pH values for standard “bes” and “tricine” buffer solutions in 30, 40, and 50 mass% dimethyl sulfoxide/water between 25 and -25°C, Cryobiology 22, 589-600, 1985; Hall, M.S. and Leach, F.R., Stability of firefly luciferase in tricine buffer and in a commercial enzyme stabilizer, J.Biolumin.Chemilumin. 2, 41-44, 1988; Patton, W.F., Chung-Welch, N., Lopez, M.F., et al,, Tris-tricine and tris-borate buffer systems provide better estimates of human mesothelial cell intermediate filament protein molecular weights than the standard Tris-glycine system, Anal.Biochem. 197, 25-33, 1991; [99mTc] tricine: a useful precursor complex for the radiolabeling of hydrazinonicotinate protein conjugates, Bioconjugate Chem. 6, 635-638, 1995; Wisdom, G.B., Molecular weight determinations using polyacrylamide gel electrophoresis with tris-tricine buffers, Methods Mol.Biol. 73, 97-100, 1997; Barrett, J.A., Crocker, A.C., Damphousee, D.J.. Biological evaluation of thrombus imaging agents utilizing water soluble phospines and tricine as coligands when used to label a hydrazinonicotinamide-modified cyclic glycoprotein IIb/IIIa receptor antagonist with 99mTc, Bioconjug.Chem. 8, 155-160, 1997; Bangard, M., Behe, M., Guhlke, S., et al., Detection of somatostatin receptor-positive tumours using the new 99mTc-tricine-HYNIC-D-Phe1-Tyr3octreotide: first results in patients and comparison with 111In-D-Phe1-octreotide, Eur.J.Nucl.Med. 27, 628-637, 2000; Ramos silva, M., Paixao, J.A. , Matos Beja, A., and Alte da Veiga, L., Conformational flexibility of tricine as a chelating agent in catena-poly-[[(tricinato)copper(II)]-mu-chloro], Acta Crystallogr.C. 57, 9-11, 2001; Silva, M.R., Paixo, J.A., Beja, A., and Alte da Veiga, L., N-[Tris(hydroxymethyl)methyl]glycine(tricine), Acta Crystallogr.C.
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List of Buffers
705 LIST OF BUFFERS (Continued)
Common Name
Chemical Name
M.W
Properties and Comment
57, 421-422, 2001; Su, Z.F., He, J., Rusckowski, M., and Hnatowich, D.J., In vitro cell studies of technetium-99m-labeled RGD-HYNIC peptide, a comparison of tricine and EDDA as co-ligands, Nucl.Med.Biol. 30, 141-149, 2003; Le, Q.T. and Katunuma, N., Detection of protease inhibitors by a reverse zymography method, performed in a tris(hydroxylmethyl)aminomethane-Tricine buffer system, Anal.Biochem. 324, 237-240, 2004. Triethanolamine
tris(2-hydroxyethyl) amine
HO
149.2
Buffer; transdermal transfer reagent
OH N
OH Fitzgerald, J.W., The tris-catalyzed isomerization of potassium D-glucose 6-O-sulfate, Can.J.Biochem. 53, 906-910, 1975; Buhl, S.N., Jackson, K.Y., and Graffunder, B., Optimal reaction conditions for assaying human lactate dehydrogenase pyruvate-to-lactate at 25, 30, and 37 degrees C, Clin.Chem. 24, 261-266, 1978; Myohanen, T.A., Bouriotas, V., and Dean, P.D. Affinity chromatography of yeast alpha-glucosidase using ligand-mediated chromatography on immobilized phenylboronic acids, Biochem.J. 197, 683-688, 1981; Shinomiya, Y., Kato, N., Imazawa, M., and Miyamoto, K., Enzyme immunoassay of the myelin basic protein, J.Neurochem. 39, 1291-1296, 1982; Arita, M., Iwamori, M., Higuchi, T., and Nagai, Y., 1,1,3,3-tetramethylurea and triethanolaminme as a new useful matrix for fast atom bombardment mass spectrometry of gangliosides and neutral glycosphingolipids, J.Biochem. 93, 319-322, 1983; Cao, H. and Preiss, J., Evidence for essential arginine residues at the active site of maize branching enzymes, J.Protein Chem. 15, 291-304, 1996; Knaak, J.B., Leung, H.W., Stott, W.T., et al., Toxicology of mono-, di-, and triethanolamine, Rev.Environ. Contim.Toxicol. 149, 1-86, 1997; Liu, Q., Li, X., and Sommer, S.S., pK-matched running buffers for gel electrophoresis, Anal.Biochem. 270, 112-122, 1999; Sanger-van de Griend, C.E., Enantiomeric separation of glycyl dipeptides by capillary electrophoresis with cyclodextrins as chiral selectors, Electrophoresis 20, 3417-3424, 1999; Fang, L., Kobayashi, Y., Numajiri, S., et al., The enhancing effect of a triethanolamine-ethanol-isopropyl myristate mixed system on the skin permeation of acidic drugs, Biol.Pharm.Bull. 25, 1339-1344, 2002; Musial, W. and Kubis, A., Effect of some anionic polymers of pH of triethanolamine aqueous solutions, Polim.Med. 34, 21-29, 2004. Triethylamine
N,N-diethylethanamine
101.2
ion-pair reagent; buffer
N
Brind, J.L., Kuo, S.W., Chervinsky, K., and Orentreich, N., A new reversed phase, paired-ion thin-layer chromatographic method for steroid sulfate separations, Steroids 52, 561-570, 1988; Koves, E.M., Use of high-performance liquid chromatography-diode array detection in forensic toxicology, J.Chromatog.A 692, 103-119, 1995; Cole, S.R. and Dorsey, J.G., Cyclohexylamine additives for enhanced peptide separations in reversed phase liquid chromatography, Biomed.Chromatog. 11, 167-171, 1997; Gilar, M., and Bouvier, E.S.P., Purification of crude DNA oligonucleotides by solid-phase extraction and reversed-phase high-performance liquid chromatography, J.Chromatog.A 890, 167-177, 2000; Loos, R. and Barcelo, D., Determination of haloacetic acids in aqueous environments by solid-phase extraction followed by ion-pair liquid chromatography-electrospray ionization mass spectrometric detection, J.Chromatog.A. 938, 45-55, 2001; Gilar, M., Fountain, K.J., Budman, Y., et al., Ion-pair reversed phase high-performance liquid chromatography analysis of oligonucleotides: retention prediction, J.Chromatog.A. 958, 167-182, 2002; El-dawy, M.A., Mabrouk, M.M., and El-Barbary, F.A., Liquid chromatographic determination of fluoxetine, J.Pharm.Biomed.Anal. 30, 561-571, 2002; Yang, X., Zhang, X., Li, A., et al., Comprehensive two-dimensional separations based on capillary high-performance liquid chromatography and microchip electrophoresis, Electrophoresis 24, 1451-1457, 2003; Murphey, A.T., Brown-Augsburger, P., Yu, R.Z., et al., Development of an ion-pair reverse-phase liquid chromatographic/tandem mass spectrometry method for the determination of an 18-mer phosphorothioate oligonucleotide in mouse liver tissue, Eur.J.Mass Spectrom. 11, 209-215, 2005; Xie, G., Sueishi, Y., and Yamamoto, S., Analysis of the effects of protic, aprotic, and multi-component solvents on the fluorescence emission of naphthalene and its exciplex with triethylamine, J.Fluoresc. 15, 475-483, 2005. Tris
tris(hydroxymethyl) aminomethylmethane
121.14
Buffer
OH HO NH2
HO
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706
LIST OF BUFFERS (Continued) Common Name
Chemical Name
M.W
Properties and Comment
Bernhard, S.A., Ionization constants and heats of tris(hydroxymethyl)aminomethane and phosphate buffers, J.Biol.Chem. 218, 961-969, 1956; Rapp, R.D. and Memminger, M.M., Tris (hydroxymethyl)aminomethane as an electrophoresis buffer, Am.J.Clin.Pathol. 31, 400-403, 1959; Rodkey, F.L., Tris(hydroxymethyl)aminomethane as a standard for Kjeldahl nitrogen analysis, Clin.Chem. 10, 606-610, 1964; Oliver, R.W. and Viswanatha, T., Reaction of tris(hydroxymethyl)aminomethane with cinnamoyl imidazole and cinnamoyltrypsin, Biochim.Biophys.Acta 156, 422-425, 1968; Douzou, P., Enzymology at sub-zero temperatures, Mol.Cell.Biochem. 1, 15-27, 1973; The tris-catalyzed isomerization of potassium D-glucose 6-O-sulfate, Can.J.Biochem. 53, 906-910, 1975; Visconti, M.A. and Castrucci, A.M., Tris buffer effects on melanophore aggegrating responses, Comp.Biochem. Physiol.C 82, 501-503 1985; Stambler, B.S., Grant, A.O., Broughton, A., and Strauss, H.C., Influences of buffers on dV/dtmax recovery kinetics with lidocaine in myocardium, Am.J.Physiol. 249, H663-H671, 1985; Nakano, M. and Tauchi, H., Difference in activation by Tris(hydroxymethyl) aminomethane of Ca,Mg-ATPase activity between young and old rat skeletal muscles, Mech.Aging.Dev. 36, 287-294, 1986; Oliveira, L., Araujo-Viel, M.S., Juliano, L., and Prado, E.S., Substrate activation of porcine kallikrein N-a derivatives of arginine 4-nitroanilides, Biochemistry 26, 5032-5035, 1987; Ashworth, C.D. and Nelson, D.R., Antimicrobial potentiation of irrigation solutions containing tris-[hydroxymethyl] aminomethane-EDTA, J.Am.Vet.Med.Assoc. 197, 1513-1514, 1990; Schacker, M., Foth, H., Schluter, J., and Kahl, R., Oxidation of tris to one-carbon compounds in a radicalproducing model system, in microsomes, in hepatocytes and in rats, Free Radic.Res.Commun. 11, 339-347, 1991; Weber, R.E., Use of ionic and zwitterionic (Tris/BisTris and HEPES) buffers in studies on hemoglobin function, J.Appl.Physiol. 72, 1611-1615, 1992; Veeck, L.L., TES and Tris (TEST)-yolk buffer systems, sperm function testing, and in vitro fertilization, Fertil.Steril. 58, 484-486, 1992; Shiraishi, H., Kataoka, M., Morita, Y., and Umemoto, J., Interaction of hydroxyl radicals with tris (hydroxymethyl) aminomethane and Good’s buffers containing hydroxymethyl or hydroxyethyl residues produce formaldehyde, Free Radic.Res.Commun. 19, 315-321, 1993; Vasseur, M., Frangne, R., and Alvarado, F., Buffer-dependent pH sensitivity of the fluorescent chloride-indicator dye SPQ, Am.J.Physiol. 264, C27-C31, 1993; Niedernhofer, L.J., Riley, M., Schnez-Boutand, N., et al., Temperature dependent formation of a conjugate between tris(hydroxymethyl)aminomethane buffer and the malondialdehyde-DNA adduct pyrimidopurinone, Chem.Res.Toxicol. 10, 556-561, 1997; Trivic, S., Leskovac, V., Zeremski, J., et al., Influence of Tris(hydroxymethyl)aminomethane on kinetic mechanism of yeast alcohol dehydrogenase, J.Enzyme Inhib. 13, 57-68, 1998; Afifi, N.N., Using difference spectrophotometry to study the influence of different ions and buffer systems on drug protein binding, Drug Dev.Ind.Pharm. 25, 735-743, 1999; AbouHaider, M.G. and Ivanov, I.G., Non-enzymatic RNA hydrolysis promotedby the combined catalytic activity of buffers and magnesium ions, Z.Naturforsch. 54, 542-548, 1999; Shihabi, Z.K., Stacking of discontinuous buffers in capillary zone electrophoresis, Electrophoresis 21, 2872-2878, 2000; Stellwagen, N.C, Bossi, A., Gelfi, C., and Righetti, P.G., DNA and buffers: are theire any nointeracting, neutral pH buffers?, Anal.Biochem. 287, 167-175, 2000;Burcham, P.C., Fontaine, F.R., Petersen, D.R., and Pyke, S.M., Reactivity of Tris(hydroxymethyl) aminomethane confounds immunodetection of acrolein-adducted proteins, Chem. Res.Toxicol. 16, 1196-1201, 2003; Koval, D., Kasicka, V., and Zuskova, I., Investigation of the effect of ionic strength of Tris-acetate background electrolyte on electrophoretic mobilities of mono-, di-, and trivalent organic anions by capillary electrophoresis, Electrophoresis 26, 3221-3231, 2005; Kinoshita, T., Yamaguchi, A., and Tada, T., Tris(hydroxymethyl)aminomethane induced conformational change and crystal-packing contraction of porcine pancreatic elastase, Acta Crystallograph.Sect.F .Struct. Biol. Cryst.Commun. 62, 623-626, 2006; Qi, Z., Li, X., Sun, D., et al,, Effect of Tris on catalytic activity of MP-11, Bioelectrochemistry 68, 40-47, 2006.
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Brønsted Acidities Lewis acids
Definition: A molecule (conjugate acid) which liberates a proton in solution; the residual molecular ion is a conjugate base EH x → EH x-1 −− Acid strength depends on the strength of the E-H bond. −− Electronegativity of E influences the polarity of the E-H bond −− Energy of solvation of [EH x-1]– – small anions have more favorable solvation energies pKa values of EH x CH4 –58 NH3 39 OH2 14 SH2 7 SeH2 4 TeH2 3 FH 3 Mingo, D.M.P., Essential Trends of Inorganic Chemistry, Oxford University Press, Oxford, United Kingdom, 1998
Definition: A molecule which can accept electrons.
AlCl3 + Cl– → [AlCl4]–
Lewis Base is an electron donor, for example PR3—which becomes a “stronger” base as the electron donating properties of the R group increases as long as the Lewis acid is a simple electron acceptor and steric effects are not important.
References Blackwell, J.A. and Carr, S.W., The role of Lewis Acid-Base processes in ligand-exchange chromatography of benzoic acid derivatives on zirconium oxide, Anal.Chem. 64, 853-862, 1992. Hancock, R.D., Bartolotti, L.J., and Kaltsoyannis, N., Density functional theory-based prediction of some aqueous-phase chemistry of superheavy element 111. Roentgenium(I) is the ‘softest’ metal ion, Inorg. Chem. 45, 10780-10785, 2006.
707
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Measurement of pH Roger G. Bates and Maya Paabo
Definition of pH
Standard solutions
The following definition of pH has received the endorsement of the International Union of Pure and Applied Chemistry. 1. Operational definition. In all existing national standards the definition of pH is an operational one. The electromotive force E x of the cell:
The pH meter or other electrometric pH assembly does not, strictly speaking, measure the pH but rather indicates a difference between the pH of an unknown solution (X) and a standard solution (S), both of which are at the same temperature. The pH meter should always be standardized routinely with two reference solutions of assigned pH, chosen if possible to bracket the pH of the test solution. These standards are prepared as indicated in Table 3. For convenience, air weights of the buffer salts are given. A good grade of distilled or de-ionized water should be used; for the four solutions of highest pH, the water should be freed of dissolved carbon dioxide by boiling or purging. For a detailed discussion of the properties of the primary standard buffer solutions, the reader is referred to chapter 4 of R. G. Bates, Determination of pH, 2nd ed., (John Wiley and Sons, Inc., New York, 1973). Highly pure buffer materials should be used. These materials are obtainable commercially; they are also distributed as certified standard reference materials by the National Bureau of Standards. It should be noted that individual lots show slight variations; hence, the values certified for a particular lot may differ slightly from those given in Table 1. The use of two or more standard reference solutions may disclose small inconsistencies in the standardization of the pH meter, depending on which standards are chosen. When this is the case, the best results are often obtained by assuming linearity between E and pH between the two calibrating points bracketing the pH of the unknown.
Pt, H2 |solution X|concentrated KCl solution| reference electrode is measured and likewise the electromotive force E s of the cell: Pt, H2 |solution S|concentrated KCl solution| reference electrode both cells being at the same temperature throughout and the reference electrodes and bridge solutions being identical in the two cells. The pH of the solution X, denoted by pH(X), is then related to the pH of the solution S, denoted by pH(S), by the definition:
pH(X ) = pH(S) +
E x − Es ( RT ln 10)/F
where R denotes the gas constant, T the thermodynamic temperature, and F the faraday constant. Thus defined the quantity pH is dimensionless. To a good approximation, the hydrogen electrodes in both cells may be replaced by other hydrogen ion responsive electrodes, e.g., glass or quinhydrone. The two bridge solutions may be of any molality not less than 3.5 mol kg−1, provided they are the same (see Pure Appl. Chem., 1, 163, 1960). 2. Standards. The difference between the pH of two solutions having been defined as above, the definition of pH can be completed by assigning a value of pH at each temperature to one or more chosen solutions designated as standards. A series of pH(S) values for seven suitable standard reference solutions is given in Table 1. The constants for calculating pH(S) values over the temperature range for 0 to 95°C are given in Table 2. If the definition of pH given above is adhered to strictly, then the pH of a solution may be slightly dependent on which standard solution is used. These unavoidable deviations are caused not only by imperfections in the response of the hydrogen ion electrodes but also by variations in the liquid-junction potentials resulting from the different ionic compositions and mobilities of the several standards and from differences in the structure of the liquid–liquid boundary. In fact such variations in measured pH are usually too small to be of practical significance. Moreover, the acceptance of several standards allows the use of the following alternative definition of pH. The electromotive force E x is measured, and likewise the electromotive forces E1 and E2, of two similar cells with the solution X replaced by the standard solutions S1 and S2 such that E1 and E2 values are on either side of, and as near as possible to, E x. The pH of solution X is then obtained by assuming linearity between pH and E, that is to say
pH(X ) − pH(S1 ) E x − E1 = pH(S2 ) − pH(S1 ) E 2 − E1
This procedure is especially recommended when the hydrogenion-responsive electrode is a glass electrode.
Electrodes Although the hydrogen electrode is the ultimate standard on which the pH scale is based, in practice the convenient and versatile glass electrode is favored for the vast majority of pH measurements. New glass electrodes, or those that have been allowed to dry out, should be conditioned by soaking in water for several hours before use and after exposure to nonaqueous or dehydrating media. Some glass electrodes are designed especially for use at high temperatures, while others are best suited to low-temperature use. Special “high pH” electrodes are also available. For optimum results, careful attention should be paid to selection of the proper electrode for the problem at hand. Glass electrodes of small dimensions are of great utility when sample volumes are limited. The pH-sensitive glasses are, however, moderately soluble, and small amounts of alkali are dissolved from the glass surface by the solutions in which the electrode is immersed. For this reason, the most accurate results are obtained when the ratio of the electrode area to sample volume is small. The concentrated solution of potassium chloride that joins the reference electrode with the unknown or standard solution is reasonably effective in reducing the liquid-junction potential to small, fairly constant, values. It is important to assure that the flow of bridge solution into the test solution is neither excessive nor completely interrupted by crystallization of salt in the aperture where liquid-liquid contact is established. Temperature gradients within the pH cell are a common source of difficulty, marked by variability and inaccuracy in the 709
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Handbook of Biochemistry and Molecular Biology
710
TABLE 1 Values of pH(S) for Seven Primary Standard Solutions A
B
C
D
E
F
G
— — — — — 3.557 3.552 3.549 3.548 3.547 3.547 3.549 3.554 3.560 3.580 3.609 3.650 3.674
3.863 3.840 3.820 3.802 3.788 3.776 3.766 3.759 3.755 3.753 3.750 3.749 — — — — — —
4.003 3.999 3.998 3.999 4.002 4.008 4.015 4.024 4.030 4.035 4.047 4.060 4.075 4.091 4.126 4.164 4.205 4.227
6.984 6.951 6.923 6.900 6.881 6.865 6.853 6.844 6.840 6.838 6.834 6.833 6.834 6.836 6.845 6.859 6.877 6.886
7.534 7.500 7.472 7.448 7.429 7.413 7.400 7.389 7.384 7.380 7.373 7.367 — — — — — —
9.464 9.395 9.332 9.276 9.225 9.180 9.139 9.102 9.081 9.068 9.038 9.011 8.985 8.962 8.921 8.885 8.850 8.833
10.317 10.245 10.179 10.118 10.062 10.012 9.966 9.925 9.903 9.889 9.856 9.828 — — — — — —
t/°C 0 5 10 15 20 25 30 35 38 40 45 50 55 60 70 80 90 95
The compositions of the standard solutions are: A: KH tartrate (saturated at 25°C) B: KH2 citrate, m = 0.05 mol kg−1 C: KH phthalate, m = 0.05 mol kg−1 D: KH2PO4, m = 0.025 mol kg−1; Na2HPO4, m = 0.025 mol kg−1 E: KH2PO4, m = 0.008695 mol kg−1; Na2HPO4, m = 0.03043 mol kg−1 F: Na2B4O7, m = 0.01 mol kg−1 G: NaHCO3, m= 0.025 mol kg−1; Na2CO3, m = 0.025 mol kg−1 where m denotes molality.
TABLE 2 Values of The Constants of The Equation: pH(S) = AT +B+CT +DT 2 For Seven Primary Standard Buffer Solutions From 0 To 95°C Solution A. Tartrate B. Citrate C . Phthalate D. Phosphate E . Phosphate F. Borax G. Carbonate
Temperature Range °C 25 to 95 0 to 50 0 to 95 0 to 95 0 to 50 0 to 95 0 to 50
A −1727.96 1280.4 1678.30 3459.39 5706.61 5259.02 2557.1
B 23.7406 −4.1650 −9.8357 −21.0574 −43.9428 −33.1064 −4.2846
C −0.075947 0.012230 0.034946 0.073301 0.154785 0.114826 0.019185
Standard Deviation of The Fitted Curves
105 D 9.2873 0 −2.4804 −6.2266 −15.6745 −10.7860 0
0.0016 0.0010 0.0027 0.0017 0.0011 0.0025 0.0026
TABLE 3 Preparation of Primary Standard Buffer Solutions Standard Solution . Tartrate A B. Citrate C . Phthalate D. Phosphate E . Phosphate F. Borax . Carbonate G a b
NBS SRM No.a 188 190 185d 186Ic 186IIb 186Ic 186IIb 187a 191 192
KHC4H4O6 KH2C6H5O7 KHC8H4O4 KH2PO4 Na2HPO4 KH2PO4 Na2HPO4 Na2B4O7 · 10H2O NaHCO3 Na2CO3
Weight In Airb (g) (Satd. at 25°C) 11.41 10.12 3.388 3.533 1.179 4.302 3.80 2.092 2.640
These materials may be ordered from the Office of Standard Reference Materials, National Bureau of Standards, Washington, D.C. 20234. This weight of salt to be dissolved in water and diluted to 1 liter at 25°C to provide concentrations indicated in Table 1.
reading. Both of the electrodes, and the standard and test solutions as well, should be within a few degrees Celsius of the same temperature. For results of the highest reliability, temperature control should be provided. It is the function of the temperature compensator of the pH meter to adjust the pH-e.m.f. slope in
9168_Book.indb 710
Buffer Substance
such a manner that a difference of e.m.f. (in volts) is correctly converted to a difference of pH. This adjustment cannot compensate for inequalities of temperature through the cell or for differences between the temperature of the standard and test solutions.
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Measurement of pH
711
Techniques Electrodes and sample cups should be washed carefully with distilled or de-ionized water and gently dried with clean absorbent tissue. The electrodes are immersed in the first standard solution and the temperature compensator of the measuring instrument is set at the temperature of the solutions whose pH is to be measured. The standardization control of the instrument is adjusted until the meter is balanced at the known pH of the standard, as given in Table 1. This procedure is repeated with successive portions of the same standard until replacement causes no change in the position of balance. The electrodes are then washed once more and dried. A second standard solution is selected and the measurement repeated without altering the position of the standardization control. The pH reading of this second solution is noted and the sample replaced with a second portion of the same solution. This replacement is continued until successive readings agree within 0.02 pH unit, when the electrodes and meter may be judged to be functioning properly. It is advisable to make a final check with one of the buffers at the conclusion of a series of measurements. After the instrument is properly standardized, a portion of the test solution is placed in the sample cup and the pH reading noted. Successive portions are again used until two measurements agree within the limits imposed by the reproducibility of the measuring instrument and the temperature control. With the best meters, measurements on buffered solutions should be reproducible to 0.01 unit or even better. With water or poorly buffered solutions, values agreeing to 0.1 unit may have to be accepted. Some improvement will result if poorly buffered solutions are protected from carbon dioxide of the atmosphere during the period of the measurements.
Interpretation of pH numbers The standard values of pH given in the table of an earlier section are based on hydrogen electrode potentials as measured in cells without a liquid junction. The uncertainty of the standard values is estimated at 0.005 unit. The accuracy of the results furnished by a given pH assembly adjusted with these primary standards is, however, further limited by inconsistencies which have their origin in defects of the glass electrode response and variations in the liquid-junction potential. For these reasons, the accuracy of experimental pH numbers can be considered to be better than 0.01 unit only under unusually favorable conditions. The operational definition of pH fulfills adequately the need for an experimental scale capable of furnishing reproducible pH
numbers. The interpretation of these numbers may be of secondary importance and should only be attempted when the standard and unknown solutions are matched so closely in composition that there is good reason to believe that the liquid-junction potential remains fairly constant when the standard is replaced by the unknown. In general, this will be the case when the unknowns are aqueous solutions of simple salts of total concentration not in excess of 0.2 M with pH values between 2.5 and 11.5. When these “ideal” conditions prevail, the experimental pH can be considered to approach −log aH, where aH is the conventional hydrogen ion activity defined in a manner consistent with the convention on which the standard values of pH(S) were based Bates, R. G., J. Res. Natl. Bur. Standards, 66A, 179 (1962). All quantitative applications of pH measurements, when justifiable, should therefore be based on the approximation pH(X) ≈ −log aH = −log mHγH, where m is molality and γ is the activity coefficient.
Indicator methods Acid-base indicators have the property of altering the color of a solution in the region 1 to 2 pH units as the pH changes. They are therefore useful for pH measurements, although in general the accuracy is inferior to that obtainable by electrometric procedures. A list of suitable indicators, their pH ranges and color changes, is given in Table 4. Equal concentrations of the same indicator are added to the test solution and to each of a series of buffer solutions of known pH selected to bracket the pH of the test solution. Color comparisons are made with a colorimeter or spectrophotometer, and solutions of equal color are assumed to have the same pH. The pH of a series of suitable reference solutions can be determined in advance by electrometric methods. Alternative ly, tables of pH as a function of composition can be utilized. The compositions and pH values of a set of useful solutions covering the range pH 1 to 13 are summarized in Table 5.
References 1. Paabo, Bates, and Robinson, J. Res. Natl. Bur. Standards, 67A, 573 (1963). 2. Hetzer, Robinson, and Bates, Anal. Chem., 40, 634 (1968). 3. Paabo and Bates, J. Phys. Chem., 74, 702 (1970). 4. Hetzer, Bates, and Robinson, J. Phys. Chem., 70, 2869 (1966). 5. Bates and Robinson, Anal. Chem., 45, 420 (1973). 6. Durst and Staples, Clin. Chem., 18, 206 (1972). 7. Bates, Roy, and Robinson, Anal. Chem., 45, 1663 (1973).
TABLE 4 Acid-Base Indicators Indicator Acid cresol red Acid metacresol purple Acid thymol blue Bromophenol blue Bromocresol green Methyl red Chlorophenol red Bromocresol purple Bromothymol blue Phenol red Cresol red
pH Range
Color Change
Indicator
0.2–1.8 1.2–2.8
Red–yellow Red–yellow
Metacresol purple Thymol blue
1.2–2.8 3.0–4.6 3.8–5.4 4.4–6.0 5.2–6.8 5.2–6.8 6.0–7.6 6.8–8.4 7.2–8.8
Red–yellow Yellow–blue Yellow–blue Red–yellow Yellow–red Yellow–purple Yellow–blue Yellow–red Yellow–red
Phthalein red Tolyl red Acyl red Parazo orange Acyl blue Benzo yellow Benzo red Thymol red
pH Range
Color Change
7.6–9.2 8.0–9.6
Yellow–purple Yellow–blue
8.6–10.2 10.0–11.6 10.0–11.6 11.0–12.6 12.0–13.6 2.4–4.0 4.4–7.6 8.0–11.2
Yellow–red Red–yellow Red–yellow Yellow–orange Red–blue Red–yellow Red–blue Yellow–red
Courtesy of W. A. Taylor and Co.
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712
TABLE 5 Buffer Solutions for Indicator Measurements and pH Control 25 ml 0.2 M KCl, x ml 0.2 M HCl, DILUTED TO 100 ml pH
x
pH
x
1.00 1.10 1.20 1.30 1.40
67.0 52.8 42.5 33.6 26.6
1.50 1.60 1.70 1.80 1.90
20.7 16.2 13.0 10.2 8.1
— — —
— — —
2.00 2.10 2.20
6.5 5.1 3.9
50 ml 0.1 M KH PHTHALATE, x ml 0.1 M NaOH, DILUTED TO 100 ml pH
x
pH
x
4.10 4.20 4.30 4.40 4.50
1.3 3.0 4.7 6.6 8.7
5.10 5.20 5.30 5.40 5.50
25.5 28.8 31.6 34.1 36.6
4.60 4.70 4.80 4.90 5.00
11.1 13.6 16.5 19.4 22.6
5.60 5.70 5.80 5.90 —
38.8 40.6 42.3 43.7 —
50 ml OF A Mixture 0.1 M with Respect to Both KCl AND H3BO3, x ml 0.1 M NaOH, Diluted to 100 ml pH
x
pH
x
8.00 8.10 8.20 8.30 8.40
3.9 4.9 6.0 7.2 8.6
9.00 9.10 9.20 9.30 9.40
20.8 23.6 26.4 29.3 32.1
8.50 8.60 8.70 8.80 8.90 — — —
10.1 11.8 13.7 15.8 18.1 — — —
9.50 9.60 9.70 9.80 9.90 10.00 10.10 10.20
34.6 36.9 38.9 40.6 42.2 43.7 45.0 46.2
50 ml 0.025 M BORAX, x ml 0.1 M HCl, DILUTED TO 100 ml pH
x
pH
x
8.00 8.10 8.20 8.30 8.40
20.5 19.7 18.8 17.7 16.6
8.50 8.60 8.70 8.80 8.90
15.2 13.5 11.6 9.4 7.1
— —
— —
9.00 9.10
4.6 2.0
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pH
50 ml 0.1 M KH PHTHALATE, x ml 0.1 M HCl, DILUTED TO 100 ml x
pH
x
2.20 2.30 2.40 2.50 2.60
49.5 45.8 42.2 38.8 35.4
3.20 3.30 3.40 3.50 3.60
15.7 12.9 10.4 8.2 6.3
2.70 2.80 2.90 3.00 3.10
32.1 28.9 25.7 22.3 18.8
3.70 3.80 3.90 4.00 —
4.5 2.9 1.4 0.1 —
50 ml 0.1 M, KH2PO4 x ml 0.1 M NaOH, DILUTED TO 100 ml pH
x
pH
x
5.80 5.90 6.00 6.10 6.20
3.6 4.6 5.6 6.8 8.1
6.80 6.90 7.00 7.10 7.20
22.4 25.9 29.1 32.1 34.7
6.30 6.40 6.50 6.60 6.70
9.7 11.6 13.9 16.4 19.3
7.30 7.40 7.50 7.60 7.70
37.0 39.1 41.1 42.8 44.2
— — —
— — —
7.80 7.90 8.00
45.3 46.1 46.7
50 ml 0.1 M TRIS(HYDROXMETHYL)AMINOMETHANE, x ml 0.1 M HCl, DILUTED TO 100 ml pH
x
pH
x
7.00 7.10 7.20 7.30 7.40
46.6 45.7 44.7 43.4 42.0
8.00 8.10 8.20 8.30 8.40
29.2 26.2 22.9 19.9 17.2
7.50 7.60 7.70 7.80 7.90 —
40.3 38.5 36.6 34.5 32.0 —
8.50 8.60 8.70 8.80 8.90 9.00
14.7 12.4 10.3 8.5 7.0 5.7
50 ml 0.025 M BORAX, x ml 0.1 M NaOH, DILUTED TO 100 ml pH
x
pH
x
9.20 9.30 9.40 9.50 9.60
0.9 3.6 6.2 8.8 11.1
10.20 10.30 10.40 10.50 10.60
20.5 21.3 22.1 22.7 23.3
9.70 9.80 9.90 10.00 10.10
13.1 15.0 16.7 18.3 19.5
10.70 10.80 — — —
23.80 24.25 — — —
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Measurement of pH
713
TABLE 5 Buffer Solutions for Indicator Measurements and pH Control (Continued) 50 ml 0.05 M Na2HPO4, x 1ml 0.1 M NaOH, DILUTED TO 100 ml
50 ml 0.05 M NaHCO3, x ml 0.1 M NaOH, Diluted to 100 ml pH
x
pH
x
9.60 9.70 9.80 9.90 10.00
5.0 6.2 7.6 9.1 10.7
10.60 10.70 10.80 10.90 11.00
19.1 20.2 21.2 22.0 22.7
10.10 10.20 10.30 10.40 10.50
12.2 13.8 15.2 16.5 17.8
— — — — —
— — — — —
pH
x
pH
x
10.90 11.00 11.10 11.20 11.30
3.3 4.1 5.1 6.3 7.6
11.40 11.50 11.60 11.70 11.80
9.1 11.1 13.5 16.2 19.4
— —
— —
11.90 12.00
23.0 26.9
25 ml 0.2 M KCl, x ml 0.2 M NaOH, DILUTED TO 100 ml pH
x
pH
x
12.00 12.10 12.20 12.30 12.40 —
6.0 8.0 10.2 12.8 16.2 —
12.50 12.60 12.70 12.80 12.90 13.00
20.4 25.6 32.2 41.2 53.0 66.0
Source: Bower and Bates, J. Res. Natl. Bur. Standards, 55, 197 (1955); Bates and Bower, Anal. Chem., 28, 1322 (1956).
TABLE 6 pH Values For Miscellaneous Buffer Solutions Over A Range of Temperature t/°C Composition of the Buffer Solution Potassium dihydrogen phosphate (m) Sodium succinate (m) (1) Piperazine phosphate (m) (2) 2,2-Bis(hydroxymethyl)-2,2′-2′′-nitrilotriethanol (2m) Hydrochloric acid (m) (3) Morpholine (1.5m) Hydrochloric acid (m) (4) Tris(hydroxymethyl)amino-methane (“Tris”) (m), Tris. HCl (m) (5) Tris (m), Tris.HCl (3m) (6) N-Tris(hydroxymethyl)methyl-glycine (“Tricine”) (m), Na Tricinate (m) (7) (“Tricine”) (3m), Na Tricinate (m) (7)
0
5
10
15
20
25
30
35
40
45
50
0.005 0.015 0.025
— — —
— — —
— — —
— — —
— — —
6.251 6.162 6.109
— — —
— — —
— — —
— — —
— — —
0.02 0.05
6.580 6.589
6.515 6.525
6.453 6.463
6.394 6.404
6.338 6.348
6.284 6.294
6.234 6.243
6.185 6.195
6.140 6.149
6.097 6.058 6.106 6.066
0.02 0.04 0.06 0.08 0.10
7.000 7.029 7.050 7.067 7.082
6.905 6.932 6.953 6.969 6.983
6.812 6.839 6.859 6.876 6.889
6.722 6.748 6.767 6.783 6.796
6.635 6.662 6.681 6.696 6.710
6.551 6.577 6.595 6.610 6.623
6.469 6.495 6.513 6.528 6.540
6.390 6.415 6.434 6.448 6.460
6.312 6.336 6.353 6.367 6.378
6.237 6.262 6.280 6.294 6.306
0.10
8.963
8.828
8.702
8.579
8.458
8.343
8.231
8.120
8.013
7.908 7.806
0.05
8.946
8.774
8.614
8.461
8.313
8.173
8.036 7.904b 7.777
7.654 7.537
0.01667 8.471
8.303
8.142
7.988
7.840
7.698
7.563
7.433
7.307
7.186 7.070
0.05
—
8.485
8.375
8.271
8.175
8.079
7.988
7.902
7.817
7.740 7.663
0.02
—
8.023
7.916
7.813
7.713
7.621
7.527 7.437c 7.355
7.275 7.197
m
a
6.165 6.190 6.208 6.222 6.235
Compiled by Roger G. Bates and Maya Paabo Contribution from the National Bureau of Standards , not subjected to copyright. a b c
mol kg−1 7.851 at 37°C 7.407 at 37°C
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714
General references for pH measurement 1. Spitzer, P. and Meinrath, G., Importance of traceable pH measurement, Anal. Bioanal. Chem., 374, 765–766, 2002. 2. Baucke, F.G.K., New IUPAC recommendations on the measurement of pH — background and essentials, Anal. Bioanal. Chem., 374, 772–777, 2002.
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Handbook of Biochemistry and Molecular Biology 3. Spitzer, P. and Werner, B., Improved reliability of pH measurements, Anal. Bioanal. Chem., 374, 787–795, 2002. 4. Covington, A.K., Bates, R.G., and Durst, R.A., Definition of pH scales, standard reference values, measurement of pH and related terminology, Pure & Appl. Chem., 57, 531–542, 1985.
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Buffer Solutions No.
Name
Range of pH Value
Temperature (°C)
1.0–2.2 1.2–3.4 1.2–5.0 2.4–4.0 4.2–6.2 5.2–6.6 5.0–8.0 7.0–9.0 7.8–9.2 8.6–12.8 9.4–10.6
Room Room Room 20 20 20 20 18 20 20 20
ΔpH/K
GENERAL BUFFERS 1 2 3 4 5 6 7 8 9 10 11
KCl/HCl (Clark and Lubs)2 Glycine/HCl (Sørensen)3 Na citrate/HCl (Sørensen)3 K biphthalate/HCl (Clark and Lubs)2 K biphthalate/NaOH (Clark and Lubs)2 Na citrate/NaOH (Sørensen)3 Phosphate (Sørensen).3 Barbital-Na/HCl (Michaelis)4 Na borate/HCl (Sørensen)3 Glycine/NaOH (Sørensen)3 Na borate/NaOH (Sørensen)3
0 0 0 + 0.001 + 0.004 − 0.003 − 0.005 − 0.025 − 0.01
UNIVERSAL BUFFERS 12 13 14
Citric acid/phosphate (Mcllvaine)5 Citrate-phosphate-borate/HCl (Teorell and Stenhagen)6 Britton-Robinson7
2.2–7.8 2.0–12.0
21 20
2.6–11.8
25
At low pH:0 At high pH: − 0.02
BUFFERS FOR BIOLOGICAL MEDIA 15 16 17
Acetate (Walpole)8–10 Dimethylglutaric acid/NaOH11 Piperazine/HCl12,13
18
Tetraethylethylenediaminea,13
19 20
Tris maleate9,14 Dimethylaminoethylaminea,13
21 22 23 24 25 26
Imidazole/HCl15 Triethanolamine/HCl16 N-Dimethylaminoleucylglycine/NaOH17 Tris/HCl9 2-Amino-2-methylpropane-1,3-diol/HCl9,14 Carbonate (Delory and King)9,8
a
3.8–5.6 3.2–7.6 4.6–6.4 8.8–10.6 5.0–6.8 8.2–10.0 5.2–8.6 5.6–7.4 8.6–10.4 6.2–7.8 7.9–8.8 7.0–8.8 7.2–9.0 7.8–10.0 9.2–10.8
25 21 20 20 23 20 25 25 23 23 23 20
− 0.015 − 0.02
Can be combined with tris buffer to give a cationic universal buffer (see Semenza et al.13).
715
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716
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BUFFER SOLUTIONS (Continued) 1
2
3
4
1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8 4.0 4.2 4.4 4.6 4.8 5.0 5.2 5.4 5.6 5.8 6.0 6.2 6.4 6.6 6.8 7.0 7.2 7.4 7.6 7.8 8.0 8.2 8.4 8.6 8.8 9.0 9.2 9.4 9.6 9.8
54.2 36.0 23.2 14.7 9.3 5.9 3.8 — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — —
— 11.1 26.4 36.2 43.9 50.7 56.5 62.3 68.4 74.7 81.0 86.2 90.3 — — — —
— 9.0 17.9 23.6 27.6 30.2 32.2 34.1 36.0 37.9 39.9 42.1 44.8 47.8 51.2 55.1 60.0 66.4 74.9 85.6 100.0 — — — — — — — — — — — — — — — — — — — — — — — —
— — — — — — — 41.0 34.3 27.8 21.6 15.9 10.9 6.7 3.3 0.0 — — — — — — — — — — — — — — — — — — — — — — — — — — — — —
— — — — — — — — — — — — — — — — — — — — — — — — — — —
5
6
7
— — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — 3.0 — — 6.7 — — 11.1 — — 16.5 — — 22.6 — 99.2 28.8 87.1 98.4 34.4 78.0 97.3 39.1 70.3 95.5 42.4 64.5 92.8 45.0 60.3 88.9 46.7 57.2 83.0 — 54.8 75.4 — 53.2 65.3 — — 53.4 — — 41.3 — — 29.6 — — 19.7 — — 12.8 — — 7.4 — — 3.7 — — — — — — — — — — — — — — — — — — — — — — — — — — —
8
9
10
11
— — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — 53.3 — — — 55.0 — — — 57.6 — — — 60.8 — — — 65.2 53.0 — — 70.6 55.4 — — 75.9 58.0 — — 81.2 62.1 — — 86.2 66.9 94.7 — 90.1 73.6 92.0 — 93.2 83.5 88.4 — — 95.6 84.0 — — — 78.9 87.0 — — 73.2 75.5 — — 67.2 65.1
12
13
— — — — — — 98.8 94.5 90.0 85.1 80.3 76.0 72.0 68.4 65.1 62.0 59.1 56.4 53.7 51.2 49.0 46.9 44.7 42.4 40.0 37.4 34.5 31.4 27.9 23.5 19.0 13.8 9.8 6.8 4.6 — — — — — — — — — —
— — — — — 74.4 68.8 64.6 63.3 58.9 56.9 55.2 53.9 52.9 51.8 50.7 49.7 48.6 47.5 46.4 45.4 44.3 43.2 42.0 40.8 39.7 38.4 37.0 35.6 34.2 32.9 31.7 30.6 29.6 28.8 28.1 27.6 27.0 26.3 25.2 24.0 22.6 21.4 20.2 19.0
14
15
— — — — — — — — — — — — — — — — 1.6 — 3.6 — 5.7 — 7.8 — 9.9 — 11.7 — 13.5 10.9 15.3 16.6 17.5 23.9 19.7 33.5 21.9 44.9 24.1 56.6 26.3 67.8 28.6 76.8 31.0 84.0 33.4 89.3 35.8 — 38.3 — 40.8 — 43.3 — 45.8 — 48.3 — 50.9 — 53.4 — 55.8 — 58.2 — 60.5 — 62.8 — 65.0 — 67.2 — 69.3 — 71.3 — 73.2 — 75.1 — 77.0 — 78.8 — 80.4 —
16a
16b
17
18
19
20
21
22
23
24
25
26
pH
— — — — — — — — — — — 7.0 13.3 20.7 26.3 32.4 36.2 39.3 41.3 43.5 45.7 48.4 51.3 55.0 58.8 63.9 69.5 74.1 83.5 87.4 90.0 91.8 93.0 93.8 — — — — — — — — — — —
— — — — — — — — — — — 14.4 20.9 26.8 32.4 36.6 40.3 43.1 45.7 48.3 51.5 53.6 58.2 63.6 68.7 73.6 78.5 83.3 87.4 91.0 93.2 94.9 95.8 96.8 — — — — — — — — — — —
— — — — — — — — — — — — — — — — — — 94.3 91.5 87.8 83.6 77.6 71.8 66.5 61.8 58.2 55.5 — — — — — — — — — — — 45.5 43.2 40.0 35.8 30.8 25.0
— — — — — — — — — — — — — — — — — — — — 94.3 91.5 87.8 83.1 77.6 71.7 66.4 61.7 58.0 55.3 — — — — — — 46.4 43.9 40.9 36.8 31.8 26.2 20.4 15.2 10.8
— — — — — — — — — — — — — — — — — — — — — 3.2 5.0 7.3 9.7 12.4 15.2 17.9 20.8 22.2 23.7 25.2 26.7 28.6 31.2 33.9 36.9 39.9 42.7 — — — — — —
— — — — — — — — — — — — — — — — — — — — — — — 94.3 91.7 88.0 83.3 77.9 72.0 66.6 61.9 58.1 55.3 — — — — — 45.4 42.8 39.2 34.7 29.3 23.6 19.0
— — — — — — — — — — — — — — — — — — — — — — — — — — 43.4 40.4 36.5 31.4 25.4 19.6 14.6 10.2 6.6 — — — — — — — — — —
— — — — — — — — — — — — — — — — — — — — — — — — — — — — — — 86.2 79.6 71.3 62.0 52.0 42.0 31.9 22.5 16.0 11.7 — — — — —
— — — — — — — — — — — — — — — — — — — — — — — — — — — — — — 86.4 80.6 72.8 63.2 52.1 41.1 31.4 23.0 15.9 10.3 — — — — —
— — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — 44.7 42.0 39.3 33.7 27.9 22.9 17.3 13.0 8.8 5.3 — — — —
— — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — 43.9 41.6 38.4 34.8 30.7 23.3 17.7 13.3 9.2 5.2 4.1
— — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — 10.0 18.4 29.3 42.0
1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8 4.0 4.2 4.4 4.6 4.8 5.0 5.2 5.4 5.6 5.8 6.0 6.2 6.4 6.6 6.8 7.0 7.2 7.4 7.6 7.8 8.0 8.2 8.4 8.6 8.8 9.0 9.2 9.4 9.6 9.8
Handbook of Biochemistry and Molecular Biology
pH
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pH
1
2
3
4
5
6
7
8
9
10.0 10.2 10.4 10.6 10.8 11.0 11.2 11.4 11.6 11.8 12.0 12.2 12.4 12.6 12.8
— — — — — — — — — — — — — — —
— — — — — — — — — — — — — — —
— — — — — — — — — — — — — — —
— — — — — — — — — — — — — — —
— — — — — — — — — — — — — — —
— — — — — — — — — — — — — — —
— — — — — — — — — — — — — — —
— — — — — — — — — — — — — — —
— — — — — — — — — — — — — — —
10
11
62.5 59.6 58.8 56.4 55.7 54.1 53.6 52.3 52.2 — 51.2 — 50.4 — 49.5 — 48.7 — 47.6 — 46.0 — 43.2 — 39.1 — 31.8 — 21.4 —
12
13
14
15
16a
16b
17
18
19
20
21
22
23
24
25
26
pH
— — — — — — — — — — — — — — —
18.1 17.1 16.5 16.0 15.5 14.7 13.5 11.7 9.1 5.5 1.3 — — — —
81.8 83.1 84.3 85.4 86.5 87.8 89.3 91.3 94.5 99.0 — — — — —
— — — — — — — — — — — — — — —
— — — — — — — — — — — — — — —
— — — — — — — — — — — — — — —
19.4 14.3 10.0 6.9 — — — — — — — — — — —
7.4 — — — — — — — — — — — — — —
— — — — — — — — — — — — — — —
13.1 9.2 6.2 — — — — — — — — — — — —
— — — — — — — — — — — — — — —
— — — — — — — — — — — — — — —
— — — — — — — — — — — — — — —
— — — — — — — — — — — — — — —
2.3 — — — — — — — — — — — — — —
53.4 63.7 73.1 81.2 87.9 — — — — — — — — — —
10.0 10.2 10.4 10.6 10.8 10.0 11.2 11.4 11.6 11.8 12.0 12.2 12.4 12.6 12.8
Buffer Solutions
9168_Book.indb 717
BUFFER SOLUTIONS (Continued)
Note: The table gives the volumes x (in ml) of the stock solutions listed that are required to make up a buffer solution of the desired pH value.
717
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Handbook of Biochemistry and Molecular Biology
718
BUFFER SOLUTIONS (Continued) Stock solutions and their amount of substance concentrations or mass and/or volume contents of the solutes No
A
1 2
KCl 0.2 mol l (14.91 g l ) Glycine 0.1 mol l−1 + NaCl 0.1 mol l−1 (1 l solution contains 7.507 g glycine + 5.844 g NaCl) Disodium citrate 0.1 mol l−1 (1 l solution contains 21.01 g citric acid monohydrate + 200 ml NaOH 1 mol l−1) Potassium biphthalate 0.1 mol l−1 (20.42 g l−1) As No. 4 As No. 3 Potassium dihydrogen phosphate 1/15 mol l−1 (9.073 g l−1)
3 4 5 6 7 8 9 10 11 12 13
−1
−1
Barbital-Na 0.1 mol l−1 (20.62 g l−1) Boric acid, half-neutralized, 0.2 mol l−1 (corresponds to 0.05 mol l−1 borax solution; 1 l solution contains 12.37 g boric acid 100 ml NaOH 1 mol l−1) As No. 2 As No. 9 Citric acid 0.1 mol l−1 (citric acid monohydrate 21.01 g l−1)
B
Composition of the Buffer
HCl 0.2 mol l HCl 0.1 mol l−1
25 ml A + x ml B made up to 100 ml x ml A + (100 − x) ml B
HCl 0.1 mol l−1
x ml A + (100 − x) ml B
HCl 0.1 mol l−1 NaOH 0.1 mol l−1 NaOH 0.1 mol l−1 Disodium phosphate 1/15 mol l−1 (Na2HPO4 · 2 H2O, 11.87 g l−1) HCl 0.1 l−1 HCl 0.1 mol l−1
50 ml A + x ml B made up to 100 ml 50 ml A + x ml B made up to 100 ml x ml A + (100 − x) ml B x ml A + (100 − x) ml B
NaOH 0.1 mol l−1 NaOH 0.1 mol l−1 Disodium phosphate 0.2 mol l−1 (Na2HPO4 · 2 H2O, 35.60 g l−1) HCl 0.1 l−1
x ml A + (100 − x) ml B x ml A + (100 − x) ml B x ml A + (100 − x) ml B
NaOH 0.2 mol l−1
100 ml A + x ml B
Acetic acid 0.1 mol l−1 (6.005 g l−1)
x ml A + (100 − x) ml B
NaOH 0.2 mol l−1
−1
x ml A + (100 − x) ml B x ml A + (100 − x) ml B
20 ml A + x ml B made up to 100 1
16a
To 100 ml citric acid and 100 ml phosphoric acid solution, each equivalent to 100 ml NaOH 1 mol l−1, add 3.54 g boric acid and 343 ml NaOH 1 mol l−1and make up to 1 l of solution Citric acid, potassium hydrogen phosphate, barbital, and boric acid, all 0.02857 mol l−1 (1 l solution contains 6.004 g citric acid monohydrate, 3.888 g potassium hydrogen phosphate, 5.263 g barbital, 1.767 g boric acid) Sodium acetate 0.1 mol l−1 (1 l solution contains 8.204 g C2H3O2Na or 13.61 g C2H3O2Na · 3 H2O) Dimethylglutaric acid 0.1 mol l−1 (16.02 g l−1)
16b
Dimethylglutaric acid 0.1 mol l−1 (16.02 g l-1)
NaOH 0.2 mol l−1
17 18 19
Piperazine 1 mol l−1 (86.14 g l−1) Tetraethylethylenediamine 1 mol l−1 (172.32 g l−1) Tris acid maleate 0.2 mol l−1 [1 l solution contains 24.23 g tris(hydroxymethyl)aminomethane + 23.21 g maleic acid or 19.61 g maleic annydride] Dimethylaminoethylamine 1 mol l−1 (88 g l−1) Imidazole 0.2 mol l−1 (13.62 g l−1) Triethanolamine 0.5 mol l−1 + ethylenediamine-tetraacetic acid disodium salt (1 l solution contains 74.60 g C6H15O3N + 20 g C10H14O8N2 · Na2 · 2 H2O) N-Dimethylaminoleucylglycine 0.1 mol l−1 + NaCl 0.2 mol l−1 (1 l solution contains 24.33 g C10H20O3N2 · ½ H2O + 11.69 g NaCl) Tris 0.2 mol l−1 [tris(hydroxymethyl)aminomethane 24.23 g l−1] 2-Amino-2-methylpropane-1,3-diol 0.1 mol l−1 (10.51 g l−1) Sodium carbonate anhydrous 0.1 mol l−1 (10.60g l−1)
HCl 0.1 mol l−1 HCl 0.1 mol l−1 NaOH 0.2 mol l−1
(a) 100 ml A + x ml B made up to 1000 ml (b) 100 ml A + x ml B + 5.844 g NaCl made up to 1000 ml NaCl − 0.1 mol l−1 5 ml A + x ml B made up to 100 ml 5 ml A + x ml B made up to 100 ml 25 ml A + x ml B made up to 100 ml
HCl 0.1 mol l−1 HCl 0.1 mol l−1 HCl 0.05 mol l−1
5 ml A + x ml B made up to 100 ml 25 ml A + x ml B made up to 100 ml 10 ml A + x ml B made up to 100 ml
14
15
20 21 22 23 24 25 26
NaOH 1 mol l−1 100 ml made up to x ml A + (100−x) ml B 1 l with solution A HCl 0.1 mol l−1 HCl 0.1 mol l−1 Sodium bicarbonate 0.1 mol l−1 (8.401 g l−1)
25 ml A + x ml B made up to 100 ml 50 ml A + x ml B made up to 100 ml x ml A + (100−x) ml B
Note: When not otherwise specified, both stock and buffer solutions should be made up with distilled water free of CO2. Only standard reagents should be used. If there is any doubt as to the purity or water content of solutions, their amount of substance concentration must be checked by titration. The volumes x (in ml) of stock solutions required to make up a buffer solution of the desired pH value are given in the table on the next page. From Lenter, C, Ed., Geigy Scientific Tables, 8th ed., volume 3, Ciba-Geigy, Basel, 1984, pages 58–60. With permission.
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Amine Buffers Useful for Biological Research Norman Good All of these amines are highly polar, water-soluble substances. Their advantages and disadvantages must be determined empirically for each biological reaction system. For best buffering performance they should be used at pH’s close to the
Chemical Name 2-(N-Morpholino)ethanesulfonic acid Bis(2-hydroxyethyl)imino-tris-(hydroxymethyl)methane N-(2-Acetamido)iminodiacetic acid Piperazine-N,N′-bis(2-ethanesulfonic acid) 1,3-Bis[tris(hydroxymethyl)methylamino] propane N-(Acetamido)-2-aminoethanesulfonic acid 3-(N-Morpholino)propanesulfonic acid N,N′-Bis(2-hydroxyethyl)-2-amino-ethanesulfonic acid N-Tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid N-2-Hydroxyethylpiperazine-N′-ethanesulfonic acid N-2-Hydroxyethylpiperazine-N′-propanesulfonic acid N-Tris(hydroxymethyl)methylglycine Tris(hydroxymethyl)aminomethane N,N-Bis(2-hydroxyethyl)glycine Glycylglycine N-Tris(hydroxymethyl)methyl-3-amino-propanesulfonic acid 1,3-Bis[tris(hydroxymethyl)-methylamino]propane Glycine
pKa, preferably within ± 0.5 pH units of the pKa and never more than ± 1.0 unit from the pKa. Note that the pKa’s, and therefore the pH’s of buffered solutions, change with temperature in the manner indicated.
Trivial Name or Acronym MES Bistris ADAa PIPES Bistrispropane ACES MOPS BES TES HEPESb HEPPSb Tricinea Tris Bicinea Glycylglycinea TAPS Bistrispropane Glycinea
Structure (HOCH2CH2)2fiN—C≡(CH2OH)3 (HOCH2)3≡C—NH(CH2)3 NH—C≡(CH2 OH)3 H2NCOCH2 N+H2CH2CH2SO3– (HOCH2CH2)2fiN+HCH2CH2SO3− (HOCH2)3≡C—N+H2CH2CH2SO3− (HOCH2)3≡C—N+H2CH2COO− (HOCH2)3≡CNH2 (HOCH2CH2)2fiN+HCH2COO− H3N+CH2CONHCH2COO− (HOCH2)3≡C—N+H2(CH2)3SO3− (HOCH2)3≡C—NH(CH2)3NH—C≡(CH2OH)3 H3N+CH2COO−
pKa at 20°C
ΔpKa/°C
6.15 6.5 6.6 6.8 6.8 (9.0) 6.9 7.15 7.15 7.5 7.55 8.1 8.15 8.3 8.35 8.4 8.55 9.0 (6.8) 9.9
− 0.011 — − 0.011 − 0.0085 — − 0.020 − 0.013 − 0.016 − 0.020 − 0.014 − 0.015 − 0.021 − 0.031 − 0.018 − 0.028 − 0.027 — —
Compiled by Norman Good. a b
These substances may bind certain di- and polyvalent cations and therefore they may sometimes be useful for providing constant, low level concentrations of free heavy metal ions (heavy metal buffering). These substances interfere with and preclude the Folin protein assay.
For further information on these and other buffers, see Good and Izawa, in Methods in Enzymology, Part B, Vol. 24, Pietro, Ed., Academic Press, New York, 1972, 53.
719
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Preparation of Buffers for Use in Enzyme Studies* G. Gomori The buffers described in this section are suitable for use either in enzymatic or histochemical studies. The accuracy of the tables is within ± 0.05 pH at 23°. In most cases the pH values will not be off by more than ± 0.12 pH even at 37° and at molarities slightly different from those given (usually 0.05 M). The methods of preparation described are not necessarily identical with those of the original authors. The titration curves of the majority of the buffers recommended have been redetermined by the writer. The buffers are arranged in the order of ascending pH range. For more complete data on phosphate and acetate buffers over a wide range of concentrations, see Vol. I [10].* *From Gomori, in Methods in Enzymology, Vol. 1, Colowick and Kaplan, Eds., Academic Press, New York, 1955, 138. With permission.
TABLE 3 Phthalate-Hydrochloric Acid Buffer* x
pH
x
pH
46.7 39.6 33.0 26.4 20.3
2.2 2.4 2.6 2.8 3.0
14.7 9.9 6.0 2.63
3.2 3.4 3.6 3.8
*
Stock solutions
A: 0.2 M solution of potassium acid phthalate (40.84 g in 1,000 ml) B: 0.2 M HCl 50 ml of A + x ml of B, diluted to a total of 200 ml Reference 1. Clark and Lubs, J. Bacteriol., 2, 1 (1917).
TABLE 1 Hydrochloric AcidPotassium Chloride Buffer*
TABLE 4 Aconitate Buffer*
x
pH
x
pH
x
pH
97.0 78.0 64.5 51.0 41.5 33.3 26.3 20.6 16.6 13.2 10.6 8.4 6.7
1.0 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2.0 2.1 2.2
15.0 21.0 28.0 36.0 44.0 52.0 60.0 68.0 76.0
2.5 2.7 2.9 3.1 3.3 3.5 3.7 3.9 4.1
83.0 90.0 97.0 103.0 108.0 113.0 119.0 126.0
4.3 4.5 4.7 4.9 5.1 5.3 5.5 5.7
* Stock solutions
A: 0.5 M solution of aconitic acid (87.05 g in 1,000 ml) B: 0.2 M NaOH 20 ml of A + x ml of B, diluted to a total of 200 ml
* Stock solutions
A: 0.2 M solution of KCl (14.91 g in 1,000 ml) B: 0.2 M HCl 50 ml of A + x ml of B, diluted to a total of 200 ml
1. Clark and Lubs, J. Bacteriol., 2, 1 (1917).
TABLE 2 Glycine-HCL Buffer* x
pH
x
pH
5.0 6.4 8.2 11.4
3.6 3.4 3.2 3.0
16.8 24.2 32.4 44.0
2.8 2.6 2.4 2.2
* Stock solutions
A: 0.2 M solution of glycine (15.01 g in 1,000 ml) B: 0.2 M HCl 50 ml of A + x ml of B, diluted to a total of 200 ml
1. Sørensen, Biochem. Z., 21, 131 (1909); 22, 352 (1909).
1. Gomori, unpublished data.
TABLE 5 Citrate Buffer*
Reference
Reference
Reference
x
y
pH
46.5 43.7 40.0 37.0 35.0 33.0 31.5 28.0 25.5 23.0 20.5 18.0 16.0 13.7 11.8 9.5 7.2
3.5 6.3 10.0 13.0 15.0 17.0 18.5 22.0 24.5 27.0 29.5 32.0 34.0 36.3 38.2 41.5 42.8
3.0 3.2 3.4 3.6 3.8 4.0 4.2 4.4 4.6 4.8 5.0 5.2 5.4 5.6 5.8 6.0 6.2
* Stock solutions
721
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Handbook of Biochemistry and Molecular Biology
722 A: 0.1 M solution of citric acid (21.01 g in 1,000 ml) B: 0.1 M solution of sodium citrate (29.41 g C6H5O7Na3 · 2H2O in 1,000 ml; the use of the salt with 5½ H2O is not recommended). x ml of A + y ml of B, diluted to a total of 100 ml Reference
B: 0.2 M solution of dibasic sodium phosphate (53.65 g of Na2HPO4 · 7H2O or 71.7 g of Na2HPO4 · 12H2O in 1,000 ml) x ml of A + y ml of B, diluted to a total of 100 ml Reference 1. McIlvaine, J. Biol. Chem., 49, 183 (1921).
1. Lillie, Histopathologic Technique, Blakiston, Philadelphia and Toronto, 1948.
TABLE 6 Acetate Buffer* x
y
pH
46.3 44.0 41.0 36.8 30.5 25.5 20.0 14.8 10.5 8.8 4.8
3.7 6.0 9.0 13.2 19.5 24.5 30.0 35.2 39.5 41.2 45.2
3.6 3.8 4.0 4.2 4.4 4.6 4.8 5.0 5.2 5.4 5.6
x
pH
x
pH
7.5 10.0 13.3 16.7 20.0 23.5
3.8 4.0 4.2 4.4 4.6 4.8
26.7 30.3 34.2 37.5 40.7 43.5
5.0 5.2 5.4 5.6 5.8 6.0
* Stock solutions
A: 0.2 M solution of succinic acid (23.6 g in 1,000 ml) B: 0.2 M NaOH 25 ml of A + x ml of B, diluted to a total of 100 ml Reference
* Stock solutions
1. Gomori, unpublished, data.
A: 0.2 M solution of acetic acid (11.55 ml in 1,000 ml) B: 0.2 M solution of sodium acetate (16.4 g of C2H3O2Na or 27.2 g of C2H3O2Na · 3H2O in 1,000 ml) x ml of A + y ml of B, diluted to a total of 100 ml Reference 1. Walpole, J. Chem. Soc., 105, 2501 (1914).
TABLE 9 Phthalate-Sodium Hydroxide Buffer* x
pH
x
pH
3.7 7.5 12.2 17.7 23.9
4.2 4.4 4.6 4.8 5.0
30.0 35.5 39.8 43.0 45.5
5.2 5.4 5.6 5.8 6.0
* Stock solutions
TABLE 7 Citrate-Phosphate Buffer* x
y
pH
44.6 42.2 39.8 37.7 35.9 33.9 32.3 30.7 29.4 27.8 26.7 25.2 24.3 23.3 22.2 21.0 19.7 17.9 16.9 15.4 13.6 9.1 6.5
5.4 7.8 10.2 12.3 14.1 16.1 17.7 19.3 20.6 22.2 23.3 24.8 25.7 26.7 27.8 29.0 30.3 32.1 33.1 34.6 36.4 40.9 43.6
2.6 2.8 3.0 3.2 3.4 3.6 3.8 4.0 4.2 4.4 4.6 4.8 5.0 5.2 5.4 5.6 5.8 6.0 6.2 6.4 6.6 6.8 7.0
* Stock solutions
A: 0.1 M solution of citric acid (19.21 g in 1,000 ml)
9168_Book.indb 722
TABLE 8 Succinate Buffer*
A: 0.2 M solution of potassium acid phthalate (40.84 g in 100 ml) B: 0.2 M NaOH 50 ml of A + x ml of B, diluted to a total of 200 ml Reference 1. Clark and Lubs, J. Bacteriol., 2, 1 (1917).
TABLE 10 Maleate Buffer* x
pH
x
pH
7.2 10.5 15.3 20.8 26.9
5.2 5.4 5.6 5.8 6.0
33.0 38.0 41.6 44.4
6.2 6.4 6.6 6.8
*
Stock solutions
A: 0.2 M solution of acid sodium maleate (8 g of NaOH + 23.2 g of maleic acid or 19.6 g of maleic anhydride in 1,000 ml) B: 0.2 M NaOH 50 ml of A + x ml of B, diluted to a total of 200 ml Reference 1. Temple, J. Am. Chem. Soc., 51, 1754 (1929).
4/16/10 1:26 PM
Preparation of Buffers for Use in Enzyme Studies TABLE 11 Cacodylate Buffer* x
pH
x
pH
2.7 4.2 6.3 9.3 13.3 18.3 23.8
7.4 7.2 7.0 6.8 6.6 6.4 6.2
29.6 34.8 39.2 43.0 45.0 47.0
6.0 5.8 5.6 5.4 5.2 5.0
723 A: 0.2 M solution of Tris acid maleate (24.2 g. of tris(hydroxymethyl)aminomethane + 23.2 g of maleic acid or 19.6 g of maleic anhydride in 1,000 ml) B: 0.2 M NaOH 50 ml of A + x ml of B, diluted to a total of 200 ml †A buffer-grade Tris can be obtained from the Sigma Chemical Co., St. Louis, MO., or From Matheson Coleman & Bell, East Rutherford, NJ.
Reference
* Stock solutions
1. Gomori, Proc. Soc. Exp. Biol. Med., 68, 354 (1948).
A: 0.2 M solution of sodium cacodylate (42.8 g of Na(CH3)2 AsO2 · 3H2O in 1,000 ml) B: 0.2 M HCl 50 ml of A + x ml of B, diluted to a total of 200 ml
TABLE 14 Barbital Buffer*†
Reference 1. Plumel, Bull. Soc. Chim. Biol., 30, 129 (1949).
TABLE 12 Phosphate Buffer* x
y
pH
x
y
pH
93.5 92.0 90.0 87.7 85.0 81.5 77.5 73.5 68.5 62.5 56.5 51.0
6.5 8.0 10.0 12.3 15.0 18.5 22.5 26.5 31.5 37.5 43.5 49.0
5.7 5.8 5.9 6.0 6.1 6.2 6.3 6.4 6.5 6.6 6.7 6.8
45.0 39.0 33.0 28.0 23.0 19.0 16.0 13.0 10.5 8.5 7.0 5.3
55.0 61.0 67.0 72.0 77.0 81.0 84.0 87.0 90.5 91.5 93.0 94.7
6.9 7.0 7.1 7.2 7.3 7.4 7.5 7.6 7.7 7.8 7.9 8.0
1.5 2.5 4.0 6.0 9.0 12.7 17.5 22.5 27.5 32.5 39.0 43.0 45.0
9.2 9.0 8.8 8.6 8.4 8.2 8.0 7.8 7.6 7.4 7.2 7.0 6.8
* Stock solutions
A: 0.2 M solution of sodium barbital (veronal) (41.2 g in 1,000 ml) B: 0.2 M HCl 50 ml of A + x ml of B, diluted to a total of 200 ml Solutions more concentrated than 0.05 M may crystallize on standing, especially in the cold.
A: 0.2 M solution of monobasic sodium phosphate (27.8 g in 1,000 ml) B: 0.2 M solution of dibasic sodium phosphate (53.65 g of Na2HPO4 · 7H2O or 71.7 g of Na2HPO4 · 12H2O in 1,000 ml) x ml of A + y ml of B, diluted to a total of 200 ml
Reference 1. Michaelis, J. Biol. Chem., 87, 33 (1930).
TABLE 15 Tris(Hydroxymethyl)Aminomethane (Tris) Buffer*†
Reference 1. Sørensen, Biochem. Z., 21, 131 (1909); 22, 352 (1909).
TABLE 13 Tris(Hydroxymethyl)AminomethaneMaleate (Tris-Maleate) Buffer*† x
pH
x
pH
7.0 10.8 15.5 20.5 26.0 31.5 37.0 42.5 45.0
5.2 5.4 5.6 5.8 6.0 6.2 6.4 6.6 6.8
48.0 51.0 54.0 58.0 63.5 69.0 75.0 81.0 86.5
7.0 7.2 7.4 7.6 7.8 8.0 8.2 8.4 8.6
9168_Book.indb 723
pH
†
* Stock solutions
* Stock solutions
x
x
pH
5.0 8.1 12.2 16.5 21.9 26.8 32.5 38.4 41.4 44.2
9.0 8.8 8.6 8.4 8.2 8.0 7.8 7.6 7.4 7.2
* Stock solutions
A: 0.2 M solution of tris(hydroxymethyl)aminomethane (24.2 g in 1,000 ml) B: 0.2 M HCl 50 ml of A + x ml of B, diluted to a total of 200 ml A buffer-grade Tris can be obtained from the Sigma Chemical Co., St. Louis, MO., or from Matheson Coleman & Bell, East Rutherford, NJ. †
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Handbook of Biochemistry and Molecular Biology
724 TABLE 16 Boric Acid-Borax Buffer* x
pH
2.0 3.1 4.9 7.3 11.5 17.5
7.6 7.8 8.0 8.2 8.4 8.6
x
TABLE 19 Borax-NaOH Buffer*
pH
22.5 30.0 42.5 59.0 83.0 115.0
8.7 8.8 8.9 9.0 9.1 9.2
* Stock solutions
A: 0.2 M solution of boric acid (12.4 g in 1,000 ml) B: 0.05 M solution of borax (19.05 g in 1,000 ml; 0.2 M in terms of sodium borate) 50 ml of A + x ml of B, diluted to a total of 200 ml Reference 1. Holmes, Anat. Rec., 86, 163 (1943).
TABLE 17 2-Amino-2-Methyl-1,3-Propanediol (Ammediol) Buffer* x
pH
x
pH
2.0 3.7 5.7 8.5 12.5 16.7
10.0 9.8 9.6 9.4 9.2 9.0
22.0 29.5 34.0 37.7 41.0 43.5
8.8 8.6 8.4 8.2 8.0 7.8
* Stock solutions
A: 0.2 M solution of 2-amino-2-methyl-l,3-propanediol (21.03 g in 1,000 ml) B: 0.2 M HCl 50 ml of A + x ml of B, diluted to a total of 200 ml Reference 1. Gomori, Proc. Soc. Exp. Biol. Med., 62, 33 (1946).
TABLE 18 Glycine-NaOH Buffer* x 4.0 6.0 8.8 12.0 16.8
pH
x
8.6 8.8 9.0 9.2 9.4
22.4 27.2 32.0 38.6 45.5
pH 9.6 9.8 10.0 10.4 10.6
* Stock solutions
A: 0.2 M solution of glycine (15.01 g in 1,000 ml) B: 0.2 M NaOH 50 ml of A + x ml of B, diluted to a total of 200 ml. Reference 1. Sørensen, Biochem. Z., 21, 131 (1909); 22, 352 (1909).
9168_Book.indb 724
x
pH
0.0 7.0 11.0 17.6 23.0 29.0 34.0 38.6 43.0 46.0
9.28 9.35 9.4 9.5 9.6 9.7 9.8 9.9 10.0 10.1
* Stock solutions
A: 0.05 M solution of borax (19.05 g in 1,000 ml; 0.02 M in terms of sodium borate) B: 0.2 M NaOH 50 ml of A + x ml of B, diluted to a total of 200 ml Reference 1. Clark and Lubs, J. Bacteriol., 2, 1 (1917).
TABLE 20 Carbonate-Bicarbonate Buffer* x
y
pH
4.0 7.5 9.5 13.0 16.0 19.5 22.0 25.0 27.5 30.0 33.0 35.5 38.5 40.5 42.5 45.0
46.0 42.5 40.5 37.0 34.0 30.5 28.0 25.0 22.5 20.0 17.0 14.5 11.5 9.5 7.5 5.0
9.2 9.3 9.4 9.5 9.6 9.7 9.8 9.9 10.0 10.1 10.2 10.3 10.4 10.5 10.6 10.7
* Stock solutions
A: 0.2 M solution of anhydrous sodium carbonate (21.2 g in 1,000 ml) B: 0.2 M solution of sodium bicarbonate (16.8 g in 1,000 ml) x ml of A + y ml of B, diluted to a total of 200 Reference 1. Delory and King, Biochem. J., 39, 245 (1945).
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9168_Book.indb 725
Buffer for Acrylamide Gels (Single-Gel Systems) Buffer Solutions In Gel Buffer Ions
In Electrode Vessels M
pH
0.09 0.070 0.007 0.010 0.070 0.007 0.0016 0.006 0.022 0.017 0.05 0.068 0.025
Tris Glycine Tris borate
Potassium acetate
0.010 0.005 0.1 0.02
9.2 8.7 2.9
Tris glycine Veronal Tris Tris
0.37 0.05 0.083 0.37
9.5 8.4 8.9 8.8
a b
M
pH
Characteristics of System
Apparatus Typea
__
VP
Serum1
Albumin band too narrow for acurate quantitation Mobilities are low in this buffer —
VP
Serum1
VP VP
Serum1
Rapid; cooled in petroleum ether
FP
Lactic dehydrogenase isoenzymes3
12 M urea in gel
VP FP
Myosin subunits4 Ribosomal proteins5
Sample unstabilized
D
Serum6
Preliminary soaking of gel possible Sucrose used to stabilize sample
VP D
Serum7 Histones8
Sucrose use to stabilize sample — 7 M urea in gel Sample concentrates as it enters gel
D FP D D
Serum12 Lactic dehydrogenase isoenzymes3 a-Crystallin14 Serum15
8.6
Veronal Tris EDTA Borateb Tris EDTA Ca lactate Tris Veronal Na lactate Acetate Tris Citrate Phenol:acetic acid: water 2:1:1
{
Buffer Ions
8.7 8.65 8.5 5.7 7.2
Same as in gel
Borateb
0.05 Same as in gel
Tris Glycine
0.04 0.022 Same as in gel 0.3 0.3 0.37 — Same as in gel 0.05
Valine, anode) Glycine, cathode) Tris glycine — Borateb
{
8.6
9.2 4.0 (acetic) 9.5 — 9.2
—
Examples of Materials Investigated
D, Disc; FP, flat plate, in the horizontal; VP, vertical plate. Molarity based on weight of orthoboric acid, (H4BO3) taken.
From Electrophoresis of Proteins in Polyacrylamide and Starch Gels. Gordon, A. H., Ed., (Laboratory Techniques in Biochemistry and Molecular Biology, Work, T. S. and Work, E., Gen. Eds.), North-Holland, New York, 1971, 40. With permission of Elsevier Science Publishers.
725
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726
9168_Book.indb 726
Buffer for Acrylamide Gels With More Than One Layer Buffer Solutions In Gel Gel Layer
Sample (large pore) Spacer (large pore)
Small-pore gel
Same gel layers as above Spacer (large pore) Small-pore gel
Spacer (large pore) Small-pore gel
a b
M
pH
Buffer Ions Tris Glycine
Tris Chloride Tris Chloride Tris Chloride and buffers
0.062 0.06 0.062 0.06 0.37 0.06
6.7
Tris Chloride Tris Chloride Tris H3PO4 Tris Chloride Acetic acid KOH Acetic acid KOH Acetic acid KOH
0.075 0.075 0.046 0.075 0.049 0.026 0.37 0.06 0.062 0.06 0.062 0.06 0.75 0.12
6.7
6.7
M
Ref.
pH
Characteristics of System
Apparatus Typea
0.005 0.038
8.3
The sample is set in the upper-the most gel and concentrates in spacer gel; the actual separation occurs in the bottom gel
D
9
Tris Glycine
0.05 0.38
8.3
Same as Ref. 9 except that improved resolution is obtained if as above carried out at 4-5°C
D
10
Tris Glycine
0.05 0.38
8.3
Same as Ref. 9 except that sample gel is omitted; the sample is stabilized with sucrose
D
2
Tris Glycine
0.025 0.19
8.4
Same as Ref. 9 except that sample gel is omitted; the sample is stabilized with acrylamide monomer
VP
11
Acetic acid β-Alanine
0.14 0.35
4.5
An acid gel suitable for separation of bases
D
13
8.9
8.7 6.7 8.9 6.8 6.8 4.3
D: disc; FP; flat plate, in the horizontal); VP: vertical plate. Molarity based on weight of orthoboric acid, (H4BO3) taken. For additional buffers see Starch Gels. All buffers recommended for starch gel can be used in acrylamide gels.
From Electrophoresis of Proteins in Polyacrylamide and Starch Gels, Gordon, A. H., Ed., (Laboratory Techniques in Biochemistry and Molecular Biology, Work, T. S. and Work, E., Gen. Eds.), North-Holland, New York, 1971. 41. With permission of Elsevier Science Publishers.
Handbook of Biochemistry and Molecular Biology
Sample (large pore) Spacer (large pore) Small-pore gel
Buffer Ions
In Electrode Vessels
4/16/10 1:26 PM
Buffer for Acrylamide Gels (Single-Gel Systems)
References For Acrylamide Gel Tables 1. Ferris, T. G., Easterling, R. E., and Budd, R. E., Anal. Biochem. 8, 477, 1964. 2. Pun, J. Y. and Lombarozo, L., Anal. Biochem., 9, 9, 1964. 3. Jensen, K., Scand. J. Clin. Lab. Invest., 17, 192, 1965. 4. Small, P. A., Harington, W. F., and Keilley, W. W., Biochim. Biophys. Acta., 49, 462, 1961. 5. Work, T. S., J. Mol. Biol., 10, 544, 1964. 6. Matson, C. F., Anal. Biochem., 13, 294, 1965. 7. Lorber, A., J. Lab. Clin. Med., 64, 133, 1964.
9168_Book.indb 727
727 8. Shepherd, G. R. and Gurley, L. R., Anal. Biochem., 14, 356, 1966. 9. Davies, B. J., Ann. N.Y. Acad. Sci., 121, 404, 1964. 10. Pastewka, J. V., Neass,A. T., and Peacock, A. C., Clin. Chim. Acta, 14, 219, 1966). 11. Ritchie, R. F., Harter, J. G., and Bayles, T. B., J. Lab, Clin. Med., 68, 842, 1966. 12. Hjerten S., Jerstedt S., and Tiselius A., Anal. Biochem., 11, 219, 1965. 13. Reisfeld R. A., Lewis U. J., and Williams D. E., Nature, 195, 281, 1962. 14. Bloemendal H., Bout W. S., Jongkind J. F., and Wisse J. H., Exp. Eye Res., 1,300, 1962. 15. Gordon A. H. and Louis L. N., Anal. Biochem., 21, 190, 1967.
4/16/10 1:26 PM
Starch Gels TABLE 1 Starch Gels: Buffer Solutions In Gel
In Electrode Vessels
Buffer Ions
M
pH
Buffer Ions
Borate Tris Citratea
0.025 0.076 0.005
8.5 8.6
Borate Borate
0.3 0.3
Tris Boratea EDTA Tris Malatea EDTA MgCl2 Glycine Citratea Boratea Tris Lithium Tris HCl Acetate
0.045 0.025 0.002 0.1 0.01 0.001 0.001 0.05 0.0027 0.0076 0.0144 0.0020 0.03 — 0.1
Tris Borate EDTA Tris Malate EDTA MgCl2 Borate — Borate — Lithium Tris HCl —
0.013 0.075 0.006 0.01 0.1 0.01 0.01 0.3 — 0.38 — 0.10 0.05 — —
a,b
a b
8.4 7.4 8.9 8.0 8.4 5.0
M
Materials Investigated (Examples Only)
pH
Results
8.5 8.5
Serum Serum,2,3 diphtheria toxin,2 horseradish peroxidase,2 haptoglobins,5 and transferins6 Hemoglobins,4 human and animal sera,7 staphylococcal enterotoxin B16
8.4
Hemoglobins12-14 Serum17
7.4
Hemolysates invested for phosphoglucomutase20
8.9
Myeloma g-globulin8
1
Citrate borate boundary is seen as a brown band The electrophoresis is over in less time compared to borate only Hb A and F are separated
Five bands
Anterior pituitary hormones15 Tissue dehydrogenases21 The enzymes were recovered from the gel
Pepsin and gastricsin23
Molarities based on weight of orthoboric (H4BO3) or other acid taken. When no cation is given, the acids have been adjusted to the pH shown by addition of NaOH. For optimum results, use borate concentration recommended for each individual batch of starch.
From Gordon, A. H., Ed., Electrophoresis of Proteins in Polyacrylamide and Starch Gels (in Laboratory Techniques in Biochemistry and Molecular Biology, Work, T. S. and Work, E., Gen. Eds.), North-Holland, New York, 1971, 100. With permission of Elsevier Science Publishers.
TABLE 2 Starch Gels Containing Urea Buffer Solutions Buffer Solutions In Gel Buffer Ions
In Electrode Vessels
M
pH
Urea M
Formate
0.05
3.0
8
Aluminum lactate Glycine
0.05
3.1
3
0.035
7—8
Boratea Tris Citrate Acetate
0.025 0.076 0.005 0.0365
Buffer Ions
Materials Investigated (Examples Only)
M
pH
Formate
0.2
2—3
8
Aluminum lactate Borate
g-Myeloma, heavy chains,10 antibodies18 Gliadin11
0.3
8.2
8.5 8.6
8 7
Borate Borate
0.3 0.3
8.5 8.6
5.6
6
g-Globulin heavy and light Light chains well separated chains9 Haptoglobins19 Without prior reduction urea has no effect Urea treatment of a-crystallin, without a-and b-casein22.25 reduction, leads to formation of many bands At least 14 bands E. coli ribosomal proteins24
—
—
—
Results Slightly sharper bands were obtained if alkylation was done as well as reduction
Molarities based on weight of orthoboric (H4BO3) or other acid taken. When no cation is given, the acids have been adjusted to the pH shown by addition of NaOH. b For optimum results, use borate concentration recommended for each individual batch of starch. a
From Gordon, A. H., Ed., Electrophoresis of Proteins in Polyacrylamide and Starch Gels (in Laboratory Techniques in Biochemistry and Molecular Biology, Work, T. S. and Work, E., Gen. Eds.), North-Holland, New York, 1971. 101. with permission of Elsevier Science Publishers.
729
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Handbook of Biochemistry and Molecular Biology
730
References to Tables 1 and 2
1. 2. 3. 4. 5.
6. 7. 8. 9. 10. 11. 12. 13.
9168_Book.indb 730
Smithies, O., Biophys. J., 61, 629, 1955. Poulik, M.D., Nature, 180, 1477, 1957. Poulik, M. D., J. Immunol., 82, 502, 1959. De Grouchy, J., Rev. Fr. Etud. Clin. Biol., 3, 877, 1958. Giblett, E. R., Motulsky, A. G., and Frazer, G. R., Am. J. Human Genet., 18, 553, 1966. Harris, H., Robson, E. B., and Siniscalco, M., Nature, 182, 452, 1958. Krotski, W. A., Benjamin, D. C., and Weimer, H. E., Can. J. Biochem., 44, 545, 1966. Askonas, I., Biochem. J., 79, 33, 1961. Cohen, S. and Porter, R. R., Biochem. J., 90, 278, 1964. Edelman, G. M. and Poulik, M. D., J. Exp. Med., 113, 861, 1961. Woychick, J. H., Boundy, J. A., and Dimler, R. J., Arch. Biochem. Biophys., 94, 477, 1961. Winterhalter, K. H. and Huehns, E. R., J Biol. Chem., 239, 3699, 1964. Huehns, E. R., Dance, N., Beaven, G. H., Keil, J. V., Hecht, F., and Motulsky, A. G., Nature, 201, 1095, 1964.
14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25.
Chernoff, A. I. and Pettit, N. M., Blood, 25, 646, 1965. Ferguson, K. A. and Wallace, A. L. C., Nature, 190, 629, 1961. Baird-Parker, A. C. and Jospeh, R. L., Nature, 202, 570, 1964 Poulik, M. D., in Methods of Biochemical Analysis, Vol. 14, Glick, D., Ed., Interscience, 1966, 455. Edelman, G. M., Benacerraf, B., and Ovary, Z., J. Exp. Med., 118, 229, 1963. Smithies, O. and Connell, G. E., in Biochemistry of Human Genetics, Churchill, London, 1959, 178. Spencer, N., Hopkinson, D. A., and Harris, H., Nature, 204, 742, 1964. Tsao, M. U., Arch. Biochem. Biophys., 90, 234, 1960. Wake, R. G. and Baldwin, R. L., Biochem. Biophys. Acta, 47, 227, 1961. Tang, J., Wolf, S., Caputto, R., and Trucco, R. E., J. Biol. Chem., 234, 1174, 1959. Waller, J. P. and Harris, J. I., Proc. Nat. Acad. Sci. U.S.A., 47, 18, 1961. Bloemendal, H., Bout, W. S., Jonkind, J. F., and Wisse, J. H., Nature, 193, 437, 1962.
4/16/10 1:26 PM
Indicators for Volumetric Work and pH Determinations Indicator Methyl violet 6B Metacresol purple (acid range) Metanil yellow p-Xylenol blue (acid range) Thymol blue (acid range) Tropaeolin OO Quinaldine red Benzopurpurine 4B Methyl violet 6B 2,4-Dinitrophenol
Acid Color
pH Range
Basic Color
Tetra and pentamethylated p-rosaniline hydrochloride m-Cresolsulfonphthalein
Y
0.1–1.5
B
pH: 0.25% water
R
0.5–2.5
Y
4-Phenylamino-azobenzene-3′-sulfonic acid 1,4-Dimethyl-5hydroxybenzenesulfonphthalein Thymolsulfonphthalein
R
1.2–2.3
Y
pH: 0.10 g. in 13.6 ml 0.02 N NaOH, diluted to 250 ml. with water pH: 0.25% in ethanol
R
1.2–2.8
Y
pH: 0.04% in ethanol
R
1.2–2.8
Y
Sodium p-diphenylamino-azobenzenesulfonate 2-(p-Dimethylaminostyryl)quinoline ethiodide Ditolyl-diazo-bis-a-naphthyl-amine-4sulfonic acid Tetra and pentamethylated p-rosaniline hydrochloride
R
1.4–2.6
Y
pH: 0.1 g. in 10.75 ml. 0.02 N NaOH, diluted to 250 ml. with water pH: 0.1% in water Vol.: 1% in water
C
1.4–3.2
R
Vol.: 0.1% in ethanol
B-V
1.3–4.0
R
pH, vol.: 0.1% in water
B
1.5–3.2
V
pH, vol.: 0.25% in water
C
2.6–4.0
Y
pH, vol.: 0.1 g. in 5 ml. ethanol, diluted to 100 ml. with water pH, vol.: 0.05% in ethanol pH: 0.1 g. in 7.45 ml. 0.02 N NaOH, diluted to 250 ml. with water pH: 0.1 g. in 5.00 ml. 0.02 N NaOH, diluted to 250 ml. with water Vol.: 0.1 g. in 7.35 ml. 0.02 N NaOH, diluted to 100 ml. with water
Chemical Name
Preparation
Methyl yellow Bromphenol blue
p-Dimethylaminoazobenzene Tetrabromophenolsulfonphthalein
R Y
2.9–4.0 3.0–4.6
Y B
Tetrabromophenol blue
Tetrabromophenol-tetrabromosulfonphthalein Disodium 4,4′-bis(2-amino-1-naphthylazo)-2,2′stilbenedisulfonate Diphenyl-diazo-bis-1-naphthylamine-4sodium sulfonate 4′-Dimethylaminoazobenzene-4-sodium sulfonate Dibromodichlorophenolsulfonphthalein
Y
3.0–4.6
B
B-P
3.0–4.6
R
B
3.0–5.2
R
pH: 0.1% in water
R
3.1–4.4
Y
Vol.: 0.1% in water
Y
3.2–4.8
B
R R Y
3.5–5.5 3.7–5.0 3.7–5.2
Y Y V
pH: 0.1 g. in 8.6 ml. 0.02 N NaOH, diluted to 250 ml. with water Vol.: 0.04% in ethanol Vol.: 0.1% in ethanol Vol.: 0.1% in ethanol pH, vol.: 1% in water
Y
3.8–5.4
B
C
4.0–5.8
Y
R
4.2–6.2
Y
R R R R
4.4–6.2 4.5–8.3 4.5–8.3 4.8–6.2
B B B V
Direct purple Congo red Methyl orange Brom-chlorphenol blue p-Ethoxychrysoidine a-Naphthyl red Sodium alizarinsulfonate Bromcresol green
4′-Ethoxy-2,4-diaminoazobenzene Dihydroxyanthraquinone sodium sulfonate Tetrabromo-m-cresolsulfonphthalein
2,5-Dinitrophenol Methyl red Lacmoid Azolitmin Litmus Cochineal
4′-Dimethylaminoazobenzene-2carboxylic acid
Complex hydroxyanthraquinone derivative
Hematoxylin Chlorphenol red
Dichlorophenolsulfonphthalein
Y Y
5.0–6.0 5.0–6.6
V R
Bromcresol purple
Dibromo-o-cresolsulfonphthalein
Y
5.2–6.8
Pu
Bromphenol red
Dibromophenolsulfonphthalein
Y
5.2–7.0
R
Alizarin
1,2-Dihydroxyanthraquinone
Y
5.5–6.8
R
pH: 0.10 g. in 7.15 ml. 0.02 N NaOH, diluted to 250 ml. with water pH, vol.: 0.10 g. in 20 ml. Ethanol, then dilute to 100 ml. with water pH: 0.10 g. in 18.6 ml. 0.02 N NaOH, diluted to 250 ml. with water Vol.: 0.1% in ethanol Vol.: 0.5% in ethanol Vol.: 0.5% in water Vol.: 0.5% in water Vol.: Triturate 1 g. with 20 ml. Ethanol and 60 ml. water, let stand 4 days, and filter Vol.: 0.5% in ethanol. pH: 0.1 g. in 11.8 ml. 0.02 N NaOH, diluted to 250 ml. with water Vol.: 0.04% in ethanol pH: 0.1 g. in 9.25 ml. 0.02 N NaOH, diluted to 250 ml with water Vol.: 0.02% in ethanol pH: 0.l g. in 9.75 ml. 0.02 N NaOH, diluted to 250 ml. with water Vol.: 0.04% in ethanol Vol.: 0.1% in ethanol
731
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Handbook of Biochemistry and Molecular Biology
732
INDICATORS FOR VOLUMETRIC WORK AND pH DETERMINATIONS (Continued)
Indicator Dibromophenoltetrabromophenolsulfonphthalein p-Nitrophenol Bromothymol blue Indo-oxine Cucumin Quinoline blue Phenol red Neutral red Rosolic acid aurin; corallin Cresol red a-Naphtholphthalein Metacresol purple (alkaline range) Ethylbis-2,4dinitrophenylacetate Tropaeolin OOO No. 1 Thymol blue (alkaline range) p-Xylenol blue o-Cresolphthalein a-Naphtholbenzein Phenolphthalein Thymolphthalein Nile blue A Alizarin yellow GG Alizarin yellow R Poirrer’s blue C4B Tropaeolin O Nitramine 1,3,5-Trinitrobenzene Indigo carmine
Acid Color
pH Range
Basic Color
Y
5.6–7.2
Pu
C Y
5.6–7.6 6.0–7.6
Y B
R
6.0–8.0
B
Y C Y
6.0–8.0 6.6–8.6 6.8–8.4
Br-R B R
R
6.8–8.0
Y
o-Cresolsulfonphthalein
Y Y
6.8–8.2 7.2–8.8
R R
m-Cresolsulfonphthalein
P Y
7.3–8.7 7.4–9.0
G P
C
7.5–9.1
B
Y
7.6–8.9
R
Y
8.0–9.6
B
Y
8.0–9.6
B
pH: 0.l g. in 10.75 ml. 0.02 N NaOH, diluted to 250 ml. with water Vol.: 0.1% in ethanol pH, vol.: 0.04% in elhanol
C Y C C B
8.2–9.8 8.5–9.8 8.2–10 9.3–10.5 10–11
R G R B P
pH, vol.: 0.04% in ethanol pH, vol.: 1% in ethanol Vol.: 1% in ethanol pH, vol.: 0.1% in ethanol Vol.: 0.1% in water
Y
10–12
L
pH, vol.: 0.1% in 50% ethanol
Y
10.2–12.0
R
pH, vol.: 0.1% in water
B Y C C B
11–13 11–13 10.8–13 11.5–14 11.6–14
R O Br O Y
pH: 0.2% in water pH: 0.1% in water pH: 0.1% in 70% ethanol pH: 0.1% in ethanol pH: 0.25% in 50% ethanol
Chemical Name
Dibromothymolsulfonphthalein 5,8-Quinolinequinone-8-hydroxy-5quinolyl-5-imide Cyanine Phenolsulfonphthalein 2-Methyl-3-amino-6dimethylaminophenazine
Sodium a-naphtholazobenzene-sulfonate Thymolsulfonphthalein 1,4-Dimethyl-5hydroxybenzenesulfonphthalein 3,3-Bis(p-hydroxyphenyl)-phthalide Aminonaphthodiethylaminophenoxazine sulfate 3-Carboxy-4-hydroxy-3′nitroazobenzene 3-Carboxy-4-hydroxy-4′nitroazobenzene sodium salt p-Benzenesulfonic acid-azoresorcinol Picrylnitromethylamine Sodium indigodisulfonate
Preparation pH: 0.1 g. in 1.21 ml. 0.1 N NaOH, diluted to 250 ml. with water pH, vol.: 0.25% in water pH: 0.1 g. in 8 ml. 0.02 N NaOH, diluted to 250 ml. with water Vol.: 0.1% in 50% ethanol Vol.: 0.05% in ethanol Vol: saturated aq. soln. Vol.: 1% in ethanol pH: 0.1 g. in 14.20 ml. 0.02 N NaOH, diluted to 250 ml. with water Vol.: 0.1% in ethanol pH, vol.: 0.1 g. in 70 ml. ethanol, diluted to 100 ml. with water pH, vol.: 1% in 50% ethanol pH: 0.1 g. in 13.1 ml. 0.02 N NaOH, diluted to 250 ml. with water Vol.: 0.1% in ethanol pH. vol.: 0.1% in 50% ethanol pH: 0.1 g. in 13.1 ml. 0.02 N NaOH, diluted to 250 ml. with water Vol.: 0.1% in ethanol Vol.: saturated soln. in equal volumes of acetone and ethanol Vol.: 0.1% in water
Note: The indicator colors are abbreviated as follows: B, blue; Br, brown; C, colorless; G, green; L, lilac; O, orange; P, pink; Pu, purple; R, red; V, violet; and Y, yellow.
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Indicators for Volumetric Work and pH Determinations
733
Mixed Indicators Composition
Solvent
Transition pH
Acid Color
Transition Color
Basic Color
Dimethyl yellow, 0.05% + Methylene blue, 0.05% Methyl orange, 0.02% + Xylene cyanole FF, 0.28% Methyl yellow, 0.08% + Methylene blue, 0.004% Methyl orange, 0.1% + Indigocarmine, 0.25% Bromcresol green, 0.1% + Methyl orange, 0.02% Bromcresol green, 0.075% + Methyl red, 0.05% Methyl red, 0.1% + Methylene blue, 0.05% Bromcresol green, 0.05% + Chlorphenol red, 0.05% Bromcresol purple, 0.05% + Bromthymol blue, 0.05% Neutral red, 0.05% + Methylene blue, 0.05% Bromthymol blue, 0.05% + Phenol red, 0.05% Cresol red, 0.025% + Thymol blue, 0.15% Phenolphthalein, 0.033% + Methyl green, 0.067% Phenolphthalein, 0.075% + Thymol blue, 0.025% Phenolphthalein, 0.067% + Naphtholphthalein, 0.033% Phenolphthalein, 0.033% + Nile blue, 0.133% Alizarin yellow, 0.033% + Nile blue, 0.133%
alc. 50% alc. alc. aq. aq. alc. alc. aq. aq. alc. aq. aq. alc. 50% alc. 50% alc. alc. alc.
3.2 3.9 3.9 4.1 4.3 5.1 5.4 6.1 6.7 7.0 7.5 8.3 8.9 9.0 9.6 10.0 10.8
Blue–violet Red Pink Violet Orange Wine–red Red–violet Yellow–green Yellow Violet–blue Yellow Yellow Green Yellow Pale rose Blue Green
— Gray Straw–pink Gray Light green — Dirty blue — Violet Violet–blue Violet Rose Gray–blue Green — Violet —
Green Green Yellow–green Yellow–green Dark green Green Green Blue–violet Violet–blue Green Dark violet Violet Violet Violet Violet Red Red–brown
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Acid and Base Indicators The following is a brief list of some acid-base indicators (a more comprehensive listing is available in the CRC Handbook of Chemistry and Physics, CRC Press, Boca Raton, FL, USA, Section 8). There is extensive use of acid-base indicators in the
measurement of intracellular pH, as indicators for enzymecatalyzed reactions, and in the measurement of material transfer across membranes and detection of changes in solid matrices.
Some Acid-Base Indicators (pH Indicators) Indicator Dye
Acid Color
Basic Color
pKa
pH Range
Cresol Red (I) (o-cresolsulfonephthalein) Crystal Violet Thymol Blue (I) (thymolsulfonephthalein) Cresol Purple (metacresol purple; m-cresolpurple) Bromophenol Blue Congo Red Methyl Red Neutral Red Phenol Red (phenolsulfonphthalein) Cresol Red (II) Cresol Purple (II) Thymol Blue (II) Phenolphthalein Nile Blue Nitramine (picrymethylnitramine)
Red
Yellow
Green Red
Blue Yellow
1.6
0.0 – 2.0 1.2 – 2.8
Red
Yellow
1.5
1.2 – 2.8
Yellow Blue/Violet Red Red Yellow
Blue Red Yellow Yellow Red
4.1
3.0 – 4.6 3.0 – 5.2 4.4 – 6.3 6.8 – 8.0 6.8 – 8.4
Yellow Yellow Yellow Colorless Blue Colorless
Purple Purple Blue Red Red Orange-Brown
0.2 – 1.8
5.1 7.4 8.0 8.4 8.3 9.2 9.6 10.0
7.3 – 8.8 7.4 – 9.0 8.0 – 9.6 8.3 – 10.0 9.0 – 10.4 10.8 – 12.8
Acid-Base Indicators in Organic Solvents pKa Indicator Thymol Blue (I) Cresol Red(I) Bromophenol Blue Neutral Red Phenol Red Cresol Purple
H2O
Dimethylformamide
1.7 4.1 7.4 8.0 8.3
15.4 15.2
2-Propanol 5.0 4.3 8.8 7.2 15.4
735
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Handbook of Biochemistry and Molecular Biology
736
References for the use of acid-base indicators Hammett, L.P. and Deyrup, A.J., A series of simple basic indicators. I. The acidity functions of mixtures of sulfuric and perchloric acids with water, J.Amer.Chem.Soc. 54, 2721-2739, 1932 Gardner, K.J., Use of acid-base indicator for quantitative paper chromatography of sugars, Nature 176, 929-930, 1955 Meikle, R.W., Paper chromatography of 2-halogenated carboxylic acids: N,N-dimethyl-p-phenylazoaniline as an acid-base indicator reagent, Nature 196, 61, 1962 Kolthoff, I.M., Bhowmik, S., and Chantooni, M.K., Acid-base indicator properties of sulfonephthaleins and benzeins in acetonitrile, Proc. Nat.Acad.Sci.USA 56, 13701376, 1966 Chance, B. and Scarpa, A., Acid-base indicator for the measurement of rapid changes in hydrogen ion concentration, Methods Enzymol. 24, 336-342, 1972 Benkovic, P.A., Hegazi, M., Cunningham, B.A., and Benkovic, S.J., Investigation of the pre-steady state kinetics of fructose bisphosphatase by employment of an indicator method, Biochemistry 18, 830860, 1979 Smith, M.A. and Thompson, R.A., A method for the estimation of the activity of the inhibitor of the first component of complement, J.Clin. Pathol. 33, 167-170, 1980 Kogure, K., Alonso, O.F., and Martinez, E., A topographical measurement of brain pH, Brain Res. 195, 95-109, 1980 Kiernan, J.A, Chromoxane cyanine R. I. Physical and chemical properties of the dye and of some its iron complexes, J.Microsc. 143, 13-23, 1984 Paradiso, A.M., Tsien, R.Y., and Machen, T.E., Na+-H+ exchange in gastric glands as measured with a cytoplasmic-trapped, fluorescent pH indicator, Proc.Nat.Acad.Sci.USA 81, 7436-7440, 1984 Mera, S.L. and Davies, J.D., Differential Congo red staining: the effects of pH, non-aqueous solvents and the substrate, Histochem.J. 16, 195210, 1984 Horie, K., Hagihara, H., Wada, A., and Fukutome, H., A highly sensitive photometric method for proton release or uptake: difference photometry, Anal.Biochem. 137, 80-87, 1984 Krchnak, V., Vagner, J., and Lebl, M., Noninvasive continuous monitoring of solid-phase peptide synthesis by acid-base indicator, Int.J.Pept. Protein Res. 32, 415-416, 1988 Rosenberg, R.M., Herreid, R.M., Piazza, G.J., and O’Leary, M.N., Indicator assay for amino acid decarboxylases, Anal.Biochem. 181, 55-65, 1989 Weiner, I.D. and Hamm, L.L., Use of fluorescent dye BCECF to measure intracellular pH in cortical collecting tubule, Am.J.Physiol(Renal, Fluid, Electrolyte Physiol.), 256, F957-F964, 1989 Bassnett, S., Reinisch, L., and Beebe, D.C., Intracellular pH measurement using single excitation-dual emission fluorescence ratios, Am.J.Physiol.(Cell Physiol.) 258, C171-C178, 1990 Anderson, R.E, Bjorkman, D. and McGreavy, J.M., Alteration of gastric surface cell pH regulation by sodium taurocholate, J.Surg.Res. 50, 65-71, 1991 Tortorello, M.L., Trotter, K.M., Angelos, S.M., et al., Microtiter plate assays for the measurement of phage adsorption and infection in Lactococcus and Enterococcus, Anal.Biochem. 192, 362-366, 1991 Raley-Sussman, K.M., Sapolsky, R.M., and Kopito, R.R., Cl– . HCO3–exchange function differs in adult and fetal rat hippocampal neurons, Brain Res. 614, 308-314, 1993 Reusch, H.P., Reusch, R., Rosskopf, D., et al., Na+/H+ exchange in human lymphocytes and platelets in chronic and subacute metabolic acidosis, J.Clin.Invest. 92, 858-865, 1993 Mehta, V.D., Kulkarni, P.V., Mason, R.P., et al., 6-Fluoropyridoxal: a novel probe of cellular pH using 19F NMR spectroscopy, FEBS Lett. 349, 234-238, 1994 Optiz, N., Merten, E., and Acker, H., Evidence for redistribution-associated intracellular pH shifts of the pH-sensitive fluoroprobe carboxy SNARF-1, Pflugers Arch. 427, 332-342, 1994 Webb, B., Frame, J., Zhao, Z., et al., Molecular entrapment of small molecules within the interior of horse spleen ferritin, Arch.Biochem. Biophys. 309, 178-183, 1994
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Zhou, Y., Marcus, E.M., Haugland, R.P., and Opas, M., Use of a new fluorescent probe, seminaphthofluorescein-calcein, for determination of intracellular pH by simulataneous dual-emission imaging laser scanning confocal microscopy, J.Cell Physiol. 164, 9-16, 1995 Scheef, C.A., Oelkrug, D., and Schmidt, P.C., Surface acidity of solid pharmaceutical excipients III. Excipients for solid dosage forms, Eur.J.Pharm.Biopharm. 46, 209-213, 1998 Shao, P.G. and Bailey, L.C., Porcine insulin biodegradable polyester microspheres: stability and in vitro release characteristics, Pharm.Dev. Technol. 5, 1-9, 2000 Silver, R.B., Breton, S. and Brown, D., Potassium depletion increases proton pump (H(+)-ATPase) activity in intercalated cells of cortical collecting duct, Am.J.Physiol.Renal Physiol. 279, F195-F202, 2000 Chu, Y.I., Penland, R.L., and Wilhemus, K.R., Colorimetric indicators of microbial contamination in corneal preservation, Cornea 19, 517520, 2000 Jayaraman, S., Song, Y., and Verkman, A.S., Airway surface liquid pH in well-differentiated airway epithelial cell cultures and mouse trachea, Am.J.Physiol.Cell Physiol. 281, C1504-155, 2001 Yu, E., Pan, J., and Zhou, H.M., A direct continuous pH-spectrophotometric assay for arginine kinase activity, Protein Pept.Lett. 9, 545-552, 2002 Hur, O., Niks, D., Casino, P., and Dunn, M.F., Proton transfer in the β-reaction catalyzed by tryptophan synthase, Biochemistry 41, 999110001, 2002 Sun, C. and Berg, J.C, A review of the different techniques for solid surface acid-base characterization, Adv.Colloid Interface Sci. 105, 1510175, 2003 Li, J., Chatterjee, K., Medek, A., et al., Acid-base characterization of bromophenol blue-citrate buffer systems in the amorphous state, J.Pharm.Sci. 93, 697-712, 2004 Balderas-Hernandez, P, Rojas-Hernandez, A., Galvan, M., and RamirezSilva, M.T., Spectrophotometric study of the system Hg(II)-thymol blue-H2O and its evidence through electrochemical means, Spectrochimica Acta A Mol.Biomol.Spectrosc. 60, 569-577, 2004 Gilman, J.B. and Vaida, V., Permeability of acetic acid through organic films at the air-aqueous interface, J.Phys.Chem.AMol.Spectros.Kinet. Environ.Gen.Theory 110, 7581-7587, 2006 Sanchez-Armass, S., Sennoune, S.R., Maiti, D., et al., Spectral imaging microscopy demonstrates cytoplasmic pH oscillations in glial cells, Am.J.Physiol.Cell Physiol. 290, C524-C538, 2006
General acid-base indicators Widmer, M., Titrimety, in Encyclopedia of Analytical Chemistry, ed. R.A. Meyers, John Wiley & Sons, Ltd., Chichester, UK, pps. 13624-13636, 2002 Encyclopedia of Analytical Sciences, ed. A. Townshend, Academic Press, London, 1995 Butler, J.N., Ionic Equilibrium. Solubility and pH Calculations, John Wiley & Sons, New York, NY, USA, 1998 Westcott, G.C., pH Measurements, Academic Press, New York, NY, USA, 1978 Kotyk, A. and Slavík, J., Intracellular pH and Its Measurement, CRC Press, Boca Raton, FL, USA, 1989 Britton, H.T.S., Hydrogen Ions. Their Determination and Importance in Pure and Industrial Chemistry, D.Van Nostrand, New York, NY, USA, 1932 Kolthoft, I.M.(trans. Rosenblum, C.), Acid-Base Indicators(Säure-Basen Indicatoren), MacMillan Company, New York, NY, USA, 1937 Kolthoff, I.M. and Laitinen, H.A., pH and Electro Titration. The Colorimetric and Potentiometric Determination of pH, Potentiometry, Conductometry, and Voltometry(Polarography). Outline of Electrometric Titration, John Wiley & Sons, Inc., New York, NY, USA, 1941 Webber, R.B., The Book of pH, George Newnes Ltd., London, UK, 1957 Fritz, J.S., Acid-Base Titrations in Nonaqueous Solvents, G.Frederich Smith Chemical Company, Columbus, Ohio, 1952 Clark, W.M. The Determination of Hydrogen Ions, 2nd edn., Williams & Wilkins, Baltimore, MD, USA, 1927
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Radhuraman, B., Gustavson, G, van Hal, R.E.G., et al., Extended-range spectroscopic pH measurement using optimized mixtures of dyes, Appl.Spectrosc. 60, 1461-1468, 2006
Velthuys, B.R., A third site of proton translocation in green plant photosynthetic electron transport, Proc.Nat.Acad.Sci.USA 75, 6031-6034, 1978
Non-aqueous titration
Crystal Violet
Kolade, Y.T., Adegbolagun, O.M., Idowu, O.S. et al., Comparative determination of halofantrine tablets by titrimetry, spectrophotometry and liquid chromatography, Afr.J.Med.Med.Sci. 35, 79-84, 2006 Mera, S.L. and Davies, J.D., Differential congo red staining: the effects of pH, non-aqueous solvents and the substrate, Histochem.J. 16, 195210, 1984
Cresol Purple Schindler, J.F., Naranjo, P.A., Honaberger, D.A., et al., Haloalkane dehalogenase: Steady-state kinetics and halide inhibition, Biochemistry 38, 5772-5778, 1999
Phenolphthalein King, B.F., Liu, M., Townsend-Nicholson, A., et al., Antagonism of ATP responses at P2X receptor subtypes by the pH indicator dye, phenol red, Br.J.Pharmacol. 145, 313-322, 2005 Riccio, M.L., Rossolini, G.M., Lombardi, G., et al., Expression cloning of different bacterial phosphatase-encoding genes by Histochemical screening of genomic libraries onto an indicator medium containing phenolphthalein diphosphate and methyl green, J.Appl.Microbiol. 82, 177185, 1997 Gerber, H., Colorimetric determination of alkaline phosphatase as indicator of mammalian feces in corn meal: collaborative study, J.Assoc.Off. Anal.Chem. 69, 496-498, 1986 50a. Khalifab, R.G., The carbon dioxide hydration activity of carbonic anhydrase. I. Stop-flow kinetic studies on the native human isoenzymes B and C, J.Biol.Chem. 246, 2561-2573, 1971
Cresol Red Borucki, B., Davanthan, S., Otto, H., et al., Kinetics of proton uptake and dye binding by photoactive yellow protein in wild type and the E46Q and E46A mutants, Biochemistry 41, 10026-10037, 2002 Actis, L.A., Smoot, J.C., Baracin, C.E., and Findlay, R.H., Comparison of differential plating media and two chromatographic techniques for the detection of histamine production in bacteria, J.Microbiol. Methods 39, 79-90, 1999 Jeronimo, P.C., Araujo, A.N., Montenegro, M.C., et al., Flow-through solgel optical biosensor for the colorimetric determination of acetazolamide, Analyst 130, 1190-1197, 2005 Nakamura, N. and Amao, Y., Optical sensor for carbon dioxide combining colorimetric change of a pH indicator and a reference luminescent dye, Anal.Bioanal.Chem. 376, 642-646, 2003 Yu, Z., Pan, J., and Zhou, H.M., A direct continuous pH-spectrophotometric assay for arginine kinase activity, Protein Pept.Lett. 9, 545-552, 2002 58a. Caselli, M., Mangone, A., Paoliollo, P., and Traini, A., Determination of the acid dissociation constant of bromocresol green and cresol red in water/AOT/isooctane reverse micelles by multiple linear regression and extended principal component analysis, Ann.Chim. 92, 501512, 2002 Actis, L.A., Smoot, J.C, Barancin, C.E., and Findlay, R.H., Comparison of differential plating media and two chromatographic techniques for the detection of histamine production in bacteria, J.Microbiol. Methods 39, 79-90, 1999 Grabner, R., Influence of cationic amphiphilic drugs on the phosophatidylcholine hydrolysis by phospholipase A2, Biochem.Pharmacol. 36, 1063-1067, 1987 Horie, K., Hagihara, H., Wada, A., and Fukutome, H., A highly sensitive photometric method for proton release or uptake: difference photometry, Anal.Biochem. 137, 80-87, 1984
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Kolade, Y.T., Adegboagun, O.M. Iodwu, O.S., et al., Comparative determination of halofantrine tablets by titrimetry, spectrophometry and liquid chromatography, Afr.J.Med.Med.Sci. 35, 79-84, 2006 Bornscheuer, U.T., Altenbuchner, J., and Meyer, J.H., Directed evolution of an esterase for the stereoselective resolution of a key intermediate in the synthesis of epothilones, Biotechnol.Bioeng. 58, 554-559, 1998 Nakayasu, H., Crystal violet as an indicator dye for nonequilibrium pH gradient
Congo Red Mera, S.L., and Davies, J.D., Differential Congo red staining: the effects of pH, non-aqueous solvents and the substrate, Histochem.J. 16, 195210, 1984 Schneider, R.L., Chung, E.B., Leffall, L.D., Jr., and Syphax, B., Delineation of the canine gastric antrum with pH probe and dye indicator, J.Natl. Med.Assoc. 63, 202-204, 1971 Xu, S., Kramer, M., and Haag, R., pH-Responsive dendritic core-shell architectures as amphiphilic nanocarriers for polar drugs, J.Drug Target. 14, 367-374, 2006 Sekine, H., Iijima, K., Koike, T., et al., Regional differences in the recovery of gastric acid secretion after Helicobacter pylori eradication: evaluations with Congo red chromoendoscopy, Gastrointest.Endosc. 64, 686-690, 2006 Parrish, N.M., Ko, C.G., Dick, J.D., et al., Growth, Congo Red agar colony morphotypes and antibiotic susceptibility testing of Mycobacterium avium subspecies paratuberculosis, Clin.Med.Res. 2, 107-114, 2004
Thymol Blue Balderas-Hernandez, P., Rojas-Hernandez, A., Galvan, M., and RamirezSilva, M.T., Spectrophotometric study of the system Hg(II)-thymol blue-H2O and its evidence through electrochemical means, Spectrochim.Acta A Mol.Biomol.Spectrosc. 60, 569-577, 2004 Nakamura, N. and Amao, Y., Optical sensor for carbon dioxide combining colorimetric change of a pH indicator and a reference luminescent dye, Anal.Bioanal.Chem. 376, 642-646, 2003 Dowding, C.E., Borda, M.J., Fey, M.V., and Sparks, D.L., A new method for gaining insight into the chemistry of drying mineral surfaces using ATR-FTIR, J.Colloid Interface Sci. 292, 148-151, 2005 Saika, P.M., Bora, M., and Dutta, R.K., Acid-base equilibrium of anionic dyes partially bound to micelles of nonionic surfactants, J.Colloid Interface Sci. 285, 382-387, 2005
Bromophenol Blue Govindarajan, R., Chatterjee, K., Gatlin, L., et al., Impact of freeze-drying on ionization of sulfonphthalein probe molecule in trehalose-citrate systems, J.Pharm.Sci. 95, 1498-1510, 2006 Suzuki, Y., Theoretical analysis concerning the characterization of a dyebinding method for determining serum protein based on protein error of pH indicator: effect of buffer concentration of the color reagent on the color development, Anal.Sci. 21, 83-88, 2005 Li, J., Chatterjee, K., Medek, A. et al., Acid-base characteristics of bromophenol blue-citrate buffer system in the amorphous state, J.Pharm. Sci. 93, 697-712, 2004 Shao, P.G. and Bailey, L.C., Porcine insulin biodegradable polyester microspheres: stability and in vitro release characteristics, Pharm.Dev. Technol. 5, 1-9, 2000 Koren, R. and Hammes, G.G., A kinetic study of protein-protein interactions, Biochemistry 15, 1165-1171, 1976
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Methyl Red Katsuda, T., Ooshima, H., Azuma, M., and Kato, J., New detection method for hydrogen gas for screening hydrogen-producing microorganisms using water-soluble Wilkinson’s catalyst derivative, J.Biosci.Bioeng. 102, 220-226, 2006 Benedict, J.B., Cohen, D.E., Lovell, S., et al., What is syncrystallization? States of the pH indicator methyl red in crystals of phthalic acid, J.Am.Chem.Soc. 128, 5548-5559, 2006 Pelechova, J., Petrova, L., Ujcova, E. and Martinkova, L., Selection of a hyperproducing strain of Aspergillus niger for biosynthesis of citric acid on unusual carbon substrates, Folia Microbiol. 35, 138-142, 1990
Phenol Red Govindarajan, R., Chatterjee, K., Gatlin, L., et al., Impact of freeze-drying on ionization of sulfonphthalein probe molecule in trehalose-citrate systems, J.Pharm.Sci. 95, 1498-1510, 2006 Chu, A., Morris, K., Greenberg, R. and Zhou, D. Stimulus induced pH changes in retinal implants, Conf.Proc.IEEE Eng.Med.Biol.Soc. 6, 4160-4162, 2004 Deng, C. and Chen ,R.R., A pH-sensitive assy for galactosyltransferase, Anal.Biochem. 330, 219-226, 2004 Still, K., Reading, L., and Scutt, A., Effects of phenol red on CRU-f differentiation and formation, Calcif.Tissue Int. 73, 173-179, 2003
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Hur, O., Nik, D., Casino, P., and Dunn, M.F., Proton transfers in the β-reaction catalyzed by tryptophan synthase, Biochemistry 41, 999110001, 2002 Oh, K.H., Nam, S.H., and Kim, H.S., Directed evolution of N-carbamyl-Damino acid amidohydrolase for simultaneous improvement of oxidative and thermal stability, Biotechnol.Prog. 18, 413-417, 2002 Girard, P., Jordan, M., Tsao, M. and Wurm, F.M., Small-scale bioreactor system for process development and optimization, Biochem.Eng.J. 7, 117-119, 2001 Jarrett, J.T, Choi, C.Y. and Matthews, R.G., Changes in protonation associated with substrate binding and Cob(I)alamin formation in cobalamin-dependent methionine synthase, Biochemistry 36, 15739-15748, 1997 Ahmed, Z. and Connor, J.A., Intracellular pH changes induced by calcium influx during electrical activity in molluscan neurons, J.Gen.Physiol. 75, 403-426, 1980 Connor, J.A. and Ahmed, Z., Diffusion of ions and indicator dyes in neural cytoplasm, Cell.Mol.Neurobiol. 4, 53-66, 1984 Clark, A.M. and Perrin, D.D., A re-investigation of the question of activators of carbonic anhydrase, Biochem.J. 48, 495-502, 1951
Nile Blue Lie, C.-W., Shulok, J.R., Wong, Y.-K., et al., Photosensitization, uptake, and retention of phenoxazine Nile Blue derivatives in human bladder carcinoma cells, Cancer Res. 51, 1109-1114, 1991
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Specific Gravity of Liquids Specific gravity and density are not identical although the abbreviation “d” is frequently used to designate specific gravity. Specific gravity and density are numerically equal when water is the standard of reference for specific gravity and g/ml is the unit designation for density. The numerical value for specific gravity is usually written with a superscript (indicating the temperature of the liquid) and a subscript (indicating the temperature of the liquid to 25 which it is referred), thus d 25 4 1.724 or sp. gr. 1.724 4 . When
these are omitted in this table, the specific gravity at 20°C referred to water at 4°C is intended. When the standard of reference is not specified, for liquids and solids, it is understood to be water. Water is most dense at 4°C, hence the sp. gr. of a liquid with reference to water will be higher at all other temperatures than it is at 4°C. To obtain the sp. gr. with reference to water at the same 20 25 temperature as the liquid, multiply the sp. gr. of 15 4 , 4 , or 4 by 1.001, 1.002, or 1.003, respectively.
(Items listed in the order of increasing specific gravities) Liquid n-Pentane n-Hexane 1-Butyne Dimethylamine Isoprene n-Heptane 2-Butyne 1,5-Hexadiene Isopropylamine Butylamine, tertiary Triethylboron Ethylamine Diethylamine 2,4-Hexadiene Diethyl ether n-Nonane Triethylamine Butylamine, secondary Isopropyl ether Ethyl methyl ether 2,4-Heptadiene Isobutylamine Propyl ether Methyl propyl ether Dipropylamine Ethyl n-propyl ether n-Butylamine Undecane N,N-Dimethylamylamine Ethyl isopropyl ether Isoamylamine Cyclopentane Butyl ethyl ether Isohexylamine Isobutyl ether Allylamine n-Amylamine Butyl methyl ether Allyl ether ether n-Dodecane Dibutylamine n-Butyl ether Cyclopentene n-Heptylamine Cyclohexane n-Octylamine Isoamyl ether
Specific Gravity 0.626 0.660 0.66804 0.68004 0.681 0.684 0.68825 0.688 0.69415 4 0.696 0.69623 0.70604 0.71118 4 0.711 0.713 0.716 0.72325 4 0.724 0.726 0.72604 0.73321.5 4 0.72425 4 0.736 0.738 0.738 0.739 0.740 0.741 0.743 0.74504 0.751 0.751 0.752 0.75825 4 0.76115 4 0.761 0.761 0.76404 0.765 0.76604 0.767 0.76920 20 0.774 0.777 0.778 0.77920 20 0.78115 15
Liquid Propionitrile Acetonitrile n-Butyl ether Isopropyl alcohol Isovaleronitrile Butyl alcohol, tertiary Methanol, anhydrous Acetone Isobutyraldehyde Acrylonitrile Ethyl alcohol, anhydrous Valeronitrile Isovaleraldehyde n-Propyl alcohol Allyl ether Ethyl methyl ketone Isobutyl alcohol Propionaldehyde Butyl alcohol, secondary Amyl alcohol, tertiary Methyl propyl ketone n-Butyl alcohol Cycloheptane Cyclohexene Isoamyl alcohol Ethyl propyl ketone pri-n-Amyl alcohol Heptyl ether Diethyl ketone Ethyl alcohol, 95 per cent Butyraldehyde Dipropyl ketone Ethyl butyl ketone n-Hexyl methyl ketone 1-Hexanol 3-Hexanol Isoamyl alcohol, secondary Pinacolin Amyl methyl ketone Cycloheptene n-Octyl alcohol 2-Undecanone Light Liquid Petrolatum 2-Hexanol n-Decyl alcohol n-Undecylaldehyde Butyl methyl ketone
Specific Gravity 0.783 0.78325 25 0.78404 0.785 0.788 0.789 0.791 0.792 0.794 0.797 0.79815.56 15.56 0.801 0.80317 4 0.804 18 0.8050 0.805 0.80615 4 0.807 0.808 0.809 0.809 0.810 0.810 0.810 0.81315 4 0.81321.8 4 0.814 0.81504 0.81619 4 15.56 0.81615.56 0.817 0.817 0.818 0.819 0.819 0.819 0.819 0.82104 0.82215 4 0.823 0.825 0.826 0.828–0.88025 25 0.82904 0.829 0.830 0.83004
739
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SPECIFIC GRAVITY OF LIQUIDS (Continued) Liquid 1-Undecanol Acrolein Orange oil Bitter orange oil Butyl chloride, tertiary Rose oil Lemon oil Amyl ether ketone n-Amyl nitrite Rectified turpentine oil Dwarf pine needle oil Allyl alcohol Mesityl oxide Myristica oil p-Cymene dl-Pinene Isopropyl chloride 2-Diethylaminoethanol Liquid Petrolatum Piperidine Cumene Coriander oil Orange flower oil Phytol m-Xylene Toluene Ethyl benzene m-Cymene Isoamyl acetate Isopropyl acetate Isobutyl nitrite Butyl chloride, secondary Octyl acetate Isobutyl acetate Isoamyl nitrite Bergamot oil Lavender oil o-Cymene Benzene Amyl acetate Geraniol n-Amyl chloride Isobutyl chloride n-Butyl chloride Pine needle oil Citronella oil 2-Dimethylaminoethanol n-Propyl acetate 1-Menthol Propyl chloride Isoamyl chloride Rosemary oil Oleic acid Isodurene Peppermint oil o-Xylene Ethyl nitrite Caraway oil Ethyl acetate Linoleic acid
9168_Book.indb 740
Specific Gravity 0.833 0.841 0.842–0.84625 25 0.845–0.85125 25 0.84715 4 0.848–0.8633015 0.849–085525 25 0.85004 0.853 0.853–0.86225 25 0.853–0.87125 25 0.854 0.854 0.854–0.91025 25 0.857 0.858 0.859 0.86025 25 0.860–0.90525 25 0.861 0.863 0.863–0.87525 25 0.863–0.88025 25 0.86404 0.864 0.866 0.867 0.870 0.87025 4 0.870 0.87020 20 0.871 0.87320 20 0.875 0.87525 25 0.875–0.88025 25 0.875–0.88825 25 0.876 0.87915 4 0.87920 20 0.88116 4 0.883 0.88315 0.884 0.884–0.88615 15 0.885–0.91225 25 0.887 0.887 0.89015 15 0.89020 20 0.893 0.894–0.91225 25 0.89518 4 0.89604 0.896–0.90825 25 0.897 0.90015 15 0.900–0.91025 25 0.902 0.903 23 4
Liquid Cubeb oil Eucalyptus oil Diethyl Carbitol Styrene Undecylenic acid Olive oil Expressed almond oil Persic oil Thyme oil n-Butyl nitrite Peanut oil Mustard oil Corn oil Methyl propionate Glycerin trioleate Cottonseed oil Sesame oil Spearmint oil Cardamom oil Coconut oil Cod liver oil Halibut liver oil Eucalyptol Ethyl format Soya oil Linseed oil Pine oil Methyl acetate Cellosolve Ionone N,N-Diethylaniline Furan Allyl chloride Valeric acid Castor oil Cyclohexanone Pyrrole Cyclopentanone Cyclopentanol Isobutyric acid 2-Picoline Chenopodium oil Myrcia oil Cycloheptanone Fennel oil Dimethylaniline 4-Picoline 3-Picoline Indan Methyl cellosolve Phenetole Vitamin K1 Tetralin Carvacrol Pyridine Anise oil Ethyl urethan Benzylamine Benzyl acetone m-Toluidine
Specific Gravity 0.905–0.92525 25 0.905–0.92525 25 0.907 0.907 0.90825 4 0.910–0.91525 25 0.910–0.91525 25 0.910–0.92325 25 0.910–0.93525 25 0 0.911 4 0.912–0.92025 25 0.914–0.91615 15 0.914–0.92125 25 0.915 0.915 0.915–0.92125 25 0.916–0.92125 25 0.917–0.93425 25 0.917–0.94725 25 0.918–0.92325 25 0.918–0.92725 25 0.920–0.93025 25 0.921–0.92325 25 0.92425 4 0.924–0.92715 15 0.925–0.93525 25 25 0.927–0.94025 0.928 0.930 0.933–0.93725 25 0.935 0.937 0.938 0.942 0.945–0.96525 25 0.948 0.948 0.948 0.949 0.949 0.95015 4 0.950–0.98025 25 0.950–0.99025 25 0.951 0.953–0.97325 25 0.956 0.95715 4 0.96115 4 0.965 0.966 0.967 0.96725 25 0.970 0.976 0.97825 4 0.978–0.98825 25 0.981 0.98319 4 0.98923 17 0.989
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Specific Gravity of Liquids
741 SPECIFIC GRAVITY OF LIQUIDS (Continued)
Liquid Carbitol Dimethyl glyoxal Isoamyl benzoate Paraldehyde Anisole Isoamyl nitrate Morpholine Water Water Water Isobutyl benzoate o-Toluidine Indene Nicotine Benzonitrile Hydrazine Ethanolamine Pimenta oil Aniline Sparteine Phenylethyl alcohol Dibenzylamine Chloroacetal Acetophenone 1,4-Dioxane m-Cresol Glycerol tributyrate Propylene glycol Phlorol Butyl nitrate, secondary Bitter almond oil Clove oil Ethyl succinate Benzyl ether Benzyl alcohol Cinnamon oil o-Cresol n-Butyl phthalate n-Butyl nitrate Acetic acid (glacial) Benzaldehyde Ethyl benzoate Ethyl malonate Benzyl acetate Allyl benzoate n-Propyl nitrate Succinaldehyde Thiophene Methyl carbonate Eugenol p-Chlorotoluene m-Chlorotoluene Diethyl maleate Benzofuran o-Chlorotoluene Acetic anhydride o-Anisidine Methyl benzoate Quinoline m-Anisidine
9168_Book.indb 741
Specific Gravity 0.990 0.99015 15 0.99319 4 0.994 0.995 0.99622 4 0.999 0.997004 0.999920 20 1.00004.08 4.08 1.00215 4 1.004 1.006 1.009 1.01015 15 1.01115 4 1.018 1.018–1.04825 25 1.022 1.023 1.02415 4 1.026 1.02616 4 1.03315 15 1.034 1.034 1.035 1.03625 4 1.03712 1.03804 1.038–1.06025 25 1.038–1.06025 25 1.040 1.043 1.04525 4 1.045–1.06325 25 1.047 1.047 1.04804 1.04925 25 1.05015 4 1.05115 4 1.055 1.05716 4 1.05815 15 1.058 1.064 1.064 1.06517 4 1.066 1.070 1.072 1.07415 15 1.07815 15 1.082 1.08715 4 1.092 1.09415 4 1.095 1.096
Liquid Diethanolamine Benzyl chloride Aldol Acetyl chloride Ethyl nitrate Chlorobenzene Polyethylene Glycol 400 Cinnamaldehyde Benzyl benzoate Diethylene glycol Anisaldehyde Diethyl phthalate Triethanolamine Polyethylene Glycol 300 Furfuryl alcohol Nitromethane Formamide Ethyl salicylate m-Nitrotoluene Ethyl chloroacetate Furfural Glycerol triacetate o-Nitrotoluene Salicylaldehyde Methyl salicylate Dimethyl phthalate Nitrobenzene Isoamyl bromide Benzoyl chloride sym.-Dichloroethyl ether Butyl bromide, tertiary Formic acid Methyl chloroacetate Amyl bromide Lactic acid (dl) .uns.-Ethylene dichloride Butyl bromide, secondary Glycerol Carbon disulfide n-Butyl bromide Isobutyl bromide sym.-Dichloroethylene o-Dichlorobenzene Isopropyl bromide Ethylsulfuric acid Methylene chloride n-Propyl bromide m-Xylyl bromide Benzotrichloride Ethyl trichloroacetate Allyl bromide Ethyl bromide Benzyl bromide Hydrogen peroxide, anhydrous Trichloroethylene Chloroform Bromobenzene Chloral Trichloroethanol Dichloroacetic acid
Specific Gravity 1.097 1.10318 4 1.103 1.105 1.105 1.107 1.110–1.14025 25 1.11215 4 1.11825 4 1.11820 20 1.123 1.12325 4 1.124 1.124–1.13025 25 1.130 1.130 1.134 1.13615 4 1.157 1.159 1.160 1.161 1.163 1.167 1.18425 25 1.18925 25 1.20515 4 1.21015 4 1.21915 15 1.222 1.222 1.22615 4 1.23820 20 1.24604 1.24915 4 1.252 1.258 1.260 1.263 1.26925 4 1.27215 4 1.29115 4 1.30720 20 1.310 1.31617 4 1.33515 4 1.353 1.37123 4 1.38015 4 1.383 1.398 1.430 1.43822 0 1.46504 1.465 1.49815 15 1.49915 15 1.512 1.55020 20 1.563
4/16/10 1:26 PM
Handbook of Biochemistry and Molecular Biology
742
SPECIFIC GRAVITY OF LIQUIDS (Continued) Liquid Benzoyl bromide Glycerophosphoric acid Nitroglycerin Carbon tetrachloride Tetrachloroethane Tetrachloroethylene Chloropicrin Diphosgene Thionyl chloride Acetyl bromide Isopropyl iodide
Specific Gravity 1.570 1.590 1.592 1.595 1.600 1.63115 4 1.651 1.65314 4 1.65510.4 1.66316 4 1.703 15 4 19 4 25 4
Liquid Ethyl iodide Ethylene bromide Ethylene dibromide Methyl iodide Bromal Methylene bromide Bromoform Tetrabromoethane Methylene iodide Mercury
Specific Gravity 1.933 2.17025 4 2.17225 25 2.251 2.30015 4 2.495 2.890 2.964 3.325 13.546
Reprinted from The Merck Index (1960), 7th ed., Merck and Co., Rahway, N.J., pp. 1532–1535, with permission of the copyright owner.
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Viscosity and Density Tables SUCROSE IN WATER, 0.0°C Sucrose %
Density g/ml
Sucrose %
Densitya g/ml
Viscosityb cP
Sucrose %
Densitya g/ml
Viscosityb cP
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23
1.0004 1.0043 1.0082 1.0122 1.0162 1.0203 1.0244 1.0285 1.0326 1.0368 1.0411 1.0453 1.0496 1.0539 1.0583 1.0627 1.0671 1.0716 1.0760 1.0806 1.0852 1.0898 1.0944 1.0991
1.780 1.830 1.884 1.941 2.002 2.066 2.135 2.208 2.286 2.369 2.458 2.552 2.653 2.761 2.877 3.001 3.134 3.277 3.430 3.596 3.774 3.967 4.175 4.401
24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47
1.1037 1.1085 1.1133 1.1181 1.1229 1.1278 1.1327 1.1376 1.1426 1.1476 1.1527 1.1578 1.1629 1.1680 1.1732 1.1784 1.1836 1.1889 1.1942 1.1996 1.2050 1.2104 1.2159 1.2213
4.646 4.912 5.202 5.519 5.866 6.246 6.665 7.126 7.635 8.201 8.829 9.530 10.31 11.20 12.19 13.31 14.58 16.03 17.69 19.59 21.78 24.31 27.24 30.66
48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70
1.2269 1.2324 1.2380 1.2436 1.2493 1.2550 1.2607 1.2665 1.2723 1.2781 1.2840 1.2899 1.2958 1.3018 1.3078 1.3138 1.3199 1.3260 1.3321 1.3383 1.3445 1.3507 1.3570
34.57 39.23 44.74 51.29 59.11 68.52 79.92 93.85 111.0 132.3 158.9 192.6 235.7 291.4 364.2 460.6 589.9 766.0 1010. 1352. 1842. 2556. 3621.
Sucrosé %
Densitya g/ml
Viscosityb cP
Sucrosé %
Densitya g/ml
Viscosityb cP
Sucrosé %
Densitya g/ml
Viscosityb cP
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23
1.0004 1.0043 1.0082 1.0121 1.0161 1.0201 1.0241 1.0282 1.0323 1.0365 1.0406 1.0448 1.0491 1.0534 1.0577 1.0620 1.0664 1.0708 1.0753 1.0798 1.0843 1.0889 1.0934 1.0981
1.516 1.558 1.603 1.650 1.700 1.753 1.809 1.869 1.933 2.001 2.073 2.150 2.232 2.319 2.413 2.513 2.621 2.736 2.859 2.992 3.135 3.290 3.456 3.636
24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47
1.1027 1.1074 1.1122 1.1169 1.1218 1.1266 1.1315 1.1364 1.1413 1.1463 1.1513 1.1563 1.1614 1.1665 1.1717 1.1768 1.1820 1.1873 1.1926 1.1979 1.2033 1.2087 1.2140 1.2195
3.831 4.042 4.272 4.523 4.796 5.094 5.422 5.781 6.177 6.614 7.099 7.637 8.236 8.905 9.656 10.50 11.45 12.53 13.76 15.16 16.76 18.60 20.73 23.18
48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70
1.2250 1.2306 1.2361 1.2417 1.2474 1.2530 1.2587 1.2645 1.2702 1.2760 1.2819 1.2877 1.2936 1.2996 1.3056 1.3116 1.3176 1.3237 1.3298 1.3360 1.3422 1.3484 1.3546
25.97 29.28 33.16 37.73 43.16 49.62 57.39 66.79 78.24 92.30 109.7 131.4 158.9 194.0 239.1 297.9 375.6 479.4 620.3 814.3 1086. 1473. 2034.
a
Viscosity cP
b
Sucrose in Water, 5.0°C
743
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Handbook of Biochemistry and Molecular Biology
744
VISCOSITY AND DENSITY TABLES (Continued) Sucrose in Water, 10.0°C Sucrosé %
Density g/ml
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23
1.0002 1.0040 1.0079 1.0118 1.0157 1.0196 1.0236 1.0277 1.0317 1.0358 1.0400 1.0442 1.0484 1.0526 1.0569 1.0612 1.0655 1.0699 1.0743 1.0788 1.0833 1.0878 1.0924 1.0969
Sucrosé %
Density g/ml
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23
0.9996 1.0034 1.0073 1.0111 1.0150 1.0189 1.0229 1.0269 1.0309 1.0350 1.0391 1.0432 1.0474 1.0516 1.0558 1.0601 1.0644 1.0688 1.0732 1.0776 1.0820 1.0865 1.0910 1.0956
a
Viscosity cP 1.308 1.343 1.380 1.420 1.462 1.506 1.553 1.603 1.655 1.711 1.771 1.835 1.902 1.974 2.051 2.134 2.222 2.316 2.417 2.525 2.642 2.767 2.903 3.049
b
Sucrosé %
Densitya g/ml
Viscosityb cP
Sucrosé %
Densitya g/ml
Viscosityb cP
24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47
1.1016 1.1062 1.1109 1.1157 1.1204 1.1252 1.1300 1.1349 1.1398 1.1448 1.1498 1.1548 1.1598 1.1649 1.1700 1.1752 1.1803 1.1856 1.1908 1.1961 1.2014 1.2068 1.2122 1.2176
3.206 3.377 3.562 3.763 3.982 4.220 4.481 4.767 5.080 5.424 5.805 6.225 6.692 7.211 7.790 8.438 9.167 9.988 10.92 11.97 13.17 14.54 16.11 17.92
48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70
1.2231 1.2286 1.2341 1.2397 1.2453 1.2510 1.2566 1.2623 1.2681 1.2739 1.2797 1.2855 1.2914 1.2973 1.3033 1.3093 1.3153 1.3214 1.3275 1.3336 1.3398 1.3460 1.3522
19.96 22.37 25.17 28.45 32.32 36.89 42.34 48.87 56.75 66.35 78.11 92.65 110.8 133.6 162.7 200.0 248.6 312.5 397.7 512.9 671.1 891.7 1205.
Sucrose in Water, 15.0°C
9168_Book.indb 744
a
Viscosity cP 1.140 1.170 1.202 1.235 1.271 1.308 1.348 1.390 1.434 1.481 1.531 1.584 1.640 1.701 1.765 1.833 1.906 1.985 2.068 2.158 2.255 2.358 2.470 2.590
b
Sucrosé %
Densitya g/ml
Viscosityb cP
Sucrosé %
Densitya g/ml
Viscosityb cP
24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47
1.1002 1.1048 1.1095 1.1142 1.1189 1.1237 1.1285 1.1334 1.1382 1.1432 1.1481 1.1531 1.1581 1.1632 1.1682 1.1734 1.1785 1.1837 1.1889 1.1942 1.1995 1.2048 1.2102 1.2156
2.719 2.859 3.010 3.174 3.352 3.546 3.757 3.987 4.239 4.515 4.818 5.153 5.522 5.932 6.386 6.894 7.461 8.097 8.813 9.621 10.54 11.58 12.77 14.13
48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70
1.2211 1.2265 1.2320 1.2376 1.2432 1.2488 1.2544 1.2601 1.2658 1.2716 1.2774 1.2832 1.2891 1.2950 1.3009 1.3069 1.3129 1.3189 1.3250 1.3311 1.3373 1.3434 1.3497
15.65 17.44 19.52 21.93 24.75 28.06 31.98 36.64 42.22 48.95 57.12 67.12 79.48 94.85 114.2 138.7 170.2 211.0 264.6 336.0 432.2 563.8 746.7
4/16/10 1:26 PM
Viscosity and Density Tables
745 VISCOSITY AND DENSITY TABLES (Continued) Sucrose in Water, 20.0°C
Sucrosé %
Density g/ml
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23
0.9988 1.0026 1.0064 1.0102 1.0140 1.0179 1.0219 1.0258 1.0298 1.0339 1.0380 1.0421 1.0462 1.0504 1.0546 1.0588 1.0631 1.0674 1.0718 1.0762 1.0806 1.0851 1.0896 1.0941
a
Viscosity cP
b
1.004 1.030 1.057 1.086 1.116 1.148 1.181 1.217 1.255 1.295 1.337 1.382 1.430 1.480 1.535 1.592 1.654 1.720 1.790 1.865 1.946 2.032 2.125 2.225
Sucrosé %
Densitya g/ml
Viscosityb cP
Sucrosé %
Densitya g/ml
Viscosityb cP
24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47
1.0987 1.1033 1.1079 1.1126 1.1173 1.1220 1.1268 1.1316 1.1365 1.1414 1.1463 1.1513 1.1563 1.1613 1.1663 1.1714 1.1766 1.1817 1.1870 1.1922 1.1975 1.2028 1.2081 1.2135
2.333 2.449 2.575 2.710 2.857 3.016 3.189 3.378 3.583 3.808 4.053 4.323 4.621 4.948 5.311 5.714 6.163 6.664 7.226 7.857 8.570 9.376 10.29 11.33
48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70
1.2189 1.2244 1.2299 1.2354 1.2409 1.2465 1.2522 1.2578 1.2635 1.2693 1.2750 1.2808 1.2867 1.2926 1.2985 1.3044 1.3104 1.3164 1.3225 1.3286 1.3347 1.3408 1.3470
12.50 13.86 15.42 17.23 19.33 21.79 24.67 28.07 32.11 36.96 42.78 49.85 58.50 69.15 82.39 99.01 120.1 147.0 182.0 227.8 288.5 370.2 481.8
Sucrose in Water, 25.0° C Sucrosé %
Densitya g/ml
Viscosityb cP
Sucrosé %
Densitya g/ml
Viscosityb cP
Sucrosé %
Densitya g/ml
Viscosityb cP
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23
0.9977 1.0014 1.0052 1.0090 1.0128 1.0167 1.0206 1.0246 1.0285 1.0325 1.0366 1.0407 1.0448 1.0489 1.0531 1.0574 1.0616 1.0659 1.0702 1.0746 1.0790 1.0835 1.0879 1.0924
0.8913 0.9139 0.9376 0.9625 0.9886 1.016 1.045 1.076 1.108 1.142 1.179 1.217 1.258 1.301 1.347 1.396 1.449 1.505 1.564 1.628 1.696 1.770 1.848 1.933
24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47
1.0970 1.1016 1.1062 1.1108 1.1155 1.1202 1.1250 1.1298 1.1346 1.1395 1.1444 1.1493 1.1543 1.1593 1.1643 1.1694 1.1745 1.1797 1.1848 1.1901 1.1953 1.2006 1.2059 1.2113
2.023 2.121 2.226 2.339 2.462 2.595 2.739 2.895 3.064 3.249 3.451 3.672 3.914 4.181 4.475 4.799 5.160 5.560 6.008 6.509 7.072 7.706 8.423 9.236
48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70
1.2167 1.2221 1.2276 1.2331 1.2386 1.2442 1.2498 1.2554 1.2611 1.2668 1.2726 1.2784 1.2842 1.2901 1.2959 1.3019 1.3078 1.3138 1.3199 1.3259 1.3320 1.3382 1.3444
10.14 11.19 12.39 13.77 15.37 17.22 19.39 21.93 24.92 28.48 32.73 37.85 44.04 51.61 60.93 72.50 86.99 105.3 128.8 159.1 198.8 251.4 322.0
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Handbook of Biochemistry and Molecular Biology
746
VISCOSITY AND DENSITY TABLES (Continued) Sucrose in Water, 30.0°C Sucrosé %
Densitya g/ml
Viscosityb cP
Sucrosé %
Densitya g/ml
Viscosityb cP
Sucrosé %
Densitya g/ml
Viscosityb cP
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23
0.9963 1.0000 1.0038 1.0075 1.0113 1.0152 1.0191 1.0230 1.0270 1.0310 1.0350 1.0391 1.0432 1.0473 1.0515 1.0557 1.0599 1.0642 1.0685 1.0728 1.0772 1.0816 1.0861 1.0906
0.7978 0.8176 0.8384 0.8601 0.8830 0.9069 0.9322 0.9588 0.9868 1.016 1.048 1.081 1.116 1.154 1.193 1.235 1.280 1.328 1.380 1.434 1.493 1.555 1.622 1.694
24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47
1.0951 1.0997 1.1043 1.1089 1.1136 1.1183 1.1230 1.1278 1.1326 1.1374 1.1423 1.1472 1.1522 1.1572 1.1622 1.1672 1.1723 1.1775 1.1826 1.1878 1.1931 1.1983 1.2036 1.2090
1.771 1.854 1.943 2.039 2.143 2.255 2.376 2.506 2.648 2.802 2.970 3.153 3.353 3.572 3.813 4.079 4.372 4.697 5.058 5.461 5.912 6.418 6.988 7.632
48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70
1.2144 1.2198 1.2252 1.2307 1.2362 1.2418 1.2474 1.2530 1.2586 1.2643 1.2701 1.2758 1.2816 1.2875 1.2933 1.2992 1.3052 1.3112 1.3172 1.3232 1.3293 1.3354 1.3416
8.344 9.168 10.10 11.18 12.42 13.84 15.50 17.43 19.69 22.36 25.52 29.30 33.84 39.34 46.05 54.30 64.53 77.35 93.54 114.2 140.9 175.8 222.0
Compiled by Norman G. Anderson based on equations developed by E. J. Barber in J. Nat. Cancer Inst. Monograph, 21, 219 (1966). a b
Original data were stated to a precision of about 1 part in 10,000. Maximum deviation from original data is 7 parts in 10,000. Precision of original data was between 1 part in 1,000 and 1 part in 10,000. Maximum deviation from original data is 4 parts in 1,000 in the range covered in this set of tables.
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A Listing of Log P Values, Water Solubility, and Molecular Weight for Some Selected Chemicalsa Compound Acetamide Acetic acid Acetic anhydride Acetoacetic acid Acetoin Acetone Acetophenone N-Acetylcysteinamide N-Acetylcysteine N-Aceylmethionine Acetylsalicylic acid Acridine Acrolein Acrylamide Adenine Adenosine Alanine Aldosterone 9-Aminoacridine 4-Aminobenzoic acid (p-aminobenzoic acid; PABA) 4-Aminobutyric acid (γ-aminobutyric acid; GABA) 6-Aminohexanoic acid (ε-aminocaproic acid) Ammonium picrate Aniline Anisole ANS (1-amino-2-naphthalenesulfonic acid) Anthracene Arabinose Arginine Ascorbic acid Asparagine Aspartic acid Barbital (5,5-diethylbarbituric acid) Barbituric acid Benzamide Benzamidine Benzene Benzoic acid Betaine Biuret (imidodicarbonic acid) Bromoacetic acid 2-Bromopropionic acid 2,3-Butanediol 2,3-Butanedione Butyl urea 3-Butyl hydroxy urea Cacodylic acid Carbon tetrachloride Cholesterol Chloroacetamide Chloroacetic anhydride Chloroacetyl chloride Chloroform 6-Chloroindole p-Chloromercuribenzoic acid Chlorosuccinic acid Cholic acid Citric acid Congo red
M.W.
Log Pb
Water Solubility(gm/L)c
59.07 60.05 102.09 102.1 88.11 58.08 120.15 162.21
–1.26 –0.17 –0.58 –0.98 –0.36 –0.24 1.58 –0.29 –0.64 –0.49 1.19 3.40 –0.01 –0.67 –0.09 –1.05 –2.96 1.08 2.74 1.03 –3.17 –2.95 –1.40 0.9 2.11 –0.97 4.45 –3.02 –4.20 –1.64 –3.82 –3.89 0.65 –1.47 0.64 0.65 2.13 1.87 –4.93
2.25 × 103 10 × 103 1.2 × 102 1 × 103 1 × 103 1 × 103 6.13 5.8
180.16 179.22 56.06 71.08 135.13 267.25 89.09 194.23 151.17 103.12 131.18 246.14 222.25 150.13 174.20 176.13 132.12 133.10 184.20 128.1 121.14 120.16 78.11 122.12 117.15 103.08 138.95 152.98 90.12 86.09 116.16 132.16 138.00 153.82 386.67 93.51 170.98 112.94 119.38 151.60 357.16 152.54 405.58 192.13 696.68
0.41 0.92` –0.36 –1.34 0.41 0.32 0.36 2.83 8.74 –0.53 –0.07 –0.22 1.97 3.25 1.48 –0.57 2.02 –1.72 2.63
4.6 0.03 2.13 × 102 6.4 × 102 1.0 8.2 1.7 × 102 0.02 9.89 1.3 × 103 5.05 × 102 1.6 × 102 2.23 1 × 103 1.82 × 102 1 × 103 29.4 5.0 7 13.5 27.9 0.002 3.4 6.11 × 102 1.5 93 29.9 7.6 × 102 2 × 102 46.3 23.5 2 × 103 0.8 0.9 90 68 1.6 × 102 8 0.1 0.3 1.8 × 102 0.2 5.92 × 102 1.2 × 102
747
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Handbook of Biochemistry and Molecular Biology
748
A LISTING OF LOG P VALUES, WATER SOLUBILITY, AND MOLECULAR WEIGHT FOR SOME SELECTED CHEMICALSa (Continued) Compound Corticosterone Cortisone Creatine Creatinine Crotonaldehyde (2-butenal) Cyanoacetic acid Cyanogen Cyanuric acid Cyclohexanone Cysteine Cystine Cytidine Cytosine Deoxycholic acid Deoxycorticosterone Dexamethasone Diazomethane Dichloromethane Dicumarol Diethyl ether (ethyl ether; ether) Diethylsuberate Diethylsulfone N,N-Diethyl urea Dihydroxyacetone Diketene Dimethylformamide Dimethylguanidine Dimethylsulfoxide Dimethylphthalate 1,4-Dinitrobenzene 2,4-Dinitrophenol EDTA EDTA, sodium salt Ethanol (ethyl alcohol) N-Hydroxy-1-ethylurea N-Ethylnicotinamide N-Hydroxyurea Estradiol N-Ethylthiourea Ethylurea Ethylene glycol Ethylene oxide Fluorescein Fluoroacetone Folic acid Formaldehyde Formic acid Galactose Glucose Glutamic acid Glutamine Glycerol Glycine Glyoxal Glyoxylic acid Guanidine Guanine Guanosine Hexanal Hydroxyproline
9168_Book.indb 748
M.W.
132.14 113.12 70.09 85.06 52.04 129.08 121.16 240.30 243.22 111.10 392.58 42.04 336.30 74.1 230.31 122.19 116.2 88.11 84.08 87.13 78.13
292.25 360.17 46.07 104.11 150.18 104.11 104.17 88.11 2.07 44.05 333.32 76.07 441.41 30.03 48.03 180.16 180.16 147.10 146.15 92.10 75.10 58.04 74.04 59.07 151.13 283.25 100.16 131.13
Log Pb 1.94 2.88 –3.72 –1.76 0.60 –0.76 0.07 0.61 0.81 –2.49 –5.08 –2.51 –1.73 3.50 2.88 2.01 2.00 1.2 2.07 0.9 3.35 –0.59 0.1 –0.49 –0.39 –1.04 –0.95 –1.35 1.56 1.47 1.55 –3.86 –13.17 –0.31 –0.10 0.31 –0.76 2.69 –0.21 –0.74 –1.36 –0.30 3.35 –0.39 –2.00 0.35 –0.54 –2.43 –1.88 –3.69 –3.64 –1.76 –3.21 –1.66 –1.40 –1.63 –0.91 –1.90 1.78 –3.17
Water Solubility(gm/L)c
13.3 80 1.8 × 102 7.7 × 102 1.2 × 102 2 1.1 × 102 0.2 1.8 × 102 8 0.04 2 0.1 0.7 1.4 × 102 4 16.2 5.3 × 102 1.6 1 × 103
1 1 × 103 1 × 10+3 7 41.2 24 26.4 1 × 103 1 x103 0.05 286 0.002 400 1 × 103 683 1.2 × 103 8.6 41 1 × 103 2.5 × 102 1 × 103 1 × 103 1.8 2.1 0.7 6 395
4/16/10 1:26 PM
A Listing of Log P Values, Water Solubility, and Molecular Weight for Some Selected Chemicals
749
A LISTING OF LOG P VALUES, WATER SOLUBILITY, AND MOLECULAR WEIGHT FOR SOME SELECTED CHEMICALSa (Continued) Compound Hydroxyurea Imidazole Indole Inositol Iodoacetamide Isoleucine Isopropanol Lactic acid Lactose Leucine Linoleic acid Lysine Maleic anhydride Maltose Mannitol Mercaptoacetic acid 2-Mercaptobenzoic acid Methane Methanol Methionine Methotrexate Methylene blue N-Methyl glycine 5-Methylindole Methyl isocyanate Methylmalonic acid Methyl methacrylate Methylmethane sulfonate Methyl thiocyanate N-Methyl thiourea Methyl urea Naphthalene Nicotinic acid Ornithine Orotic acid Oxalic acid Oxindole Palmitic acid Paraldehyde Pentobarbital Phenol Phenylalanine Phosgene Proline Prostaglandin E2 Propylamine Propylene oxide Pyridine Pyridoxal Pyridoxal-5-phosphate Pyridoxine Pyruvic acid Ribose Sarin Serine Sorbic acid Sorbitol Stearic acid Succinic anhydride Succinimide
9168_Book.indb 749
M.W.
Log Pb
76.06 68.08 117.15 180.16 184.96 131.18 60.10 90.08 342.30 131.18 280.45 146.19 98.06 342.30 182.17 92.12 154.19 16.04 32.04 149.21 454.45 319.86 89.09 131.18 57.05 118.09 86.09 110.13 73.12 119.21 74.08 128.17 123.11 132.16 156.10 90.06 133.15 256.43 132.16 226.28 94.11 165.19 98.02 115.13 352.48 59.11 58.08 79.10 203.63 247.15 169.18 88.06 150.13 140.10 105.09 112.13 182.17 284.49 100.07 99.09
–1.80 –0.08 2.14 –2.08 –0.19 –1.70 0.05 –0.72 –5.43 –1.52 7.05 –3.05 1.62 –5.43 –3.10 0.09 2.39 1.09 –0.77 –1.87 –1.85 5.85 –2.78 2.68 0.79 –0.83 0.80 –0.66 0.73 –0.69 –1.40 3.29 0.36 –4.22 –0.83 –2.22 1.16 7.17 0.67 2.10 1.46 –1.52 –0.71 –2.54 2.82 0.48 0.03 0.65 –3.32 0.37 –0.77 –1.24 –2.32 0.72 –3.07 1.33 –2.20 8.23 0.81 –0.85
Water Solubility(gm/L)c 224 160 4 143 76 34 1 × 103 1 × 103 195 22 0.00004 1 × 103 5 780 216 1 × 103 0.7 0.002 1 × 103 57 2.6 44 300 0.5 29 680 49 1 × 103 32 240 100 220 18 1 × 103 2 9 0.0008 112 0.7 83 22 475 131 0.006 1 × 103 595 1 × 103 500 20 282 1 × 103 1 × 103 425 2 3 × 103 0.03 24 196
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A LISTING OF LOG P VALUES, WATER SOLUBILITY, AND MOLECULAR WEIGHT FOR SOME SELECTED CHEMICALSa (Continued) Compound Sucrose Testosterone Tetrahydrofuran Threonine Toluene 2,4,6-Trinitrobenzene Tryptophan Urea Valine
M.W.
Log Pb
Water Solubility(gm/L)c
342.30 288.43 72.11 119.12 92.14 257.12 204.23 60.06 117.15
–3.70 3.32 0.46 –2.94 2.73 0.23 –1.06 –2.11 –2.26
2.12 × 103 0.03 1 × 103 97 0.5 21 12 545 60
a
Adapted from Handbook of Physical Properties of Organic Chemicals, ed. P.H. Howard and W.M. Meylan, CRC Press, Boca Raton, FL, 1997
b
Log P = log
[Concentration in 1-octanol ] [concentration in water]
See above Howard and Meylan and following for discussion of log P (log of partitioning coefficient for a substance between 1-octanol and water. c Solubility values taken from various literature sources and in some cases are approximations
General references Chuman, H., Mori, A., and Tanaka, H., Prediction of the 1-octanol/H2O partition coefficient, Log P, by Ab Initio calculations: hydrogenbonding effect of organic solutes on Log P, Analyt.Sci. 18, 1015-1020, 2002. Hansch, C. and Leo, A., Exploring QSAR. Fundamentals and Applications in Chemistry and Biology, American Chemical Society, Washington, DC, 1995 Uttamsingh, V., Keller, D.A., and Anders, M.W., Acylase I-catalyzed deacetylation of N-acetyl-L-cysteine and S-Alkyl-N-acetyl-L-cysteines, Chem.Res.Toxicol. 11, 800-809, 1998 Yalkowsky, S.H. and He, Y., Handbook of Aqueous Solubility Data, CRC Press, Boca Raton, Florida, 2003
9168_Book.indb 750
Halling, P.J., Thermodynamic predictions for biocatalysis in nonconventional media: theory, tests, and recommendations for experimental design and analysis, Enzyme Microb.Technol. 16, 178-206, 1994 Abrahams, M.H., Du, C.M., and Platts, J.A., Lipophilicity of the nitrophenols, J.Org.Chem. 65, 7114-7718, 2000 Lipinski, C.A., Lombardo, F., Dominy, B.W., and Feeney, P.J., Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings, Adv.Drug.Deliv.Rev. 46, 3-26, 2001 Valko, K., Du, C.M., Bevan, C., Reynolds, D.P., and Abraham, M.H., Rapid method for the estimation of octanol/water partition coefficient (Log Poct) from gradient RP-HPLC retention and a hydrogen bond acidity term (∑a2H), Curr.Medicin.Chem. 8, 1137-1146, 2001 Avdeef, A., Physicochemical profiling (solubility, permeability and charge state), Curr.Top.Med.Chem. 1, 277-351, 2001
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Chemicals Commonly Used in Biochemistry and Molecular Biology and Their Properties Common Name
Chemical Name
Acetaldehyde
Acetaldehyde, Ethanal
44.05
Properties and Comment Manufacturing intermediate; modification of amino groups; toxic chemical; first product in detoxification of ethanol.
OH
O + H3C
M.W.
H
Acetaldehyde
H2O
H
H3C OH
gem-diol form (approximately 60%)
Burton, R.M. and Stadtman, E.R., The oxidation of acetaldehyde to acetyl coenzyme A, J. Biol. Chem. 202, 873–890, 1953; Gruber, M. and Wesselius, J.C., Nature of the inhibition of yeast carboxylase by acetaldehyde, Biochim. Biophys. Acta 57, 171–173, 1962; Holzer, H., da Fonseca-Wollheim, F., Kohlhaw, G., and Woenckhaus, C.W., Active forms of acetaldehyde, pyruvate, and glycolic aldehyde, Ann. N.Y. Acad. Sci. 98, 453–465, 1962; Brooks, P.J. and Theruvathu, J.A., DNA adducts from acetaldehyde: implications for alcohol-related carcinogenesis, Alcohol 35, 187–193, 2005; Tyulina, O.V., Prokopieva, V.D., Boldyrev, A.A., and Johnson, P., Erthyrocyte and plasma protein modification in alcoholism: a possible role of acetaldehyde, Biochim. Biophys. Acta 1762, 558–563, 2006; Pluskota-Karwatka, D., Pawlowicz, A.J., and Kronberg, L., Formation of malonaldehyde-acetaldehyde conjugate adducts in calf thymus DNA, Chem. Res. Toxicol. 19, 921–926, 2006. Acetic Acid
Acetic Acid, Glacial
60.05
Solvent (particular use in the extraction of collagen from tissue), buffer component (used in urea-acetic acid electrophoresis). Use in endoscopy as mucous-resolving agent.
O
H3C
OH
Banfield, A.G., Age changes in the acetic acid-soluble collagen in human skin, Arch. Pathol. 68, 680–684, 1959; Steven, F.S. and Tristram, G.R., The denaturation of acetic acid-soluble calf-skin collagen. Changes in optical rotation, viscosity, and susceptibility towards enzymes during serial denaturation in solutions of urea, Biochem. J. 85, 207–210, 1962; Neumark, T. and Marot, I., The formation of acetic-acid soluble collagen under polarization and electron microscrope, Acta Histochem. 23, 71–79, 1966; Valfleteren, J.R., Sequential two-dimensional and acetic acid/urea/Triton X-100 gel electrophoresis of proteins, Anal. Biochem. 177, 388–391, 1989; Smith, B.J., Acetic acid-urea polyacrylamide gel electrophoresis of proteins, Methods Mol.Biol. 32, 39–47, 1994; Banfield, W.G., MacKay, C.M., and Brindley, D.C., Quantitative changes in acetic acid-extractable collagen of hamster skin related to anatomical site and age, Gerontologia 12, 231–236, 1996; Lian, J.B., Morris, S., Faris, B. et al., The effects of acetic acid and pepsin on the crosslinkages and ultrastructure of corneal collagen, Biochim. Biophys. Acta. 328, 193–204, 1973; Canto, M.I., Chromoendoscopy and magnifying endoscopy for Barrett’s esophagus, Clin.Gastroenterol.Hepatol. 3 (7 Suppl. 1), S12–S15, 2005; Sionkowska, A., Flash photolysis and pulse radiolysis studies on collagen Type I in acetic acid solution, J. Photochem. Photobiol. B 84, 38–45, 2006.
751
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Chemicals Commonly Used in Biochemistry and Molecular Biology and Their Properties (Continued) Common Name
Chemical Name
Acetic Anhydride
H3C
Acetic Anhydride
O
M.W. 102.07
Properties and Comment Protein modification (trace labeling of amino groups); modification of amino groups and hydroxyl groups.
CH3
C
C
O
O
Jencks, W.P., Barley, F., Barnett, R., and Gilchrest, M., The free energy of hydrolysis of acetic anhydride, J. Am. Chem. Soc. 88, 4464–4467, 1966; Cromwell, L.D. and Stark, G.D., Determination of the carboxyl termini of proteins with ammonium thiocyanate and acetic anhydride, with direct identification of the thiohydantoins, Biochemistry 8, 4735–4740, 1969; Montelaro, R.C. and Rueckert, R.R., Radiolabeling of proteins and viruses in vitro by acetylation with radioactive acetic anhydride, J. Biol. Chem. 250, 1413–1421, 1975; Valente, A.J. and Walton, K.W., The binding of acetic anhydride- and citraconic anhydride-modified human low-density lipoprotein to mouse peritoneal macrophages. The evidence for separate binding sites, Biochim. Biophys. Acta 792, 16–24, 1984; Fojo, A.T., Reuben, P.M., Whitney, P.L., and Awad, W.M., Jr., Effect of glycerol on protein acetylation by acetic anhydride, Arch. Biochem. Biophys. 240, 43–50, 1985; Buechler, J.A., Vedvick, T.A., and Taylor, S.S., Differential labeling of the catalytic subunit of cAMP-dependent protein kinase with acetic anhydride: substrate-induced conformational changes, Biochemistry 28, 3018–3024, 1989; Baker, G.B., Coutts, R.T., and Holt, A., Derivatization with acetic anhydride: applications to the analysis of biogenic amines and psychiatric drugs by gas chromatography and mass spectrometry, J. Pharmacol. Toxicol. Methods 31, 141–148, 1994; Ohta, H., Ruan, F., Hakomori, S., and Igarashi, Y., Quantification of free Sphingosine in cultured cells by acetylation with radioactive acetic anhydride, Anal. Biochem. 222, 489–494, 1994; Yadav, S.P., Brew, K., and Puett, D., Holoprotein formation of human chorionic gonadotropin: differential trace labeling with acetic anhydride, Mol. Endocrinol. 8, 1547–1558, 1994; Miyazaki, K. and Tsugita, A., C-terminal sequencing method for peptides and proteins by the reaction with a vapor of perfluoric acid in acetic anhydride, Proteomics 4, 11–19, 2004. Acetone
Dimethyl Ketone; 2-propanone
58.08
Solvent, protein purification (acetone powders); rare reaction with amino groups.
O
H3C
CH3
La Du, B., Jr. and Greenberg, D.M., The tyrosine oxidation system of liver. I. Extracts of rat liver acetone powder, J. Biol. Chem. 190, 245–255, 1951; Korn, E.D. and Payza, A.N., The degradation of heparin by bacterial enzymes. II. Acetone powder extracts, J. Biol. Chem. 223, 859–864, 1956; Ohtsuki, K., Taguchi, K., Sato, K., and Kawabata, M., Purification of ginger proteases by DEAE-Sepharose and isoelectric focusing, Biochim. Biophys. Acta 1243, 181–184, 1995; Selden, L.A., Kinosian, H.J., Estes, J.E., and Gershman, L.C., Crosslinked dimers with nucleating activity in actin prepared from muscle acetone powder, Biochemistry 39, 64–74, 2000; Abadir, W.F., Nakhla, V., and Chong, F., Removal of superglue from the external ear using acetone: case report and literature review, J. Laryngol. Otol. 109, 1219–1221, 1995; Jones, A.W., Elimination half-life of acetone in humans: case reports and review of the literature, J. Anal. Toxicol. 24, 8–10, 2000; Huang, L.P. and Guo, P., Use of acetone to attain highly active and soluble DNA packaging protein Gp16 of Phi29 for ATPase assay, Virology 312, 449–457, 2003; Paska, C., Bogi, K., Szilak, L. et al., Effect of formalin, acetone, and RNAlater fixatives on tissue preservation and different size amplicons by real-time PCR from paraffin-embedded tissues, Diagn. Mol. Pathol. 13, 234–240, 2004; Kuksis, A., Ravandi, A., and Schneider, M., Covalent binding of acetone to aminophospholipids in vitro and in vivo, Ann. N.Y. Acad. Sci. 1043, 417–439, 2005; Perera, A., Sokolic, F., Almasy, L. et al., On the evaluation of the Kirkwood–Buff integrals of aqueous acetone mixtures, J. Chem. Physics 123, 23503, 2005; Zhou, J., Tao, G., Liu, Q. et al., Equilibrium yields of mono- and di-lauroyl mannoses through lipasecatalyzed condensation in acetone in the presence of molecular sieves, Biotechnol. Lett. 28, 395–400, 2006. Acetonitrile
Ethenenitrile, Methyl Cyanide
41.05
Chromatography solvent, general solvent.
C H3C
9168_Book.indb 752
N
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753
Chemicals Commonly Used in Biochemistry and Molecular Biology and Their Properties (Continued) Common Name
Chemical Name
M.W.
Properties and Comment
Hodgkinson, S.C. and Lowry, P.J., Hydrophobic-interaction chromatography and anion-exchange chromatography in the presence of acetonitrile. A two-step purification method for human prolactin, Biochem. J. 199, 619–627, 1981; Wolf-Coporda, A., Plavsic, F., and Vrhovac, B., Determination of biological equivalence of two atenolol preparations, Int. J. Clin. Pharmacol. Ther. Toxicol. 25, 567–571, 1987; Fischer, U., Zeitschel, U., and Jakubke, H.D., Chymotrypsin-catalyzed peptide synthesis in an acetonitrile-water-system: studies on the efficiency of nucleophiles, Biomed. Biochim. Acta 50, S131–S135, 1991; Haas, R. and Rosenberry, T.L., Protein denaturation by addition and removal of acetonitrile: application to tryptic digestion of acetylcholinesterase, Anal. Biochem. 224, 425–427, 1995; Joansson, A., Mosbach, K., and Mansson, M.O., Horse liver alcohol dehydrogenase can accept NADP+ as coenzyme in high concentrations of acetonitrile, Eur. J. Biochem. 227, 551–555, 1995; Barbosa, J., Sanz-Nebot, V., and Toro, I., Solvatochromic parameter values and pH in acetonitrile-water mixtures. Optimization of mobile phase for the separation of peptides by high-performance liquid chromatography, J. Chromatog. A 725, 249–260, 1996; Barbosa, J., Hernandez-Cassou, S., Sanz-Nebot, V., and Toro, I., Variation of acidity constants of peptides in acetonitrile-water mixtures with solvent composition: effect of preferential salvation, J. Pept. Res. 50, 14–24, 1997; Badock, V., Steinhusen, U., Bommert, K., and Otto, A., Prefractionation of protein samples for proteome analysis using reversed-phase high-performance liquid chromatography, Electrophoresis 22, 2856–2864, 2001; Yoshida, T., Peptide separation by hydrophilic-interaction chromatography: a review, J. Biochem. Biophys. Methods 60, 265–280, 2004: Kamau, P. and Jordan, R.B., Complex formation constants for the aqueous copper(I)-acetonitrile system by a simple general method, Inorg. Chem. 40, 3879–3883, 2001; Nagy, P.I. and Erhardt, P.W., Monte Carlo simulations of the solution structure of simple alcohols in water-acetonitrile mixtures, J. Phys. Chem. B Condens. Matter Mater. Surf. Interfaces Biophys. 109, 5855–5872, 2005; Kutt, A., Leito, I., Kaljurand, I. et al., A comprehensive self-consistent spectrophotometric acidity scale of neutral Bronstad acids in acetonitrile, J. Org. Chem. 71, 2829–2938, 2006. Acetyl Chloride
Ethanoyl Chloride
78.50
Acetylating agent.
O
H3C
Cl
Hallaq, Y., Becker, T.C., Manno, C.S., and Laposata, M., Use of acetyl chloride/methanol for assumed selective methylation of plasma nonesterified fatty acids results in significant methylation of esterified fatty acids, Lipids 28, 355–360, 1993; Shenoy, N.R., Shively, J.E., and Bailey, J.M., Studies in C-terminal sequencing: new reagents for the synthesis of peptidylthiohydantoins, J. Protein Chem. 12, 195–205, 1993; Bosscher, G., Meetsma, A., and van De Grampel, J.C., Novel organo-substituted cyclophosphazenes via reaction of a monohydro cyclophosphazene and acetyl chloride, Inorg. Chem. 35, 6646–6650, 1996; Mo, B., Li, J., and Liang, S., A method for preparation of amino acid thiohydantoins from free amino acids activated by acetyl chloride for development of protein C-terminal sequencing, Anal. Biochem. 249, 207–211, 1997; Studer, J., Purdie, N., and Krouse, J.A., Friedel–Crafts acylation as a quality control assay for steroids, Appl. Spectros. 57, 791–796, 2003. Acetylcysteine
N-acetyl-L-cysteine
Mild reducing agent for clinical chemistry (creatine kinase); therapeutic use for aminoacetophen intoxication; some other claimed indications.
O
O NH H3C
163.2
CH
C
OH
CH2 SH
Szasz, G., Gruber, W., and Bernt, E., Creatine kinase in serum. I. Determination of optimum reaction conditions, Clin. Chem. 22, 650–656, 1976; Holdiness, M.R., Clinical pharmacokinetics of N-acetylcysteine, Clin. Pharmacokinet. 20, 123–134, 1991; Kelley, G.S., Clinical applications of N-acetylcysteine, Altern. Med. Rev. 3, 114–127, 1998; Schumann, G., Bonora, R., Ceriotti, F. et al., IFCC primary reference procedures for the measurement of catalytic activity concentrations of enzymes at 37°C. Part 2. Reference procedure for the measurement of catalytic concentration of creatine kinase, Clin. Chem. Lab. Med. 40, 635–642, 2002; Zafarullah, M., Li, W.Q., Sylvester, J., and Ahmad, M., Molecular mechanisms of N-acetylcysteine actions, Cell. Mol. Life Sci. 60, 6–20, 2003; Marzullo, L., An update of N-acetylcysteine treatment for acute aminoacetophen toxicity in children, Curr. Opin. Pediatr. 17, 239–245, 2005; Aitio, M.L., N-acetylcysteine — passé-partout or much ado about nothing? Br. J. Clin. Pharmacol. 61, 5–15, 2006.
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Chemicals Commonly Used in Biochemistry and Molecular Biology and Their Properties (Continued) Common Name N-Acetylimidazole
Chemical Name
M.W.
1-acetyl-1H-imidazole
110.12
Properties and Comment Reagent for modification of tyrosyl residues in proteins.
H3C O N
N Lundblad, R.L., Chemical Reagents for Protein Modification, CRC Press, Boca Raton, FL, 2004; Gorbunoff, M.J., Exposure of tyrosine residues in proteins. 3. The reaction of cyanuric fluoride and N-acetylimidazole with ovalbumin, chymotrypsinogen, and trypsinogen, Biochemistry 44, 719–725, 1969; Houston, L.L. and Walsh, K.A., The transient inactivation of trypsin by mild acetylation with N-acetylimidazole, Biochemistry 9, 156–166, 1970; Shifrin, S. and Solis, B.G., Reaction of N-acetylimidazole with L-asparaginase, Mol. Pharmacol. 8, 561–564, 1972; Ota, Y., Nakamura, H., and Samejima, T., The change of stability and activity of thermolysin by acetylation with N-acetylimidazole, J. Biochem. 72, 521–527, 1972; Kasai, H., Takahashi, K., and Ando, T., Chemical modification of tyrosine residues in ribonuclease T1 with N-acetylimidazole and p-diazobenzenesulfonic acid, J. Biochem. 81, 1751–1758, 1977; Zhao, X., Gorewit, R.C., and Currie, W.B., Effects of N-acetylimidazole on oxytocin binding in bovine mammary tissue, J. Recept. Res. 10, 287–298, 1990; Wells, I. and Marnett, L.J., Acetylation of prostaglandin endoperoxide synthase by N-acetylimidazole: comparison to acetylation by aspirin, Biochemistry 31, 9520–9525, 1992; Cymes, G.D., Iglesias, M.M., and Wolfenstein-Todel, C., Chemical modification of ovine prolactin with N-acetylimidazole, Int. J. Pept. Protein Res. 42, 33–38, 1993; Zhang, F., Gao, J., Weng, J. et al., Structural and functional differences of three groups of tyrosine residues by acetylation of N-acetylimidazole in manganese-stabilizing protein, Biochemistry 44, 719–725, 2005. Acetylsalicylic Acid
2-(acetoxy)benzoic Acid; Aspirin
180.16
Analgesic, anti-inflammatory; mild acetylating agent.
O
CH3
O
HO
O
Hawkins, D., Pinckard, R.N., and Farr, R.S., Acetylation of human serum albumin by acetylsalicylic acid, Science 160, 780–781, 1968; Kalatzis, E., Reactions of aminoacetophen in pharmaceutical dosage forms: its proposed acetylation by acetylsalicylic acid, J. Pharm. Sci. 59, 193–196, 1970; Pinckard, R.N., Hawkins, D., and Farr, R.S., The inhibitory effect of salicylate on the actylation of human albumin by acetylsalicylic acid, Arthritis Rheum. 13, 361–368, 1970; Van Der Ouderaa, F.J., Buytenhek, M., Nugteren, D.H., and Van Dorp, D.A., Acetylation of prostaglandin endoperoxide synthetase with acetylsalicylic acid, Eur. J. Biochem. 109, 1–8, 1980; Rainsford, K.D., Schweitzer, A., and Brune, K., Distribution of the acetyl compared with the salicyl moiety of acetylsalicylic acid. Acetylation of macromolecules in organs wherein side effects are manifest, Biochem. Pharmacol. 32, 1301–1308, 1983; Liu, L.R. and Parrott, E.L., Solid-state reaction between sulfadiazine and acetylsalicyclic acid, J. Pharm. Sci. 80, 564–566, 1991; Minchin, R.F., Ilett, K.F., Teitel, C.H. et al., Direct O-acetylation of N-hydroxy arylamines by acetylsalicylic acid to form carcinogen-DNA adducts, Carcinogenesis 13, 663–667, 1992. Acrylamide
2-propenamide
71.08
Monomer unit of polyacrylamide in gels, hydrogels, hard polymers; environmental carcinogen; fluorescence quencher.
NH 2 H 2C O
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Chemicals Commonly Used in Biochemistry and Molecular Biology and Their Properties (Continued) Common Name
Chemical Name
M.W.
Properties and Comment
Eftink, M.R. and Ghiron, C.A., Fluorescence quenching studies with proteins, Anal. Biochem. 114, 199–227, 1981; Dearfield, K.L., Abernathy, C.O., Ottley, M.S. et al., Acrylamide: its metabolism, developmental and reproductive effects, Mutat. Res. 195, 45–77, 1988; Williams, L.R., Staining nucleic acids and proteins in electrophoresis gels, Biotech. Histochem. 76, 127–132, 2001; Hamden, M., Bordini, E., Galvani, M., and Righetti, P.G., Protein alkylation by acrylamide, its N-substituted derivatives and crosslinkers and its relevance to proteomics: a matrix-assisted laser desorption/ ionization-time of flight-mass spectrometry study, Electrophoresis 22, 1633–1644, 2001; Cioni, P. and Strambini, G.B., Tryptophan phosphorescence and pressure effects on protein structure, Biochim. Biophys. Acta 1595, 116–130, 2002; Taeymans, D., Wood, J., Ashby, P. et al., A review of acrylamide: an industry perspective on research, analysis, formation, and control, Crit. Rev. Food Sci. Nutr. 44, 323–347, 2004; Rice, J.M., The carcinogenicity of acrylamide, Mutat. Res. 580, 3–20, 2005; Besaratinia, A. and Pfeifer, G.P., DNA adduction and mutagenic properties of acrylamide, Mutat. Res. 580, 31–40, 2005; Hoenicke, K. and Gaterman, R., Studies on the stability of acrylamide in food during storage, J. AOAC Int. 88, 268–273, 2005; Castle, L. and Ericksson, S., Analytical methods used to measure acrylamide concentrations in foods, J. AOAC Int. 88, 274–284, 2005; Stadler, R.H., Acrylamide formation in different foods and potential strategies for reduction, Adv. Exp. Med. Biol. 561, 157–169, 2005; Lopachin, R.M. and Decaprio, A.P., Protein adduct formation as a molecular mechanism in neurotoxicity, Toxicol. Sci. 86, 214–225, 2005. Gamma (γ)-aminobutyric Acid (GABA)
4-aminobutanoic acid
103.12
Neurotransmitter.
O H 2N OH Mandel, P. and DeFeudis, F.V., Eds., GABA—Biochemistry and CNS Functions, Plenum Press, New York, 1979; Costa, E. and Di Chiara, G., GABA and Benzodiazepine Receptors, Raven Press, New York, 1981; Racagni, G. and Donoso, A.O., GABA and Endocrine Function, Raven Press, New York, 1986; Squires, R.F., GABA and Benzodiazepine Receptors, CRC Press, Boca Raton, FL, 1988; Martin, D.L. and Olsen, R.W., GABA in the Nervous System: The View at Fifty Years, Lippincott, Williams & Wilkins, Philadelphia, PA, 2000. Amiloride
3,5-diamino-N-(aminoiminomethyl)-6chloropyrazinecarboxamide O
229.63
Sodium ion channel blocker.
NH
N
Cl
NH2
N
H2N
NH2
Benos, D.J., A molecular probe of sodium transport in tissues and cells, Am. J. Physiol. 242, C131–C145, 1982; Garty, H., Molecular properties of epithelial, amiloride-blockable Na+ channels, FASEB J. 8, 522–528, 1994; Barbry, P. and Lazdunski, M., Structure and regulation of the amiloridesensitive epithelial sodium channel, Ion Channels 4, 115–167, 1996; Kleyman, T.R., Sheng, S., Kosari, F., and Kieber-Emmons, T., Mechanism of action of amiloride: a molecular perspective, Semin. Nephrol. 19, 524–532, 1999; Alvarez de la Rosa, D., Canessa, C.M., Fyfe, G.K., and Zhang, P., Structure and regulation of amiloride-sensitive sodium channels, Annu. Rev. Physiol. 62, 573–594, 2000; Haddad, J.J., Amiloride and the regulation of NF-κ β: an unsung crosstalk and missing link between fluid dynamics and oxidative stress-related inflammation — controversy or pseudocontroversy, Biochem. Biophys. Res. Commun. 327, 373–381, 2005. 2-Aminopyridine
N
9168_Book.indb 755
α-aminopyridine
94.12
Precursor for synthesis of pharmaceuticals and reagents; used to derivatize carbohydrates for analysis; blocker of K+ channels.
NH2
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Chemicals Commonly Used in Biochemistry and Molecular Biology and Their Properties (Continued) Common Name
Chemical Name
M.W.
Properties and Comment
Hase, S., Hara, S., and Matsushima, Y., Tagging of sugars with a fluorescent compound, 2-aminopyridine, J. Biochem. 85, 217–220, 1979; Hase, S., Ibuki, T., and Ikenaka, T., Reexamination of the pyridylamination used for fluorescence labeling of oligosaccharides and its application to glycoproteins, J. Biochem. 95, 197–203, 1984; Chen, C. and Zheng, X., Development of the new antimalarial drug pyronaridine: a review, Biomed. Environ. Sci. 5, 149–160, 1992; Hase, S., Analysis of sugar chains by pyridylamination, Methods Mol. Biol. 14, 69–80, 1993; Oefner, P.J. and Chiesa, C., Capillary electrophoresis of carbohydrates, Glycobiology 4, 397–412, 1994; Dyukova, V.I., Shilova, N.V., Galanina, O.E. et al., Design of carbohydrate multiarrays, Biochim. Biophys. Acta 1760, 603–609, 2006; Takegawa, Y., Deguchi, K., Keira, T. et al., Separation of isomeric 2-aminopyridine derivatized N-glycans and N-glycopeptides of human serum immunoglobulin G by using a zwitterionic type of hydrophilicinteraction chromatography, J. Chromatog. A 1113, 177–181, 2006; Suzuki, S., Fujimori, T., and Yodoshi, M., Recovery of free oligosaccharides from derivatives labeled by reductive amination, Anal. Biochem. 354, 94–103, 2006; Caballero, N.A., Melendez, F.J., Munoz-Caro, C., and Nino, A., Theoretical prediction of relative and absolute pK(a) values of aminopyridine, Biophys. Chem., 124, 155–160, 2006. Ammonium Bicarbonate
Acid Ammonium Carbonate
79.06
Volatile buffer salt.
H O-
O H
N
+
H
C
H
OH
Gibbons, G.R., Page, J.D., and Chaney, S.G., Treatment of DNA with ammonium bicarbonate or thiourea can lead to underestimation of platinum-DNA monoadducts, Cancer Chemother. Pharmacol. 29, 112–116, 1991; Sorenson, S.B., Sorenson, T.L., and Breddam, K., Fragmentation of protein by S. aureus strain V8 protease. Ammonium bicarbonate strongly inhibits the enzyme but does not improve the selectivity for glutamic acid, FEBS Lett. 294, 195–197, 1991; Fichtinger-Schepman, A.M., van Dijk-Knijnenburg, H.C., Dijt, F.J. et al., Effects of thiourea and ammonium bicarbonate on the formation and stability of bifunctional cisplatinin-DNA adducts: consequences for the accurate quantification of adducts in (cellular) DNA, J. Inorg. Biochem. 58, 177–191, 1995; Overcashier, D.E., Brooks, D.A., Costantino, H.R., and Hus, C.C., Preparation of excipient-free recombinant human tissue-type plasminogen activator by lyophilization from ammonium bicarbonate solution: an investigation of the two-stage sublimation process, J. Pharm. Sci. 86, 455–459, 1997. ANS
1-anilino-8naphthalenenesulfonate
299.4
Fluorescent probe for protein conformation; considered a hydrophobic probe; study of molten globules.
OH NH O
S
O
Ferguson, R.N., Edelhoch, H., Saroff, H.A. et al., Negative cooperativity in the binding of thyroxine to human serum prealbumin. Preparation of tritium-labeled 8-anilino-1-naphthalenesulfonic acid, Biochemistry 14, 282–289, 1975; Ogasahara, K., Koike, K., Hamada, M., and Hiraoka, T., Interaction of hydrophobic probes with the apoenzyme of pig heart lipoamide dehydrogenase, J. Biochem. 79, 967–975, 1976; De Campos Vidal, B., The use of the fluorescence probe 8-anilinonaphthalene sulfate (ANS) for collagen and elastin histochemistry, J. Histochem. Cytochem. 26, 196–201, 1978; Royer, C.A., Fluorescence spectroscopy, Methods Mol. Biol. 40, 65–89, 1995; Celej, M.S., Dassie, S.A., Freire, E. et al., Ligand-induced thermostability in proteins: thermodynamic analysis of ANS-albumin interaction, Biochim. Biophys. Acta 1750, 122–133, 2005; Banerjee, T. and Kishore, N., Binding of 8-anilinonaphthalene sulfonate to dimeric and tetrameric concanavalin A: energetics and its implications on saccharide binding studied by isothermal titration calorimetry and spectroscopy, J. Phys. Chem. B Condens. Matter Mater. Surf. Interfaces Biophys. 110, 7022–7028, 2006; Sahu, K., Mondal, S.K., Ghosh, S. et al., Temperature dependence of salvation dynamics and anisotropy decay in a protein: ANS in bovine serum albumin, J. Chem. Phys. 124, 124909, 2006; Wang, G., Gao, Y., and Geng, M.L., Analysis of heterogeneous fluorescence decays in proteins. Using fluorescence lifetime of 8-anilino-1-naphthalenesulfonate to probe apomyoglobin unfolding at equilibrium, Biochim. Biophys. Acta 1760, 1125–1137, 2006; Greene, L.H., Wijesinha-Bettoni, R., and Redfield, C., Characterization of the molten globule of human serum retinolbinding protein using NMR spectroscopy, Biochemistry 45, 9475–9484, 2006.
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757
Chemicals Commonly Used in Biochemistry and Molecular Biology and Their Properties (Continued) Common Name
Chemical Name
Arachidonic Acid
5,8,11,14(all cis)eicosatetraenoic acid
M.W. 304.5
Properties and Comment Essential fatty acid; precursor of prostaglandins, thromboxanes, and leukotrienes.
COOH
COOH Moncada, S. and Vane, J.R., Interaction between anti-inflammatory drugs and inflammatory mediators. A reference to products of arachidonic acid metabolism, Agents Actions Suppl. 3, 141–149, 1977; Moncada, S. and Higgs, E.A., Metabolism of arachidonic acid, Ann. N.Y. Acad. Sci. 522, 454–463, 1988; Piomelli, D., Arachidonic acid in cell signaling, Curr. Opin. Cell Biol. 5, 274–280, 1993; Janssen-Timmen, U., Tomic, I., Specht, E. et al., The arachidonic acid cascade, eicosanoids, and signal transduction, Ann. N.Y. Acad. Sci. 733, 325–334, 1994; Wang, X. and Stocco, D.M., Cyclic AMP and arachidonic acid: a tale of two pathways, Mol. Cell. Endocrinol. 158, 7–12, 1999; Brash, A.R., Arachidonic acid as a bioactive molecule, J. Clin. Invest. 107, 1339–1345, 2001; Luo, M., Flamand, N., and Brock, T.G., Metabolism of arachidonic acid to eicosanoids within the nucleus, Biochim. Biophys. Acta 1761, 618–625, 2006; Balboa, M.A. and Balsinde, J., Oxidative stress and arachidonic acid mobilization, Biochim. Biophys. Acta 1761, 385–391, 2006. Ascorbic Acid HO
Vitamin C; 3-oxo-L-gulofuranolactone HO
OH
H2C
CH
O
O O
HO OH Ascorbic acid
Nutrition, antioxidant (reducing agent); possible antimicrobial function.
OH
H2C
CH
176.13
O Dehydroascorbic acid
Barnes, M.J. and Kodicek, E., Biological hydroxylations and ascorbic acid with special regard to collagen metabolism, Vitam. Horm. 30, 1–43, 1972; Leibovitz, B. and Siegel, B.V., Ascorbic acid and the immune response, Adv. Exp. Med. Biol. 135, 1–25, 1981; Englard, S. and Seifter, S., The biochemical functions of ascorbic acid, Annu. Rev. Nutr. 6, 365–406, 1986; Levine, M. and Hartzell, W., Ascorbic acid: the concept of optimum requirements, Ann. N.Y. Acad. Sci. 498, 424–444, 1987; Padh, H., Cellular functions of ascorbic acid, Biochem. Cell Biol. 68, 1166–1173, 1990; Meister, A., On the antioxidant effects of ascorbic acid and glutathione, Biochem. Pharmacol. 44, 1905–1915, 1992; Wolf, G., Uptake of ascorbic acid by human neutrophils, Nutr. Rev. 51, 337–338, 1993; Kimoto, E., Terada, S., and Yamaguchi, T., Analysis of ascorbic acid, dehydroascorbic acid, and transformation products by ion-pairing high-performance liquid chromatography with multiwavelength ultraviolet and electrochemical detection, Methods Enzymol. 279, 3–12, 1997; May, J.M., How does ascorbic acid prevent endothelial dysfunction? Free Rad. Biol. Med. 28, 1421–1429, 2000; Smirnoff, N. and Wheeler, G.L., Ascorbic acid in plants: biosynthesis and function, Crit. Rev. Biochem. Mol. Biol. 35, 291–314, 2000; Arrigoni, O. and De Tullio, M.C., Ascorbic acid: much more than just an antioxidant, Biochim. Biophys. Acta 1569, 1–9, 2002; Akyon, Y., Effect of antioxidant on the immune response of Helicobacter pyrlori, Clin. Microbiol. Infect. 8, 438–441, 2002; Takanaga, H., MacKenzie, B., and Hediger, M.A., Sodium-dependent ascorbic acid transporter family SLC23, Pflügers Arch. 447, 677–682, 2004. Benzaldehyde
Benzoic Aldehyde; Essential Oil of Almond
106.12
Intermediate in manufacture of pharmaceuticals, flavors; reacts with amino groups, semicarbidizide.
O
H C
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758
Chemicals Commonly Used in Biochemistry and Molecular Biology and Their Properties (Continued) Common Name
Chemical Name
M.W.
Properties and Comment
Chalmers, R.M., Keen, J.N., and Fewson, C.A., Comparison of benzyl alcohol dehydrogenases and benzaldehyde dehydrogenases from the benzyl alcohol and mandelate pathways in Acinetobacter calcoaceticus and the TOL-plasmid-encoded toluene pathway in Pseudomonas putida. N-terminal amino acid sequences, amino acid composition, and immunological cross-reactions, Biochem. J. 273, 99–107, 1991; Pettersen, E.O., Larsen, R.O., Borretzen, B. et al., Increased effect of benzaldehyde by exchanging the hydrogen in the formyl group with deuterium, Anticancer Res. 11, 369–373, 1991; Nierop Groot, M.N. and de Bont, J.A.M., Conversion of phenylalanine to benzaldehyde initiated by an amino-transferase in Lactobacillus plantarum, Appl. Environ. Microbiol. 64, 3009–3013, 1998; Podyminogin, M.A., Lukhtanov, E.A., and Reed, M.W., Attachment of benzaldehyde-modified oligodeoxynucleotide probes to semicarbazide-coated glass, Nucleic Acids Res. 29, 5090–5098, 2001; Kurchan, A.N. and Kutateladze, A.G., Amino acid-based dithiazines: synthesis and photofragmentation of their benzaldehyde adducts, Org. Lett. 4, 4129–4131, 2002; Kneen, M.M., Pogozheva, I.D., Kenyon, G.L., and McLeish, M.J., Exploring the active site of benzaldehyde lyase by modeling and mutagenesis, Biochim. Biophys. Acta 1753, 263–271, 2005; Mosbacher, T.G., Mueller, M., and Schultz, G.E., Structure and mechanism of the ThDP-dependent benzaldehyde lyase from Pseudomonas fluorescens, FEBS J. 272, 6067–6076, 2005; Sudareva, N.N. and Chubarova, E.V., Time-dependent conversion of benzyl alcohol to benzaldehyde and benzoic acid in aqueous solution, J. Pharm. Biomed. Anal. 41, 1380–1385, 2006. Benzamidine HCl
H 2N
156.61
+
Inhibitor of trypticlike serine proteases.
Cl -
NH 2
Ensinck, J.W., Shepard, C., Dudl, R.J., and Williams, R.H., Use of benzamidine as a proteolytic inhibitor in the radio-immunoassay of glucagon in plasma, J. Clin. Endocrinol. Metab. 35, 463–467, 1972; Bode, W. and Schwager, P., The refined crystal structure of bovine beta-trypsin at 1.8 Å resolution. II. Crystallographic refinement, calcium-binding site, benzamidine-binding site and active site at pH 7.0., J. Mol. Biol. 98, 693–717, 1975; Nastruzzi, C., Feriotto, G., Barbieri, R. et al., Differential effects of benzamidine derivatives on the expression of c-myc and HLA-DR alpha genes in a human B-lymphoid tumor cell line, Cancer Lett. 38, 297–305, 1988; Clement, B., Schmitt, S., and Zimmerman, M., Enzymatic reduction of benzamidoxime to benzamidine, Arch. Pharm. 321, 955–956, 1988; Clement, B., Immel, M., Schmitt, S., and Steinman, U., Biotransformation of benzamidine and benzamidoxime in vivo, Arch. Pharm. 326, 807–812, 1993; Renatus, M., Bode, W., Huber, R. et al., Structural and functional analysis of benzamidine-based inhibitors in complex with trypsin: implications for the inhibition of factor Xa, tPA, and urokinase, J. Med. Chem. 41, 5445–5456, 1998; Henriques, R.S., Fonseca, N., and Ramos, M.J., On the modeling of snake venom serine proteinase interactions with benzamidinebased thrombin inhibitors, Protein Sci. 13, 2355–2369, 2004; Gustavsson, J., Farenmark, J., and Johansson, B.L., Quantitative determination of the ligand content in benzamidine Sepharose• 4 Fast Flow media with ion-pair chromatography, J. Chromatog. A 1070, 103–109, 2005. Benzene
Benzene
78.11
Solvent; a xenobiotic.
Lovley, D.R., Anaerobic benzene degradation, Biodegradation 11, 107–116, 2000; Snyder, R., Xenobiotic metabolism and the mechanism(s) of benzene toxicity, Drug Metab. Rev. 36, 531–547, 2004; Rana, S.V. and Verma, Y., Biochemical toxicity of benzene, J. Environ. Biol. 26, 157–168, 2005; Lin, Y.S., McKelvey, W., Waidyanatha, S., and Rappaport, S.M., Variability of albumin adducts of 1,4-benzoquinone, a toxic metabolite of benzene, in human volunteers, Biomarkers 11, 14–27, 2006; Baron, M. and Kowalewski, V.J., The liquid water-benzene system, J. Phys. Chem. A Mol. Spectrosc. Kinet. Environ. Gen. Theory 100, 7122–7129, 2006; Chambers, D.M., McElprang, D.O., Waterhouse, M.G., and Blount, B.C., An improved approach for accurate quantitation of benzene, toluene, ethylbenzene, xylene, and styrene in blood, Anal. Chem. 78, 5375–5383, 2006. Benzidine
H2N
9168_Book.indb 758
p-benzidine; 184.24 (1,1′-biphenyl)-4,4′-diamine
Precursor for azo dyes; mutagenic agent; forensic analysis for bloodstains based on reactivity with hemoglobin.
NH2
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Chemicals Commonly Used in Biochemistry and Molecular Biology and Their Properties (Continued) Common Name
Chemical Name
M.W.
Properties and Comment
Ahlquist, D.A. and Schwartz, S., Use of leuco-dyes in the quantitative colorimetric microdetermination of hemoglobin and other heme compounds, Clin. Chem. 21, 362–369, 1975; Josephy, P.D., Benzidine: mechanisms of oxidative activation and mutagensis, Fed. Proc. 45, 2465–2470, 1986; Choudhary, G., Human health perspectives on environmental exposure to benzidine: a review, Chemosphere 32, 267–291, 1996; Madeira, P., Nunes, M.R., Borges, C. et al., Benzidine photodegradation: a mass spectrometry and UV spectroscopy combined study, Rapid Commun. Mass Spectrom. 19, 2015–2020, 2005; Saitoh, T., Yoshida, S., and Ichikawa, J., Naphthalene-1,8-diylbis(diphenylmethylium) as an organic two-electron oxidant: benzidine synthesis via oxidative self-coupling of N,N-dialkylanilines, J. Org. Chem. 71, 6414–6419, 2006. BIG CHAP/Deoxy BIG CHAP
N,N-bis(3-d-gluconamidopropyl) cholamide/N,Nbis(3-d-gluconamidopropyl) deoxycholamide
878.1/ 862.1
Nonionic detergents; protein solubilization, adenovirus gene transfer enhancement.
HO
HO OH HO HO
OH O
HO HO
NH
OH OH
HN O N H3C OH CH3
O
CH3
HO
OH
Bonelli, F.S. and Jonas, A., Reaction of lecithin: cholesterol acyltransferase with a water-soluble substrate: effects of surfactants, Biochim. Biophys. Acta 1166, 92–98, 1993; Aigner, A., Jager, M., Pasternack, R. et al., Purification and characterization of cysteine-S-conjugate N-acetyltransferase from pig kidney, Biochem. J. 317, 213–218, 1996; Mechref, Y. and Eirassi, Z., Micellar electrokinetic capillary chromatography with in-situ charged micelles. 4. Evaluation of novel chiral micelles consisting of steroidal glycoside surfactant borate complexes, J. Chromatog. A 724, 285–296, 1996; Abe, S., Kunii, S., Fujita, T., and Hiraiwa, K., Detection of human seminal gamma-glutamyl transpeptidase in stains using sandwich ELISA, Forensic Sci. Int. 91, 19–28, 1998; Akutsu, Y., Nakajima-Kambe, T., Nomura, N., and Nakahara, T., Purification and properties of a polyester polyurethane-degrading enzyme form Comamonas acidovorans TB-35, Appl. Environ. Microbiol. 64, 62–67, 1998: Connor, R.J., Engler, H., Machemer, T. et al., Identification of polyamides that enhance adenovirus-mediated gene expression in the urothelium, Gene Therapy 8, 41–48, 2001; Vajdos, F.F., Ultsch, M., Schaffer, M.L. et al., Crystal structure of human insulin-like growth factor-1: detergent binding inhibits binding protein interactions, Biochemistry 40, 11022–11029, 2001; Kuball, J., Wen, S.F., Leissner, J. et al., Successful adenovirus-mediated wild-type p53 gene transfer in patients with bladder cancer by intravesical vector instillation, J. Clin. Oncol. 20, 957–965, 2002; Susasara, K.M., Xia, F., Gronke, R.S., and Cramer, S.M., Application of hydrophobic interaction displacement chromatography for an industrial protein purification, Biotechnol. Bioeng. 82, 330–339, 2003; Ishibashi, A. and Nakashima, N., Individual dissolution of single-walled carbon nanotubes in aqueous solutions of steroid or sugar compounds and their Raman and near-IR spectral properties, Chemistry, 12, 7595–7602, 2006.
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760
Chemicals Commonly Used in Biochemistry and Molecular Biology and Their Properties (Continued) Common Name
Chemical Name
Biotin
Coenzyme R
M.W. 244.31
Properties and Comment Coenzyme function in carboxylation reactions; growth factor; tight binding to avidin used for affinity interactions.
H H N O
O
HO
N H
H H Biotin
Knappe, J., Mechanism of biotin action, Annu. Rev. Biochem. 39, 757–776, 1970; Dunn, M.J., Detection of proteins on blots using the avidin-biotin system, Methods Mol. Biol. 32, 227–232, 1994; Wisdom, G.B., Enzyme and biotin labeling of antibody, Methods Mol. Biol. 32, 433–440, 1994; Wilbur, D.S., Pathare, P.M, Hamlin, D.K. et al., Development of new biotin/streptavidin reagents for pretargeting, Biomol. Eng. 16, 113–118, 1999; Jitrapakdee, S. and Wallace, J.C., The biotin enzyme family: conserved structural motifs and domain rearrangements, Curr. Protein Pept. Sci. 4, 217–229, 2003; Nikolau, B.J., Ohlrogge, J.B., and Wurtels, E.S., Plant biotin-containing carboxylases, Arch. Biochem. Biophys. 414, 211–222, 2003; Fernandez-Mejia, C., Pharmacological effects of biotin, J. Nutri. Biochem. 16, 424–427, 2005; Wilchek, M., Bayer, E.A., and Livnah, O., Essentials of biorecognition: the (strept)avidin-biotin system as a model for protein–protein and protein–ligand interactions, Immunol. Lett. 103, 27–32, 2006; Furuyama, T. and Henikoff, S., Biotin-tag affinity purification of a centromeric nucleosome assembly complex, Cell Cycle 5, 1269–1274, 2006; Streaker, E.D. and Beckett, D., Nonenzymatic biotinylation of a biotin carboxyl carrier protein: unusual reactivity of the physiological target lysine, Protein Sci. 15, 1928–1935, 2006; Raichur, A.M., Voros, J., Textor, M., and Fery, A., Adhesion of polyelectrolyte microcapsules through biotin-streptavidin specific interaction, Biomacromolecules 7, 2331–2336, 2006. For biotin switch assay, see Martinez-Ruiz, A. and Lamas, S., Detection and identification of S-nitrosylated proteins in endothelial cells, Methods Enzymol. 396, 131–139, 2005; Huang, B. and Chen, C., An ascorbate-dependent artifact that interferes with the interpretation of the biotin switch assay, Free Radic. Biol. Med. 41, 562–567, 2006; Gladwin, M.T., Wang, X., and Hogg, N., Methodological vexation about thiol oxidation versus S-nitrosation — a commentary on “An ascorbate-dependent artifact that interferes with the interpretation of the biotinswitch assay,” Free Radic. Biol. Med. 41, 557–561, 2006. Biuret
Imidodicarbonic Diamide
O
103.08
Prepared by heating urea, reaction with cupric ions in base yields red-purple (the biuret reaction); nonprotein nitrogen (NPN) nutritional source.
O
N H
H 2N
NH 2
Jensen, H.L. and Schroder, M., Urea and biuret as nitrogen sources for Rhizobium spp., J. Appl. Bacteriol. 28, 473–478, 1965; Ronca, G., Competitive inhibition of adenosine deaminase by urea, guanidine, biuret, and guanylurea, Biochim. Biophys. Acta 132, 214–216, 1967; Oltjen, R.R., Slyter, L.L., Kozak, A.S., and Williams, E.E., Jr., Evaluation of urea, biuret, urea phosphate, and uric acid as NPN sources for cattle, J. Nutr. 94, 193–202, 1968; Tsai, H.Y. and Weber, S.G., Electrochemical detection of oligopeptides through the precolumn formation of biuret complexes, J. Chromatog. 542, 345–350, 1991; Gawron, A.J. and Lunte, S.M., Optimization of the conditions for biuret complex formation for the determination of peptides by capillary electrophoresis with ultraviolet detection, Clin. Chem. 51, 1411–1419, 2000; Roth, J., O’Leary, D.J., Wade, C.G. et al., Conformational analysis of alkylated biuret and triuret: evidence for helicity and helical inversion in oligoisocyates, Org. Lett. 2, 3063–3066, 2000; Hortin, G.L., and Mellinger, B., Cross-reactivity of amino acids and other compounds in the biuret reaction: interference with urinary peptide measurements, Clin. Chem. 51, 1411–1419, 2005. Boric Acid
HO
o-boric Acid
61.83
Buffer salt, manufacturing; complexes with carbohydrates and other polyhydroxyl compounds; therapeutic use as a topic antibacterial/antifungal agent.
OH B
B(OH)3 + 2H2O
B(OH)4– + H3O+
OH
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761
Chemicals Commonly Used in Biochemistry and Molecular Biology and Their Properties (Continued) Common Name
Chemical Name
M.W.
Properties and Comment
Sciarra, J.J. and Monte Bovi, A.J., Study of the boric acid–glycerin complex. II. Formation of the complex at elevated temperature, J. Pharm. Sci. 51, 238–242, 1962; Walborg, E.F., Jr. and Lantz, R.S., Separation and quantitation of saccharides by ion-exchange chromatography utilizing boric acid–glycerol buffers, Anal. Biochem. 22, 123–133, 1968; Lerch, B. and Stegemann, H., Gel electrophoresis of proteins in borate buffer. Influence of some compounds complexing with boric acid, Anal. Biochem. 29, 76–83, 1969; Walborg, E.F., Jr., Ray, D.B., and Ohrberg, L.E., Ion-exchange chromatography of saccharides: an improved system utilizing boric acid/2,3-butanediol buffers, Anal. Biochem. 29, 433–440, 1969; Chen, F.T. and Sternberg, J.C., Characterization of proteins by capillary electrophoresis in fused-silica columns: review on serum protein anlaysis and application to immunoassays, Electrophoresis 15, 13–21, 1994; Allen, R.C. and Doktycz, M.J., Discontinuous electrophoresis revisited: a review of the process, Appl. Theor. Electrophor. 6, 1–9, 1996; Manoravi, P., Joseph, M., Sivakumar, N., and Balasubramanian, H., Determination of isotopic ratio of boron in boric acid using laser mass spectrometry, Anal. Sci. 21, 1453–1455, 2005; De Muynck, C., Beauprez, J., Soetaert, W., and Vandamme, E.J., Boric acid as a mobile phase additive for high-performance liquid chromatography separation of ribose, arabinose, and ribulose, J. Chromatog. A 1101, 115–121, 2006; Herrmannova, M., Kirvankova, L., Bartos, M., and Vytras, K., Direct simultaneous determination of eight sweeteners in foods by capillary isotachophoresis, J. Sep. Sci. 29, 1132–1137, 2006; Alencar de Queiroz, A.A., Abraham, G.A., Pires Camillo, M.A. et al., Physicochemical and antimicrobial properties of boron-complexed polyglycerol-chitosan dendrimers, J. Biomater. Sci. Polym. Ed. 17, 689–707, 2006; Ringdahl, E.N., Recurrent vulvovaginal candidiasis, Mol. Med. 103, 165–168, 2006. BNPS-Skatole
(2-[2′-nitrophenyl-sulfenyl]3-methyl-3′bromoindolenine
363.23
Tryptophan modification, peptide-bond cleavage; derived from skatole, which is also known as boar taint.
H3C Br S
NO2
N
Boulanger, P., Lemay, P., Blair, G.E., and Russell, W.C., Characterization of adenovirus protein IX, J. Gen. Virol. 44, 783–800, 1979; Russell, J., Kathendler, J., Kowalski, K. et al., The single tryptophan residue of human placental lactogen. Effects of modification and cleavage on biological activity and protein conformation, J. Biol. Chem. 256, 304–307, 1981; Moskaitis, J.E. and Campagnoni, A.T., A comparison of the dodecyl sulfate-induced precipitation of the myelin basic protein with other water-soluble proteins, Neurochem. Res. 11, 299–315, 1986; Mahboub, S., Richard, C., Delacourte, A., and Han, K.K., Applications of chemical cleavage procedures to the peptide mapping of neurofilament triplet protein bands in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Anal. Biochem. 154, 171–182, 1986; Rahali, V. and Gueguen, J., Chemical cleavage of bovine beta-lactoglobulin by BPNS-skatole for preparative purposes: comparative study of hydrolytic procedure and peptide characterization, J. Protein Chem. 18, 1–12, 1999; Swamy, N., Addo, J., Vskokovic, M.R., and Ray, R., Probing the vitamin D sterolbinding pocket of human vitamin D-binding protein with bromoacetate affinity-labeling reagents containing the affinity probe at C-3, C-6, C-11, and C-19 positions of parent vitamin D sterols, Arch. Biochem. Biophys. 373, 471–478, 2000; Celestina, F. and Suryanarayana, T., Biochemical characterization and helix-stabilizing properties of HSNP-C’ from the thermophilic archaeon Sulfolobus acidocaldarius, Biochem. Biophys. Res. Commun. 267, 614–618, 2000; Kibbey, M.M., Jameson, M.J., Eaton, E.M., and Rosenzweig, S.A., Insulinlike growth factor binding protein-2: contributions of the C-terminal domain to insulinlike growth factor-1 binding, Mol. Pharmacol. 69, 833–845, 2006. p-Bromophenacyl Bromide
2-bromo-1-(4-bromophenyl) ethanone; 4-bromophenacyl bromide
277.04
Modification of various residues in proteins: reagent for identification of carboxylic acids; phospholipase A2 inhibitor.
Br
H2C
O
Br
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Chemicals Commonly Used in Biochemistry and Molecular Biology and Their Properties (Continued) Common Name
Chemical Name
M.W.
Properties and Comment
Erlanger, B.F., Vratrsanos, S.M., Wasserman, N., and Cooper, A.G., A chemical investigation of the active center of pepsin, Biochem. Biophys. Res. Commun. 23, 243–245, 1966; Yang, C.C. and King, K., Chemical modification of the histidine residue in basic phospholipase A2 from the venom of Naja nigricollis, Biochim. Biophys. Acta. 614, 373–388, 1980; Darke, P.L., Jarvis, A.A., Deems, R.A., and Dennis, E.A., Further characterization and N-terminal sequence of cobra venom phospholipase A2, Biochim. Biophys. Acta 626, 154–161, 1980; Ackerman, S.K., Matter, L., and Douglas, S.D., Effects of acid proteinase inhibitors on human neutrophil chemotaxis and lysosomal enzyme release. II. Bromophenacyl bromide and 1,2-epoxy-3-(p-nitrophenoxy)propane, Clin. Immunol. Immunopathol. 26, 213–222, 1983; Carine, K. and Hudig, D., Assessment of a role for phospholipase A2 and arachidonic acid metabolism in human lymphocyte natural cytotoxicity, Cell Immunol. 87, 270–283, 1984; Duque, R.E., Fantone, J.C., Kramer, C. et al., Inhibition of neutrophil activation by p-bromophenacyl bromide and its effects on phospholipase A2, Br. J. Pharmacol. 88, 463–472, 1986; Zhukova, A., Gogvadze, G., and Gogvadze, V., p-bromophenacyl bromide prevents cumene hydroperoxide-induced mitochondrial permeability transition by inhibiting pyridine nucleotide oxidation, Redox Rep. 9, 117–121, 2004; Thommesen, L. and Laegreid, A., Distinct differences between TNF receptor 1- and TNR receptor 2-mediated activation of NF-κβ• J. Biochem. Mol. Biol. 38, 281–289, 2005; Yue, H.Y., Fujita, T., and Kumamoto, E., Phospholipase A2 activation by melittin enhances spontaneous glutamatergic excitatory transmission in rat substantia gelatinosa neurons, Neuroscience 135, 485–495, 2005; Costa-Junior, H.M., Hamaty, F.C., de Silva Farias, R. et al., Apoptosis-inducing factor of a cytotoxic T-cell line: involvement of a secretory phospholipase A(2), Cell Tissue Res. 324, 255–266, 2006; Marchi-Salvador, D.P., Fernandes, C.A., Amui, S.F. et al., Crystallization and preliminary X-ray diffraction analysis of a myotoxic Lys49-PLA2 from Bothrops jararacussu venom complexed with p-bromophenacyl bromide, Acta Crystallograph. Sect. F Struct. Biol. Cryst. Commun. 62, 600–603, 2006. Calcium Chloride
CaCl2
110.98
Anhydrous form as drying agent for organic solvents, variety of manufacturing uses; meat quality enhancement; therapeutic use in electrolyte replacement and bone cements; source of calcium ions for biological assays.
Barratt, J.O., Thrombin and calcium chloride in relation to coagulation, Biochem. J. 9, 511–543, 1915; Van der Meer, C., Effect of calcium chloride on choline esterase, Nature 171, 78–79, 1952; Bhat, R. and Ahluwalia, J.C., Effect of calcium chloride on the conformation of proteins. Thermodynamic studies of some model compounds, Int. J. Pept. Protein Res. 30, 145–152, 1987; Furihata, C., Sudo, K., and Matsushima, T., Calcium chloride inhibits stimulation of replicative DNA synthesis by sodium chloride in the pyloric mucosa of rat stomach, Carcinogenesis 10, 2135–2137, 1989; Ishikawa, K., Ueyama, Y., Mano, T. et al., Self-setting barrier membrane for guided tissue regeneration method: initial evaluation of alginate membrane made with sodium alginate and calcium chloride aqueous solutions, J. Biomed. Mater. Res. 47, 111–115, 1999; Vujevic, M., Vidakovic-Cifrek, Z., Tkalec, M. et al., Calcium chloride and calcium bromide aqueous solutions of technical and analytical grade in Lemna bioassay, Chemosphere 41, 1535–1542, 2000; Miyazaki, T., Ohtsuki, C., Kyomoto, M. et al., Bioactive PMMA bone cement prepared by modification with methacryloxypropyltrimethoxysilane and calcium chloride, J. Biomed. Mater. Res. A 67, 1417–1423, 2003; Harris, S.E., Huff-Lonegan, E., Lonergan, S.M. et al., Antioxidant status affects color stability and tenderness of calcium chloride-injected beef, J. Anim. Sci. 79, 666–677, 2001; Behrends, J.M., Goodson, K.J., Koohmaraie, M. et al., Beef customer satisfaction: factors affecting consumer evaluations of calcium chloride-injected top sirloin steaks when given instructions for preparation, J. Anim. Sci. 83, 2869–2875, 2005. Cetyl Pyridinium Chloride
1-hexadecylpyridinium chloride
350.01
Cationic detergent; precipitating agent and staining agent for glycosaminoglycans; antimicrobial agent.
Cl– N+
Laurent, T.C. and Scott, J.E., Molecular weight fractionation of polyanions by cetylpyridinium chloride in salt solutions, Nature 202, 661–662, 1964; Kiss, A., Linss, W., and Geyer, G., CPC-PTA section staining of acid glycans, Acta Histochem. 64, 183–186, 1979; Khan, M.Y. and Newman, S.A., An assay for heparin by decrease in color yield (DECOY) of a protein-dye-binding reaction, Anal. Biochem. 187, 124–128, 1990; Chardin, H., Septier, D., and Goldberg, M., Visualization of glycosaminoglycans in rat incisor predentin and dentin with cetylpyridinium chloride-glutaraldehyde as fixative, J. Histochem. Cytochem. 38, 885–894, 1990; Chardin, H., Gokani, J.P., Septier, D. et al., Structural variations of different oral basement membranes revealed by cationic dyes and detergent added to aldehyde fixative solution, Histochem. J. 24, 375–382, 1992; Agren, U.M., Tammi, R., and Tammi, M., A dot-blot assay of metabolically radiolabeled hyaluronan, Anal. Biochem. 217, 311–315, 1994; Maccari, F. and Volpi, N., Glycosaminoglycan blotting on nitrocellulose membranes treated with cetylpyridinium chloride afer agarose-gel electrophoretic separation, Electrophoresis 23, 3270–3277, 2002; Maccari, F. and Volpi, N., Direct and specific recognition of glycosaminoglycans by antibodies after their separation by agarose gel electrophoresis and blotting on cetylpyridinium chloride-treated nitrocellulose membranes, Electrophoresis 24, 1347–1352, 2003.
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Chemicals Commonly Used in Biochemistry and Molecular Biology and their Properties
763
Chemicals Commonly Used in Biochemistry and Molecular Biology and Their Properties (Continued) Common Name CHAPS
CH3 OH H3C CH3 CH3
H
Chemical Name
M.W.
3-[(3-cholamidopropyl)dimethylammonio]-1propanesulfonate
614.89
Properties and Comment Detergent, solubilizing agent; extensive use for the solubilization of membrane proteins.
O O–
O
N H
S
O
N+ H
H3C
CH3
OH H Hjelmeland, L.M., A nondenaturing zwitterionic detergent for membrane biochemistry: design and synthesis, Proc. Natl. Acad. Sci. USA 77, 6368–6370, 1980; Giradot, J.M. and Johnson, B.C., A new detergent for the solubilization of the vitamin K–dependent carboxylation system from liver microsomes: comparison with triton X-100, Anal. Biochem. 121, 315–320, 1982; Liscia, D.S., Alhadi, T., and Vonderhaar, B.K., Solubilization of active prolactin receptors by a nondenaturing zwitterionic detergent, J. Biol. Chem. 257, 9401–9405, 1982; Womack, M.D., Kendall, D.A., and MacDonald, R.C., Detergent effects on enzyme activity and solubilization of lipid bilayer membranes, Biochim. Biophys. Acta 733, 210–215, 1983; Klaerke, D.A. and Jorgensen, P.L., Role of Ca2+-activated K+ channel in regulation of NaCl reabsorption in thick ascending limb of Henle’s loop, Comp. Biochem. Physiol. A 90, 757–765, 1988; Kuriyama, K., Nakayasu, H., Mizutani, H. et al., Cerebral GABAB receptor: proposed mechanisms of action and purification procedures, Neurochem. Res. 18, 377–383, 1993; Koumanov, K.S., Wolf, C., and Quinn, P.J., Lipid composition of membrane domains, Subcell. Biochem. 37, 153–163, 2004. HO
Chloroform
Trichloromethane
177.38
Used for extraction of lipids, usually in combination with methanol.
Cl Cl
C
Cl
H Stevan, M.A. and Lyman, R.L., Investigations on extraction of rat plasma phospholipids, Proc. Soc. Exp. Biol. Med. 114, 16–20, 1963; Wells, M.A. and Dittmer, J.C., A microanalytical technique for the quantitative determination of twenty-four classes of brain lipids, Biochemistry 5, 3405–3418, 1966; Colacicco, G. and Rapaport, M.M., A simplified preparation of phosphatidyl inositol, J. Lipid. Res. 8, 513–515, 1967; Curtis, P.J., Solubility of mitochondrial membrane proteins in acidic organic solvents, Biochim. Biophys. Acta 183, 239–241, 1969; Privett, O.S., Dougherty, K.A., and Castell, J.D., Quantitative analysis of lipid classes, Am. J. Clin. Nutr. 24, 1265–1275, 1971; Claire, M., Jacotot, B., and Robert, L., Characterization of lipids associated with macromolecules of the intercellular matrix of human aorta, Connect. Tissue Res. 4, 61–71, 1976; St. John, L.C. and Bell, F.P., Extraction and fractionation of lipids from biological tissues, cells, organelles, and fluids, Biotechniques 7, 476–481, 1989; Dean, N.M. and Beaven, M.A., Methods for the analysis of inositol phosphates, Anal. Biochem. 183, 199–209, 1989; Singh, A.K. and Jiang, Y., Quantitative chromatographic analysis of inositol phospholipids and related compounds, J. Chromatog. B Biomed. Appl. 671, 255–280, 1995.
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764
Chemicals Commonly Used in Biochemistry and Molecular Biology and Their Properties (Continued) Common Name
Chemical Name
Cholesterol
M.W. 386.66
Properties and Comment The most common sterol in man and other higher animals. Cholesterol is essential for the synthesis of a variety of compounds including estrogens and vitamin D; also membrane component.
CH3 H3C CH3
CH3 H
CH3
H
H HO Cholesterol
Doree, C., The occurrence and distribution of cholesterol and allied bodies in the animal kingdom, Biochem. J. 4, 72–106, 1909; Heilbron, I.M., Kamm, E.D., and Morton, R.A., The absorption spectrum of cholesterol and its biological significance with reference to vitamin D. Part I: Preliminary observations, Biochem. J. 21, 78–85, 1927; Cook, R.P., Ed., Cholesterol: Chemistry, Biochemistry, and Pathology, Academic Press, New York, 1958; Vahouny, G.V. and Treadwell, C.R., Enzymatic synthesis and hydrolysis of cholesterol esters, Methods Biochem. Anal. 16, 219–272, 1968; Heftmann, E., Steroid Biochemistry, Academic Press, New York, 1970; Nestel, P.J., Cholesterol turnover in man, Adv. Lipid Res. 8, 1–39, 1970; Dennick, R.G., The intracellular organization of cholesterol biosynthesis. A review, Steroids Lipids Res. 3, 236–256, 1972; J. Polonovski, Ed., Cholesterol Metabolism and Lipolytic Enzymes, Masson Publications, New York, 1977; Gibbons, G.F., Mitrooulos, K.A., and Myant, N.B., Biochemistry of Cholesterol, Elsevier, Amsterdam, 1982; Bittman, R., Cholesterol: Its Functions and Metabolism in Biology and Medicine, Plenum Press, New York, 1997; Oram, J.P. and Heinecke, J.W., ATP-binding cassette transporter A1: a cell cholesterol exporter that protects against cardiovascular disease, Physiol. Rev. 85, 1343–1372, 2005; Holtta-Vuori, M. and Ikonen, E., Endosomal cholesterol traffic: vesicular and nonvesicular mechanisms meet, Biochem. Soc. Trans. 34, 392–394, 2006; Cuchel, M. and Rader, D.J., Macrophage reverse cholesterol transport: key to the regression of atherosclerosis? Circulation 113, 2548–2555, 2006. Cholic Acid
408.57
Component of bile; detergent.
O OH
H3C OH CH3
CH3
H
H
H HO
OH H
Schreiber, A.J. and Simon, F.R., Overview of clinical aspects of bile salt physiology, J. Pediatr. Gastroenterol. Nutr. 2, 337–345, 1983; Chiang, J.Y., Regulation of bile acid synthesis, Front. Biosci. 3, dl176–dl193, 1998; Cybulsky, M.I., Lichtman, A.H., Hajra, L., and Iiyama, K., Leukocyte adhesion molecules in atherogenesis, Clin. Chim. Acta 286, 207–218, 1999.
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765
Chemicals Commonly Used in Biochemistry and Molecular Biology and Their Properties (Continued) Common Name
Chemical Name
Citraconic Anhydride
Methylmaleic Anhydride
M.W. 112.1
Properties and Comment Reversible modification of amino groups.
CH3
O
O
O
Dixon, H.B. and Perham, R.N., Reversible blocking of amino groups with citraconic anhydride, Biochem. J. 109, 312–314, 1968; Gibbons, I. and Perham, R.N., The reaction of aldolase with 2-methylmaleic anhydride, Biochem. J. 116, 843–849, 1970; Yankeelov, J.A., Jr. and Acree, D., Methylmaleic anhydride as a reversible blocking agent during specific arginine modification, Biochem. Biophys. Res. Commun. 42, 886–891, 1971; Takahashi, K., Specific modification of arginine residues in proteins with ninhydrin, J. Biochem. 80, 1173–1176, 1976; Brinegar, A.C. and Kinsella, J.E., Reversible modification of lysine in soybean proteins, using citraconic anhydride: characterization of physical and chemical changes in soy protein isolate, the 7S globulin, and lipoxygenase, J. Agric. Food Chem. 28, 818–824, 1980; Shetty, J.K. and Kensella, J.F., Ready separation of proteins from nucleoprotein complexes by reversible modification of lysine residues, Biochem. J. 191, 269–272, 1980; Yang, H. and Frey, P.A., Dimeric cluster with a single reactive amino group, Biochemistry 23, 3863–3868, 1984; Bindels, J.G., Misdom, L.W., and Hoenders, H.J., The reaction of citraconic anhydride with bovine alpha-crystallin lysine residues. Surface probing and dissociation-reassociation studies, Biochim. Biophys. Acta 828, 255–260, 1985; Al jamal, J.A., Characterization of different reactive lysines in bovine heart mitochondrial porin, Biol. Chem. 383, 1967–1970, 2002; Kadlik, V., Strohalm, M., and Kodicek, M., Citraconylation — a simple method for high protein sequence coverage in MALDI-TOF mass spectrometry, Biochem. Biophys. Res. Commun. 305, 1091–1093, 2003. Coomassie Brilliant Blue G-250
CI Acid Blue 90
H3C CH2
H2C
Most often used for the colorimetric determination of protein.
CH3
N+
N
C H2
C H2
HO3S
854
SO3H
CH3
CH3
NH H2 C H3C
O
Bradford, M.M., A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding Anal. Biochem. 72, 248–254, 1976; Saleemuddin, M., Ahmad, H., and Husain, A., A simple, rapid, and sensitive procedure for the assay of endoproteases using Coomassie Brilliant Blue G-250, Anal. Biochem. 105, 202–206, 1980; van Wilgenburg, M.G., Werkman, E.M., van Gorkom, W.H., and Soons, J.B., Criticism of the use of Coomassie Brilliant Blue G-250 for the quantitative determination of proteins, J.Clin. Chem.Clin. Biochem. 19, 301–304, 1981; Mattoo, R.L., Ishaq, M., and Saleemuddin, M., Protein assay by Coomassie Brilliant Blue G-250-binding method is unsuitable for plant tissues rich in phenols and phenolases, Anal. Biochem. 163, 376–384, 1987; Lott, J.A., Stephan, V.A., and Pritchard, K.A., Jr., Evaluation of the Coomassie Brilliant Blue G-250 method for urinary proteins, Clin. Chem. 29, 1946–1950, 1983; Fanger, B.O., Adaptation of the Bradford protein assay to membrane-bound proteins by solubilizing in glucopyranoside detergents, Anal. Biochem. 162, 11–17, 1987; Marshall, T. and Williams, K.M., Recovery of proteins by Coomassie Brilliant Blue precipitation prior to electrophoresis, Electrophoresis 13, 887–888, 1992; Sapan, C.V., Lundblad, R.L., and Price, N.C., Colorimetric protein assay techniques, Biotechnol. Appl. Biochem. 29, 99–108, 1999.
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766
Chemicals Commonly Used in Biochemistry and Molecular Biology and Their Properties (Continued) Common Name
Chemical Name
Coomassie Brilliant Blue R-250
CI Acid Blue 83
H3C
826
Properties and Comment Most often used for the detection of proteins on solid matrices such as polyacrylamide gels.
CH3
CH2
H2C N+
N C H2
HO3S
M.W.
C H2
SO3H
NH H2 C H3C
O
Vesterberg, O., Hansen, L., and Sjosten, A., Staining of proteins after isoelectric focusing in gels by a new procedure, Biochim. Biophys. Acta 491, 160–166, 1977; Micko, S. and Schlaepfer, W.W., Metachromasy of peripheral nerve collagen on polyacrylamide gels stained with Coomassie Brilliant Blue R-250, Anal. Biochem. 88, 566–572, 1978; Osset, M., Pinol, M., Fallon, M.J. et al., Interference of the carbohydrate moiety in Coomassie Brilliant Blue R-250 protein staining, Electrophoresis 10, 271–273, 1989; Pryor, J.L., Xu, W., and Hamilton, D.W., Immunodetection after complete destaining of Coomassie blue-stained proteins on immobilon-PVDF, Anal. Biochem. 202, 100–104, 1992; Metkar, S.S., Mahajan, S.K., and Sainis, J.K., Modified procedure for nonspecific protein staining on nitrocellulose paper using Coomassie Brilliant Blue R-250, Anal. Biochem. 227, 389–391, 1995; Kundu, S.K., Robey, W.G., Nabors, P. et al., Purification of commercial Coomassie Brilliant Blue R-250 and characterization of the chromogenic fractions, Anal. Biochem. 235, 134–140, 1996; Choi, J.K., Yoon, S.H., Hong, H.Y. et al., A modified Coomassie blue staining of proteins in polyacrylamide gels with Bismark brown R, Anal. Biochem. 236, 82–84, 1996; Moritz, R.L., Eddes, J.S., Reid, G.E., and Simpson, R.J., S- pyridylethylation of intact polyacrylamide gels and in situ digestion of electrophoretically separated proteins: a rapid mass spectrometric method for identifying cysteine-containing peptides, Electrophoresis 17, 907–917, 1996; Choi, J.K. and Yoo, G.S., Fast protein staining in sodium dodecyl sulfate polyacrylamide gel using counter ion-dyes, Coomassie Brilliant Blue R-250, and neutral red, Arch. Pharm. Res. 25, 704–708, 2002; Bonar, E., Dubin, A., Bierczynska-Krzysik, A. et al., Identification of major cellular proteins synthesized in response to interleukin-1 and interleukin-6 in human hepatoma HepG2 cells, Cytokine 33, 111–117, 2006. Cy 2
714
O
Fluorescent label used in proteomics and gene expression; use for internal standard.
O N
N+
O
O N O O
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767
Chemicals Commonly Used in Biochemistry and Molecular Biology and Their Properties (Continued) Common Name
Chemical Name
M.W.
Properties and Comment
Tonge, R., Shaw, J., Middleton, B. et al., Validation and development of fluorescence two-dimensional differential gel electrophoresis proteomics technology, Proteomics 1, 377–396, 2001; Chan, H.L., Gharbi, S., Gaffney, P.R. et al., Proteomic analysis of redox- and ErbB2-dependent changes in mammary luminal epithelial cells using cysteine- and lysine-labeling two-dimensional difference gel electrophoresis, Proteomics 5, 2908–2926, 2005; Misek, D.E., Kuick, R., Wang, H. et al., A wide range of protein isoforms in serum and plasma uncovered by a quantitative intact protein analysis system, Proteomics 5, 3343–3352, 2005; Doutette, P., Navet, R., Gerkens, P. et al., Steatosis-induced proteomic changes in liver mitochondria evidenced by two-dimensional differential in-gel electrophoresis, J. Proteome Res. 4, 2024–2031, 2005. Cy 3
911.0
Fluorescent label used in proteomics and gene expression; in combination with Cy 5 is used for FRET-based assays.
OH O
O S
CH3
N
CH3
CH3
OH
O S
H3C
O
N+ O
N
O O
O
O O
O N
O Brismar, H. and Ulfake, B., Fluorescence lifetime measurements in confocal microscopy of neurons labeled with multiple fluorophores, Nat. Biotechnol. 15, 373–377, 1997; Strohmaier, A.R., Porwol, T., Acker, H., and Spiess, E., Tomography of cells by confocal laser scanning microscopy and computer-assisted three-dimensional image reconstruction: localization of cathepsin B in tumor cells penetrating collagen gels in vitro, J. Histochem. Cytochem. 45, 975–983, 1997; Alexandre, I., Hamels, S., Dufour, S. et al., Colorimetric silver detection of DNA microarrays, Anal. Biochem. 295, 1–8, 2001; Shaw, J., Rowlinson, R., Nickson, J. et al., Evaluation of saturation labeling two-dimensional difference gel electrophoresis fluorescent dyes, Proteomics 3, 1181–1195, 2003.
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768
Chemicals Commonly Used in Biochemistry and Molecular Biology and Their Properties (Continued) Common Name
Chemical Name
M.W.
Cy 5
937.1
Properties and Comment Fluorescent label used in proteomics and gene expression; also used in histochemistry.
OH O
O S
CH3
CH3
N
OH
O
CH3
S
H3C
N+
O
N
O
O O
O O O
O N
O Uchihara, T., Nakamura, A., Nagaoka, U. et al., Dual enhancement of double immunofluorescent signals by CARD: participation of ubiquitin during formation of neurofibrillary tangles, Histochem. Cell Biol. 114, 447–451, 2000; Duthie, R.S., Kalve, I.M., Samols, S.B. et al., Novel cyanine dye-based dideoxynucleoside triphosphates for DNA sequencing, Bioconjug. Chem. 13, 699–706, 2002; Graves, E.E., Yessayan., D., Turner, G. et al., Validation of in vivo fluorochrome concentrations measured using fluorescence molecular tomography, J. Biomed. Opt. 10, 44019, 2005; Lapeyre, M., Leprince, J., Massonneau, M. et al., Aryldithioethyloxycarbonyl (Ardec): a new family of amine-protecting groups removable under mild reducing conditions and their applications to peptide synthesis, Chemistry 12, 3655–3671, 2006; Tang, X., Morris, S.L., Langone, J.J., and Bockstahler, L.E., Simple and effective method for generating single-stranded DNA targets and probes, Biotechniques 40, 759–763, 2006. 4-HCCA; Cinnamate 189.2 Used as matrix substance for MALDI; α-Cyano-4-hydroxycinnamic Acid transport inhibitor and enzyme inhibitor. O H C
C C
OH
C HO
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N
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769
Chemicals Commonly Used in Biochemistry and Molecular Biology and Their Properties (Continued) Common Name
Chemical Name
M.W.
Properties and Comment
Gobom, J., Schuerenberg, M., Mueller, M. et al., α-cyano-4-hydroxycinnamic acid affinity sample preparation. A protocol for MALDI-MS peptide analysis in proteomics, Anal. Chem. 73, 434–438, 2001; Zhu, X. and Papayannopoulos, I.A., Improvement in the detection of low concentration protein digests on a MALDI TOF/TOF workstation by reducing α-cyano-4-hydroxycinnamic acid adduct ions, J. Biomol. Tech. 14, 298–307, 2003; Neubert, H., Halket, J.M., Fernandez Ocana, M., and Patel, R.K., MALDI post-source decay and LIFT-TOF/TOF investigation of α-cyano-4hydroxycinnamic acid cluster interferences, J. Am. Soc. Mass Spectrom. 15, 336–343, 2004; Kobayashi, T., Kawai, H., Suzuki, T. et al., Improved sensitivity for insulin in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry by premixing α-cyano-4-hydroxycinnamic acid with transferrin, Rapid Commun. Mass Spectrom. 18, 1156–1160, 2004; Pshenichnyuk, S.A. and Asfandiarov, N.L., The role of free electrons in MALDI: electron capture by molecules of α-cyano-4-hydroxycinnamic acid, Eur. J. Mass Spectrom. 10, 477–486, 2004; Bogan, M.J., Bakhoum, S.F., and Agnes, G.R., Promotion of α-cyano-4-hydroxycinnamic acid and peptide cocrystallization within levitated droplets with net charge, J. Am. Soc. Mass Spectrom. 16, 254–262, 2005. As enzyme inhibitor: Clarke, P.D., Clift, D.L., Dooledeniya, M. et al., Effects of α-cyano-4-hydroxycinnamic acid on fatigue and recovery of isolated mouse muscle, J. Muscle Res. Cell Motil. 16, 611–617, 1995; Del Prete, E., Lutz, T.A., and Scharrer, E., Inhibition of glucose oxidation by α-cyano-4-hydroxycinnamic acid stimulates feeding in rats, Physiol. Behav. 80, 489–498, 2004; Briski, K.P. and Patil, G.D., Induction of Fox immunoreactivity labeling in rat forebrain metabolic loci by caudal fourth ventricular infusion of the monocarboxylate transporter inhibitor, α-cyano-4-hydroxycinnamic acid, Neuroendocrinology 82, 49–57, 2005. Cyanogen
C2N2; Ethanedinitrile
C
53.03
Protein crosslinking at salt bridges.
N
N
C
Ghenbot, G., Emge, T., and Day, R.A., Identification of the sites of modification of bovine carbonic anhydrase II (BCA II) by the salt bridge reagent cyanogen, C2N2, Biochim. Biophys. Acta 1161, 59–65, 1993; Karagozler, A.A., Ghenbot, G., and Day, R.A., Cyanogen as a selective probe for carbonic anhydrase hydrolase, Biopolymers 33, 687–692, 1993; Winters, M.S. and Day, R.A., Identification of amino acid residues participating in intermolecular salt bridges between self-associating proteins, Anal. Biochem. 309, 48–59, 2002; Winters, M.S. and Day, R.A., Detecting protein– protein interactions in the intact cell of Bacillus subtilis(ATCC 6633), J. Bacteriol. 185, 4268–4275, 2003. Cyanogen Bromide
Br
CNBr; Bromide Cyanide
105.9
Protein modification; cleavage of peptide bonds; coupled nucleophiles to polyhydroxyl matrices; environmental toxicon derived from monobromamine and cyanide.
N C
Hofmann, T., The purification and properties of fragments of trypsinogen obtained by cyanogen bromide cleavage, Biochemistry 3, 356–364, 1964; Chu, R.C. and Yasunobu, K.T., The reaction of cyanogen bromide and N-bromosuccinimide with some cytochromes C, Biochim. Biophys. Acta 89, 148–149, 1964; Inglis, A.S. and Edman, P., Mechanism of cyanogen bromide reaction with methionine in peptides and proteins. I. Formation of imidate and methyl thiocyanate, Anal. Biochem. 37, 73–80, 1970; Kagedal, L. and Akerstrom, S., Binding of covalent proteins to polysaccharides by cyanogen bromide and organic cyanates. I. Preparation of soluble glycine-, insulin- and ampicillin-dextran, Acta Chem. Scand. 25, 1855–1899, 1971; Sipe, J.D. and Schaefer, F.V., Preparation of solid-phase immunosorbents by coupling human serum proteins to cyanogen bromide–activated agarose, Appl. Microbiol. 25, 880–884, 1973; March, S.C., Parikh, I., and Cuatrecasas, P., A simplified method for cyanogen bromide activation of agarose for affinity chromatography, Anal. Biochem. 60, 149–152, 1974; Boulware, D.W., Goldsworthy, P.D., Nardella, F.A., and Mannik, M., Cyanogen bromide cleaves Fc fragments of pooled human IgG at both methionine and tryptophan residues, Mol. Immunol. 22, 1317–1322, 1985; Jaggi, K.S. and Gangal, S.V., Monitoring of active groups of cyanogen bromide-activated paper discs used as allergosorbent, Int. Arch. Allergy Appl. Immunol. 89, 311–313, 1989; Villa, S., De Fazio, G., and Canosi, U., Cyanogen bromide cleavage at methionine residues of polypeptides containing disulfide bonds, Anal. Biochem. 177, 161–164, 1989; Luo, K.X., Hurley, T.R., and Sefton, B.M., Cyanogen bromide cleavage and proteolytic peptide mapping of proteins immobilized to membranes, Methods Enzymol. 201, 149–152, 1991; Jennissen, H.P., Cyanogen bromide and tresyl chloride chemistry revisited: the special reactivity of agarose as a chromatographic and biomaterial support for immobilizing novel chemical groups, J. Mol. Recognit. 8, 116–124, 1995; Kaiser, R. and Metzka, L., Enhancement of cyanogen bromide cleavage yields for methionyl-serine and methionyl-threonine peptide bonds, Anal. Biochem. 266, 1–8, 1999; Kraft, P., Mills, J., and Dratz, E., Mass spectrometric analysis of cyanogen bromide fragments of integral membrane proteins at the picomole level: application to rhodopsin, Anal. Biochem. 292, 76–86, 2001; Kuhn, K., Thompson, A., Prinz, T. et al., Isolation of N-terminal protein sequence tags from cyanogen bromide-cleaved proteins as a novel approach to investigate hydrophobic proteins, J. Proteome Res. 2, 598–609, 2003; Macmillan, D. and Arham, L., Cyanogen bromide cleavage generates fragments suitable for expressed protein and glycoprotein ligation, J. Am. Chem. Soc. 126, 9530–9531, 2004; Lei, H., Minear, R.A., and Marinas, B.J., Cyanogen bromide formation from the reactions of monobromamine and dibromamine with cyanide ions, Environ. Sci. Technol. 40, 2559–2564, 2006.
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Chemicals Commonly Used in Biochemistry and Molecular Biology and Their Properties (Continued) Common Name
Chemical Name
Cyanuric Chloride
2,4,6-trichloro-1,3,5-triazine
M.W. 184.41
Properties and Comment Coupling of carbohydrates to proteins; more recently for coupling of nucleic acid to microarray platforms.
Cl
N
Cl
N
Cl
N
Gray, B.M., ELISA methodology for polysaccharide antigens: protein coupling of polysaccharides for adsorption to plastic tubes, J. Immunol. Methods 28, 187–192, 1979: Horak, D., Rittich, B., Safar, J. et al., Properties of RNase A immobilized on magnetic poly(2-hydroxyethyl methacrylate) microspheres, Biotechnol. Prog. 17, 447–452, 2001; Lee, P.H., Sawan, S.P., Modrusan, Z. et al., An efficient binding chemistry for glass polynucleotide microarrays, Bioconjug. Chem. 13, 97–103, 2002; Steinberg, G., Stromsborg, K., Thomas, L. et al., Strategies for covalent attachment of DNA to beads, Biopolymers 73, 597–605, 2004; Abuknesha, R.A., Luk, C.Y., Griffith, H.H. et al., Efficient labeling of antibodies with horseradish peroxidase using cyanuric chloride, J. Immunol. Methods 306, 211–217, 2005. 1,2-Cyclohexylenedinitrilotetraacetic acid
COOH
CDTA
Chelating agent suggested to have specificity for manganese ions; weaker for other metal ions such as ferric.
COOH COOH N N
COOH
Tandon, S.K. and Singh, J., Removal of manganese by chelating agents from brain and liver of manganese, Toxicology 5, 237–241, 1975; Hazell, A.S., Normandin, L., Norenberg, M.D., Kennedy, G., and Yi, J.H., Alzheimer type II astrocyte changes following sub-acute exposure to manganese, Neurosci. Lett., 396, 167–171, 2006; Hassler, C.S. and Twiss, M.R., Bioavailability of iron sensed by a phytoplanktonic Fe-bioreporter, Environ. Sci. Tech. 40, 2544–2551, 2006. Dansyl Chloride
5-(dimethylamino)-1naphthalenesulfonyl chloride
269.8
Fluorescent label for proteins; amino acid analysis.
Cl O
S
O
N H3C
CH3
Hill, R.D. and Laing, R.R., Specific reaction of dansyl chloride with one lysine residue in rennin, Biochim. Biophys. Acta 132, 188–190, 1967; Chen, R.F., Fluorescent protein-dye conjugates. I. Heterogeneity of sites on serum albumin labeled by dansyl chloride, Arch. Biochem. Biophys. 128, 163–175, 1968; Chen, R.F., Dansyl-labeled protein modified with dansyl chloride: activity effects and fluorescence properties, Anal. Biochem. 25, 412–416, 1968; Brown, C.S. and Cunningham, L.W., Reaction of reactive sulfydryl groups of creatine kinase with dansyl chloride, Biochemistry 9, 3878–3885, 1970; Hsieh, W.T. and Matthews, K.S., Lactose repressor protein modified with dansyl chloride: activity effects and fluorescence properties, Biochemistry 34, 3043–3049, 1985;
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Chemicals Commonly Used in Biochemistry and Molecular Biology and Their Properties (Continued) Common Name
Chemical Name
M.W.
Properties and Comment
Scouten, W.H., van den Tweel, W., Kranenburg, H., and Dekker, M., Colored sulfonyl chloride as an activated agent for hydroxylic matrices, Methods Enzymol. 135, 79–84, 1987; Martin, M.A., Lin, B., Del Castillo, B., The use of fluorescent probes in pharmaceutical analysis, J. Pharm. Biomed. Anal. 6, 573–583, 1988; Walker, J.M., The dansyl method for identifying N-terminal amino acids, Methods Mol. Biol. 32, 321–328, 1994; Walker, J.M., The dansyl-Edman method for peptide sequencing, Methods Mol. Biol. 32, 329–334, 1994; Pin, S. and Royer, C.A., High-pressure fluorescence methods for observing subunit dissociation in hemoglobin, Methods Enzymol. 323, 42–55, 1994; Rangarajan, B., Coons, L.S., and Scarnton, A.B., Characterization of hydrogels using luminescence spectroscopy, Biomaterials 17, 649–661, 1996; Kang, X., Xiao, J., Huang, X., and Gu, X., Optimization of dansyl derivatization and chromatographic conditions in the determination of neuroactive amino acids of biological samples, Clin. Chim. Acta 366, 352–356, 2006. DCC
N,N ′-dicyclohexylcarbodiimide
N
C
206.33
Activates carboxyl groups to react with hydroxyl groups to form esters and with amines to form an amide bond; used to modify ion-transporting ATPases. Lack of water solubility has presented challenges.
N
Chau, A.S. and Terry, K., Analysis of pesticides by chemical derivatization. I. A new procedure for the formation of 2-chloroethyl esters of ten herbicidal acids, J. Assoc. Off. Anal. Chem. 58, 1294–1301, 1975; Patel, L. and Kaback, H.R., The role of the carbodiimide-reactive component of the adenosine-5′-triphosphatase complex in the proton permeability of Escherichia coli membrane vesicles, Biochemistry 15, 2741–2746, 1976; Esch, F.S., Bohlen, P., Otsuka, A.S. et al., Inactivation of the bovine mitochondrial F1-ATPase with dicyclohexyl[14C]carbodiimide leads to the modification of a specific glutamic acid residue in the beta subunit, J. Biol. Chem. 256, 9084–9089, 1981; Hsu, C.M. and Rosen, B.P., Characterization of the catalytic subunit of an anion pump, J. Biol. Chem. 264, 17349–17354, 1989; Gurdag, S., Khandare, J., Stapels, S. et al., Activity of dendrimer-methotrexate conjugates on methotrexate-sensitive and -resistant cell lines, Bioconjug. Chem. 17, 275–283, 2006; Vgenopoulou, I., Gemperli, A.C., and Steuber, J., Specific modification of a Na+ binding site in NADH: quinone oxidoreductase from Klebsiella pneumoniae with dicyclohexylcarbodiimide, J. Bacteriol. 188, 3264–3272, 2006; Ferguson, S.A., Keis, S., and Cook, G.M., Biochemical and molecular characterization of a Na+-translocating F1Fo-ATPase from the thermophilic bacterium Clostridium paradoxum, J. Bacteriol. 188, 5045–5054, 2006. Deoxycholic Acid
Desoxycholic Acid
392.57
Detergent, nanoparticles.
O OH
H3C OH CH3
CH3
H
H
H HO H Akare, S. and Martinez, J.D., Bile acid-induced hydrophobicity-dependent membrane alterations, Biochim. Biophys. Acta 1735, 59–67, 2005; Chae, S.Y., Son, S., Lee, M. et al., Deoxycholic acid-conjugated chitosan oligosaccharide nanoparticles for efficient gene carrier, J. Control. Release 109, 330–344, 2005; Dall’Agnol, M., Bernstein, C., Bernstein, H. et al., Identification of S-nitrosylated proteins after chronic exposure of colon epithelial cells to deoxycholate, Proteomics 6, 1654–1662, 2006; Dotis, J., Simitsopoulou, M., Dalakiouridou, M. et al. Effects of lipid formulations of amphotericin B on activity of human monocytes against Aspirgillus fumigatus, Antimicrob. Agents Chemother. 128, 3490–3491, 2006; Darragh, J., Hunter, M., Pohler, E. et al., The calcium-binding domain of the stress protein SEP53 is required for survival in response to deoxycholic acidmediated injury, FEBS J. 273, 1930–1947, 2006.
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Chemicals Commonly Used in Biochemistry and Molecular Biology and Their Properties (Continued) Common Name
Chemical Name
Deuterium Oxide D2O
“Heavy Water”
M.W. 20.03
Properties and Comment Structural studies in proteins, enzyme kinetics; in vivo studies of metabolic flux.
Cohen, A.H., Wilkinson, R.R., and Fisher, H.F., Location of deuterium oxide solvent isotope effects in the glutamate dehydrogenase reaction, J. Biol. Chem. 250, 5343–5246, 1975; Rosenberry, T.L., Catalysis by acetylcholinesterase: evidence that the rate-limiting step for acylation with certain substrates precedes general acid-base catalysis, Proc. Natl. Acad. Sci. USA 72, 3834–3838, 1975; Viggiano, G., Ho, N.T., and Ho, C., Proton nuclear magnetic resonance and biochemical studies of oxygenation of human adult hemoglobin in deuterium oxide, Biochemistry 18, 5238–5247, 1979; Bonnete, F., Madern, D., and Zaccai, G., Stability against denaturation mechanisms in halophilic malate dehydrogenase “adapt” to solvent conditions, J. Mol. Biol. 244, 436–447, 1994; Thompson, J.F., Bush, K.J., and Nance, S.L., Pancreatic lipase activity in deuterium oxide, Proc. Soc. Exp. Biol. Med. 122, 502–505, 1996; Dufner, D. and Previs, S.F., Measuring in vivo metabolism using heavy water, Curr. Opin. Clin. Nutr. Metab. Care 6, 511–517, 2003; O’Donnell, A.H., Yao, X., and Byers, L.D., Solvent isotope effects on alpha-glucosidase, Biochem. Biophys. Acta 1703, 63–67, 2004; Hellerstein, M.K. and Murphy, E., Stable isotope-mass spectrometric measurements of molecular fluxes in vivo: emerging applications in drug development, Curr. Opin. Mol. Ther. 6, 249–264, 2004; Mazon, H., Marcillat, O., Forest, E., and Vial, C., Local dynamics measured by hydrogen/ deuterium exchange and mass spectrometry of the creatine kinase digested by two proteases, Biochimie 87, 1101–1110, 2005; Carmieli, R., Papo, N., Zimmerman, H. et al., Utilizing ESEEM spectrscopy to locate the position of specific regions of membrane-active peptides within model membranes, Biophys. J. 90, 492–505, 2006. DFP
Diisopropylphosphorofluoridate; Isofluorophate
184.15
Classic cholinesterase inhibitor; inhibitor of serine proteases, some nonspecific reaction tyrosine.
CH3
O
H3C O H3C
P
O
F CH3
Baker, B.R., Factors in the design of active-site-directed irreversible inhibitors, J. Pharm. Sci. 53, 347–364, 1964; Dixon, G.H. and Schachter, H., The chemical modification of chymotrypsin, Can. J. Biochem. Physiol. 42, 695–714, 1964; Singer, S.J., Covalent labeling active site, Adv. Protein Chem. 22, 1–54, 1967; Kassell, B. and Kay, J., Zymogens of proteolytic enzymes, Science 180, 1022–1027, 1973; Fujino, T., Watanabe, K., Beppu, M. et al., Identification of oxidized protein hydrolase of human erythrocytes as acylpeptide hydrolase, Biochim. Biophys. Acta 1478, 102–112, 2000; Manco, G., Camardello, L., Febbraio, F. et al., Homology modeling and identification of serine 160 as nucleophile as the active site in a thermostable carboxylesterase from the archeon Archaeoglobus fulgidus, Protein Eng. 13, 197–200, 2000; Gopal, S., Rastogi, V., Ashman, W., and Mulbry, W., Mutagenesis of organophosphorous hydrolase to enhance hydrolysis of the nerve agent VX, Biochem. Biophys. Res. Commun. 279, 516–519, 2000; Yeung, D.T., Lenz, D.E., and Cerasoli, D.M., Analysis of active-site amino acid residues of human serum paraoxanse using competitive substrates, FEBS J. 272, 2225–2230, 2005; D’Souza, C.A., Wood, D.D., She, Y.M., and Moscarello, M.A., Autocatalytic cleavage of myelin basic protein: an alternative to molecular mimicry, Biochemistry 44, 12905–12913, 2005. Dichloromethane
Methylene Chloride
84.9
Lipid solvent; isolation of sterols, frequently used in combination with methanol.
H2 C Cl
Cl
Bouillon, R., Kerkhove, P.V., and De Moor, P., Measurement of 25-hydroxyvitamin D3 in serum, Clin. Chem. 22, 364–368, 1976; Redhwi, A.A., Anderson, D.C., and Smith, G.N., A simple method for the isolation of vitamin D metabolites from plasma extracts, Steroids 39, 149–154, 1982; Scholtz, R., Wackett, L.P., Egli, C. et al., Dichloromethane dehalogenase with improved catalytic activity isolated form a fast-growing dichloromethane-utilizing bacterium, J. Bacteriol. 170, 5698–5704, 1988; Russo, M.V., Goretti, G., and Liberti, A., Direct headspace gas chromatographic determination of dichloromethane in decaffeinated green and roasted coffee, J. Chromatog. 465, 429–433, 1989; Shimizu, M., Kamchi, S., Nishii, Y., and Yamada, S., Synthesis of a reagent for fluorescence-labeling of vitamin D and its use in assaying vitamin D metabolites, Anal. Biochem. 194, 77–81, 1991; Rodriguez-Palmero, M., de la Presa-Owens, S., Castellote-Bargallo, A.I. et al., Determination of sterol content in different food samples by capillary gas chromatography, J. Chromatog. A 672, 267–272, 1994; Raghuvanshi, R.S., Goyal, S., Singh, O., and Panda, A.K., Stabilization of dichloromethane-induced protein denaturation during microencapsulation, Pharm. Dev. Technol. 3, 269–276, 1998; El Jaber-Vazdekis, N., Gutierrez-Nicolas, F., Ravelo, A.G., and Zarate, R., Studies on tropane alkaloid extraction by volatile organic solvents: dichloromethane vs. chloroform, Phytochem. Anal. 17, 107–113, 2006.
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773
Chemicals Commonly Used in Biochemistry and Molecular Biology and Their Properties (Continued) Common Name
Chemical Name
Diethyldithiocarbamate
Ditiocarb; Dithiocarb; DTC
M.W. 171.3 (Na)
Properties and Comment Chelating agent with particular affinity for Pb, Cu, Zn, Ni; colorimetric determination of Cu.
H3C Na+ S–
N
H3C
S Matsuba, Y. and Takahashi, Y., Spectrophotometric determination of copper with N,N,N′,N′-tetraethylthiuram disulfide and an application of this method for studies on subcellular distribution of copper in rat brains, Anal. Biochem. 36, 182–191, 1970, Koutensky, J., Eybl, V., Koutenska, M. et al., Influence of sodium diethyldithiocarbamate on the toxicity and distribution of copper in mice, Eur. J. Pharmacol. 14, 389–392, 1971; Xu, H. and Mitchell, C.L., Chelation of zinc by diethyldithiocarbamate facilitates bursting induced by mixed antidromic plus orthodromic activation of mossy fibers in hippocampal slices, Brain Res. 624, 162–170, 1993; Liu, J., Shigenaga, M.K., Yan, L.J. et al., Antioxidant activity of diethyldithiocarbamate, Free Radic. Res. 24, 461–472, 1996; Zhang, Y., Wade, K.L., Prestera, T., and Talalav, P., Quantitative determination of isothiocyanates, dithiocarbamates, carbon disulfide, and related thiocarbonyl compounds by cyclocondensation with 1,2-benzenedithiol, Anal. Biochem. 239, 160–167, 1996; Shoener, D.F., Olsen, M.A., Cummings, P.G., and Basic, C., Electrospray ionization of neutral metal dithiocarbamate complexes using in-source oxidation, J. Mass Spectrom. 34, 1069–1078, 1999; Turner, B.J., Lopes, E.C., and Cheema, S.S., Inducible superoxide dismutase 1 aggregation in transgenic amyotrophic lateral sclerosis mouse fibroblasts, J. Cell Biochem. 91, 1074–1084, 2004; Xu, K.Y. and Kuppusamy, P., Dual effects of copper-zinc superoxide dismutase, Biochem. Biophys. Res. Commun. 336, 1190–1193, 2005; Jiang, X., Sun, S., Liang, A. et al., Luminescence properties of metal(II)diethyldithiocarbamate chelate complex particles and its analytical application, J. Fluoresc. 15, 859–864, 2005; Wang, J.S. and Chiu, K.H., Mass balance of metal species in supercritical fluid extraction using sodium diethyldithio-carbamate and dibuylammonium dibutyldithiocarbamate, Anal. Sci. 22, 363–369, 2006. Diethylpyrocarbonate (DEPC)
O
O
H3C
Ethoxyformic Anhydride
162.1
Reagent for modification of proteins and DNA; used as a sterilizing agent; RNAse inhibitor for RNA purification; preservative for wine and fruit fluids.
O
O
O
CH3
Wolf, B., Lesnaw, J.A., and Reichmann, M.E., A mechanism of the irreversible inactivation of bovine pancreatic ribonuclease by diethylpyrocarbonate. A general reaction of diethylpyrocarbonate with proteins, Eur. J. Biochem. 13, 519–525, 1970; Splittstoesser, D.F. and Wilkison, M., Some factors affecting the activity of diethylpyrocarbonate as a sterilant, Appl. Microbiol. 25, 853–857, 1973; Fedorcsak, I., Ehrenberg, L., and Solymosy, F., Diethylpyrocarbonate does not degrade RNA, Biochem. Biophys. Res. Commun. 65, 490–496, 1975; Berger, S.L., Diethylpyrocarbonate: an examination of its properties in buffered solutions with a new assay technique, Anal. Biochem. 67, 428–437, 1975; Lloyd, A.G. and Drake, J.J., Problems posed by essential food preservatives, Br. Med. Bull. 31, 214–219, 1975; Ehrenberg, L., Fedorcsak, I., and Solymosy, F., Diethylpyrocarbonate in nucleic acid research, Prog. Nucleic Acid Res. Mol. Biol. 16, 189–262, 1976; Saluz, H.P. and Jost, J.P., Approaches to characterize protein–DNA interactions in vivo, Crit. Rev. Eurkaryot. Gene Expr. 3, 1–29, 1993; Bailly, C. and Waring, M.J., Diethylpyrocarbonate and osmium tetroxide as probes for drug-induced changes in DNA conformation in vitro, Methods Mol. Biol. 90, 51–59, 1997; Mabic, S. and Kano, I., Impact of purified water quality on molecular biology experiments, Clin. Chem. Lab. Med. 41, 486–491, 2003; Colleluori, D.M., Reczkowski, R.S., Emig, F.A. et al., Probing the role of the hyper-reactive histidine residue of argininase, Arch. Biochem. Biophys. 444, 15–26, 2005; Wu, S.N. and Chang, H.D., Diethylpyrocarbonate, a histidine-modifying agent, directly stimulates activity of ATP-sensitive potassium channels in pituitary GH(3) cells, Biochem. Pharmacol. 71, 615–623, 2006. Dimedone
5,5-dimethyl-1,3cyclohexanedione
140.18
Originally described as reagent for assay of aldehydes; used as a specific modifier of sulfenic acid.
O H3C
CH3
9168_Book.indb 773
O
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Chemicals Commonly Used in Biochemistry and Molecular Biology and Their Properties (Continued) Common Name
Chemical Name
M.W.
Properties and Comment
Bulmer, D., Dimedone as an aldehyde-blocking reagent to facilitate the histochemical determination of glycogen, Stain Technol. 34, 95–98, 1959; Sawicki, E. and Carnes, R.A., Spectrophotofluorimetric determination of aldehydes with dimedone and other reagents, Mikrochim. Acta 1, 95–98, 1968; Benitez, L.V. and Allison, W.S., The inactivation of the acyl phosphatase activity catalyzed by the sulfenic acid form of glyceraldehyde 3-phosphate dehydrogenase by dimedone and olefins, J. Biol. Chem. 249, 6234–6243, 1974; Huszti, Z. and Tyihak, E., Formation of formaldehyde from S-adenosyl-L-[methyl-3H]methionine during enzymic transmethylation of histamine, FEBS Lett. 209, 362–366, 1986; Sardi, E. and Tyihak, E., Sample determination of formaldehyde in dimedone adduct form in biological samples by high-performance liquid chromatography, Biomed. Chromatog. 8, 313–314, 1994; Demaster, A.G., Quast, B.J., Redfern, B., and Nagasawa, H.T., Reaction of nitric oxide with the free sulfhydryl group of human serum albumin yields a sulfenic acid and nitrous oxide, Biochemistry 34, 14494–14949, 1995; Rozylo, T.K., Siembida, R., and Tyihak, E., Measurement of formaldehyde as dimedone adduct and potential formaldehyde precursors in hard tissues of human teeth by overpressurized layer chromatography, Biomed. Chromatog. 13, 513–515, 1999; Percival, M.D., Ouellet, M., Campagnolo, C. et al., Inhibition of cathepsin K by nitric oxide donors: evidence for the formation of mixed disulfides and a sulfenic acid, Biochemistry 38, 13574–13583, 1999; Carballal, S., Radi, R., Kirk, M.C. et al., Sulfenic acid formation in human serum albumin by hydrogen peroxide and peroxynitrite, Biochemistry 42, 9906–9914, 2003; Poole, L.B., Zeng, B.-B., Knaggs, S.A., Yakuba, M., and King, S.B., Synthesis of chemical probes to map sulfenic acid modifications on proteins, Bioconjugate Chem. 16, 1624–1628, 2005; Kaiserov, K., Srivastava, S., Hoetker, J.D. et al., Redox activation of aldose reductase in the ischemic heart, J. Biol. Chem. 281, 15110–15120, 2006. Dimethylformamide (DMF)
N,N-dimethylformamide
73.09
Solvent.
O CH3 H
N CH3
Eliezer, N. and Silberberg, A., Structure of branched poly-alpha-amino acids in dimethylformamide. I. Light scattering, Biopolymers 5, 95–104, 1967; Bonner, O.D., Bednarek, J.M., and Arisman, R.K., Heat capacities of ureas and water in water and dimethylformamide, J. Am. Chem. Soc. 99, 2898–2902, 1977; Sasson, S. and Notides, A.C., The effects of dimethylformamide on the interaction of the estrogen receptor with estradiol, J. Steroid Biochem. 29, 491–495, 1988; Jeffers, R.J., Feng, R.Q., Fowlkes, J.B. et al., Dimethylformamide as an enhancer of cavitation-induced cell lysis in vitro, J. Acoust. Soc. Am. 97, 669–676, 1995; You, L. and Arnold, F.H., Directed evolution of subtilisin E in Bacillus subtilis to enhance total activity in aqueous dimethylformamide, Protein Eng. 9, 77–83, 1996; Szabo, P.T. and Kele, Z., Electrospray mass spectrometry of hydrophobic compounds using dimethyl sulfoxide and dimethylformamide, Rapid Commun. Mass Spectrom. 15, 2415–2419, 2001; Nishida, Y., Shingu, Y., Dohi, H., and Kobayashi, K., One-pot alpha-glycosylation method using Appel agents in N,N-dimethylformamide, Org. Lett. 5, 2377–2380, 2003; Shingu, Y., Miyachi, A., Miura, Y. et al., One-pot alpha-glycosylation pathway via the generation in situ of alphaglycopyranosyl imidates I N,N-dimethylformamide, Carbohydr. Res. 340, 2236–2244, 2005; Porras, S.P. and Kenndler, E., Capillary electrophoresis in N,N-dimethylformamide, Electrophoresis 26, 3279–3291, 2005; Wei, Q., Zhang, H., Duan, C. et al., High sensitive fluorophotometric determination of nucleic acids with pyronine G sensitized by N,N-dimethylformamide, Ann. Chim. 96, 273–284, 2006. Dimethyl Suberimidate (DMS)
Crosslinking agent.
NH2+ H3C
O O
CH3 NH2+
Davies, G.E. and Stark, G.R., Use of dimethyl suberimidate, a crosslinking reagent, in studying the subunit structure of oligomeric proteins, Proc. Natl. Acad. Sci. USA 66, 651–656, 1970; Hassell, J. and Hand, A.R., Tissue fixation with diimidoesters as an alternative to aldehydes. I. Comparison of crosslinking and ultrastructure obtained with dimethylsuberimidate and glutaraldehyde, J. Histochem. Cytochem. 22, 223–229, 1974; Thomas, J.O., Chemical crosslinking of histones, Methods Enzymol. 170, 549–571, 1989; Roth, M.R., Avery, R.B., and Welti, R., Crosslinking of phosphatidylethanolamine neighbors with dimethylsuberimidate is sensitive to the lipid phase, Biochim. Biophys. Acta 986, 217–224, 1989; Redl, B., Walleczek, J., Soffler-Meilicke, M., and Stoffler, G., Immunoblotting analysis of protein–protein crosslinks within the 50S ribosomal subunit of Escherichia coli. A study using dimethylsuberimidate as crosslinking reagent, Eur. J. Biochem. 181, 351–256, 1989; Konig, S., Hubner, G., and Schellenberger, A., Crosslinking of pyruvate decarboxylase-characterization of the native and substrate-activated enzyme states, Biomed. Biochim. Acta 49, 465–471, 1990; Chen, J.C., von Lintig, F.C., Jones, S.B. et al., High-efficiency solid-phase capture using glass beads bonded to microcentrifuge tubes: immunoprecipitation of proteins from cell extracts and assessment of ras activation, Anal. Biochem. 302, 298–304, 2002; Dufes, C., Muller, J.M., Couet, W. et al., Anticancer drug delivery with transferrin-targeted polymeric chitosan vesicles, Pharm. Res. 21, 101–107, 2004; Levchenko, V. and Jackson, V., Histone release during transcription: NAP1 forms a complex with H2A and H2B and facilitates a topologically dependent release of H3 and H4 from the nucleosome, Biochemistry 43, 2358–2372, 2004; Jastrzebska, M., Barwinski, B., Mroz, I. et al., Atomic force microscopy investigation of chemically stabilized pericardium tissue, Eur. Phys. J. E 16, 381–388, 2005.
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Chemicals Commonly Used in Biochemistry and Molecular Biology and Their Properties (Continued) Common Name
Chemical Name
Dimethyl Sulfate
126.1
O S
O H3C
M.W.
Properties and Comment Methylating agent; methylation of nucleic acids; used for a process called footprinting to identify sites of protein–nucleic acid interaction.
CH3 O
O
Nielsen, P.E., In vivo footprinting: studies of protein–DNA interactions in gene regulation, Bioessay 11, 152–155, 1989; Saluz, H.P. and Jost, J.P., Approaches to characterize protein–DNA interactions in vivo, Crit. Rev. Eurkaryot. Gene Expr. 3, 1–29, 1993; Saluz, H.P. and Jost, J.P., In vivo DNA footprinting by linear amplification, Methods Mol. Biol. 31, 317–329, 1994; Paul, A.L. and Ferl, R.J., In vivo footprinting of protein–DNA interactions, Methods Cell Biol. 49, 391–400, 1995; Gregory, P.D., Barbaric, S., and Horz, W., Analyzing chromatin structure and transcription factor binding in yeast, Methods 15, 295–302, 1998; Simpson, R.T., In vivo to analyze chromatin structrure, Curr. Opin. Genet. Dev. 9, 225–229, 1999; Nawrocki, A.R., Goldring, C.E., Kostadinova, R.M. et al., In vivo footprinting of the human 11β-hydroxysteroid dehydro-genase type 2 promoter: evidence for cell-specific regulation by Sp1 and Sp3, J. Biol. Chem. 277, 14647–14656, 2002; McGarry, K.C., Ryan, V.T., Grimwade, J.E., and Leonard, A.C., Two discriminatory binding sites in the Escherichia coli replication origin are required for DNA stand opening by initiator DnaA-ATP, Proc. Natl. Acad. Sci. USA 101, 2811–2816, 2004; Kellersberger, K.A., Yu, E., Kruppa, G.H. et al., Two-down characterization of nucleic acids modified by structural probes using high-resolution tandem mass spectrometry and automated data interpretation, Anal. Chem. 76, 2438–2445, 2004; Matthews, D.H., Disney, M.D., Childs, J.L. et al., Incorporating chemical modification constraints into a dynamic programming algorithm for prediction of RNA secondary structure, Proc. Natl. Acad. Sci. USA 101, 7287–7292, 2004; Forstemann, K. and Lingner, J., Telomerase limits the extent of base pairing between template RNA and temomeric DNA, EMBO Rep. 6, 361–366, 2005; Kore, A.R. and Parmar, G., An industrial process for selective synthesis of 7-methyl guanosine 5′-diphosphate: versatile synthon for synthesis of mRNA cap analogues, Nucleosides Nucleotides Nucleic Acids 25, 337–340, 2006. Dioxane
1,4-diethylene Dioxide
88.1
Solvent.
O
O Sideri, C.N. and Osol, A., A note on the purification of dioxane for use in preparing nonaqueous titrants, J. Am. Pharm. Am. Pharm. Assoc. 42, 586, 1953; Martel, R.W. and Kraus, C.A., The association of ions in dioxane-water mixtures at 25 degrees, Proc. Natl. Acad. Sci. USA 41, 9–20, 1955; Mercier, P.L. and Kraus, C.A, The ion-pair equilibrium of electrolyte solutions in dioxane-water mixtures, Proc. Natl. Acad. Sci. USA 41, 1033–1041, 1995; Inagami, T., and Sturtevant, J.M., The trypsin-catalyzed hydrolysis of benzoyl-L-arginine ethyl ester. I. The kinetics in dioxane-water mixtures, Biochim. Biophys. Acta 38, 64–79, 1980; Zaeklj, A. and Gros, M., Electrophoresis of lipoprotein, prestained with Sudan Black B, dissolved in a mixture of dioxane and ethylene glycol, Clin. Chim. Acta 5, 947, 1960; Krasner, J. and McMenamy, R.H., The binding of indole compounds to bovine plasma albumin. Effects of potassium chloride, urea, dioxane, and glycine, J. Biol. Chem. 241, 4186–4196, 1966; Smith, R.R. and Canady, W.J., Solvation effects upon the thermodynamic substrate activity: correlation with the kinetics of enzyme-catalyzed reactions. II. More complex interactions of alpha-chymotrypsin with dioxane and acetone which are also competitive inhibitors, Biophys. Chem. 43, 189–195, 1992; Forti, F.L., Goissis, G., and Plepis, A.M., Modifications on collagen structures promoted by 1,4-dioxane improve thermal and biological properties of bovine pericardium as a biomaterial, J. Biomater. Appl. 20, 267–285, 2006. Dithiothreitol
1,4-dithiothreitol; DTT; Cleland’s Reagent; threo-2,3-dihydroxy-1,4dithiolbutane
SH H2C
OH
9168_Book.indb 775
H2C
CH2 CH
Reducing agent.
S
SH
CH HO
154.3
S
CH HO
CH2 CH OH
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Chemicals Commonly Used in Biochemistry and Molecular Biology and Their Properties (Continued) Common Name
Chemical Name
M.W.
Properties and Comment
Cleland, W.W., Dithiothreitol, a new protective reagent for SH groups, Biochemistry 3, 480–482, 1964; Gorin, G., Fulford, R., and Deonier, R.C., Reaction of lysozyme with dithiothreitol and other mercaptans, Experientia 24, 26–27, 1968; Stanton, M. and Viswantha, T., Reduction of chymotryptin A by dithiothreitol, Can. J. Biochem. 49, 1233–1235, 1971; Warren, W.A., Activation of serum creatine kinase by dithiothreitol, Clin. Chem. 18, 473–475, 1972; Hase, S. and Walter, R., Symmetrical disulfide bonds as S-protecting groups and their cleavage by dithiothreitol: synthesis of oxytocin with high biological activity, Int. J. Pept. Protein Res. 5, 283–288, 1973; Fleisch, J.H., Krzan, M.C., and Titus, E., Alterations in pharmacologic receptor activity by dithiothreitol, Am. J. Physiol. 227, 1243–1248, 1974; Olsen, J. and Davis, L., The oxidation of dithiothreitol by peroxidases and oxygen, Biochim. Biophys. Acta. 445, 324–329, 1976; Chao, L.P., Spectrophotometric determination of choline acetyltransferase in the presence of dithiothreitol, Anal. Biochem. 85, 20–24, 1978; Fukada, H. and Takahashi, K., Calorimetric study of the oxidation of dithiothreitol, J. Biochem. 87, 1105–1110, 1980; Alliegro, M.C., Effects of dithiothreitol on protein activity unrelated to thiol-disulfide exchange: for consideration in the analysis of protein function with Cleland’s reagent, Anal. Biochem. 282, 102–106, 2000; Rhee, S.S. and Burke, D.H., Tris(2-carboxyethyl)phosphine stabilization of RNA: comparison with dithiothreitol for use with nucleic acid and thiophosphoryl chemistry, Anal. Biochem. 325, 137–143, 2004; Pan, J.C., Cheng, Y., Hui, E.F., and Zhou, H.M., Implications of the role of reactive cysteine in arginine kinase: reactivation kinetics of 5,5′-dithiobis-(2-nitrobenzoic acid)-modified arginine kinase reactivated by dithiothreitol, Biochem. Biophys. Res. Commun. 317, 539–544, 2004; Thaxton, C.S., Hill, H.D., Georganopoulou, D.G. et al., A bio-barcode assay based upon dithiothreitol-induced oligonucleotide release, Anal. Chem. 77, 8174–8178, 2005. DMSO
Dimethylsulfoxide
78.13
Solvent; suggested therapeutic use; effect on cellular function; cyropreservative.
O S H3C
CH3
Huggins, C.E., Reversible agglomeration used to remove dimethylsulfoxide from large volumes of frozen blood, Science 139, 504–505, 1963; Yehle, A.V. and Doe, R.H., Stabilization of Bacillus subtilis phage with dimethylsulfoxide, Can. J. Microbiol. 11, 745–746, 1965; Fowler, A.V. and Zabin, I., Effects of dimethylsulfoxide on the lactose operon of Escherichia coli, J. Bacteriol. 92, 353–357, 1966; Williams, A.E. and Vinograd, J., The buoyant behavior of RNA and DNA in cesium sulfate solutions containing dimethylsulfoxide, Biochim. Biophys. Acta 228, 423–439, 1971; Levine, W.G., The effect of dimethylsulfoxide on the binding of 3-methylcholanthrene to rat liver fractions, Res. Commum. Chem. Pathol. Pharmacol. 4, 511–518, 1972; Fink, A.L, The trypsin-catalyzed hydrolysis of N-alpha-benzoyl-L-lysine p-nitrophenyl ester in dimethylsulfoxide at subzero temperatures, J. Biol. Chem. 249, 5072–5932, 1974; Hutton, J.R. and Wetmur, J.G., Activity of endonuclease S1 in denaturing solvents: dimethylsulfoxide, dimethylformamide, formamide, and formaldehyde, Biochem. Biophys. Res. Commun. 66, 942–948, 1975; Gal, A., De Groot, N., and Hochberg, A.A., The effect of dimethylsulfoxide on ribosomal fractions from rat liver, FEBS Lett. 94, 25–27, 1978; Barnett, R.E., The effects of dimethylsulfoxide and glycerol on Na+, K+-ATPase, and membrane structure, Cryobiology 15, 227–229, 1978; Borzini, P., Assali, G., Riva, M.R. et al., Platelet cryopreservation using dimethylsulfoxide/polyethylene glycol/sugar mixture as cryopreserving solution, Vox Sang. 64, 248–249, 1993; West, R.T., Garza, L.A., II, Winchester, W.R., and Walmsley, J.A., Conformation, hydrogen bonding, and aggregate formation of guanosine 5′-monophosphate and guanosine in dimethylsulfoxide, Nucleic Acids Res. 22, 5128–5134, 1994; Bhattacharjya, S. and Balarma, P., Effects of organic solvents on protein structures; observation of a structured helical core in hen egg-white lysozyme in aqueous dimethylsulfoxide, Proteins 29, 492–507, 1997; Simala-Grant, J.L. and Weiner, J.H., Modulation of the substrate specificity of Escherichia coli dimethylsulfoxide reductase, Eur. J. Biochem. 251, 510–515, 1998; Tsuzuki, W., Ue, A., and Kitamura, Y., Effect of dimethylsulfoxide on hydrolysis of lipase, Biosci. Biotechnol. Biochem. 65, 2078–2082, 2001; Pedersen, N.R., Halling, P.J., Pedersen, L.H. et al., Efficient transesterification of sucrose catalyzed by the metalloprotease thermolysin in dimethylsulfoxide, FEBS Lett. 519, 181–184, 2002; Fan, C., Lu, J., Zhang, W., and Li, G., Enhanced electron-transfer reactivity of cytochrome b5 by dimethylsulfoxide and N,N′-dimethylformamide, Anal. Sci. 18, 1031–1033, 2002; Tait, M.A. and Hik, D.S., Is dimethylsulfoxide a reliable solvent for extracting chlorophyll under field conditions? Photosynth. Res. 78, 87–91, 2003; Malinin, G.I. and Malinin, T.I., Effects of dimethylsulfoxide on the ultrastructure of fixed cells, Biotech. Histochem. 79, 65–69, 2004; Clapisson, G., Salinas, C., Malacher, P. et al., Cryopreservation with hydroxyethylstarch (HES) + dimethylsulfoxide (DMSO) gives better results than DMSO alone, Bull. Cancer 91, E97–E102, 2004. EDC 1-ethyl-(3-dimethylamino 191.7 Water-soluble carbodiimide for the propyl)carbodiimide; (HCl) modification of carboxyl groups in proteins; N-(3-dimethylaminozero-length crosslinking proteins; activation propyl)-N′-ethylof carboxyl groups for amidation reactions, carbodiimide as for the coupling of amino-nucleotides to matrices for DNA microarrays. H3C N H3C N
9168_Book.indb 776
C
N
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777
Chemicals Commonly Used in Biochemistry and Molecular Biology and Their Properties (Continued) Common Name
Chemical Name
M.W.
Properties and Comment
Lin, T.Y. and Koshland, D.E., Jr., Carboxyl group modification and the activity of lysozyme, J. Biol. Chem. 244, 505–508, 1969; Carraway, K.L., Spoerl, P., and Koshland, D.E., Jr., Carboxyl group modification in chymotrypsin and chymotrypsinogen, J. Mol. Biol. 42, 133–137, 1969; Yamada, H., Imoto, T., Fujita, K. et al., Selective modification of aspartic acid-101 in lysozyme by carbodiimide reaction, Biochemistry 20, 4836–4842, 1981; Buisson, M. and Reboud, A.M., Carbodiimide-induced protein-RNA crosslinking in mammalian subunits, FEBS Lett. 148, 247–250, 1982; Millett, F., Darley-Usmar, V., and Capaldi, R.A., Cytochrome c is crosslinked to subunit II of cytochrome c oxidase by a water-soluble carbodiimide, Biochemistry 21, 3857–3862, 1982; Chen, S.C., Fluorometric determination of carbodiimides with trans-aconitic acid, Anal. Biochem. 132, 272–275, 1983; Davis, L.E., Roth, S.A., and Anderson, B., Antisera specificities to 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide adducts of proteins, Immunology 53, 435–441, 1984; Ueda, T., Yamada, H., and Imoto, T., Highly controlled carbodiimide reaction for the modification of lysozyme. Modification of Leu129 or As119, Protein Eng. 1, 189–193, 1987; Ghosh, M.K., Kildsig, D.O., and Mitra, A.K., Preparation and characterization of methotrexate-immunoglobulin conjugates, Drug. Des. Deliv. 4, 13–25, 1989; Grabarek, Z. and Gergely, J., Zero-length crosslinking procedure with the use of active esters, Anal. Biochem. 185, 131–135, 1990; Gilles, M.A., Hudson, A.Q., and Borders, C.L., Jr., Stability of water-soluble carbodiimides in aqueous solutions, Anal. Biochem. 184, 244–248, 1990; Soinila, S., Mpitsos, G.J., and Soinila, J., Immunohistochemistry of enkephalins: model studies on hapten-carrier conjugates and fixation methods, J. Histochem. Cytochem. 40, 231–239, 1992; Soper, S.A., Hashimoto, M., Situma, C. et al., Fabrication of DNA microarrays onto polymer substrates using UV modification protocols with integration into microfluidic platforms for the sensing of low-abundant DNA point mutations, Methods 37, 103–113, 2005. EDTA
Ethylenediaminetetraacetic acid
292.24
Chelating agent; some metal ion-EDTA complexes (i.e., Fe2+-EDTA) function as chemical nucleases.
COOH HOOC
N COOH
N COOH
Flaschka, H.A., EDTA Titrations: An Introduction to Theory and Practice, Pergammon Press, Oxford, UK, 1964; West, T.S., Complexometry with EDTA and Related Reagents, BDH Chemicals Ltd., Poole (Dorset), UK, 1969; Pribil, R., Analytical Applications of EDTA and Related Compounds, Pergammon Press, Oxford, UK, 1972; Papavassiliou, A.G., Chemical nucleases as probes for studying DNA–protein interactions, Biochem. J. 305, 345–357, 1995; Martell, A.E., and Hancock, R.D., Metal Complexes in Aqueous Solutions, Plenum Press, New York, 1996; Loizos, N. and Darst, S.A, Mapping protein–ligand interactions by footprinting, a radical idea, Structure 6, 691–695, 1998; Franklin, S.J., Lanthanide-mediated DNA hydrolysis, Curr. Opin. Chem. Biol. 5, 201–208, 2001; Heyduk, T., Baichoo, N., and Henduk, E., Hydroxyl radical footprinting of proteins using metal ion complexes, Met. Ions Biol. Syst. 38, 255–287, 2001; Orlikowsky, T.W., Neunhoeffer, F., Goelz, R. et al., Evaluation of IL-8-concentrations in plasma and lyszed EDTA-blood in healthy neonates and those with suspected early onset bacterial infection, Pediatr. Res. 56, 804–809, 2004; Matt, T., Martinez-Yamout, M.A., Dyson, H.J., and Wright, P.E., The CBP/p300 TAZ1 domain in its native state is not a binding partner of MDM2, Biochem. J. 381, 685–691, 2004; Nyborg, J.K. and Peersen, O.B., That zincing feeling: the effects of EDTA on the behavior of zinc-binding transcriptional regulators, Biochem. J. 381, e3–e4, 2004; Haberz, P., Rodriguez-Castanada, F., Junker, J. et al., Two new chiral EDTA-based metal chelates for weak alignment of proteins in solution, Org. Lett. 8, 1275–1278, 2006. Ellman’s Reagent
5,5′-dithiobis[2-nitrobenzoic] acid
396.35
Reagent for determination of sulfydryl groups/ disulfide bonds.
NO2 HOOC
S S
COOH NO2
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Chemicals Commonly Used in Biochemistry and Molecular Biology and Their Properties (Continued) Common Name
Chemical Name
M.W.
Properties and Comment
Ellman, G.L., Tissue sulfydryl groups, Arch. Biochem. Biophys. 82, 70–77, 1959; Boyne, A.F. and Ellman, G.L., A methodology for analysis of tissue sulfydryl components, Anal. Biochem. 46, 639–653, 1972; Brocklehurst, K., Kierstan, M., and Little, G., The reaction of papain with Ellman’s reagent (5,5′-dithiobis-(2-nitrobenzoate), Biochem. J. 128, 811–816, 1972; Weitzman, P.D., A critical reexamination of the reaction of sulfite with DTNB, Anal. Biochem. 64, 628–630, 1975; Hull, H.H., Chang, R., and Kaplan, L.J., On the location of the sulfhydryl group in bovine plasma albumin, Biochim. Biophys. Acta 400, 132–136, 1975; Banas, T., Banas, B., and Wolny, M., Kinetic studies of the reactivity of the sulfydryl groups of glyceraldehyde-3-phosphate dehydrogenase, Eur. J. Biochem. 68, 313–319, 1976; der Terrossian, E. and Kassab, R., Preparation and properties of S-cyano derivatives of creatine kinase, Eur. J. Biochem. 70, 623–628, 1976; Riddles, P.W., Blakeley, R.L., and Zerner, B., Ellman’s reagent: 5,5′-dithiobis(2-nitrobenzoic acid) — a reexamination, Anal. Biochem. 94, 75–81, 1979; Luthra, N.P., Dunlap, R.B., and Odom, J.D., Characterization of a new sulfydryl group reagent: 6, 6′- diselenobis-(3-nitrobenzoic acid), a selenium analog of Ellman’s reagent, Anal. Biochem. 117, 94–102, 1981; Di Simplicio, P., Tiezzi, A., Moscatelli, A. et al., The SH-SS exchange reaction between the Ellman’s reagent and proteincontaining SH groups as a method for determining conformational states: tubulin, Ital. J. Biochem. 38, 83–90, 1989; Woodward, J., Tate, J., Herrmann, P.C., and Evans, B.R., Comparison of Ellman’s reagent with N-(1-pyrenyl)maleimide for the determination of free sulfydryl groups in reduced cellobiohydrolase I from Trichoderma reesei, J. Biochem. Biophys. Methods 26, 121–129, 1993; Berlich, M., Menge, S., Bruns, I. et al., Coumarins give misleading absorbance with Ellman’s reagent suggestive of thiol conjugates, Analyst 127, 333–336, 2002; Riener, C.K., Kada, G., and Gruber, H.J., Quick measurement of protein sulfhydryls of Ellman’s reagents and with 4,4′-dithiopyridine, Anal. Bio. Anal. Chem. 373, 266–276, 2002; Zhu, J., Dhimitruka, I., and Pei, D., 5-(2-aminoethyl)dithio-2-nitrobenzoate as a more base-stable alternative to Ellman’s reagent, Org. Lett. 6, 3809–3812, 2004; Owusu-Apenten, R., Colorimetric analysis of protein sulfhydryl groups in milk: applications and processing effects, Crit. Rev. Food Sci. Nutr. 45, 1–23, 2005. Ethanolamine
H2 C H2N
Glycinol
61.08
Buffer component; component of a phospholipid (phosphatidyl ethanolamine, PE).
OH C H2
Vance, D.E. and Ridgway, N.D., The methylation of phosophatidylethanolamine, Prog. Lipid Res. 27, 61–79, 1988; Louwagie, M., Rabilloud, T., and Garin, J., Use of ethanolamine for sample stacking in capillary electrophoresis, Electrophoresis 19, 2440–2444, 1998; de Nogales, V., Ruiz, R., Roses. M. et al., Background electrolytes in 50% methanol/water for the determination of acidity constants of basic drugs by capillary zone electrophoresis, J. Chromatog. A 1123, 113–120, 2006. Ethidium Bromide
394.31
NH2
H2N
– N+ Br
CH3
Sela, I., Fluorescence of nucleic acids with ethidium bromide: an indication of the configurative state of nucleic acids, Biochim. Biophys. Acta 190, 216–219, 1969; Le Pecq, J.B., Use of ethidium bromide for separation and determination of nucleic acids of various conformational forms and measurement of their associated enzymes, Methods Biochem. Anal. 20, 41–86, 1971; Borst, P., Ethidium DNA agarose gel electrophoresis: how it started, IUBMB Life 57, 745–747, 2005. Ethyl Alcohol
Ethanol
46.07
Solvent; used to adjust solvent polarity; use in plasma protein fractionation.
H2 C H3C
9168_Book.indb 778
OH
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779
Chemicals Commonly Used in Biochemistry and Molecular Biology and Their Properties (Continued) Common Name
Chemical Name
M.W.
Properties and Comment
Dufour, E., Bertrand-Harb, C., and Haertle, T., Reversible effects of medium dielectric constant on structural transformation of beta-lactoglobulin and its retinol binding, Biopolymers 33, 589–598, 1993; Escalera, J.B., Bustamante, P., and Martin, A., Predicting the solubility of drugs in solvent mixtures: multiple solubility maxima and the chameleonic effect, J. Pharm. Pharmcol. 46, 172–176, 1994; Gratzer, P.F., Pereira, C.A., and Lee, J.M., Solvent environment modulates effects of glutaraldehyde crosslinking on tissue-derived biomaterials, J. Biomed. Mater. Res. 31, 533–543, 1996; Sepulveda, M.R. and Mata, A.M., The interaction of ethanol with reconstituted synaptosomal plasma membrane Ca2+, Biochim. Biophys. Acta 1665, 75–80, 2004; Ramos, A.S. and Techert, S., Influence of the water structure on the acetylcholinesterase efficiency, Biophys. J. 89, 1990–2003, 2005; Wehbi, Z., Perez, M.D., and Dalgalarrondo, M., Study of ethanol-induced conformation changes of holo and apo alpha-lactalbumin by spectroscopy anad limited proteolysis, Mol. Nutr. Food Res. 50, 34–43, 2006; Sasahara, K. and Nitta, K., Effect of ethanol on folding of hen egg-white lysozyme under acidic condition, Proteins 63, 127–135, 2006; Perham, M., Liao, J., and Wittung-Stafshede, P., Differential effects of alcohol on conformational switchovers in alpha-helical and beta-sheet protein models, Biochemistry 45, 7740–7749, 2006; Pena, M.A., Reillo, A., Escalera, B., and Bustamante, P., Solubility parameter of drugs for predicting the solubility profile type within a wide polarity range in solvent mixtures, Int. J. Pharm. 321, 155–161, 2006; Jenke, D., Odufu, A., and Poss, M., The effect of solvent polarity on the accumulation of leachables from pharmaceutical product containers, Eur. J. Pharm. Sci. 27, 133–142, 2006. Ethylene Glycol
1,2-ethanediol
62.07
Solvent/cosolvent; increases viscosity (visogenic osmolyte); perturbant; cryopreservative.
OH CH H
OH CH H
Tanford, C., Buckley, C.E., III, De, P.K., and Lively, E.P., Effect of ethylene glycol on the conformation of gamma-globulin and beta-lactoglobulin, J. Biol. Chem. 237, 1168–1171, 1962; Kay, C.M. and Brahms, J., The influence of ethylene glycol on the enzymatic adenosine triphosphatase activity and molecular conformation of fibrous muscle proteins, J. Biol. Chem. 238, 2945–2949, 1963; Narayan, K.A., The interaction of ethylene glycol with rat-serum lipoproteins, Biochim. Biophys. Acta 137, 22–30, 1968; Bello, J., The state of the tyrosines of bovine pancreatic ribonuclease in ethylene glycol and glycerol, Biochemistry 8, 4535–4541, 1969; Lowe, C.R. and Mosbach, K., Biospecific affinity chromatography in aqueous-organic cosolvent mixtures. The effect of ethylene glycol on the binding of lactate dehydrogenase to an immobilized-AMP analogue, Eur. J. Biochem. 52, 99–105, 1975; Ghrunyk, B.A. and Matthews, C.R., Role of diffusion in the folding of the alpha subunit of tryptophan synthase from Escherichia coli, Biochemistry 29, 2149–2154, 1990; Silow, M. and Oliveberg, M., High concentrations of viscogens decrease the protein folding rate constant by prematurely collapsing the coil, J. Mol. Biol. 326, 263–271, 2003; Naseem, F. and Khan, R.H., Effect of ethylene glycol and polyethylene glycol on the acid-unfolded state of trypsinogen, J. Protein Chem. 22, 677–682, 2003; Hubalek, Z., Protectants used in the cyropreservation of microorganisms, Cryobiology 46, 205–229, 2003; Menezo, Y.J., Blastocyst freezing, Eur. J. Obstet. Gynecol. Reprod. Biol. 155 (Suppl. 1), S12–S15, 2004; Khodarahmi, R. and Yazdanparast, R., Refolding of chemically denatured alpha-amylase in dilution additive mode, Biochim. Biophys. Acta. 1674, 175–181, 2004; Zheng, M., Li, Z., and Huang, X., Ethylene glycol monolayer protected nanoparticles: synthesis, characterization, and interactions with biological molecules, Langmuir 20, 4226–4235, 2004; Bonincontro, A., Cinelli, S., Onori, G., and Stravato, A., Dielectric behavior of lysozyme and ferricytochrome-c in water/ ethylene-glycol solutions, Biophys. J. 86, 1118–1123, 2004; Kozer, N. and Schreiber, G., Effect of crowding on protein–protein association rates: fundamental differences between low and high mass crowding agents, J. Mol. Biol. 336, 763–774, 2004; Levin, I., Meiri, G., Peretz, M. et al., The ternary complex of Pseudomonas aeruginosa dehydrogenase with NADH and ethylene glycol, Protein Sci. 13, 1547–1556, 2004; Stupishina, E.A., Khamidullin, R.N., Vylegzhanina, N.N. et al., Ethylene glycol and the thermostability of trypsin in a reverse micelle system, Biochemistry 71, 533–537, 2006; Nordstrom, L.J., Clark, C.A., Andersen, B. et al., Effect of ethylene glycol, urea, and N-methylated glycines on DNA thermal stability: the role of DNA base pair composition and hydration, Biochemistry 45, 9604–9614, 2006. Ethyleneimine
Aziridine
43.07
Modification of sulfhydryl groups to produce amine functions; alkylating agent; reacts with carboxyl groups at acid pH; monomer unit for polyethylene amine, a versatile polymer.
H2C NH H2C
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Chemicals Commonly Used in Biochemistry and Molecular Biology and Their Properties (Continued) Common Name
Chemical Name
M.W.
Properties and Comment
Raftery, M.A. and Cole, R.D., On the aminoethylation of proteins, J. Biol. Chem. 241, 3457–3461, 1966; Fishbein, L., Detection and thin-layer chromatography of derivatives of ethyleneimine. I. N-carbamoyl and aziridines, J. Chromatog. 26, 522–526, 1967; Yamada, H., Imoto, T., and Noshita, S., Modification of catalytic groups in lysozyme with ethyleneimine, Biochemistry 21, 2187–2192, 1982; Okazaki, K., Yamada, H., and Imoto, T., A convenient S-2-aminoethylation of cysteinyl residues in reduced proteins, Anal. Biochem. 149, 516–520, 1985; Hemminki, K., Reactions of ethyleneimine with guanosine and deoxyguanosine, Chem. Biol. Interact. 48, 249–260, 1984; Whitney, P.L., Powell, J.T., and Sanford, G.L., Oxidation and chemical modification of lung beta-galactosidase-specific lectin, Biochem. J. 238, 683–689, 1986; Simpson, D.M., Elliston, J.F., and Katzenellenbogen, J.A., Desmethylnafoxidine aziridine: an electrophilic affinity label for the estrogen receptor with high efficiency and selectivity, J. Steroid Biochem. 28, 233–245, 1987; Musser, S.M., Pan, S.S., Egorin, M.J. et al., Alkylation of DNA with aziridine produced during the hydrolysis of N,N′,N′′-triethylenethiophosphoramide, Chem. Res. Toxicol. 5, 95–99, 1992; Thorwirth, S., Muller, H.S., and Winnewisser, G., The millimeter- and submillimeter-wave spectrum and the dipole moment of ethyleneimine, J. Mol. Spectroso. 199, 116–123, 2000; Burrage, T., Kramer, E., and Brown, F., Inactivation of viruses by aziridines, Dev. Biol. (Basel) 102, 131–139, 2000; Brown, F., Inactivation of viruses by aziridines, Vaccine 20, 322–327, 2001; Sasaki, S., Active oligonucleotides incorporating alkylating agent as potential sequence- and base-selective modifier of gene expression, Eur. J. Pharm. Sci. 13, 43–51, 2001; Hou, X.L., Fan, R.H., and Dai, L.X., Tributylphosphine: a remarkable promoting reagent for the ring-opening reaction of aziridines, J. Org. Chem. 67, 5295–5300, 2002; Thevis, M., Loo, R.R.O., and Loo, J.A., In-gel derivatization of proteins for cysteine-specific cleavages and their analysis by mass spectrometry, J. Proteome Res. 2, 163–172, 2003; Sasaki, M., Dalili, S., and Yudin, A.K., N-arylation of aziridines, J. Org. Chem. 68, 2045–2047, 2003; Gao, G.Y., Harden, J.D., and Zhang, J.P., Cobalt-catalyzed efficient aziridination of alkenes, Org. Lett. 7, 3191–3193, 2005; Hopkins, C.E., Hernandez, G., Lee, J.P., and Tolan, D.R., Aminoethylation in model peptides reveals conditions for maximizing thiol specificity, Arch. Biochem. Biophys. 443, 1–10, 2005; Li, C. and Gershon, P.D., pK(a) of the mRNA cap-specific 2′-O-methyltransferase catalytic lysine by HSQC NMR detection of a two-carbon probe, Biochemistry 45, 907–917, 2006; Vicik R., Helten, H., Schirmeister, T., and Engels, B., Rational design of aziridine-containing cysteine protease inhibitors with improved potency: studies on inhibition mechanism, ChemMedChem, 1, 1021–1028, 2006. Ethylene Oxide
Oxirane
44.05
Sterilizing agent; starting material for ethylene glycol and other products such as nonionic surfactants.
H2C O H2C Windmueller, H.G., Ackerman, C.J., and Engel, R.W., Reaction of ethylene oxide with histidine, methionine, and cysteine, J. Biol. Chem. 234, 895–899, 1959; Starbuck, W.C. and Busch, H., Hydroxyethylation of amino acids in plasma albumin with ethylene oxide, Biochim. Biophys. Acta 78, 594–605, 1963; Guengerich, F.P., Geiger, L.E., Hogy, L.L., and Wright,. P.L., In vitro metabolism of acrylonitrile to 2-cyanoethylene oxide, reaction with glutathione, and irreversible binding to proteins and nucleic acids, Cancer Res. 41, 4925–4933, 1981; Peter, H., Schwarz, M., Mathiasch, B. et al., A note on synthesis and reactivity towards DNA of glycidonitrile, the epoxide of acrylonitrile, Carcinogenesis 4, 235–237, 1983; Grammer, L.C. and Patterson, R., IgE against ethylene oxide-altered human serum albumin (ETO-HAS) as an etiologic agent in allergic reactions of hemodialysis patients, Artif. Organs 11, 97–99, 1987; Bolt, H.M., Peter, H., and Fost, U., Analysis of macromolecular ethylene oxide adducts, Int. Arch. Occup. Environ. Health 60, 141–144, 1988; Young, T.L., Habraken, Y., Ludlum, D.B., and Santella, R.M., Development of monoclonal antibodies recognizing 7-(2-hydroxyethyl) guanine and imidazole ring-opened 7-(2-hydroxyethyl) guanine, Carcinogenesis 11, 1685–1689, 1990; Walker, V.E., Fennell, T.R., Boucheron, J.A. et al., Macromolecular adducts of ethylene oxide: a literature review and a time-course study on the formation of 7-(2-hydroxyethyl)guanine following exposure of rats by inhalation, Mutat. Res. 233, 151–164, 1990; Framer, P.B., Bailey, E., Naylor, S. et al., Identification of endogenous electrophiles by means of mass spectrometric determination of protein and DNA adducts, Environ. Health Perspect. 99, 19–24, 1993; Tornqvist, M. and Kautianinen, A., Adducted proteins for identification of endogenous electrophiles, Environ. Health Perspect. 99, 39–44, 1993; Galaev, I. Yu. and Mattiasson, B., Thermoreactive water-soluble polymers, nonionic surfactants, and hydrogels as reagents in biotechnology, Enzyme Microb. Technol. 15, 354–366, 1993; Segerback, D., DNA alkylation by ethylene oxide and mono-substituted expoxides, IARC Sci. Publ. 125, 37–47, 1994; Phillips, D.H. and Farmer, P.B., Evidence for DNA and protein binding by styrene and styrene oxide, Crit. Rev. Toxicol. 24 (Suppl.), S35–S46, 1994; Marczynski, B., Marek, W., and Baur, X., Ethylene oxide as a major factor in DNA and RNA evolution, Med. Hypotheses 44, 97–100, 1995; Mosely, G.A. and Gillis, J.R., Factors affecting tailing in ethylene oxide sterilization part 1: when tailing is an artifact… and scientific deficiencies in ISO 11135 and EN 550, PDA J. Pharm. Sci. Technol. 58, 81–95, 2004. N-Ethylmaleimide
1-ethyl-1H-pyrrole-2,5dione
125.13
Modification of sulfhydryl groups; basic building block for a number of reagents. Mechanism different from alkylating agent in that reaction involves a Michael addition.
O CH3 N
O
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Chemicals Commonly Used in Biochemistry and Molecular Biology and Their Properties (Continued) Common Name
Chemical Name
M.W.
Properties and Comment
Lundblad, R.L., Chemical Reagent for Protein Modification, 3rd ed., CRC Press, Boca Raton, FL, 2004; Bowes, T.J. and Gupta, R.S., Induction of mitochondrial fusion by cysteine-alkylators ethyacrynic acid and N-ethylmaleimide, J. Cell Physiol. 202, 796–804, 2005; Engberts, J.B., Fernandez, E., Garcia-Rio, L., and Leis, J.R., Water in oil microemulsions as reaction media for a Diels–Alder reaction between N-ethylmaleimide and cyclopentadiene, J. Org. Chem. 71, 4111–4117, 2006; Engberts, J.B., Fernandez, E., Garcia-Rio, L., and Leis, J.R, AOT-based microemulsions accelerate the 1,3-cycloaddition of benzonitrile oxide to N-ethylmaleimide, J. Org. Chem. 71, 6118–6123, 2006; de Jong, K. and Kuypers, F.A., Sulphydryl modifications alter scramblase activity in murine sickle cell disease, Br. J. Haematol. 133, 427–432, 2006; Martin, H.G., Henley, J.M., and Meyer, G., Novel putative targets of N-ethylmaleimide sensitive fusion proteins (NSF) and alpha/beta soluble NSF attachment proteins (SNAPs) include the Pak-binding nucleotide exchange factor betaPIX, J. Cell. Biochem., 99, 1203–1215, 2006; Carrasco, M.R., Silva, O., Rawls, K.A. et al., Chemoselective alkylation of N-alkylaminooxy-containing peptides, Org. Lett. 8, 3529–3532, 2006; Pobbati, A.V., Stein, A., and Fasshauer, D., N- to C-terminal SNARE complex assembly promotes rapid membrane fusion, Science 313, 673–676, 2006; Mollinedo, F., Calafat, J., Janssen, H. et al., Combinatorial SNARE complexes modulate the secretion of cytoplasmic granules in human neutrophils, J. Immunol. 177, 2831–2841, 2006. Formaldehyde
Methanal
Formaldehyde O
Tissue fixation; protein modification; zero-length crosslinking; protein–nucleic acid interactions.
OH +
H
30.03
H2O
H
H
H OH gem-diol form
“Paraformaldehyde” O H2C
CH2
O
O C H2
And higher polymers
Feldman, M.Y., Reactions of nucleic acids and nucleoproteins with formaldehyde, Prog. Nucleic Acid Res. Mol. Biol. 13, 1–49, 1973; Russell, A.D. and Hopwood, D., The biological uses and importance of glutaraldehyde, Prog. Med. Chem. 13, 271–301, 1976; Means, G.E., Reductive alkylation of amino groups, Methods Enzymol. 47, 469–478, 1977; Winkelhake, J.L., Effects of chemical modification of antibodies on their clearance for the circulation. Addition of simple aliphatic compounds by reductive alkylation and carbodiimide-promoted amide formation, J. Biol. Chem. 252, 1865–1868, 1977; Yamazaki, Y. and Suzuki, H., A new method of chemical modification of N6-amino group in adenine nucleotides with formaldehyde and a thiol and its application to preparing immobilized ADP and ATP, Eur. J. Biochem. 92, 197–207, 1978; Geoghegan, K.F., Cabacungan, J.C., Dixon, H.B., and Feeney, R.E., Alternative reducing agents for reductive methylation of amino groups in proteins, Int. J. Pept. Protein Res. 17, 345–352, 1981; Kunkel, G.R., Mehradian, M., and Martinson, H.G., Contact-site crosslinking agents, Mol. Cell. Biochem. 34, 3–13, 1981; Fox, C.H., Johnson, F.B., Whiting, J., and Roller, P.P., Formaldehyde fixation, J. Histochem. Cytochem. 33, 845–853, 1985; Conaway, C.C., Whysner, J., Verna, L.K., and Williams, G.M., Formaldehyde mechanistic data and risk assessment: endogenous protection from DNA adduct formation, Pharmacol. Ther. 71, 29–55, 1996; Masuda, N., Ohnishi, T., Kawamoto, S. et al., Analysis of chemical modifications of RNA from formalin-fixed samples and optimization of molecular biology applications for such samples, Nucleic Acids Res. 27, 4436–4443, 1999; Micard, V., Belamri, R., Morel, M., and Guilbert, S., Properties of chemically and physically treated wheat gluten films, J. Agric. Food Chem. 48, 2948–2953, 2000; Taylor, I.A. and Webb, M., Chemical modification of lysine by reductive methylation. A probe for residues involved in DNA binding, Methods Mol. Biol. 148, 301–314, 2001; Perzyna, A., Marty, C., Facopre, M. et al., Formaldehyde-induced DNA crosslink of indolizino[1,2-b]quinolines derived from the A-D rings of camptothecin, J. Med. Chem. 45, 5809–5812, 2002; Yurimoto, H., Hirai, R., Matsuno, N. et al., HxlR, a member of the DUF24 protein family, is a DNA-binding protein that acts as a positive regulator of the formaldehyde-inducible hx1AB operon in Bacillus subtilis, Mol. Microbiol. 57, 511–519, 2005. Formic Acid
Methanoic Acid
46.03
Solvent; buffer component.
O
H
9168_Book.indb 781
OH
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Chemicals Commonly Used in Biochemistry and Molecular Biology and Their Properties (Continued) Common Name
Chemical Name
M.W.
Properties and Comment
Sarkar, P.B., Decomposition of formic acid by periodate, Nature 168, 122–123, 1951; Hass, P., Reactions of formic acid and its salts, Nature 167, 325, 1951; Smillie, L.B. and Neurath, H., Reversible inactivation of trypsin by anhydrous formic acid, J. Biol. Chem 234, 355–359, 1959; Hynninen, P.H. and Ellfolk, N., Use of the aqueous formic acid-chloroform-dimethylformamide solvent system for the purification of porphyrins and hemins, Acta Chem. Scand. 27, 1795–1806, 1973; Heukeshoven, J. and Dernick, R., Reversed-phase high-performance liquid chromatography of virus proteins and other large hydrophobic proteins in formic acid-containing solvents, J. Chromatog. 252, 241–254, 1982; Tarr, G.E. and Crabb, J.W., Reverse-phase high-performance liquid chromatography of hydrophobic proteins and fragments thereof, Anal. Biochem. 131, 99–107, 1983; Heukeshoven, J. and Dernick, R., Characterization of a solvent system for separation of water-insoluble poliovirus proteins by reversed-phase high-performance liquid chromatography, J. Chromatog. 326, 91–101, 1985; De Caballos, M.L., Taylor, M.D., and Jenner, P., Isocratic reverse-phase HPLC separation and RIA used in the analysis of neuropeptides in brain tissue, Neuropeptides 20, 201–209, 1991; Poll, D.J. and Harding, D.R., Formic acid as a milder alternative to trifluoroacetic acid and phosphoric acid in two-dimensional peptide mapping, J. Chromatog. 469, 231–239, 1989; Klunk W.E. and Pettegrew, J.W., Alzheimer’s beta-amyloid protein is covalently modified when dissolved in formic acid, J. Neurochem. 54, 2050–2056, 1990; Erdjument-Bromage, H., Lui, M., Lacomis, L. et al., Examination of the micro-tip reversed phase liquid chromatographic extraction of peptide pools for mass spectrometric analysis, J. Chromatog. A 826, 167–181, 1998; Duewel, H.S. and Honek, J.F., CNBr/formic acid reactions of methionine- and trifluoromethionine-containing lambda lysozyme: probing chemical and positional reactivity and formylation side reactions of mass spectrometry, J. Protein Chem. 17, 337–350, 1998; Kaiser, R. and Metzka, L., Enhancement of cyanogen bromide cleavage yields for methionyl-serine and methionyl-threonine peptide bonds, Anal. Biochem. 266, 1–8, 1999; Rodriguez, J.C., Wong, L., and Jennings, P.A., The solvent in CNBr cleavage reactions determines the fragmentation efficiency of ketosteroid isomerase fusion proteins used in the production of recombinant peptides, Protein Expr. Purif. 28, 224–231, 2003; Zu, Y., Zhao, C., Li, C., and Zhang, L., A rapid and sensitive LC-MS/MS method for determination of coenzyme Q10 in tobacco (Nicotiana tabacum L.) leaves, J. Sep. Sci. 29, 1607–1612, 2006; Kalovidouris, M., Michalea, S., Robola, N. et al., Ultra-performance liquid chromatography/tandem mass spectrometry method for the determination of lercaidipine in human plasma, Rapid Commun. Mass Spectrom., 20, 2939–2946, 2006; Wang, P.G., Wei, J.S., Kim, G. et al., Validation and application of a high-performance liquid chromatography-tandem mass spectrometric method for simultaneous quantification of lopinavir and ritonavir in human plasma using semi-automated 96-well liquid–liquid chromatography, J. Chromatog. A, 1130, 302–307, 2006. Glutaraldehyde
Pentanedial
H2 C
H C
H2 C
100.12
Protein modification; tissue fixation; sterilization agent approved by regulatory agencies; use with albumin as surgical sealant.
H
C H2
C
O
O Glutaraldehyde Aldol condensation/dehydraion
O
H2 C
H
H
C
C
C
C
O
H2 C C H2
H
C H
H2 C
C H2
H2 C
C H
H
C H2
C
O
O Protein-NH2
Protein O
H2 C
H
H2 C
H N C
HC
CH
C
CH
C H2 O
H2 C C H
C H2
H2 C
H C
C H2
O
NH Protein O
H2 C
H
H2 C C H2
O
9168_Book.indb 782
H
C
C
O
CH CH NH
Protein
H
H2 C
CH C H2
CH
H2 C C H2
H C O
HN Protein
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783
Chemicals Commonly Used in Biochemistry and Molecular Biology and Their Properties (Continued) Common Name
Chemical Name
M.W.
Properties and Comment
Hopwood, D., Theoretical and practical aspects of glutaraldehyde fixation, Histochem. J., 4, 267–303, 1972; Hassell, J. and Hand, A.R., Tissue fixation with diimidoesters as an alternative to aldehydes. I. Comparison of crosslinking and ultrastructure obtained with dimethylsubserimidate and glutaraldehyde, J. Histochem. Cytochem. 22, 223–229, 1974; Russell, A.D. and Hopwood, D., The biological uses and importance of glutaraldehyde, Prog. Med. Chem. 13, 271–301, 1976; Woodroof, E.A., Use of glutaraldehyde and formaldehyde to process tissue heart valves, J. Bioeng. 2, 1–9, 1978; Heumann, H.G., Microwave-stimulated glutaraldehyde and osmium tetroxide fixation of plant tissue: ultrastructural preservation in seconds, Histochemistry 97, 341–347, 1992; Abbott, L., The use and effects of glutaraldehyde: a review, Occup. Health 47, 238–239, 1995; Jayakrishnan, A. and Jameela, S.R., Glutaraldehyde as a fixative in bioprosthesis and drug delivery matrices, Biomaterials 17, 471–484, 1996; Tagliaferro, P., Tandler, C.J., Ramos, A.J. et al., Immunofluorescence and glutaraldehyde fixation. A new procedure base on the Schiff-quenching method, J. Neurosci. Methods 77, 191–197, 1997; Cohen, R.J., Beales, M.P., and McNeal, J.E., Prostate secretory granules in normal and neoplastic prostate glands: a diagnostic aid to needle biopsy, Hum. Pathol. 31, 1515–1519, 2000; Chae, H.J., Kim, E.Y., and In, M., Improved immobilization yields by addition of protecting agents in glutaraldehyde-induced immobilization of protease, J. Biosci. Bioeng. 89, 377–379, 2000; Nimni, M.E., Glutaraldehyde fixation revisited, J. Long Term Eff. Med. Implants 11, 151–161, 2001; Fujiwara, K., Tanabe, T., Yabuchi, M. et al., A monoclonal antibody against the glutaraldehyde-conjugated polyamine, putrescine: application to immunocytochemistry, Histochem. Cell Biol. 115, 471–477, 2001; Chao, H.H. and Torchiana, D.F., Bioglue: albumin/glutaraldehyde sealant in cardiac surgergy, J. Card. Surg. 18, 500–503, 2003; Migneault, I., Dartiguenave, C., Bertrand, M.J., and Waldron, K.C., Glutaraldehyde: behavior in aqueous solution, reaction with proteins, and application to enzyme crosslinking, Biotechniques 37, 790–796, 2004; Jearanaikoon, S. and Abraham-Peskir, J.V., An x-ray microscopy perspective on the effect of glutaraldehyde fixation on cells, J. Microsc. 218, 185–192, 2005; Buehler, P.W., Boykins, R.A., Jia, Y. et al., Structural and functional characterization of glutaraldehyde-polymerized bovine hemoglobin and its isolated fractions, Anal. Chem. 77, 3466–3478, 2005; Kim, S.S., Lim, S.H., Cho, S.W. et al., Tissue engineering of heart valves by recellularization of glutaraldehyde-fixed porcine values using bone marrow-derived cells, Exp. Mol. Med. 38, 273–283, 2006. Glutathione
γ-GluCysGly O
H N
Reducing agent; intermediate in phase II detoxification of xenobiotics.
O
OH
HN
O
307.32
SH
NH2
HO O Arias, I.M. and Jakoby, W.B., Glutathione, Metabolism and Function, Raven Press, New York, 1976; Meister, A., Glutamate, Glutamine, Glutathione, and Related Compounds, Academic Press, Orlando, FL, 1985; Sies, H. and Ketterer, B., Glutathione Conjugation: Mechanisms and Biological Significance, Academic Press, London, UK, 1988; Tsumoto, K., Shinoki, K., Kondo, H. et al., Highly efficient recovery of functional single-chain Fv fragments from inclusion bodies overexpressed in Escherichia coli by controlled introduction of oxidizing reagent — application to a human single-chain Fv fragment, J. Immunol. Methods 219, 119–129, 1998; Jiang, X., Ookubo, Y., Fujii, I. et al., Expression of Fab fragment of catalytic antibody 6D9 in an Escherichia coli in vitro coupled transcription/translation system, FEBS Lett. 514, 290–294, 2002; Sun, X.X., Vinci, C., Makmura, L. et al., Formation of disulfide bond in p53 correlates with inhibition of DNA binding and tetramerization, Antioxid. Redox Signal. 5, 655–665, 2003; Sies, H. and Packer, L., Eds., Glutathione Transferases and Gamma-Glutamyl Transpeptidases, Elsevier, Amsterdam, 2005; Smith, A.D. and Dawson, H., Glutathione is required for efficient production of infectious picornativur virions, Virology, 353, 258–267, 2006. Glycine
Aminoacetic Acid
75.07
Buffer component; protein-precipitating agent, excipient for pharmaceutical formulation.
NH2 CH H3C
OH C O
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Chemicals Commonly Used in Biochemistry and Molecular Biology and Their Properties (Continued) Common Name
Chemical Name
M.W.
Properties and Comment
Sarquis, J.L. and Adams, E.T., Jr., The temperature-dependent self-association of beta-lactoglobulin C in glycine buffers, Arch. Biochem. Biophys. 163, 442–452, 1974; Poduslo, J.F., Glycoprotein molecular-weight estimation using sodium dodecyl suflate-pore gradient electrophoresis: comparison of Tris-glycine and Tris-borate-EDTA buffer systems, Anal. Biochem. 114, 131–139, 1981; Patton, W.F., Chung-Welch, N., Lopez, M.F. et al., Tris-tricine and Tris-borate buffer systems provide better estimates of human mesothelial cell intermediate filament protein molecular weights than the standard Tris-glycine system, Anal. Biochem. 197, 25–33, 1991; Trasltas, G. and Ford, C.H., Cell membrane antigen-antibody complex dissociation by the widely used glycine-HC1 method: an unreliable procedure for studying antibody internalization, Immunol. Invest. 22, 1–12, 1993; Nail, S.L., Jiang, S., Chongprasert, S., and Knopp, S.A., Fundamentals of freeze-drying, Pharm. Biotechnol. 14, 281–360, 2002; Pyne, A., Chatterjee, K., and Suryanarayanan, R., Solute crystallization in mannitol-glycine systems — implications on protein stabilization in freeze-dried formulations, J. Pharm. Sci. 92, 2272–2283, 2003; Hasui, K., Takatsuka, T., Sakamoto, R. et al., Double immunostaining with glycine treatment, J. Histochem. Cytochem. 51, 1169–1176, 2003; Hachmann, J.P. and Amshey, J.W., Models of protein modification in Tris-glycine and neutral pH Bis-Tris gels during electrophoresis: effect of gel pH, Anal. Biochem. 342, 237–245, 2005. Glyoxal
Ethanedial
58.04
Modification of proteins and nucleic acids; model for glycation reaction; fluorescent derivates formed with tryptophan.
O C
H
H O Nakaya, K., Takenaka, O., Horinishi, H., and Shibata, K., Reactions of glyoxal with nucleic acids. Nucleotides and their component bases, Biochim. Biophys. Acta 161, 23–31, 1968; Canella, M. and Sodini, G., The reaction of horse-liver alcohol dehydrogenase with glyoxal, Eur. J. Biochem. 59, 119–125, 1975; Kai, M., Kojima, E., Okhura, Y., and Iwaski, M., High-performance liquid chromatography of N-terminal tryptophan-containing peptides with precolumn fluorescence derivatization with glyoxal, J. Chromatog. A. 653, 235–250, 1993; Murata-Kamiya, N., Kamiya, H., Kayi, H., and Kasai, H., Glyoxal, a major product of DNA oxidation, induces mutations at G:C sites on a shuttle vector plasmid replicated in mammalian cells, Nucleic Acids Res. 25, 1897–1902, 1997; Leng, F., Graves, D., and Chaires, J.B., Chemical crosslinking of ethidium to DNA by glyoxal, Biochim. Biophys. Acta 1442, 71–81, 1998; Thrornalley, P.J., Langborg, A., and Minhas, H.S., Formation of glyoxal, methylglyoxal, and 3-deoxyglucosone in the glycation of proteins by glucose, Biochem. J. 344, 109–116, 1999; Sady, C., Jiang, C.L., Chellan, P. et al., Maillard reactions by alpha-oxoaldehydes: detection of glyoxal-modified proteins, Biochim. Biophys. Acta 1481, 255–264, 2000; Olsen, R., Molander P., Ovrebo, S. et al., Reaction of glyoxal with 2′-deoxyguanosine, 2′-deoxyadenosine, 2′-deoxycytidine, cytidine, thymidine, and calf thymus DNA: identification of the DNA adducts, Chem. Res. Toxicol. 18, 730–739, 2005; Manini, P., La Pietra, P., Panzella, L. et al., Glyoxal formation by Fenton-induced degradation of carbohydrates and related compounds, Carbohydr. Res. 341, 1828–1833, 2006. Guanidine Guanidine Hydrochloride (GuCl) Guanidine Thiocyanate (GTIC)
Aminomethanamidine
NH
Chaotropic agent; guanidine hydrochloride use for study of protein denaturation; GTIC is considered to be more effective than GuCl; GTIC used for nucleic acid extraction.
NH2+
C H2N
59.07 95.53 118.16
NH2
pKa ~ 12.5
C H2N
NH2
Hill, R.L., Schwartz, H.C., and Smith, E.L., The effect of urea and guanidine hydrochloride on activity and optical rotation of crystalline papain, J. Biol. Chem. 234, 572–576, 1959; Appella, E. and Markert, C.L., Dissociation of lactate dehydrogenase into subunits with guanidine hydrochloride, Biochem. Biophys. Res. Commun. 6, 171–176, 1961; von Hippel, P.H. and Wong, K.-Y., On the conformational stability of globular proteins. The effects of various electrolytes and nonelectrolytes on the thermal transition ribonuclease transition, J. Biol. Chem. 240, 3909–3923, 1965; Katz, S., Partial molar volume and conformational changes produced by the denaturation of albumin by guanidine hydrochloride, Biochim. Biophys. Acta 154, 468–477, 1968; Shortle, D., Guanidine hydrochloride denaturation studies of mutant forms of staphylococcal nuclease, J. Cell Biochem. 30, 281–289, 1986; Lippke, J.A., Strzempko, M.N., Rai, F.F. et al., Isolation of intact high-molecular-weight DNA by using guanidine isothiocyanate, Appl. Environ. Microbiol. 53, 2588–2589, 1987; Alberti, S. and Fornaro, M., Higher transfection efficiency of genomic DNA purified with a guanidinium thiocyanate–based procedure, Nucleic Acids Res. 18, 351–353, 1990; Shirley, B.A., Urea and guanidine hydrochloride denaturation curves, Methods Mol. Biol. 40, 177–190, 1995; Cota, E. and Clarke, J., Folding of beta-sandwich proteins: three-state transition of a fibronectin type III module, Protein Sci. 9, 112–120, 2000; Kok, T., Wati, S., Bayly, B. et al., Comparison of six nucleic
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Chemicals Commonly Used in Biochemistry and Molecular Biology and Their Properties (Continued) Common Name
Chemical Name
M.W.
Properties and Comment
acid extraction methods for detection of viral DNA or RNA sequences in four different non-serum specimen types, J. Clin. Virol. 16, 59–63, 2000; Salamanca, S., Villegas, V., Vendrell, J. et al., The unfolding pathway of leech carboxypeptidase inhibitor, J. Biol. Chem. 277, 17538–17543, 2002; Bhuyan, A.K., Protein stabilization by urea and guanidine hydrochloride, Biochemistry 41, 13386–13394, 2002; Jankowska, E., Wiczk, W., and Grzonka, Z., Thermal and guanidine hydrochloride-induced denaturation of human cystatin C, Eur. Biophys. J. 33, 454–461, 2004; Fuertes, M.A., Perez, J.M., and Alonso, C., Small amounts of urea and guanidine hydrochloride can be detected by a far-UV spectrophotometric method in dialyzed protein solutions, J. Biochem. Biophys. Methods 59, 209–216, 2004; Berlinck, R.G., Natural guanidine derivatives, Nat. Prod. Rep. 22, 516–550, 2005; Rashid, F., Sharma, S., and Bano, B., Comparison of guanidine hydrochloride (GdnHCl) and urea denaturation on inactivation and unfolding of human placental cystatin (HPC), Biophys. J. 91, 686–693, 2006; Nolan, R.L. and Teller, J.K., Diethylamine extraction of proteins and peptides isolated with a mono-phasic solution of phenol and guanidine isothiocyanate, J. Biochem. Biophys. Methods 68, 127–131, 2006. Hydrazine
N2H4
32.05
Reducing agent; modification of aldehydes and carbohydrates; hydrazinolysis used for release of carbohydrates from protein; derivatives such as dinitrophenyl-hydrazine used for analysis of carbonyl groups in oxidized proteins; detection of acetyl and formyl groups in proteins.
Schmer, G. and Kreil, G., Micro method for detection of formyl and acetyl groups in proteins, Anal. Biochem. 29, 186–192, 1969; Gershoni, J.M., Bayer, E.A., and Wilchek, M., Blot analyses of glycoconjugates: enzyme-hydrazine — a novel reagent for the detection of aldehydes, Anal. Biochem. 146, 59–63, 1985; O’Neill, R.A., Enzymatic release of oligosaccharides from glycoproteins for chromatographic and electrophoretic analysis, J. Chromatog. A 720, 201–215, 1996; Routier, F.H., Hounsell, E.F., and Rudd, P.M., Quantitation of the oligosaccharides of human serum IgG from patients with rheumatoid arthritis: a critical evaluation of different methods, J. Immunol. Methods 213, 113–130, 1998; Robinson, C.E., Keshavarzian, A., Pasco, D.S. et al., Determination of protein carbonyl groups by immunoblotting, Anal. Biochem. 266, 48–57, 1999; Merry, A.H., Neville, D.C., Royle, L. et al., Recovery of intact 2-aminobenzamide-labeled O-glycans released from glycoproteins by hydrazinolysis, Anal. Biochem. 304, 91–99, 2002; Vinograd, E., Lindner, B., and Seltmann, G., Lipopolysaccharides from Serratia maracescens possess one or two 4-amino-4-deoxy-L-arabinopyranose 1-phosphate residues in the lipid A and D-glycero-D-talo-Oct-ulopyranosonic acid in the inner core region, Chemistry 12, 6692–6700, 2006. Hydrogen Peroxide Hydroxylamine 8-Hydroxyquinoline
H2O2 H3NO 8-quinolinol
34.02 33.03 145.16
Oxidizing agent; bacteriocidal agent. Metal chelator.
1,3-diazole
69.08
Buffer component.
Traut’s Reagent (earlier as methyl-4-mercaptobutyrimidate)
137.63
Introduction of sulfhydryl group by modification of amino group; sulfhydryl groups could then be oxidized to form cystine, which served as cleavable protein crosslink.
N OH Imidazole H N
N 2-Iminothiolane
S NH
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Chemicals Commonly Used in Biochemistry and Molecular Biology and Their Properties (Continued) Common Name
Chemical Name
M.W.
Properties and Comment
Traut, R.R., Bollen, A., Sun, T.-T. et al., Methyl-4-mercaptobutyrimidate as a cleavable crosslinking reagent and its application to the Escherichia coli 30S ribosome, Biochemistry 12, 3266–3273, 1973; Schram, H.J. and Dulffer, T., The use of 2-iminothiolane as a protein crosslinking reagent, Hoppe Seylers Z. Physiol.Chem. 358, 137–139, 1977; Jue, R., Lambert, J.M., Pierce, L.R., and Traut, R.R., Addition of sulfhydryl groups Escherichia coli ribosomes by protein modification with 2-iminothiolane (methyl 4-mercaptobutyrimidate), Biochemistry 17, 5399–5406, 1978; Lambert, J.M., Jue, R., and Traut, R.R., Disulfide crosslinking of Escherichia coli ribosomal proteins with 2-iminothiolane (methyl 4-mercaptobutyrimidate): evidence that the crosslinked protein pairs are formed in the intact ribosomal subunit, Biochemistry 17, 5406–5416, 1978; Alagon, A.C. and King, T.P., Activation of polysaccharides with 2-iminothiolane and its use, Biochemistry 19, 4341–4345, 1980; Tolan, D.R. and Traut, R.R., Protein topography of the 40 S ribosomal subunit from rabbit reticulocytes shown by crosslinking with 2-iminothiolane, J. Biol. Chem. 256, 10129–10136, 1981; Boileau, G., Butler, P., Hershey, J.W., and Traut, R.R., Direct crosslinks between initiation factors 1, 2, and 3 and ribosomal proteins promoted by 2-iminothiolane, Biochemistry 22, 3162–3170, 1983; Kyriatsoulis, A., Maly, P., Greuer, B. et al., RNA-protein crosslinking in Escherichia coli ribosomal subunits: localization of sites on 16S RNA which are crosslinked to proteins S17 and S21 by treatment with 2-iminothiolane, Nucleic Acids Res. 14, 1171–1186, 1986; Uchiumi, T., Kikuchi, M., and Ogata, K., Crosslinking study on protein neighborhoods at the subunit interface of rat liver ribosomes with 2-iminothiolane, J. Biol. Chem. 261, 9663–9667, 1986; McCall, M.J., Diril, H., and Meares, C.F., Simplified method for conjugating macrocyclic bifunctional chelating agents to antibodies via 2-iminothiolane, Bioconjug. Chem. 1, 222–226, 1990; Tarentino, A.L., Phelan, A.W., and Plummer, T.H., Jr., 2-iminothiolane: a reagent for the introduction of sulphydryl groups into oligosaccharides derived from asaparagine-linked glycans, Glycobiology 3, 279–285, 1993; Singh, R., Kats, L., Blattler, W.A., and Lambert, J.M., Formation of N-substituted 2-iminothiolanes when amino groups in proteins and peptides are modified by 2-iminothiolanes, Anal. Biochem. 236, 114–125, 1996; Hosono, M.N., Hosono, M., Mishra, A.K. et al., Rhenium-188-labeled anti-neural cell adhesion molecule antibodies with 2-iminothiolane modification for targeting small-cell lung cancer, Ann. Nucl. Med. 14, 173–179, 2000; Mokotoff, M., Mocarski, Y.M., Gentsch, B.L. et al., Caution in the use of 2-iminothiolane (Traut’s reagent) as a crosslinking agent for peptides. The formation of N-peptidyl-2-iminothiolanes with bombesin (BN) antagonists (D-trp 6-leu13-ψ[CH 2NH]-Phe 14BN 6-14 and D-trp- glntrp-NH2, J. Pept. Res. 57, 383–389, 2001; Kuzuhara, A., Protein structural changes in keratin fibers induced by chemical modification using 2-iminothiolane hydrochloride: a Raman spectroscopic investigation, Biopolymers 79, 173–184, 2005. Indole 2,3-benzopyrrole 117.15
N H Indole-3-acetic Acid
Indoleacetic Acid; Heteroauxin H2 C
175.19
Plant growth regulator.
O C OH
N H Kawaguchi, M. and Syono, K., The excessive production of indole-3-acetic and its significance in studies of the biosynthesis of this regulator of plant growth and development, Plant Cell Physiol. 37, 1043–1048, 1996; Normanly, J. and Bartel, B., Redundancy as a way of life-IAA metabolism, Curr. Opin. Plant Biol. 2, 207–213, 1999; Leyser, O., Auxin signaling: the beginning, the middle, and the end, Curr. Opin. Plant Biol. 4, 382–386, 2001; Ljung, K., Hull, A.K., Kowalczyk, M. et al., Biosynthesis, conjugation, catabolism, and homeostasis of indole-3-acetic acid in Arabidopsis thaliana, Plant Mol. Biol. 49, 249–272, 2002; Kawano, T. Roles of the reactive oxygen species-generating peroxidase reactions in plant defense and growth induction, Plant Cell Rep. 21, 829–837, 2003; Aloni, R., Aloni, E., Langhans, M., and Ullrich, C.I., Role of cytokine and auxin in shaping root architecture: regulating vascular differentiation, laterial root initiation, root apical dominance, and root gravitropism, Ann. Bot. 97, 882–893, 2006. Iodoacetamide
2-iodoacetamide
184.96
Alkylating agents that react with a variety of nucleophiles in proteins and nucleic acids. Reaction is more rapid than the bromo or chloro derivatives.
O I C H2
9168_Book.indb 786
NH2
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787
Chemicals Commonly Used in Biochemistry and Molecular Biology and Their Properties (Continued) Common Name
Chemical Name
Iodoacetic Acid
M.W.
Properties and Comment
185.95
O I OH
C H2
The amide is neutral and is not susceptible to either positive or negative influence from locally charged groups; iodoacetamide is frequently used to modify sulfhydryl groups as part of reduction and carboxymethylation prior to structural analysis. Crestfield, A.M., Moore, S., and Stein, W.H., The preparation and enzymatic hydrolysis of reduced and S-carboxymethylated proteins, J. Biol. Chem. 238, 622–627, 1963; Watts, D.C., Rabin, B.R., and Crook, E.M., The reaction of iodoacetate and iodoacetamide with proteins as determined with a silver/silver iodide electrode, Biochim. Biophys. Acta 48, 380–388, 1961; Inagami, T., The alkylation of the active site of trypsin with iodoacetamide in the presence of alkylguanidines, J. Biol. Chem. 240, PC3453–PC3455, 1965; Fruchter, R.G. and Crestfield, A.M., The specific alkylation by iodoacetamide of histidine-12 in the active site of ribonuclease, J. Biol. Chem. 242, 5807–5812, 1967; Takahashi, K., The structure and function of ribonuclease T. X. Reactions of iodoacetate, iodoacetamide, and related alkylating reagents with ribonuclease T, J. Biochem. 68, 517–527, 1970; Whitney, P.L., Inhibition and modification of human carbonic anhydrase B with bromoacetate and iodoacetate, Eur. J. Biochem. 16, 126–135, 1970; Harada, M. and Irie, M., Alkylation of ribonuclease from Aspirgillus saitoi with iodoacetate and iodoacetamide, J. Biochem. 73, 705–716, 1973; Halasz, P. and Polgar, L., Effect of the immediate microenvironment on the reactivity of the essential SH group of papain, Eur. J. Biochem. 71, 571–575, 1976; Franzen, J.S., Ishman, P., and Feingold, D.S., Half-of-the-sites reactivity of bovine liver uridine diphosphoglucose dehydrogenase toward iodoacetate and iodoacetamide, Biochemistry 15, 5665–5671, 1976; David, M., Rasched, I.R., and Sund, H., Studies of glutamate dehydrogenase. Methionione-169: the preferentially carboxymethylated residue, Eur. J. Biochem. 74, 379–385, 1977; Ohgi, K., Watanabe, H., Emman, K. et al., Alkylation of a ribonuclease from Streptomyces erthreus with iodoacetate and iodoacetamide, J. Biochem. 90, 113–123, 1981; Dahl, K.H. and McKinley-McKee, J.S., Enzymatic catalysis in the affinity labeling of liver alcohol dehydrogenase with haloacids, Eur. J. Biochem. 118, 507–513, 1981; Syvertsen, C. and McKinley-McKee, J.S., Binding of ligands to the catalytic zinc ion in horse liver alcohol dehydrogenase, Arch. Biochem. Biophys. 228, 159–169, 1984; Communi, D. and Erneux, C., Identification of an active site cysteine residue in type Ins(1,4,5)P35-phosphatase by chemical modification and site-directed mutagenesis, Biochem. J. 320, 181–186, 1996; Sarkany, Z., Skern, T., and Polgar, L., Characterization of the active site thiol group of rhinovirus 21 proteinase, FEBS Lett. 481, 289–292, 2000; Lundblad, R.L., Chemical Reagents for Protein Modification, CRC Press, Boca Raton, FL, 2004. Isatoic Anhydride
3,1-benzoxazine-2,4(1H)dione
163.13
Fluorescent reagents for amines and sulfydryl groups; amine scavenger.
O
O
N H
O
Gelb, M.H. and Abeles, R.H., Substituted isatoic anhydrides: selective inactivators of trypsinlike serine proteases, J. Med. Chem. 29, 585–589, 1986; Gravett, P.S., Viljoen, C.C., and Oosthuizen, M.M., Inactivation of arginine esterase E-1 of Bitis gabonica venom by irreversible inhibitors including a water-soluble carbodiimide, a chloromethyl ketone, and isatoic anhydride, Int. J. Biochem. 23, 1101–1110, 1991; Servillo, L., Balestrieri, C., Quagliuolo, L. et al., tRNA fluorescent labeling at 3′ end including an aminoacyl-tRNA-like behavior, Eur. J. Biochem. 213, 583–589, 1993; Churchich, J.E., Fluorescence properties of o-aminobenzoyl-labeled proteins, Anal. Biochem. 213, 229–233, 1993; Brown, A.D. and Powers, J.C., Rates of thrombin acylation and deacylation upon reaction with low molecular weight acylating agents, carbamylating agents, and carbonylating agents, Bioorg. Med. Chem. 3, 1091–1097, 1995; Matos, M.A., Miranda, M.S., Morais, V.M., and Liebman, J.F., Are isatin and isatoic anhydride antiaromatic and aromatic, respectively? A combined experimental and theoretic investigation, Org. Biomol. Chem. 1, 2566–2571, 2003; Matos, M.A., Miranda, M.S., Morais, V.M., and Liebman, J.F., The energetics of isomeric benzoxazine diones: isatoic anhydride revisited, Org. Biomol. Chem. 2, 1647–1650, 2004; Raturi, A., Vascratsis, P.O., Seslija, D. et al., A direct, continuous, sensitive assay for protein disulphide-isomerase based on fluorescence self-quenching, Biochem. J. 391, 351–357, 2005; Zhang, W., Lu, Y., and Nagashima, T., Plate-to-plate fluorous solid-phase extraction for solution-phase parallel synthesis, J. Comb. Chem. 7, 893–897, 2005. Isoamyl Alcohol
Isopentyl Alcohol; 3-methyl-1-butanol
88.15
Solvent.
CH3 H2 C
CH H3C
9168_Book.indb 787
C H2
OH
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788
Chemicals Commonly Used in Biochemistry and Molecular Biology and Their Properties (Continued) Common Name
Chemical Name
Isopropanol
2-propanol
M.W. 60.10
Properties and Comment Solvent; precipitation agent for purification of plasmid DNA; reagent in stability test for identification of abnormal hemoglobins.
CH3 CH H3C
OH
Brosious, E.M., Morrison, B.Y., and Schmidt, R.M., Effects of hemoglobin F levels, KCN, and storage on the isopropanol precipitation test for unstable hemoglobins, Am. J. Clin. Pathol. 66, 878–882, 1976; Bensinger, T.A. and Beutler, E., Instability of the oxy form of sickle hemoglobin and of methemoglobin in isopropanol, Am. J. Clin. Pathol. 67, 180–183, 1977; Acree, W.E., Jr. and Bertrand, G.L., A cholesterol-isopropanol gel, Nature 269, 450, 1977; Naoum, P.C. Teixeira, U.A., de Abreu Machado, P.E., and Michelin, O.C., The denaturation of human oxyhemoglobin A, A2, and S by isopropanol/buffer method, Rev. Bras. Pesqui. Med. Biol. 11, 241–244, 1978; Ali, M.A., Quinlan, A., and Wong, S.C., Identification of hemoglobin E by the isopropanol solubility test, Clin. Biochem. 13, 146–148, 1980; Horer, O.L. and Enache, C., 2-propanol dependent RNA absorbances, Virologie 34, 257–272, 1983; De Vendittis, E., Masullo, M., and Bocchini, V., The elongation factor G carries a catalytic site for GTP hydrolysis, which is revealed by using 2-propanol in the absence of ribosomes, J. Biol. Chem. 261, 4445–4450, 1986; Wang, L., Hirayasu, K., Ishizawa, M., and Kobayashi, Y., Purification of genomic DNA from human whole blood by isopropanol-fractionation with concentrated NaI and SDS, Nucleic Acids Res. 22, 1774–1775, 1994; Dalhus, B. and Gorbitz, C.H., Glycyl-L-leucyl-L-tyrosine dehydrate 2-propanol solvate, Acta Crystallogr. C 52, 2087–2090, 1996; Freitas, S.S., Santos, J.A., and Prazeres, D.M., Optimization of isopropanol and ammonium sulfate precipitation steps in the purification of plasmid DNA, Biotechnol. Prog. 22, 1179–1186, 2006; Halano, B., Kubo, D., and Tagaya, H., Study on the reactivity of diarylmethane derivatives in supercritical alcohols media: reduction of diarylmethanols and diaryl ketones to diarylmethanes using supercritical 2-propanol, Chem. Pharm. Bull. 54, 1304–1307, 2006. Isopropyl-β-D-thiogalactoside
OH OH
IPTG, Isopropyl-β-Dthiogalactopyroa-noside
238.3
“Gratuitous” inducer of the lac operon.
H3C H
O
CH
CH3
H S
HO H H
OH H
Cho, S., Scharpf, S., Franko, M., and Vermeulen, C.W., Effect of isopropyl-β-D-galactoside concentration on the level of lac-operon induction in steady state Escherichia coli, Biochem. Biophys. Res. Commun. 128, 1268–1273, 1985; Carlsson, U., Ferskgard, P.O., and Svensson, S.C., A simple and efficient synthesis of the induced IPTG made for inexpensive heterologous protein production using the lac-promoter, Protein Eng. 4, 1019–1020, 1991; Donovan, R.S., Robinson, C.W., and Glick, B.R., Review: optimizing inducer and culture conditions for expression of foreign proteins under control of the lac promoter, J. Ind. Microbiol. 16, 145–154, 1996; Hansen, L.H., Knudsen, S., and Sorensen, S.J., The effect of the lacy gene on the induction of IPTG-inducible promoters, studied in Escherichia coli and Pseudomonas fluorescens, Curr. Microbiol. 36, 341–347, 1998; Teich, A., Lin, H.Y., Andersson, L. et al., Amplification of ColE1 related plasmids in recombinant cultures of Escherichia coli after IPTG induction, J. Biotechnol. 64, 197–210, 1998; Ren, A. and Schaefer, T.S., Isopropyl-β-D-thiogalactoside (IPTG)-inducible tyrosine phosphorylation of proteins in E. coli, Biotechniques 31, 1254–1258, 2001; Ko, K.S., Kruse, J., and Pohl, N.L., Synthesis of isobutryl-Cgalactoside (IBCG) as an isopropylthiogalactoside (IPTG) substitute for increased induction of protein expression, Org. Lett. 5, 1781–1783, 2003; Intasai, N., Arooncharus, P., Kasinrerk, W., and Tayapiwatana, C., Construction of high-density display of CD147 ectodomain on VCSM13 phage via gpVIII: effects of temperature, IPTG, and helper phage infection-period, Protein Expr. Purif. 32, 323–331, 2003; Faulkner, E., Barrett, M., Okor, S. et al., Use of fed-batch cultivation for achieving high cell densities for the pilot-scale production of a recombinant protein (phenylalanine dehydrogenase) in Escherichia coli, Biotechnol. Prog. 22, 889–897, 2006; Gardete, S., de Laencastre, H., and Tomasz, A., A link in transcription between the native pbpG and the acquired mecA gene in a strain of Staphylococcus aureus, Microbiology 152, 2549–2558, 2006; Hewitt, C.J., Onyeaka, H., Lewis, G. et al., A comparison of high cell density fed-batch fermentations involving both induced and noninduced recombinant Escherichia coli under well-mixed small-scale and simulated poorly mixed large-scale conditions, Biotechnol. Bioeng., in press, 2006; Picaud, S., Olsson, M.E., and Brodelius, P.E., Improved conditions for production of recombinant plant sesquiterpene synthases in Escherichia coli, Protein Expr. Purif., in press, 2006.
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789
Chemicals Commonly Used in Biochemistry and Molecular Biology and Their Properties (Continued) Common Name
Chemical Name
Maleic Anhydride
O
2,5-furandione
M.W. 98.06
Properties and Comment Modification of amino groups in proteins. The dimethyl derivative (dimethylmaleic anhydride) is used for ribosome dissociation; monomer for polymer.
O
O
Giese, R.W. and Vallee, B.L., Metallocenes. A novel class of reagents for protein modification. I. Maleic anhydride-iron tetracarbonyl, J. Am. Chem. Soc. 94, 6199–6200, 1972; Cantrell, M. and Craven, G.R., Chemical inactivation of Escherichia coli 30 S ribosomes with maleic anhydride: identification of the proteins involved in polyuridylic acid binding, J. Mol. Biol. 115, 389–402, 1977; Jordano, J., Montero, F., and Palacian, E., Relaxation of chromatin structure upon removal of histones H2A and H2B, FEBS Lett. 172, 70–74, 1984; Jordano, J., Montero, F., and Palacian, E., Rearrangement of nucleosomal components by modification of histone amino groups. Structural role of lysine residues, Biochemistry 23, 4280–4284, 1984; Palacian, E., Gonzalez, P.J., Pineiro, M., and Hernandez, F., Dicarboxylic acid anhydrides as dissociating agents of protein-containing structures, Mol. Cell. Biochem. 97, 101–111, 1990; Paetzel, M., Strynadka, N.C., Tschantz, W.R. et al., Use of site-directed chemical modification to study an essential lysine in Escherichia coli leader peptidase, J. Biol. Chem. 272, 9994–10003, 1997; Wink, M.R., Buffon, A., Bonan, C.D. et al., Effect of protein-modifying reagents on ecto-apyrase from rat brain, Int. J. Biochem. Cell Biol. 32, 105–113, 2000. 2-Mercaptoethanol
H2 C HS
β-mercaptoethanol
78.13
Reducing agent; used frequently in the reduction and alkylation of proteins for structural analysis and for preservation of oxidation-sensitive enzymes.
OH C H2
Geren, C.R., Olomon, C.M., Jones, T.T., and Ebner, D.E., 2-mercaptoethanol as a substrate for liver alcohol dehydrogenase, Arch. Biochem. Biophys. 179, 415–419, 1977; Opitz, H.G., Lemke, H, and Hewlett, G., Activation of T-cells by a macrophage or 2-mercaptoethanol-activated serum factor is essential for induction of a primary immune response to heterologous red cells in vitro, Immunol. Rev. 40, 53–77, 1978; Burger, M., An absolute requirement for 2-mercaptoethanol in the in vitro primary immune response in the absence of serum, Immunology 37, 669–671, 1979; Nealon, D.A., Pettit, S.M., and Henderson, A.R., Diluent pH and the stability of the thiol group in monothioglycerol, N-acetyl-L-cysteine, and 2-mercaptoethanol, Clin. Chem. 27, 505–506, 1981; Dahl, K.H. and McKinley-McKee, J.S., Enzymatic catalysis in the affinity labeling of liver alcohol dehydrogenase with haloacids, Eur. J. Biochem. 118, 507–513, 1981; Righetti, P.G., Tudor, G., and Glanazza, E., Effect of 2-mercaptoethanol on pH gradients in isoelectric focusing, J. Biochem. Biophys. Methods 6, 219–227, 1982; Soderberg, L.S. and Yeh, N.H., T-cells and the anti-trinitrophenyl antibody response to fetal calf serum and 2-mercaptoethanol, Proc. Soc. Exp. Biol. Med. 174, 107–113, 1983; Ochs, D., Protein contaminants of sodium dodecyl sulfate-polyacrylamide gels, Anal. Biochem. 135, 470–474, 1983; Schaefer, W.H., Harris, T.M., and Guengerich, F.P., Reaction of the model thiol 2-mercaptoethanol and glutathione with methylvinylmaleimide, a Michael acceptor with extended conjugation, Arch. Biochem. Biophys. 257, 186–193, 1987; Obiri, N. and Pruett, S.B., The role of thiols in lymphocyte responses: effect of 2-mercaptoethanol on interleukin 2 production, Immunobiology 176, 440–449, 1988; Gourgerot-Pocidalo, M.A., Fay, M., Roche, Y., and Chollet-Martin, S., Mechanisms by which oxidative injury inhibits the proliferative response of human lymphocytes to PHA. Effect of the thiol compound 2-mercaptoethanol, Immunology 64, 281–288, 1988; Fong, T.C. and Makinodan, T., Preferential enhancement by 2-mercaptoethanol of IL-2 responsiveness of T blast cells from old over young mice is associated with potentiated protein kinase C translocation, Immunol. Lett. 20, 149–154, 1989; De Graan, P.N., Moritz, A., de Wit, M., and Gispen, W.H., Purification of B-50 by 2-mercaptoethanol extraction from rat brain synaptosomal plasma membranes, Neurochem. Res. 18, 875–881, 1993; Carrithers, S.L. and Hoffman, J.L., Sequential methylation of 2-mercaptoenthanol to the dimethyl sulfonium ion, 2-(dimethylthio)ethanol, in vivo and in vitro, Biochem. Pharmacol. 48, 1017–1024, 1994; Paul-Pretzer, K. and Parness, J., Elimination of keratin contaminant from 2-mercaptoethanol, Anal. Biochem. 289, 98–99, 2001; Adebiyi, A.P., Jin, D.H, Ogawa, T., and Muramoto, K., Acid hydrolysis of protein in a microcapillary tube for the recovery of tryptophan, Biosci. Biotechnol. Biochem. 69, 255–257, 2005; Adams, B., Lowpetch, K., Throndycroft, F. et al., Stereochemistry of reactions of the inhibitor/substrates L- and D-β-chloroalanine with β-mercaptoethanol catalyzed by L-aspartate aminotransferase and D-amino acid amino-transferase, respectively, Org. Biomol. Chem. 3, 3357–3364, 2005; Layeyre, M., Leprince, J., Massonneau, M. et al., Aryldithioethyloxycarbonyl (Ardec): a new family of amine-protecting groups removable under mild reducing conditions and their applications to peptide synthesis, Chemistry 12, 3655–3671, 2006; Okun, I., Malarchuk, S., Dubrovskaya, E. et al., Screening for caspace-3 inhibitors: effect of a reducing agent on the identified hit chemotypes, J. Biomol. Screen. 11, 694–703, 2006; Aminian, M., Sivam, S., Lee, C.W. et al., Expression and purification of a trivalent pertussis toxin-diphtheria toxin-tetanus toxin fusion protein in Escherichia coli, Protein Expr. Purif. 51, 170–178, 2006.
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790
Chemicals Commonly Used in Biochemistry and Molecular Biology and Their Properties (Continued) Common Name
Chemical Name
(3-Mercaptopropyl)trimethoxysilane
HS
O
H2 C
H2 C C H2
3-(trimethoxysilyl)-1propanethiol
M.W. 196.34
Properties and Comment Introduction of reactive sulfhydryl onto glass (silane) surface.
CH3
Si O
O
CH3 CH3
Jung, S.K. and Wilson, G.S., Polymeric mercaptosilane-modified platinum electrodes for elimination of interferants in glucose biosensors, Anal. Chem. 68, 591–596, 1996; Mansur, H.S., Lobato, Z.P., Orefice, R.L. et al., Surface functionalization of porous glass networks: effects on bovine serum albumin and porcine insulin immobilization, Biomacromolecules 1, 479–497, 2000; Kumar, A., Larsson, O., Parodi, D., and Liang, Z., Silanized nucleic acids: a general platform for DNA immobilization, Nucleic Acids Res. 28, E71, 2000; Zhang, F., Kang, E.T., Neoh, K.G. et al., Surface modification of stainless steel by grafting of poly(ethylene glycol) for reduction in protein adsorption, Biomaterials 22, 1541–1548, 2001; Jia, J., Wang, B., Wu, A. et al., A method to construct a third-generation horseradish peroxidase biosensor: self-assembling gold nanoparticles to three-dimensional sol-gel network, Anal. Chem. 74, 2217–2223, 2002; Abdelghani-Jacquin, C., Abdelghani, A., Chmel, G. et al., Decorated surfaces by biofunctionalized gold beads: application to cell adhesion studies, Eur. Biophys. J. 31, 102–110, 2002; Ganesan, V. and Walcarius, A., Surfactant templated sulfonic acid functionalized silica microspheres as new efficient ion exchangers and electrode modifiers, Langmuir 20, 3632–3640, 2004; Crudden, C.M., Sateesh, M., and Lewis, R., Mercaptopropyl-modified mesoporous silica: a remarkable support for the preparation of a reusable, heterogeneous palladium catalyst for coupling to reactions, J. Am. Chem. Soc. 127, 10045–10050, 2005; Yang, L., Guihen, E., and Glennon, J.D., Alkylthiol gold nanoparticles in sol-gel-based open tabular capillary electrochromatography, J. Sep. Sci. 28, 757–766, 2005. Methanesulfonic Acid
96.11
Protein hydrolysis for amino acid analysis; deprotection during peptide synthesis; hydrolysis of protein substituents such as fatty acids.
O H3C
S
OH
O Simpson, R.J., Neuberger, M.R., and Liu, T.Y., Complete amino acid analysis of proteins from a single hydrolyzate, J. Biol. Chem. 251, 1936–1940, 1976; Kubota, M., Hirayama, T., Nagase, O., and Yajima, H., Synthesis of two peptides corresponding to an alpha-endophin and gammaendorphin by the methanesulfonic acid deprotecting procedures, Chem. Pharm. Bull. 27, 1050–1054, 1979; Yajima, H., Akaji, K., Saito, H. et al., Studies on peptides. LXXXII. Synthesis of [4-Gln]-neurotensin by the methanesulfonic acid deprotecting procedure, Chem. Pharm. Bull. 27, 2238–2242, 1979; Sakuri, J. and Nagahama, M. Tryptophan content of Clostridium perfringens epsilon toxin, Infect. Immun. 47, 260–263, 1985; Malmer, M.F. and Schroeder, L.A., Amino acid analysis by high-performance liquid chromatography with methanesulfonic acid hydrolysis and 9-fluorenylmethyl-chloroformate derivatization, J. Chromatog. 514, 227–239, 1990; Weiss, M., Manneberg, M., Juranville, J.F. et al., Effect of the hydrolysis method on the determination of the amino acid composition of proteins, J. Chromatog. A 795, 263–275, 1998; Okimura, K., Ohki, K., Nagai, S., and Sakura, N., HPLC analysis of fatty acyl-glycine in the aqueous methanesulfonic acid hydrolysates of N-terminally fatty acylated peptides, Biol. Pharm. Bull. 26, 1166–1169, 2003; Wrobel, K., Kannamkumarath, S.S., Wrobel, K., and Caruso, J.A., Hydrolysis of proteins with methanesulfonic acid for improved HPLC-ICP-MS determination of seleno-methionine in yeast and nuts, Anal. BioAnal. Chem. 375, 133–138, 2003. Methanol Methylethyl Ketone (MEK)
Methyl Alcohol 2-butanal; 2-butanone
32.04 72.11
Solvent. Solvent; with acid for cleavage of heme moiety of hemeproteins for preparation of apoproteins.
O CH3 H3C
C H2
Teale, F.W., Cleavage of haem-protein link by acid methylethylketone, Biochim. Biophys. Acta 35, 543, 1959; Tran, C.D. and Darwent, J.R., Characterization of tetrapyridylporphyrinatozinc (II) apomyoglobin complexes as a potential photosynthetic model, J. Chem. Soc. Faraday Trans. II, 82, 2315–2322, 1986.
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791
Chemicals Commonly Used in Biochemistry and Molecular Biology and Their Properties (Continued) Common Name
Chemical Name
Methylglyoxal
Pyruvaldehyde; 2-oxopropanal
M.W. 72.06
Properties and Comment Derived from oxidative modification of triose phosphate during glucose metabolism; model for glycation of proteins; reacts with amino groups in proteins and nucleic acids; involved in advanced glycation endproducts.
O H
C C
H3C
O Szabo, G., Kertesz, J.C., and Laki, K., Interaction of methylglyoxal with poly-L-lysine, Biomaterials 1, 27–29, 1980; McLaughlin, J.A., Pethig, R., and Szent-Gyorgyi, A., Spectroscopic studies of the protein-methylglyoxal adduct, Proc. Natl. Acad. Sci. USA 77, 949–951, 1980; Cooper, R.A., Metabolism of methylglyoxal in microorganisms, Annu. Rev. Microbiol. 38, 49–68, 1984; Richard, J.P., Mechanism for the formation of methylglyoxal from triosephosphates, Biochem. Soc. Trans. 21, 549–553, 1993; Riley, M.L. and Harding, J.J., The reaction of methylglyoxal with human and bovine lens proteins, Biochim. Biophys. Acta 1270, 36–43, 1995; Thornalley, P.J., Pharmacology of methylglyoxal: formation, modification of proteins and nucleic acids, and enzymatic detoxification — a role in pathogenesis and antiproliferative chemotherapy, Gen. Pharmacol. 27, 565–573, 1996; Nagaraj, R.H., Shipanova, I.N., and Faust, F.M., Protein crosslinking by the Maillard reaction. Isolation, characterization, and in vivo detection of a lysine–lysine crosslink derived from methylglyoxal, J. Biol. Chem. 271, 19338–19345, 1996; Shipanova, I.N., Glomb, M.A., and Nagaraj, R.H., Protein modification by methylglyoxal: chemical nature and synthetic mechanism of a major fluorescent adduct, Arch. Biochem. Biophys. 344, 29–34, 1997; Uchida, K., Khor, O.T., Oya, T. et al., Protein modification by a Maillard reaction intermediate methylglyoxal. Immunochemical detection of fluorescent 5-methylimidazolone derivatives in vivo, FEBS Lett. 410, 313–318, 1997; Degenhardt, T.P., Thorpe, S.R., and Baynes, J.W., Chemical modification of proteins by methylglyoxal, Cell. Mol. Biol. 44, 1139–1145, 1998; Izaguirre, G., Kikonyogo, A., and Pietruszko, R., Methylglyoxal as substrate and inhibitor of human aldehyde dehydrogenase: comparison of kinetic properties among the three isozymes, Comp. Biochem. Physiol. B Biochem. Mol. Biol. 119, 747–754, 1998; Lederer, M.O. and Klaiber, R.G., Crosslinking of proteins by Maillard processes: characterization and detection of lysine–arginine crosslinks derived from glyoxal and methylglyoxal, Bioorg. Med. Chem. 7, 2499–2507, 1999; Kalapos, M.P., Methylglyoxal in living organisms: chemistry, biochemistry, toxicology, and biological implications, Toxicol. Lett. 110, 145–175, 1999; Thornalley, P.J., Landborg, A., and Minhas, H.S., Formation of glyoxal, methylglyoxal, and 3-deoxyglucose in the glycation of proteins by glucose, Biochem. J. 344, 109–116, 1999; Nagai, R., Araki, T., Hayashi, C.M. et al., Identification of N-epsilon-(carboxyethyl)lysine, one of the methylglyoxal-derived AGE structures, in glucose-modified protein: mechanism for protein modification by reactive aldehydes, J. Chromatog. B Analyt. Technol. Biomed. Life Sci.788, 75–84, 2003. Methyl Methanethiosulfonate (MMTS)
S-methyl Methanethiosulfonate
126.2
Modification of sulfhydryl groups.
O H3C
S
S
CH3
O Smith, D.J., Maggio, E.T., and Kenyon, G.L., Simple alkanethiol groups for temporary sulfhydryl groups of enzymes, Biochemistry 14, 766–771, 1975; Nishimura, J.S., Kenyon, G.L., and Smith, D.J., Reversible modification of the sulfhydryl groups of Escherichia coli succinic thiokinase with methanethiolating reagents, 5,5′-dithio-bis(2-nitrobenzoic acid), p-hydroxymercuribenzoate, and ethylmercurithiosalicylate, Arch. Biochem. Biophys. 170, 407–430, 1977; Bloxham, D.P., The chemical reactivity of the histidine-195 residue in lactate dehydrogenase thiomethylated at the cysteine-165 residue, Biochem. J. 193, 93–97, 1981; Gavilanes, F., Peterson, D., and Schirch, L., Methyl methanethiosulfate as an active site probe of serine hydroxymethyltransferase, J. Biol. Chem. 257, 11431–11436, 1982; Daly, T.J., Olson, J.S., and Matthews, K.S., Formation of mixed disulfide adducts as cysteine-281 of the lactose repressor protein affects operator- and inducer-binding parameters, Biochemistry 25, 5468–5474, 1986; Salam, W.H. and Bloxham, D.P., Identification of subsidiary catalytic groups at the active site of β-ketoacyl-CoA thiolase by covalent modification of the protein, Biochim. Biophys. Acta 873, 321–330, 1986; Stancato, L.F., Hutchison, K.A., Chakraborti, P.K. et al., Differential effects of the reversible thiol-reactive agents arsenite and methyl methanethiosulfonate on steroid binding by the glucocorticoid receptor, Biochemistry 32, 3739–3736, 1993; Hou, L.X. and Vollmer, S., The activity of S-thiolated modified creatine kinase is due to the regeneration of free thiol at the active site, Biochim. Biophys. Acta 1205, 83–88, 1994; Jensen, P.E., Shanbhag, V.P., and Stigbrand, T., Methanethiolation of the liberated cysteine residues of human α-2-macroglobulin treated with methylamine generates a derivative with similar functional characteristics as native β-2-macroglobulin, Eur. J. Biochem. 227, 612–616, 1995; Trimboli, A.J., Quinn, G.B., Smith, E.T., and Barber, M.J., Thiol modification and site-directed mutagenesis of the flavin domain of spinach NADH: nitrate reductase, Arch. Biochem. Biophys. 331, 117–126, 1996; Quinn, K.E. and Ehrlich, B.E., Methanethiosulfonate derivatives inhibits current through the rynodine receptor/channel, J. Gen. Physiol. 109, 225–264, 1997;
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Chemicals Commonly Used in Biochemistry and Molecular Biology and Their Properties (Continued) Common Name
Chemical Name
M.W.
Properties and Comment
Hashimoto, M., Majima, E., Hatanaka, T. et al., Irreversible extrusion of the first loop facing the matrix of the bovine heart mitochondrial ADP/ATP carrier by labeling the Cys(56) residue with the SH-reagent methyl methanethiosulfonate, J. Biochem. 127, 443–449, 2000; Spelta, V., Jiang, L.H., Bailey, R.J. et al., Interaction between cysteines introduced into each transmembrane domain of the rat P2X2 receptor, Br. J. Pharmacol. 138, 131–136, 2003; Britto, P.J., Knipling, L., McPhie, P., and Wolff, J., Thiol-disulphide interchange in tubulin: kinetics and the effect on polymerization, Biochem. J. 389, 549–558, 2005; Miller, C.M., Szegedi, S.S., and Garrow, T.A., Conformation-dependent inactivation of human betaine-homocysteine S-methyltransferase by hydrogen peroxide in vitro, Biochem. J. 392, 443–448, 2005. N-Methylpyrrolidone
1-methyl-2-pyrrolidone
99.13
Polar solvent; transdermal transport of drugs.
O N CH3 Barry, B.W. and Bennett, S.L., Effect of penetration enhancers on the permeation of mannitol, hydrocortisone, and progesterone through human skin, J. Pharm. Pharmacol. 39, 535–546, 1987; Forest, M. and Fournier, A., BOP reagent for the coupling of pGlu and Boc-His(Tos) in solid phase peptide synthesis, Int. J. Pept. Protein Res. 35, 89–94, 1990; Sasaki, H., Kojima, M., Nakamura, J., and Shibasaki, J., Enhancing effect of combining two pyrrolidone vehicles on transdermal drug delivery, J. Pharm. Pharmacol. 42, 196–199, 1990; Uch, A.S., Hesse, U., and Dressman, J.B., Use of 1-methyl-pyrrolidone as a solubilizing agent for determining the uptake of poorly soluble drugs, Pharm. Res. 16, 968–971, 1999; Zhao, F. Bhanage, B.M., Shirai, M., and Arai, M., Heck reactions of iodobenzene and methyl acrylate with conventional supported palladium catalysts in the presence of organic and/or inorganic bases without ligands, Chemistry 6, 843–848, 2000; Lee, P.J., Langer, R., and Shastri, V.P., Role of n-methyl pyrrolidone in the enhancement of aqueous phase transdermal transport, J. Pharm. Sci. 94, 912–917, 2005; Tae, G., Kornfield, J.A., and Hubbell, J.A., Sustained release of human growth hormone from in situ forming hydrogels using self-assembly of fluoroalkyl-ended poly(ethylene glycol), Biomaterials 26, 5259–5266, 2005; Babu, R.J. and Pandit, J.K., Effect of penetration enhancers on the transdermal delivery of bupranolol through rat skin, Drug Deliv. 12, 165–169, 2005; Luan, X. and Bodmeier, R., In situ forming microparticle system for controlled delivery of leupolide acetate: influence of the formulation and processing parameters, Eur. J. Pharm. Sci. 27, 143–149, 2006; Lee, P.J., Ahmad, N., Langer, R. et al., Evaluation of chemical enhancers in the transdermal delivery of lidocaine, Int. J. Pharm. 308, 33–39, 2006; Ruble, G.R., Giardino, O.X., Fossceco, S.L. et al., J. Am. Assoc. Lab. Anim. Sci. 45, 25–29, 2006. NBS
N-bromosuccinimide; 1-bromo-2,5pyrrolidinedione
178
Protein modification reagent; bromination of olefins; analysis of a variety of other compounds.
Br O
N O
Sinn, H.J., Schrenk, H.H., Friedrich, E.A. et al., Radioiodination of proteins and lipoproteins using N-bromosuccinimide as oxidizing agent, Anal. Biochem. 170, 186–192, 1988; Tanemura, K., Suzuki, T., Nishida, Y. et al., A mild and efficient procedure for α-bromination of ketones using N-bromosuccinimide catalyzed by ammonium acetate, Chem. Commun. 3, 470–471, 2004; Lundblad, R.L., Chemical Reagents for Protein Modification, 3rd ed., CRC Press, Boca Raton, FL, 2004; Edens, G.J., Redox titration of antioxidant mixtures with N-bromosuccinimide as titrant: analysis by nonlinear least-squares with novel weighting function, Anal. Sci. 21, 1349–1354, 2005; Abdel-Wadood, H.M., Mohamed, H.A., and Mohamed, F.A., Spectrofluorometric determination of acetaminophen with N-bromosuccinimide, J. AOAC Int. 88, 1626–1630, 2005; Krebs, A., Starczyewska, B., Purzanowska-Tarasiewicz, H., and Sledz, J., Spectrophotometric determination of olanzapine by its oxidation with N-bromosuccinimide and cerium(IV) sulfate, Anal. Sci. 22, 829–833, 2006; Braddock, D.C., Cansell, G., Hermitage, S.A., and White, A.J., Bromoiodinanes with a I(III)-Br bond: preparation, X-ray crystallography, and reactivity as electrophilic brominating agents, Chem. Commun. 13, 1442–1444, 2006; Chen, G., Sasaki, M., Li, X., and Yudin, A.K., Strained enamines as versatile intermediates for stereocontrolled construction of nitrogen heterocycles, J. Org. Chem. 71, 6067–6073, 2006; Braddock D.C., Cansell, G., and Hermitage, S.A., Ortho-substituted iodobenzenes as novel organocatalysts for the transfer of electrophilic bromine from N-bromosuccinmide to alkenes, Chem. Commun. 23, 2483–2485, 2006.
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793
Chemicals Commonly Used in Biochemistry and Molecular Biology and Their Properties (Continued) Common Name
Chemical Name
NHS
N-hydroxysuccinimide; 1-hydroxy-2,5pyrrolidinedione
M.W. 111.1
Properties and Comment Use in preparation of active esters for modification of amino groups (with carbodiimide); structural basis for reagents for amino group modification.
O
N
OH
O Anderson, G.W., Callahan, F.M., and Zimmerman, J.E., Synthesis of N-hydroxysuccinimide esters of acyl peptides by the mixed anhydride method, J. Am. Chem. Soc. 89, 178, 1967; Lapidot, Y., Rappoport, S., and Wolman, Y., Use of esters of N-hydroxysuccinimide in the synthesis of N-acylamino acids, J. Lipid Res. 8, 142–145, 1967; Holmquist, B., Blumberg, S., and Vallee, B.L., Superactivation of neutral proteases: acylation with N-hydroxysuccinimide esters, Biochemistry 15, 4675–4680, 1976; ‘t Hoen, P.A., de Kort, F., van Ommen, G.J., and den Dunnen, J.T., Fluorescent labeling of cRNA for microarray applications, Nucleic Acids Res. 31, e20, 2003; Vogel, C.W., Preparation of immunoconjugates using antibody oligosaccharide moieties, Methods Mol. Biol. 283, 87–108, 2004; Cooper, M., Ebner, A., Briggs, M. et al., Cy3B: improving the performance of cyanine dyes, J. Fluoresc. 14, 145–150, 2004; Lundblad, R.L., Chemical Reagents for Protein Modification, 3rd ed., CRC Press, Boca Raton, FL, 2004; Zhang, R., Tang, M., Bowyer, A. et al., A novel pH- and ionic-strength-sensitive carboxy methyl dextran hydrogel, Biomaterials 26, 4677– 4683, 2005; Tyan, Y.C., Jong, S.B., Liao, J.D. et al., Proteomic profiling of erythrocyte proteins by proteolytic digestion chip and identification using two-dimensional electrospray ionization tandem mass spectrometry, J. Proteome Res. 4, 748–757, 2005; Lovrinovic, M., Spengler, M., Deutsch, C., and Niemeyer, C.M., Synthesis of covalent DNA-protein conjugates by expressed protein ligation, Mol. Biosyst. 1, 64–69, 2005; Smith, G.P., Kinetics of amine modification of proteins, Bioconjug. Chem. 17, 501–506, 2006; Yang, W.C., Mirzael, H., Liu, X., and Regnier, F.E., Enhancement of amino acid detection and quantitation by electrospray ionization mass spectrometry, Anal. Chem. 78, 4702–4708, 2006; Yu, G., Liang, J., He, Z., and Sun, M., Quantum dot-mediated detection of gamma-aminobutyric acid binding sites on the surface of living pollen protoplasts in tobacco, Chem. Biol. 13, 723–731, 2006; Adden, N., Gamble, L.J., Castner, D.G. et al., Phosphonic acid monolayers for binding of bioactive molecules to titanium surfaces, Langmuir 22, 8197–8204, 2006. Ninhydrin
1-H-indene-1,2,3-trione Monohydrate
178.14
Reagent for amino acid analysis; reagent for modification of arginine residues in proteins; reaction with amino groups and other nucleophiles such as sulfhydryl groups.
O OH
OH O Duliere, W.L., The amino-groups of the proteins of human serum. Action of formaldehyde and ninhydrin, Biochem. J. 30, 770–772, 1936; Schwartz, T.B. and Engel, F.L., A photometric ninhydrin method for the measurement of proteolysis, J. Biol. Chem. 184, 197–202, 1950; Troll, W. and Cannan, R.K., A modified photometric ninhydrin method for the analysis of amino and imino acids, J. Biol. Chem. 200, 803–811, 1953; Moore, S. and Stein, W.H., A modified ninhydrin reagent for the photometric determination of amino acids and related compounds, J. Biol. Chem. 211, 907–913, 1954; Rosen, H., A modified ninhydrin colorimetric analysis for amino acids, Arch. Biochem. Biophys. 67, 10–15, 1957; Meyer, H., The ninhydrin reactions and its analytical applications, Biochem. J. 67, 333–340, 1957; Whitaker, J.R., Ninhydrin assay in the presence of thiol compounds, Nature 189, 662–663, 1961; Grant, D.R., Reagent stability in Rosen’s ninhydrin method for analysis of amino acids, Anal. Biochem. 6, 109–110, 1963; Shapiro, R. and Agarwal, S.C., Reaction of ninhydrin with cytosine derivatives, J. Am. Chem. Soc. 90, 474–478, 1968; Moore, S., Amino acid analysis: aqueous dimethylsulfoxide as solvent for the ninhydrin reaction, J. Biol. Chem. 243, 6281–6283, 1968; McGrath, R., Protein measurement by ninhydrin determination of amino acids released by alkaline hydrolysis, Anal. Biochem. 49, 95–102, 1972; Lamothe, P.J. and McCormick, P.G., Role of hydrindantin in the determination of amino acids using ninhydrin, Anal. Chem. 45, 1906–1911, 1973; Quinn, J.R., Boisvert, J.G., and Wood, I., Semi-automated ninhydrin assay of Kjeldahl nitrogen, Anal. Biochem. 58, 609–614, 1974; Chaplin, M.R., The use of ninhydrin as a reagent for the reversible modification of arginine residues in proteins, Biochem. J. 155, 457–459, 1976; Takahashi, K., Specific modification of arginine residues in proteins with ninhydrin, J. Biochem. 80, 1173–1176, 1976; Yu, P.H. and Davis, B.A., Deuterium isotope effects in the ninhydrin reaction of primary amines, Experientia 38, 299–300, 1982; D’Aniello, A., D’Onofrio, G., Pischetola, M., and Strazzulo, L., Effect of various substances on the
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Chemicals Commonly Used in Biochemistry and Molecular Biology and Their Properties (Continued) Common Name
Chemical Name
M.W.
Properties and Comment
colorimetric amino acid–ninhydrin reaction, Anal. Biochem. 144, 610–611, 1985; Macchi, F.D., Shen, F.J., Keck, R.G., and Harris, R.J., Amino acid analysis, using postcolumn ninhydrin detection, in a biotechnology laboratory, Methods Mol. Biol. 159, 9–30, 2000; Moulin, M., Deleu, C., Larher, F.R., and Bouchereau, A., High-performance liquid chromatography determination of pipecolic acid after precolumn derivatization using domestic microwave, Anal. Biochem. 308, 320–327, 2002; Pool, C.T., Boyd, J.G., and Tam, J.P., Ninhydrin as a reversible protecting group of amino-terminal cysteine, J. Pept. Res. 63, 223–234, 2004; Schulz, M.M., Wehner, H.D., Reichert, W., and Graw, M., Ninhydrin-dyed latent fingerprints as a DNA source in a murder case, J. Clin. Forensic Med. 11, 202–204, 2004; Buchberger, W. and Ferdig, M., Improved high-performance liquid chromatographic determination of guanidine compounds by precolumn derivatization with ninhydrin and fluorescence detection, J. Sep. Sci. 27, 1309–1312, 2004; Hansen, D.B., and Joullie, M.M., The development of novel ninhydrin analogues, Chem. Soc. Rev. 34, 408–417, 2005. Nitric Acid
HNO3
63.01
Strong acid.
p-Nitroaniline (PNA)
4-nitroaniline
138.13
Signal from cleavage of chromogenic substrate.
o-nitrophenylsulfenyl Chloride
189.6
Modification of tryptophan in proteins.
O2N
NH2 2-Nitrobenzylsulfenyl Chloride Cl S NO2
Fontana, A. and Scofone, E., Sulfenyl halides as modifying reagents for peptides and proteins, Methods Enzymol. 25B, 482–494, 1972; Sanda, A. and Irie, M., Chemical modification of tryptophan residues in ribonuclease form a Rhizopus sp., J. Biochem. 87, 1079–1087, 1980; De Wolf, M.J., Fridkin, M., Epstein, M., and Kohn, L.D., Structure-function studies of cholera toxin and its A and B protomers. Modification of tryptophan residues, J. Biol. Chem. 256, 5481–5488, 1981; Mollier, P., Chwetzoff, S., Bouet, F. et al., Tryptophan 110, a residue involved in the toxic activity but in the enzymatic activity of notexin, Eur. J. Biochem. 185, 263–270, 1989; Cymes, C.D., Iglesias, M.M., and Wolfenstein-Todel, C., Selective modification of tryptophan-150 in ovine placental lactogen, Comp. Biochem. Physiol. B 106, 743–746, 1993; Kuyama, H., Watanabe, M., Toda, C. et al., An approach to quantitate proteome analysis by labeling tryptophan residues, Rapid Commun. Mass Spectrom. 17, 1642–1650, 2003; Lundblad, R.L., Chemical Reagents for Protein Modification, 3rd ed., CRC Press, Boca Raton, FL, 2004; Matsuo, E., Toda, C., Watanabe, M., et al., Selective detection of 2-nitrobenzensulfenyl-labeled peptides by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry using a novel matrix, Proteomics 6, 2042–2049, 2006; Ou, K., Kesuma, D., Ganesan, K. et al., Quantitative labeling of drug-assisted proteomic alterations by combined 2-nitrobenzenesulfenyl chloride (NBS) isotope labeling and 2DE/MS identification, J. Proteome Res. 5, 2194–2206, 2006. p-Nitrophenol
4-nitrophenol
139.11
Popular signal from indicator enzymes such as alkaline phosphatase.
n-Octanol
1-octanol; Caprylic Alcohol
130.23
Partitioning between octanol and water is used to determine lipophilicity; a factor in QSAR studies.
H2 C H3C
9168_Book.indb 794
H2 C
H2 C C H2
C H2
H2 C C H2
OH
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Chemicals Commonly Used in Biochemistry and Molecular Biology and their Properties
795
Chemicals Commonly Used in Biochemistry and Molecular Biology and Their Properties (Continued) Common Name
Chemical Name
M.W.
Properties and Comment
Marland, J.S. and Mulley, B.A., A phase-rule study of multiple-phase formation in a model emulsion system containing water, n-octanol, n-dodecane, and a non-ionic surface-active agent at 10 and 25 degrees, J. Pharm. Pharmacol. 23, 561–572, 1971; Dorsey, J.G. and Khaledi, M.G., Hydrophobicity estimations by reversed-phase liquid chromatography. Implications for biological partitioning processes, J. Chromatog. 656, 485–499, 1993; Vailaya, A. and Horvath, C., Retention in reversed-phase chromatography: partition or adsorption? J. Chromatog. 829, 1–27, 1998; Kellogg, G.E. and Abraham, D.J., Hydrophobicity: is logP(o/w) more than the sum of its parts? Eur. J. Med. Chem. 35, 651–661, 2000; van de Waterbeemd,H., Smith, D.A., and Jones, B.C., Lipophilicity in PK design: methyl, ethyl, futile, J. Comput. Aided Mol. Des. 15, 273–286, 2001; Bethod, A. and Carda-Broch, S., Determination of liquid–liquid partition coefficients by separation methods, J. Chromatog. A 1037, 3–14, 2004. Octoxynol
Nonionic detergent; surfactant.
Triton X-100™; Igepal CA-630™ (CH2CH2O)nH O
CH3 H3C
C CH3
CH3 CH3
Octoxynol, n = 5–15 Peroxynitrite Petroleum Ether
N/A
Perchloric Acid
Mixture of Pentanes and Hexanes HClO4
100.5
Oxidizing agent.
1,10-Phenanthroline Monohydrate
o-phenanthroline Hydrate
198.21
Chelating agent; inhibitor for metalloproteinases; use in design of synthetic nucleases and proteases.
N N
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796
Chemicals Commonly Used in Biochemistry and Molecular Biology and Their Properties (Continued) Common Name
Chemical Name
M.W.
Properties and Comment
Hoch, F.L., Willams, R.J., and Vallee, B.L., The role of zinc in alcohol dehydrogenases. II. The kinetics of the instantaneous reversible inactivation of yeast alcohol dehydrogenase by 1,10-phenanthroline, J. Biol. Chem. 232, 453–464, 1958; Sigman, D.S. and Chen, C.H., Chemical nucleases: new reagents in molecular biology, Annu. Rev. Biochem. 59, 207–236, 1990; Pan, C.Q., Landgraf, R., and Sigman, D.S., DNA-binding proteins as site-specific nucleases, Mol. Microbiol. 12, 335–342, 1994; Galis, Z.S., Sukhova, G.K., and Libby, P., Microscopic localization of active proteases by in situ zymography: detection of matrix metalloproteinase activity in vascular tissue, FASEB J. 9, 974–980, 1995; Papavassiliou, A.G., Chemical nucleases as probes for studying DNA–protein interactions, Biochem. J. 305, 345–357, 1995; Perrin, D.M., Mazumder, A., and Sigman, D.S., Oxidative chemical nucleases, Prog. Nucleic Acid Res. Mol. Biol. 52, 123–151, 1996; Sigman, D.S., Landgraf, R., Perrin, D.M., and Pearson, L., Nucleic acid chemistry of the cuprous complexes of 1,10-phenanthroline and derivatives, Met. Ions Biol. Syst. 33, 485–513, 1996; Cha, J., Pedersen, M.V., and Auld, D.S., Metal and pH dependence of heptapeptide catalysis by human matrilysin, Biochemistry 35, 15831–15838, 1996; Kidani, Y. and Hirose, J., Coordination chemical studies on metalloenzymes. II. Kinetic behavior of various types of chelating agents towards bovine carbonic anhydrase, J. Biochem. 81, 1383–1391, 1997; Marini, I., Bucchioni, L., Borella, P. et al., Sorbitol dehydrogenase from bovine lens: purification and properties, Arch. Biochem. Biophys. 340, 383–391, 1997; Dri, P., Gasparini, C., Menegazzi, R. et al., TNF-induced shedding of TNF receptors in human polymorphonuclear leukocytes: role of the 55-kDa TNF receptor and involvement of a membrane-bound and non-matrix metalloproteinase, J. Immunol. 165, 2165–2172, 2000; Kito, M. and Urade, R., Protease activity of 1,10-phenanthroline-copper systems, Met. Ions Biol. Syst. 38, 187–196, 2001; Winberg, J.O., Berg, E., Kolset, S.O. et al., Calcium-induced activation and truncation of promatrix metalloproteinase-9 linked to the core protein of chondroitin sulfate proteoglycans, Eur. J. Biochem. 270, 3996–4007, 2003; Butler, G.S., Tam, E.M., and Overall, C.M., The canonical methionine 392 of matrix metalloproteinase 2 (gelatinase A) is not required for catalytic efficiency or structural integrity: probing the role of the methionine-turn in the metzincin metalloprotease superfamily, J. Biol. Chem. 279, 15615– 15620, 2004; Vauquelin, G. and Vanderheyden, P.M., Metal ion modulation of cystinyl aminopeptidase, Biochem. J. 390, 351–357, 2005; Schilling, S., Cynis, H., von Bohlen, A. et al., Isolation, catalytic properties, and competitive inhibitors of the zinc-dependent murine glutaminyl cyclase, Biochemistry 44, 13415–13424, 2005; Vik, S.B. and Ishmukhametov, R.R., Structure and function of subunit a of the ATP synthase of Escherichia coli, J. Bioenerg. Biomembr. 37, 445–449, 2005. Phenol
Hydroxybenzene; Phenyl Hydroxide
94.11
Solvent; nucleic acid purification.
OH Braun, W., Burrous, J.W., and Phillips, J.H., Jr., A phenol-extracted bacterial deoxyribonucleic acid, Nature 180, 1356–1357, 1957; Habermann, V., Evidence for peptides in RNA prepared by phenol extraction, Biochim. Biophys. Acta 32, 297–298, 1959; Colter, J.S., Brown, R.A., and Ellem, K.A., Observations on the use of phenol for the isolation of deoxyribonucleic acid, Biochim. Biophys. Acta 55, 31–39, 1962; Lust, J. and Richards, V., Influence of buffers on the phenol extraction of liver microsomal ribonucleic acids, Anal. Biochem. 20, 65–76, 1967; Yamaguchi, M., Dieffenbach, C.W., Connolly, R. et al., Effect of different laboratory techniques for guanidinium-phenol-chloroform RNA extraction on A260/A280 and on accuracy of mRNA quantitation by reverse transcriptase-PCR, PCR Methods Appl. 1, 286–290, 1992; Pitera, R., Pitera, J.E., Mufti, G.J., Salisbury, J.R., and Nickoloff, J.A., Sepharose spin column chromatography. A fast, nontoxic replacement for phenol: chloroform extraction/ethanol precipitation, Mol. Biotechnol. 1, 105–108, 1994; Finnegan, M.T., Herbert, K.E., Evans, M.D., and Lunec, J., Phenol isolation of DNA yields higher levels of 8-deoxodeoxyguanosine compared to pronase E isolation, Biochem. Soc. Trans. 23, 430S, 1995; Beaulieux, F., See, D.M., Leparc-Goffart, I. et al., Use of magnetic beads versus guanidium thiocyanate-phenol-chloroform RNA extraction followed by polymerase chain reaction for the rapid, sensitive detection of enterovirus RNA, Res. Virol. 148, 11–15, 1997; Fanson, B.G., Osmack, P., and Di Bisceglie, A.M., A comparison between the phenolchloroform method of RNA extraction and the QIAamp viral RNA kit in the extraction of hepatitis C and GB virus-C/hepatitis G viral RNA from serum, J. Virol. Methods 89, 23–27, 2000; Kochl, S., Niederstratter, N., and Parson, W., DNA extraction and quantitation of forensic samples using the phenol-chloroform method and real-time PCR, Methods Mol. Biol. 297, 13–30, 2005; Izzo, V., Notomista, E., Picardi, A. et al., The thermophilic archaeon Sulfolobus solfatarius is able to grow on phenol, Res. Microbiol. 156, 677–689, 2005; Robertson, N. and Leek, R., Isolation of RNA from tumor samples: single-step guanidinium acid-phenol method, Methods Mol. Biol. 120, 55–59, 2006. Phenoxyethanol
2-phenoxyethanol
138.16
Biochemical preservative; preservative in personal care products.
HO
O
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797
Chemicals Commonly Used in Biochemistry and Molecular Biology and Their Properties (Continued) Common Name
Chemical Name
M.W.
Properties and Comment
Nakahishi, M., Wilson, A.C., and Nolan, R.A., Phenoxyethanol: protein preservative for taxonomists, Science 163, 681–683, 1969; Frolich, K.W., Anderson, L.M., Knutsen, A., and Flood, P.R., Phenoxyethanol as a nontoxic substitute for formaldehyde in long-term preservation of human anatomical specimens for dissection and demonstration purposes, Anat. Rec. 208, 271–278, 1984. Phenylglyoxal
Phenylglyoxal Hydrate
134.13
Modification of arginine residues.
H O O
Takahashi, K., The reaction of phenylglyoxal with arginine residues in proteins, J. Biol. Chem. 243, 6171–6179, 1968; Bunzli, H.F. and Bosshard, H.R., Modification of the single arginine residue in insulin with phenylglyoxal, Hoppe Seylers Z. Physiol. Chem. 352, 1180–1182, 1971; Cheung, S.T. and Fonda, M.L., Reaction of phenylglyoxal with arginine. The effect of buffers and pH, Biochem. Biophys. Res. Commun. 90, 940–947, 1979; Srivastava, A. and Modak, M.J., Phenylglyoxal as a template site-specific reagent for DNA or RNA polymerases. Selective inhibition of initiation, J. Biol. Chem. 255, 917–921, 1980; Communi, D., Lecocq, R., Vanweyenberg, V., and Erneux, C., Active site labeling of inositol 1,4,5-triphosphate 3-kinase A by phenylglyoxal, Biochem. J. 310, 109–115, 1995; Eriksson, O., Fontaine, E., and Bernardi, P., Chemical modification of arginines by 2,3-butanedione and phenylglyoxal causes closure of the mitochondrial permeability transition pore, J. Biol. Chem. 273, 12669–12674, 1998; Redowicz, M.J., Phenylglyoxal reveals phosphorylation-dependent difference in the conformation of Acanthamoeba myosin II active site, Arch. Biochem. Biophys. 384, 413–417, 2000; Kucera, I., Inhibition by phenylglyoxal of nitrate transport in Paracoccus denitrificans; a comparison with the effect of a protonophorous uncoupler, Arch. Biochem. Biophys. 409, 327–334, 2003; Johans, M., Milanesi, E., Frank, M. et al., Modification of permeability transition pore arginine(s) by phenylglyoxal derivatives in isolated mitochondria and mammalian cells. Structure-function relationship of arginine ligands, J. Biol. Chem. 280, 12130–12136, 2005. Phosgene Cl
Carbonyl Chloride; Carbon Oxychloride
98.92
Reagent for organic synthesis; preparation of derivatives for analysis.
Cl C O
Wilchek, M., Ariely, S., and Patchornik, A., The reaction of asparagine, glutamine, and derivatives with phosgene, J. Org. Chem. 33, 1258–1259, 1968; Hamilton, R.D. and Lyman, D.J., Preparation of N-carboxy-α-amino acid anhydrides by the reaction of copper(II)-amino acid complexes with phosgene, J. Org. Chem. 34, 243–244, 1969; Pohl, L.R., Bhooshan, B., Whittaker, N.F., and Krishna, G., Phosgene: a metabolite of chloroform, Biochem. Biophys. Res. Commun. 79, 684–691, 1977; Gyllenhaal, O., Derivatization of 2-amino alcohols with phosgene in aqueous media: limitations of the reaction selectivity as found in the presence of O-glucuronides of alprenolol in urine, J. Chromatog. 413, 270–276, 1987; Gyllenhaal, O. and Vessman, J., Phosgene as a derivatizing reagent prior to gas and liquid chromatography, J. Chromatog. 435, 259–269, 1988; Noort, D., Hulst, A.G., Fidder, A., et al. In vitro adduct formation of phosgene with albumin and hemoglobin in human blood, Chem. Res. Toxicol. 13, 719–726, 2000; Lemoucheux, L. Rouden, J., Ibazizene, M. et al., Debenylation of tertiary amies using phosgene or triphosgen: an efficient and rapid procedure for the preparation of carbamoyl chlorides and unsymmetrical ureas. Application in carbon-11 chemistry, J. Org. Chem. 68, 7289–7297, 2003. Picric Acid
2,4,6-trinitrophenol
229.1
Analytical reagent.
NO2
O2N
NO2 OH
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Handbook of Biochemistry and Molecular Biology
798
Chemicals Commonly Used in Biochemistry and Molecular Biology and Their Properties (Continued) Common Name
Chemical Name
M.W.
Properties and Comment
De Wesselow, O.L., The picric acid method for the estimation of sugar in blood and a comparison of this method with that of MacLean, Biochem. J. 13, 148–152, 1919; Newcomb, C., The error due to impure picric acid in creatinine estimations, Biochem. J. 18, 291–293, 1924; Davidsen, O., Fixation of proteins after agarose gel electrophoresis by means of picric acid, Clin. Chim. Acta 21, 205–209, 1968; Gisin, B.F., The monitoring of reactions in solid-phase peptide synthesis with picric acid, Anal. Chim. Acta 58, 248–249, 1972; Hancock, W.S., Battersby, J.E., and Harding, D.R., The use of picric acid as a simple monitoring procedure for automated peptide synthesis, Anal. Biochem. 69, 497–503, 1975; Vasiliades, J., Reaction of alkaline sodium picrate with creatinine: I. Kinetics and mechanism of formation of the mono-creatinine picric acid complex, Clin. Chem. 22, 1664–1671, 1976; Somogyi, P. and Takagi, H., A note on the use of picric acid-formaldehyde-glutaraldehyde fixative for correlated light and electron microscopic immunocytochemistry, Neuroscience 7, 1779–1783, 1982; Meyer, M.H., Meyer, R.A., Jr., Gray, R.W., and Irwin, R.L., Picric acid methods greatly overestimate serum creatinine in mice: more accurate results with high-performance liquid chromatography, Anal. Biochem. 144, 285–290, 1985; Knisley, K.A. and Rodkey, L.S., Direct detection of carrier ampholytes in immobilized pH gradients using picric acid precipitation, Electrophoresis 13, 220–224, 1992; Massoomi, F., Mathews, H.G., III, and Destache, C.J., Effect of seven fluoroquinolines on the determination of serum creatinine by the picric acid and enzymatic methods, Ann. Pharmacother. 27, 586–588, 1993. Polysorbate
Tween 20
Nonionic detergent; surfactant.
R2
O
R3
R R1 Polysorbates
Polyvinylpyrrolidone (PVP)
Povidone
N/A
Pharmaceutical; excipient; phosphate analysis.
n
N
N O
O
Morin, L.G. and Prox, J., New and rapid procedure for serum phosphorus using o-phenylenediamine as reductant, Clin. Chim. Acta. 46, 113–117, 1973; Ohnishi, S.T. and Gall, R.S., Characterization of the catalyzed phosphate assay, Anal. Biochem. 88, 347–356, 1978; Steige, H. and Jones, J.D., Determination of serum inorganic phosphorus using a discrete analyzer, Clin. Chim. Acta. 103, 123–127, 1980, Plaizier-Vercammen, J.A. and De Neve, R.E., Interaction of povidone with aromatic compounds. II: evaluation of ionic strength, buffer concentration, temperature, and pH by factorial analysis, J. Pharm. Sci. 70, 1252–1256, 1981; van Zanten, A.P. and Weber, J.A., Direct kinetic method for the determination of phosphate, J. Clin. Chem. Clin. Biochem. 25, 515–517, 1987; Barlow, I.M., Harrison, S.P., and Hogg, G.L., Evaluation of the Technicon Chem-1, Clin. Chem. 34, 2340–2344, 1988; Giulliano, K.A., Aqueous two-phase protein partitioning using textile dyes as affinity ligands, Anal. Biochem. 197, 333–339, 1991; Goldenheim, P.D., An appraisal of povidone-iodine and wound healing, Postgrad. Med. J., 69 (Suppl. 3), S97–S105, 1993; Vemuri, S., Yu, C.D., and Roosdorp, N., Effect of cryoprotectants on freezing, lyophilization, and storage of lyophilized recombinant alpha 1-antitrypsin formulations, PDA J. Pharm. Sci. Technol. 48, 241–246, 1994; Anchordoquy, T.J. and Carpenter, J.F., Polymers protect lactate dehydrogenase during freeze-drying by inhibiting dissociation in the frozen state, Arch. Biochem. Biophys. 332, 231–238, 1996; Fleisher, W., and Reimer, K., Povidone-iodine in antisepsis — state of the art, Dermatology 195 (Suppl. 2), 3–9, 1997; Fernandes, S., Kim, H.S., and Hatti-Kaul, R., Affinity extraction of dye- and metal ion-binding proteins in polyvinalypyrrolidone-based aqueous two-phase system, Protein Expr. Purif. 24, 460–469, 2002; D’Souza, A.J., Schowen, R.L., Borchardt, R.T. et al., Reaction of a peptide with polyvinylpyrrolidone in the solid state, J. Pharm. Sci. 92, 585–593, 2003; Kaneda, Y., Tsutsumi, Y., Yoshioka, Y. et al., The use of PVP as a polymeric carrier to improve the plasma half-life of drugs, Biomaterials 25, 3259–3266, 2004; Art, G., Combination povidone-iodine and alcohol formulations more effective, more convenient versus formulations containing either iodine or alcohol alone: a review of the literature, J. Infus. Nurs. 28, 314–320, 2005; Yoshioka, S., Aso, Y., and Miyazaki, T., Negligible contribution of molecular mobility to the degradation of insulin lyophilized with poly(vinylpyrrolidone), J. Pharm. Sci. 95, 939–943, 2006.
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Chemicals Commonly Used in Biochemistry and Molecular Biology and their Properties
799
Chemicals Commonly Used in Biochemistry and Molecular Biology and Their Properties (Continued) Common Name
Chemical Name
Pyridine
Azine
M.W. 79.10
Properties and Comment Solvent.
N Klingsberg, E. and Newkome, G.R., Eds., Pyridine and Its Derivatives, Interscience, New York, 1960; Schoefield, K., Hetero-aromatic Nitrogen Compounds; Pyrroles and Pyridines, Butterworths, London, 1967; Hurst, D.T., An Introduction to the Chemistry and Biochemistry and Pyrimidines, Purines, and Ptreridines, J. Wiley, Chichester, UK, 1980; Plunkett, A.O., Pyrrole, pyrrolidine, pyridine, piperidine, and azepine alkaloids, Nat. Prod. Rep. 11, 581–590, 1994; Kaiser, J.P., Feng, Y., and Bollag, J.M., Microbial metabolism of pyridine, quinoline, acridine, and their derivatives under aerobic and anaerobic conditions, Microbiol. Rev. 60, 483–498, 1996. Pyridoxal-5-phosphate (PLP)
Pyridoxal-5-(dihydrogen phosphate)
247.14
Selective modification of amino groups in proteins; affinity label for certain sites based on phosphate group.
O HC O HO
O
OH P OH
H3C
N
Hughes, R.C., Jenkins, W.T., and Fischer, E.H., The site of binding of pyridoxal-5′-phosphate to heart glutamic-aspartic transaminase, Proc. Natl. Acad. Sci. USA 48, 1615–1618, 1962; Finseth, R. and Sizer, I.W., Complexes of pyridoxal phosphate with amino acids, peptides, polylysine, and apotransaminase, Biochem. Biophys. Res. Commun. 26, 625–630, 1967; Pages, R.C., Benditt, E.P., and Kirkwood, C.R., Schiff base formation by the lysyl and hydroxylysyl side chains of collagen, Biochem. Biophys. Res. Commun. 33, 752–757, 1968; Whitman, W.B., Martin, M.N., and Tabita, F.R., Activation and regulation of ribulose bisphosphate carboxylase-oxygenase in the absence of small subunits, J. Biol. Chem. 254, 10184–10189, 1979; Howell, E.E. and Schray, K.J., Comparative inactivation and inhibition of the anomerase and isomerase activities of phosphoglucose isomerase, Mol. Cell. Biochem. 37, 101–107, 1981; Colanduoni, J. and Villafranca, J.J., Labeling of specific lysine residues at the active site of glutamine synthetase, J. Biol. Chem. 260, 15042–15050, 1985; Peterson, C.B., Noyes, C.M., Pecon, J.M. et al., Identification of a lysyl residue in antithrombin which is essential for heparin binding, J. Biol. Chem. 262, 8061–8065, 1987; Diffley, J.F., Affinity labeling the DNA polymerase alpha complex. Identification of subunits containing the DNA polymerase active site and an important regulatory nucleotide-binding site, J. Biol. Chem. 263, 19126–19131, 1988; Perez-Ramirez, B. and Martinez-Carrion, M., Pyridoxal phosphate as a probe of the cytoplasmic domains of transmembrane proteins: application to the nicotinic acetylcholine receptor, Biochemistry 28, 5034–5040, 1989; Valinger, Z., Engel, P.C., and Metzler, D.E., Is pyridoxal-5′-phosphate an affinity label for phosphate-binding sites in proteins? The case of bovine glutamate dehydrogenase, Biochem. J. 294, 835–839, 1993; Illy, C., Thielens, N.M., and Arlaud, G.J., Chemical characterization and location of ionic interactions involved in the assembly of the C1 complex of human complement, J. Protein Chem. 12, 771–781, 1993; Hountondji, C., Gillet, S., Schmitter, J.M. et al., Affinity labeling of Escherichia coli lysyl-tRNA synthetase with pyridoxal mono- and diphosphate, J. Biochem. 116, 502–507, 1994; Brody, S., Andersen, J.S., Kannangara, C.G. et al., Characterization of the different spectral forms of glutamate-1-semialdehyde aminotransferase by mass spectrometry, Biochemistry 34, 15918–15924, 1995; Kossekova, G., Miteva, M., and Atanasov, B., Characterization of pyridoxal phosphate as an optical label for measuring electrostatic potentials in proteins, J. Photochem. Photobiol. B 32, 71–79, 1996; Kim S.W., Lee, J., Song, M.S. et al., Essential active-site lysine of brain glutamate dehydrogenase isoproteins, J. Neurochem. 69, 418–422, 1997; Martin, D.L., Liu, H., Martin, S.B., and Wu, S.J., Structural features and regulatory properties of the brain glutamate decarboxylase, Neurochem. Int. 37, 111–119, 2000; Jaffe, M. and Bubis, J., Affinity labeling of the guanine nucleotide binding site of transducin by pyridoxal 5′-phosphate, J. Protein Chem. 21, 339–359, 2002. Sodium Borohydride
9168_Book.indb 799
NaBH4
37.83
Reducing agent for Schiff bases; reduction of aldehydes; other chemical reductions.
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Chemicals Commonly Used in Biochemistry and Molecular Biology and Their Properties (Continued) Common Name
Chemical Name
M.W.
Properties and Comment
Chaykin, S., King, L., and Watson, J.G., The reduction of DPN+ and TPN+ with sodium borohydride, Biochim. Biophys. Acta 124, 13–25, 1966; Cerutti, P. and Miller, N., Selective reduction of yeast transfer ribonucleic acid with sodium borohydride, J. Mol. Biol. 26, 55–66, 1967; Tanzer, M.L., Collagen reduction by sodium borohydride: effects of reconstitution, maturation, and lathyrism, Biochem. Biophys. Res. Commun. 32, 885–892, 1968; Phillips, T.M., Kosicki, G.W., and Schmidt, D.E., Jr., Sodium borohydride reduction of pyruvate by sodium borohydride catalyzed by pyruvate kinase, Biochim. Biophys. Acta 293, 125–133, 1973; Craig, A.S., Sodium borohydride as an aldehyde-blocking reagent for electron microscope histochemistry, Histochemistry 42, 141–144, 1974; Miles, E.W., Houck, D.R., and Floss, H.G., Stereochemistry of sodium borohydride reduction of tryptophan synthase of Escherichia coli and its amino acid Schiff ’s bases, J. Biol. Chem. 257, 14203–14210, 1982; Kumar, A., Rao, P., and Pattabiraman, T.N., A colorimetric method for the estimation of serum glycated proteins based on differential reduction of free and bound glucose by sodium borohydride, Biochem. Med. Metab. Biol. 39, 296–304, 1988; Lenz, A.G., Costabel, U., Shaltiel, S., and Levine, R.L., Determination of carbonyl groups in oxidatively modified proteins by reduction with tritiated sodium borohydride, Anal. Biochem. 177, 419–425, 1989; Yan, L.J. and Sohal, R.S., Gel electrophoresis quantiation of protein carbonyls derivatized with tritiated sodium borohydride, Anal. Biochem. 265, 176–182, 1998; Azzam, T., Eliyahu, H., Shapira, L. et al., Polysaccharide-oligoamine-based conjugates for gene delivery, J. Med. Chem. 45, 1817–1824, 2002; Purich, D.L., Use of sodium borohydride to detect acyl-phosphate linkages in enzyme reactions, Methods Enzymol. 354, 168–177, 2002; Bald, E., Chwatko, S., Glowacki, R., and Kusmierek, K., Analysis of plasma thiols by high-performance liquid chromatography with ultraviolet detection, J. Chromatog. A 1032, 109–115, 2004; Eike, J.H. and Palmer, A.F., Effect of NABH4 concentration and reaction time on physical properties of glutaraldehyde-polymerized hemoglobin, Biotechnol. Prog. 20, 946–952, 2004; Zhang, Z., Edwards, P.J., Roeske, R.W., and Guo, L., Synthesis and self-alkylation of isotope-coded affinity tag reagents, Bioconjug. Chem. 16, 458–464, 2005; Studelski, D.R., Giljum, K., McDowell, L.M., and Zhang, L., Quantitation of glycosaminoglycans by reversed-phase HPLC separation of fluorescent isoindole derivatives, Glycobiology 16, 65–72, 2006; Floor, E., Maples, A.M., Rankin, C.A. et al., A one-carbon modification of protein lysine associated with elevated oxidative stress in human substantia nigra, J. Neurochem. 97, 504–514, 2006; Kusmierek, K., Glowacki, R., and Bald, E., Analysis of urine for cysteine, cysteinylglycine, and homocysteine by high-performance liquid chromatography, Anal. BioAnal. Chem. 385, 855–860, 2006. Sodium Chloride Sodium Cholate
Salt; NaCl
58.44
Ionic strength; physiological saline.
430.55
Detergent.
Lindstrom, J., Anholt, R., Einarson, B. et al., Purification of acetylcholine receptors, reconstitution into lipid vesicles, and study of agonist-induced channel regulation, J. Biol. Chem. 255, 8340–8350, 1980; Gullick, W.J., Tzartos, S., and Lindstrom, J., Monoclonal antibodies as probes of acetylcholine receptor structure. 1. Peptide mapping, Biochemistry 20, 2173–2180, 1981; Henselman, R.A. and Cusanovich, M.A., The characterization of sodium cholate solubilized rhodopsin, Biochemistry 13, 5199–5203, 1974; Ninomiya, R., Masuoka, K., and Moroi, Y., Micelle formation of sodium chenodeoxycholate and solublization into the micelles: comparison with other unconjugated bile salts, Biochim. Biophys. Acta 1634, 116–125, 2003; Simoes, S.I., Marques, C.M., Cruz, M.E. et al., The effect of cholate on solubilization and permeability of simple and protein-loaded phosphatidylcholine/ sodium cholate-mixed aggregates designed to mediate transdermal delivery of macromolecules, Eur. J. Pharm. Biopharm. 58, 509–519, 2004; Reis, S., Moutinho, C.G., Matos, C. et al., Noninvasive methods to determine the critical micelle concentration of some bile acid salts, Anal. Biochem. 334, 117–126, 2004; Nohara, D., Kajiura, T., and Takeda, K., Determination of micelle mass by electrospray ionization mass spectrometry, J. Mass Spectrom. 40, 489–493, 2005; Guo, J., Wu., T., Ping, Q. et al., Solublization and pharmacokinetic behaviors of sodium cholate/lecithin-mixed micelles containing cyclosporine A, Drug Deliv. 12, 35–39, 2005; Bottari, E., Buonfigli, A., and Festa, M.R., Composition of sodium cholate micellar solutions, Ann. Chim. 95, 479–490, 2005; Schweitzer, B., Felippe, A.C., Dal Bo, A. et al., Sodium dodecyl sulfate promoting a cooperative association process of sodium cholate with bovine serum albumin, J. Colloid Interface Sci. 298, 457–466, 2006; Burton, M.I., Herman, M.D., Alcain, F.J., and Villalba, J.M., Stimulation of polyprenyl 4-hydroxybenzoate transferase activity by sodium cholate and 3- [(cholamidopropyl)dimethylammonio]-1-propanesulfonate, Anal. Biochem. 353, 15–21, 2006; Ishibashi, A. and Nakashima, N., Individual dissolution of single-walled carbon nanotubes in aqueous solutions of steroid of sugar compounds and their Raman and near-IR spectral properties, Chemistry, 12, 7595–7602, 2006. 62.84 Reducing agent; considered more selective Sodium Cyanoborohydride NaBH3 (CN) than NaBH4. Rosen, G.M., Use of sodium cyanoborohydride in the preparation of biologically active nitroxides, J. Med. Chem. 17, 358–360, 1974; Chauffe, L. and Friedman, M., Factors affecting cyanoborohydride reduction of aromatic Schiff ’s bases in proteins, Adv. Exp. Med. Biol. 86A, 415–424, 1977; Baues, R.J. and Gray, G.R., Lectin purification on affinity columns containing reductively aminated disaccharides, J. Biol. Chem. 252, 57–60, 1977; Jentoft, N. and Dearborn, D.G., Labeling of proteins by reductive methylation using sodium cyanoborohydride, J. Biol. Chem. 254, 4359–4365, 1979; Jentoft, N., and Dearborn, D.G., Protein labeling by reductive methylation with sodium cyanoborohydride: effect of cyanide and metal ions on the reaction, Anal. Biochem. 106, 186–190, 1980; Bunn, H.F. and Higgins, P.T., Reaction of monosaccharides with proteins: possible evolutionary significance, Science 213, 222–224, 1981; Geoghegan, K.F., Cabacungan, J.C., Dixon, H.B., and Feeney, R.E., Alternative reducing agents for reductive methylation of amino groups in proteins, Int. J. Pept. Protein Res. 17, 345–352, 1981; Habeeb, A.F., Comparative studies on radiolabeling of lysozyme by iodination and reductive methylation, J. Immunol. Methods 65, 27–39, 1983; Prakash, C. and Vijay, I.K., A new fluorescent tag for labeling of saccharides, Anal. Biochem. 128, 41–46, 1983; Acharya, A.S. and Sussman, L.G., The reversibility of the ketoamine linkages of aldoses with proteins, J. Biol. Chem. 259, 4372–4378, 1984; Climent, I., Tsai, L., and Levine, R.L., Derivatization of gamma-glutamyl semialdehyde residues in oxidized proteins by fluorescamine, Anal. Biochem. 182, 226–232, 1989; Hartmann, C. and Klinman, J.P., Reductive trapping of substrate to methylamine oxidase from Arthrobacter P1, FEBS Lett. 261, 441–444, 1990; Meunier, F. and Wilkinson, K.J., Nonperturbing fluorescent labeling of polysaccharides, Biomacromolecules 3, 858–864, 2002; Webb, M.E., Stephens, E., Smith, A.G., and Abell, C., Rapid screening by MALDI-TOF mass spectrometry to probe binding specificity at enzyme active sites, Chem. Commun. 19, 2416–2417, 2003; Sando, S., Matsui, K., Niinomi, Y. et al., Facile preparation of DNA-tagged carbohydrates, Bioorg. Med. Chem. Lett. 13, 2633–2636, 2003; Peelen, D. and Smith, L.M., Immobilization of anine-modified oligonucleotides on aldehyde-terminated alkanethiol monolayers on gold, Langmuir 21, 266–271, 2005; Mirzaei, H. and Regnier, F., Enrichment of carbonylated peptides using Girard P reagent and strong cation exchange chromatography, Anal. Chem. 78, 770–778, 2006.
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801
Chemicals Commonly Used in Biochemistry and Molecular Biology and Their Properties (Continued) Common Name Sodium Deoxycholate
Chemical Name
M.W.
Desoxycholic Acid, Sodium Salt
414.55
Properties and Comment Detergent; potential therapeutic use with adipose tissue.
Bril, C., van der Horst, D.J., Poort, S.R., and Thomas, J.B., Fractionation of spinach chloroplasts with sodium deoxycholate, Biochim. Biophys. Acta 172, 345–348, 1969; Smart, J.E. and Bonner, J., Selective dissociation of histones from chromatin by sodium deoxycholate, J. Mol. Biol. 58, 651–659, 1971; Part, M., Tarone, G., and Comoglio, P.M., Antigenic and immunogenic properties of membrane proteins solubilized by sodium desoxycholate, papain digestion, or high ionic strength, Immunochemistry 12, 9–17, 1975; Johansson, K.E. and Wbolewski, H., Crossed immunoelectrophoresis, in the presence of tween 20 or sodium deoxycholate, or purified membrane proteins from Acholeplasma laidlawii, J. Bacteriol. 136, 324–330, 1978; Lehnert, T. and Berlet, H.H., Selective inactivation of lactate dehydrogenase of rat tissues by sodium deoxycholate, Biochem. J. 177, 813–818, 1979; Suzuki, N., Kawashima, S., Deguchi, K., and Ueta, N., Low-density lipoproteins form human ascites plasma. Characterization and degradation by sodium deoxycholate, J. Biochem. 87, 1253–1256, 1980; Robern, H., The application of sodium deoxycholate and Sephacryl S-200 for the delipidation and separation of high-density lipoprotein, Experientia 38, 437–439, 1982; Nedivi, E. and Schramm, M., The beta-adrenergic receptor survives solubilization in deoxycholate while forming a stable association with the agonist, J. Biol. Chem. 259, 5803–5808, 1984; McKernan, R.M., Castro, S., Poat, J.A., and Wong, E.H., Solubilization of the N-methyl-D-aspartate receptor channel complex from rat and porcine brain, J. Neurochem. 52, 777–785, 1989; Carter, H.R. Wallace, M.A., and Fain, J.N., Activation of phospholipase C in rabbit brain membranes by carbachol in the presence of GTP gamma S: effects of biological detergents, Biochim. Biophys. Acta 1054, 129–134, 1990; Shivanna, B.D. and Rowe, E.S., Preservation of the native structure and function of Ca2+-ATPase from sarcoplasmic reticulum: solubilization and reconstitution by new short-chain phospholipid detergent 1,2-diheptanoyl-sn-phosphatidylcholine, Biochem. J. 325, 533–542, 1997; Arnold, U. and Ulbrich-Hofmann, R., Quantitative protein precipitation from guandine hydrochloride-containing solutions by sodium deoxycholate/trichloroacetic acid, Anal. Biochem. 271, 197–199, 1999; Haque, M.E., Das, A.R., and Moulik, S.P., Mixed micelles for sodium deoxycholate and polyoxyethylene sobitan monooleate (Tween 80), J. Colloid Interface Sci. 217, 1–7, 1999; Srivastava, O.P. and Srivastava, K., Characterization of a sodium deoxycholate-activable proteinase activity associated with betaA3/A1-crystallin of human lenses, Biochim. Biophys. Acta 1434, 331–346, 1999; Rotunda, A.M., Suzuki, H., Moy, R.L., and Kolodney, M.S., Detergent effects of sodium deoxycholate are a major feature of an injectable phosphatidylcholine formulation used for localized fat dissolution, Dermatol. Surg. 30, 1001–1008, 2004; Asmann, Y.W., Dong, M., and Miller, L.J., Functional characterization and purification of the secretin receptor expressed in baculovirus- infected insect cells, Regul. Pept. 123, 217–223, 2004; Ranganathan, R., Tcacenco, C.M., Rosseto, R., and Hajdu, J., Characterization of the kinetics of phospholipase C activity toward mixed micelles of sodium deoxycholate and dimyristoyl-phophatidylcholine, Biophys. Chem. 122, 79–89, 2006. Sodium Dodecylsulfate
Sodium Lauryl Sulfate, SDS
288.38
Detergent.
O S
O–
O
H3C
O Sodium dodecylsulfate, SDS, lauryl sulfate, sodium salt
Shapiro, A.L., Vinuela, E., and Maizel, J.V., Jr., Molecular weight estimation of polypeptide chains by electrophoresis in SDS-polyacrylamide gels, Biochem. Biophys. Res. Commun. 28, 815–820, 1967; Shapiro, A.L., and Maizel, J.V., Jr., Molecular weight estimation of polypeptides by SDSpolyacrylamide gel electrophoresis: further data concerning resolving power and general considerations, Anal. Biochem. 29, 505–514, 1969; Weber, K. and Osborn, M., The reliability of molecular weight determinations of dodecyl sulfate-polyacryalmide gel electrophoresis, J. Biol. Chem. 244, 4406–4412, 1969; Weber, K. and Kuter, D.J., Reversible denaturation of enzymes by sodium dodecyl sulfate, J. Biol. Chem. 246, 4504–4509, 1971; de Haen, C., Molecular weight standards for calibration of gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis: ferritin and apoferritin, Anal. Biochem. 166, 235–245, 1987; Smith, B.J., SDS polyacrylamide gel electrophoresis of proteins, Methods Mol. Biol. 32, 23–34, 1994; Guttman, A., Capillary sodium dodecyl sulfate-gel electrophoresis of proteins, Electrophoresis 17, 1333–1341, 1996; Bischoff, K.M., Shi, L., and Kennelly, P.J., The detection of enzyme activity following sodium dodecyl sulfate-polyacryalamide gel electrophoresis, Anal. Biochem. 260, 1–17, 1998; Maizel, J.V., SDS polyacrylamide gel electrophoresis, Trends Biochem. Sci. 35, 590–592, 2000; Robinson, J.M. and Vandre, D.D, Antigen retrieval in cells and tissues: enhancement with sodium dodecyl sulfate, Histochem. Cell Biol. 116, 119–130, 2001; Todorov, P.D., Kralchevsky, P.A., Denkov, N.D. et al., Kinetics of solublization of n-decane and benzene by micellar solutions of sodium dodecyl sulfate, J. Colloid Interface Sci. 245, 371–382, 2002; Zhdanov, S.A., Starov, V.M., Sobolev, V.D., and Velarde, M.G., Spreading of aqueous SDS solutions over nitrocellulose membranes, J. Colloid Interface Sci. 264, 481–489, 2003; Santos, S.F., Zanette, D., Fischer, H., and Itri, R., A systematic study of bovine serum albumin (BSA) and sodium dodecyl sulfate (SDS) interactions by surface tension and small angle X-ray scattering, J. Colloid Interface Sci. 262, 400–408, 2003; Biswas, A. and Das, K.P., SDS-induced structural changes in alpha-crystallin and its effect on refolding, Protein J. 23, 529–538, 2004; Jing, P., Kaneta, T., and Imasaka, T., On-line concentration of a protein using denaturation by sodium dodecyl sulfate, Anal. Sci. 21, 37–42, 2005; Choi, N.S., Hahm, J.H., Maeng, P.J., and Kim, S.H., Comparative study of enzyme activity and stability of bovine and human plasmins in electrophoretic reagents, β-mercaptoethanol, DTT, SDS, Triton X-100, and urea, J. Biochem. Mol. Biol. 38, 177–181, 2005; Miles, A.P. and Saul, A., Quantifying recombinant proteins and their degradation products using SDS-PAGE and scanning laser densitometry, Methods Mol. Biol. 308, 349–356, 2005; Thongngam, M. and McClements, D.J., Influence of pH, ionic strength, and temperature on self-association and interactions of sodium dodecyl sulfate in the absence and presence of chitosan, Langmuir 21, 79–86, 2005; Romani, A.P., Gehlen, M.H., and Itri, R., Surfactantpolymer aggregates formed by sodium dodecyl sulfate, poly(N-vinyl-2-pyrrolidone), and poly(ethylene glycol), Langmuir 21, 1271–1233, 2005; Gudiksen, K.L., Gitlin, I., and Whitesides, G.M., Differentiation of proteins based on characteristic patterns of association and denaturation in solutions of SDS, Proc. Natl. Acad. Sci. USA 103, 7968–7972, 2006; Freitas, A.A., Paulo, L., Macanita, A.L, and Quina, F.H., Acid-base equilibria and dynamics in sodium dodecyl sulfate micelles: geminate recombination and effect of charge stabilization, Langmuir 22, 7986–7893, 2006.
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Handbook of Biochemistry and Molecular Biology
802
Chemicals Commonly Used in Biochemistry and Molecular Biology and Their Properties (Continued) Common Name
Chemical Name
Sodium Metabisulfite
Sodium Bisulfite
M.W. 190.1
Properties and Comment Mild reducing agent; converts unmethylated cytosine residues to uracil residues (DNA methylation).
Miller, R.F., Small, G., and Norris, L.C., Studies on the effect of sodium bisulfite on the stability of vitamin E, J. Nutr. 55, 81–95, 1955; Hayatsu, H., Wataya, Y., Kai, K., and Iida, S., Reaction of sodium bisulfite with uracil, cytosine, and their derivatives, Biochemistry 9, 2858–2865, 1970; Seno, T., Conversion of Escherichia coli tRNATrp to glutamine-accepting tRNA by chemical modification with sodium bisulfite, FEBS Lett. 51, 325–329, 1975; Tasheva, B. and Dessev, G., Artifacts in sodium dodecyl sulfate-polyacrylamide gel electrophoresis due to 2-mercaptoethanol, Anal. Biochem. 129, 98–102, 1983; Draper, D.E., Attachment of reporter groups to specific, selected cytidine residues in RNA using a bisulfite-catalyzed transamination reaction, Nucleic Acids Res. 12, 989–1002, 1984; Oakeley, E.J., DNA methylation analysis: a review of current methodologies, Pharmacol. Ther. 84, 389–400, 1999; Geisler, J.P., Manahan, K.J., and Geisler, H.E., Evaluation of DNA methylation in the human genome: why examine it and what method to use, Eur. J. Gynaecol. Oncol. 25, 19–24, 2004; Thomassin, H., Kress, C., and Grange, T., MethylQuant: a sensitive method for quantifying methylation of specific cytosines within the genome, Nucleic Acids Res. 32, e168, 2004; Derks, S., Lentjes, M.H., Mellebrekers, D.M. et al., Methylation-specific PCR unraveled, Cell. Oncol. 26, 291–299, 2004; Galm, O. and Herman, J.G., Methylation-specific polymerase chain reaction, Methods Mol. Biol. 113, 279–291, 2005; Ogino, S., Kawasaki, T., Brahmandam, M. et al., Precision and performance characteristics of bisulfite conversion and real-time PCR (MethylLight) for quantitative DNA methylation analysis, J. Mol. Diagn. 8, 209–217, 2006; Yang, I., Park, I.Y., Jang, S.M. et al., Rapid quantitation of DNA methylation through dNMP analysis following bisulfite PCR, Nucleic Acids Res. 34, e61, 2006; Wischnewski, F., Pantel, K., and Schwazenbach, H., Promoter demethylation and histone acetylation mediate gene expression of MAGE-A1, -A2, -A3, and -A12 in human cancer cells, Mol. Cancer Res. 4, 339–349, 2006; Zhou, Y., Lum, J.M., Yeo, G.H. et al., Simplified molecular diagnosis of fragile X syndrome by fluorescent methylation-specific PCR and GeneScan analysis, Clin. Chem. 52, 1492–1500, 2006. Succinic Anhydride
O
O
Butanedioic Anhydride; 2,5-diketotetra-hydrofuran
100.1
Protein modification; dissociation of protein complexes.
O
Habeeb, A.F., Cassidy, H.G., and Singer, S.J., Molecular structural effects produced in proteins by reaction with succinic anhydride, Biochim. Biophys. Acta 29, 587–593, 1958; Hass, L.F., Aldolase dissociation into subunits by reaction with succinic anhydride, Biochemistry 3, 535–541, 1964; Scanu, A., Pollard, H., and Reader, W., Properties of human serum low-density lipoproteins after modification by succinic anhydride, J. Lipid Res. 9, 342–349, 1968; Vasilets, I.M., Moshkov, K.A., and Kushner, V.P., Dissociation of human ceruloplasmin into subunits under the action of alkali and succinic anhydride, Mol. Biol. 6, 193–199, 1972; Tedeschi, H., Kinnally, K.W., and Mannella, C.A., Properties of channels in mitochondrial outer membrane, J. Bioenerg. Biomembr. 21, 451–459, 1989; Palacian, E., Gonzalez, P.J., Pineiro, M., and Hernandez, F., Dicarboxylic acid anhydrides as dissociating agents of protein-containing structures, Mol. Cell. Biochem. 97, 101–111, 1990; Pavliakova, D., Chu, C., Bystricky, S. et al., Treatment with succinic anhydride improves the immunogenicity of Shigella flexneri type 2a O-specific polysaccharide-protein conjugates in mice, Infect. Immun. 67, 5526–5529, 1999; Ferretti, V., Gilli, P., and Gavezzotti, A., X-ray diffraction and molecular simulation study of the crystalline and liquid states of succinic anhydride, Chemistry 8, 1710–1718, 2002. Sucrose
342.30
Osmolyte; density gradient centrifugation.
CH2OH O
CH2OH
O OH
HO O
OH OH
CH2OH OH
Cann, J.R., Coombs, R.O., Howlett, G.J. et al., Effects of molecular crowding on protein self-association: a potential source of error in sedimentation coefficients obtained by zonal ultracentrifugation in a sucrose gradient, Biochemistry 33, 10185–10190, 1994; Camacho-Vanegas, O., Lorein, F., and Amaldi, F., Flat absorbance background for sucrose gradients, Anal. Biochem. 228, 172–173, 1995; Ben-Zeev, O. and Doolittle, M.H., Determining lipase subunit structure by sucrose gradient centrifugation, Methods Mol. Biol. 109, 257–266, 1999; Lustig, A., Engel, A., Tsiotis, G. et al., Molecular weight determination of membrane proteins by sedimentation equilibrium at the sucrose of nycodenz-adjusted density of the hydrated detergent micelle, Biochim. Biophys. Acta 1464, 199–206, 2000; Kim, Y.S., Jones, L.A., Dong, A. et al., Effects of sucrose on conformational equilibria and fluctuations within the native-state ensemble of proteins, Protein Sci. 12, 1252–1261, 2003; Srinivas, K.A., Chandresekar, G., Srivastava, R., and Puvanakrishna, R., A novel protocol for the subcellular fractionation of C3A hepatoma cells using sucrose-density gradient centrifugation, J. Biochem. Biophys. Methods 60, 23–27, 2004; Richter, W., Determining the subunit structure of phosphodiesterase using gel filtration and sucrose-density gradient centrifugation, Methods Mol. Biol. 307, 167–180, 2005; Cioni, P., Bramanti, E., and Strambini, G.B., Effects of sucrose on the internal dynamics of azurin, Biophys. J. 88, 4213–4222, 2005; Desplats, P., Folco, E. and Salerno, G.L., Sucrose may play an additional role to that of an osmolyte in Synechocystis sp. PCC 6803 salt-shocked cells, Plant Physiol. Biochem. 43, 133–138, 2005; Chen, L., Ferreira, J.A., Costa, S.M. et al., Compaction of ribosomal protein S6 by sucrose occurs only under native conditions, Biochemistry 21, 2189–2199, 2006.
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803
Chemicals Commonly Used in Biochemistry and Molecular Biology and Their Properties (Continued) Common Name
Chemical Name
Sulfuric Acid
H2SO4
Tetrabutylammonium Chloride
M.W. 98.1 277.9
Properties and Comment Strong acid; component of piranha solution with hydrogen peroxide. Ion-pair reagent for extraction and HPLC.
H3C CH3
Cl–
N+
H3C CH3
Walseth, T.F., Graff, G., Moos, M.C., Jr., and Goldberg, N.D., Separation of 5′-ribonucleoside monophosphates by ion-pair reverse-phase highperformance liquid chromatography, Anal. Biochem. 107, 240–245, 1980; Ozkul, A. and Oztunc, A., Determination of naprotiline hydrochloride in tables by ion-pair extraction using bromthymol blue, Pharmzie 55, 321–322, 2000; Cecchi, T., Extended thermodynamic approach to ion interaction chromatography. Influence of the chain length of the solute ion; a chromatographic method for the determination of ion-pairing constants, J. Sep. Sci 28, 549–554, 2005; Pistos, C., Tsantili-Kakoulidou, A., and Koupparis, M., Investigation of the retention/pH profile of zwitterionic fluoroquinolones in reversed-phase and ion-interaction high-performance liquid chromatography, J. Pharm. Biomed. Anal. 39, 438–443, 2005; Choi, M.M., Douglas, A.D., and Murray, R.W., Ion-pair chromatographic separation of water-soluble gold monolayer-protected clusters, Anal. Chem. 78, 2779–2785, 2006; Saradhi, U.V., Prarbhakar, S., Reddy, T.J., and Vairamani, M., Ion-pair solid-phase extraction and gas chromatography mass spectrometric determination of acidic hydrolysis products of chemical warfare agents from aqueous samples, J. Chromatog. A, 1129, 9–13, 2006. Tetrahydrofuran
Trimethylene Oxide
72.1
Solvent; template for combinatorial chemistry.
O
Leuty, S.J., Rapid dehydration of plant tissues for paraffin embedding; tetrahydrofuran vs. t-butanol, Stain Technol. 44, 103–104, 1969; Tandler, C.J. and Fiszer de Plazas, S., The use of tetrahydrofuran for delipidation and water solubilization of brain proteolipid proteins, Life Sci. 17, 1407–1410, 1975; Dressman, J.B., Himmelstein, K.J., and Higuchi, T., Diffusion of phenol in the presence of a complexing agent, tetrahydrofuran, J. Pharm. Sci. 72, 12–17, 1983; Diaz, R.S., Regueiro, P., Monreal, J., and Tandler, C.J., Selective extraction, solubilization, and reversed-phase highperformance liquid chromatography separation of the main proteins from myelin using tetrahydrofuran/water mixtures, J. Neurosci. Res. 29, 114–120, 1991; Santa, T., Koga, D., and Imai, K., Reversed-phase high-performance liquid chromatography of fullerenes with tetrahydrofuran-water as a mobile phase and sensitive ultraviolet or electrochemical detection, Biomed. Chromatogr. 9, 110–111, 1995; Lee, J., Kang, J.H., Lee, S.Y. et al., Protein kinase C ligands based on tetrahydrofuran templates containing a new set of phorbol ester pharmacophores, J. Med. Chem. 42, 4129–4139, 1999; Edwards, A.A., Ichihara, O., Murfin, S. et al., Tetrahydrofuran-based amino acids as library scaffolds, J. Comb. Chem. 6, 230–238, 2004; Baron, C.P., Refsgaard, H.H., Skibsted, L.H., and Andersen, M.L., Oxidation of bovine serum albumin initiated by the Fenton reaction — effect of EDTA, tert-butylhydroperoxide, and tetrahydrofuran, Free Radic. Res. 40, 409–417, 2006; Bowron, D.T., Finney, J.L., and Soper, A.K., The structure of liquid tetrahydrofuran, J. Am. Chem. Soc. 128, 5119–5126, 2006; Hermida, S.A., Possari, E.P., Souza, D.B. et al., 2′-deoxyguanosine, 2′-deoxycytidine, and 2′-deoxyadenosine adducts resulting from the reaction of tetrahydrofuran with DNA bases, Chem. Res. Toxicol. 19, 927–936, 2006; Li, A.C., Li, Y., Guirguis, M.S., Advantages of using tetrahydrofuran-water as mobile phases in the quantitation of cyclosporine A in monkey and rat plasma by liquid chromatography-tandem mass spectrometry, J. Pharm. Biomed. Anal. 43, 277–284, 2007.
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Chemicals Commonly Used in Biochemistry and Molecular Biology and Their Properties (Continued) Common Name
Chemical Name
Tetraphenylphosphonium Bromide
M.W. 419.3
Properties and Comment Membrane-permeable probe; determination of metal ions.
Br+
P+
Boxman, A.W., Barts, P.W., and Borst-Pauwels, G.W., Some characteristics of tetraphenylphosphonium uptake into Saccharomyces cerevisiae, Biochim. Biophys. Acta 686, 13–18, 1982; Flewelling, R.F. and Hubbell, W.L., Hydrophobic ion interactions with membranes. Thermodynamic analysis of tetraphenylphosphonium binding to vesicles, Biophys. J. 49, 531–540, 1986; Prasad, R. and Hofer, M., Tetraphenylphosphonium is an indicator of negative membrane potential in Candida albicans, Biochim. Biophys. Acta 861, 377–380, 1986; Aiuchi, T., Matsunada, M., Nakaya, K., and Nakamura, Y., Calculation of membrane potential in synaptosomes with use of a lipophilic cation (tetraphenylphosphonium), Chem. Pharm. Bull. 37, 3333–3337, 1989; Nhujak T. and Goodall, D.M., Comparison of binding of tetraphenylborate and tetraphenylphosphonium ion to cyclodextrins studied by capillary electrophoresis, Electrophoresis 22, 117–122, 2001; Yasuda, K., Ohmizo, C., and Katsu, T., Potassium and tetraphenylphosphonium ion-selective electrodes for monitoring changes in the permeability of bacterial outer and cytoplasmic membranes, J. Microbiol. Methods 54, 111–115, 2003; Min, J.J., Biswal, S., Deroose, C., and Gambhir, S.S., Tetraphenylphosphonium as a novel molecular probe for imaging tumors, J. Nucl. Med. 45, 636–643, 2004. Thionyl Chloride
Sulfurous Oxychloride
118.97
Preparation of acyl chlorides.
Rodin, R.L. and Gershon, H., Photochemical alpha-chlorination of fatty acid chlorides by thionyl chloride, J. Org. Chem. 38, 3919–3921, 1973; DuVal, G., Swaisgood, H.E., and Horton, H.R, Preparation and characterization of thionyl chloride-activated succinamidopropyl-glass as a covalent immobilization matrix, J. Appl. Biochem. 6, 240–250, 1984; Molnar-Perl, I., Pinter-Szakacs, M., and Fabian-Vonsik, V., Esterification of amino acids with thionyl chloride acidified butanols for their gas chromatographic analysis, J. Chromatog. 390, 434–438, 1987; Stabel, T.J., Casele, E.S., Swaisgood, H.E., and Horton, H.R., Anti-IgG immobilized controlled pore glass. Thionyl chloride-activated succinamidopropyl-gas as a covalent immobization matrix, Appl. Biochem. Biotechnol. 36, 87–96, 1992; Chamoulaud, G. and Belanger, D., Chemical modification of the surface of a sulfonated membrane by formation of a sulfonamide bond, Langmuir 20, 4989–4895, 2004; Porjazoska, A.,Yilmaz, O.K., Baysal, K. et al., Synthesis and characterization of poly(ethylene glycol)-poly(D,L-lactide-co-glycolide) poly(ethylene glycol) tri-block co-polymers modified with collagen: a model surface suitable for cell interaction, J. Biomater. Sci. Polym. Ed. 17, 323–340, 2006; Gao, C., Jin, Z.Q., Kong, H. et al., Polyurea-functionalized multiwalled carbon nanotubes: synthesis, morphology, and Ramam spectroscopy, J. Phys. Chem. B 109, 11925–11932, 2005; Chen, G.X., Kim, H.S., Park, B.H., and Yoon, J.S., Controlled functionalization of multiwalled carbon nanotubes with various molecularweight poly(L-lactic acid), J. Phys. Chem. B 109, 22237–22243, 2005. Thiophosgene
CSCl2
115
Thiourea
Thiocarbamide
76.12
Chaotropic agent; useful for membrane proteins; will react with haloacetyl derivatives such as iodoacetamide; protease inhibitor.
S
H2N
9168_Book.indb 804
NH2
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Chemicals Commonly Used in Biochemistry and Molecular Biology and their Properties
805
Chemicals Commonly Used in Biochemistry and Molecular Biology and Their Properties (Continued) Common Name
Chemical Name
M.W.
Properties and Comment
Maloof, F. and Soodak, M., Cleavage of disulfide bonds in thyroid tissue by thiourea, J. Biol. Chem. 236, 1689–1692, 1961; Gerfast, J.A., Automated analysis for thiourea and its derivatives in biological fluids, Anal. Biochem. 15, 358–360, 1966; Lippe, C., Urea and thiourea permeabilities of phospholipid and cholesterol bilayer membranes, J. Mol. Biol. 39, 588–590, 1966; Carlsson, J., Kierstan, M.P., and Brocklehurst, K., Reactions of L-ergothioneine and some other aminothiones with 2,2′- and 4,4′-dipyridyl disulphides and of L-ergothioneine with iodoacetamide, 2-mercaptoimidazoles, and 4-thioypyridones, thiourea, and thioacetamide as highly reactive neutral sulphur nucleophiles, Biochem. J. 139, 221–235, 1974; Filipski, J., Kohn K.W., Prather, R., and Bonner, W.M., Thiourea reverses crosslinks and restores biological activity in DNA treated with dichlorodiaminoplatinum (II), Science 204, 181–183, 1979; Wasil, M., Halliwell, B., Grootveld, M. et al., The specificity of thiourea, dimethylthiourea, and dimethyl sulphoxide as scavengers of hydroxyl radicals. Their protection of alpha-1-antiproteinase against inactivation by hypochlorous acid, Biochem. J. 243, 867–870, 1987; Doona, C.J. and Stanbury, D.M., Equilibrium and redox kinetics of copper(II)–thiourea complexes, Inorg. Chem. 35, 3210–3216, 1996; Rabilloud, T., Use of thiourea to increase the solubility of membrane proteins in two-dimensional electrophoresis, Electrophoresis 19, 758–760, 1998; Musante, L., Candiano, G., and Ghiggeri, G.M., Resolution of fibronectin and other uncharacterized proteins by two-dimensional polyacrylamide electrophoresis with thiourea, J. Chromatog. B 705, 351–356, 1998; Nagy, E., Mihalik, R., Hrabak, A. et al., Apoptosis inhibitory effect of the isothiourea compound, tri-(2-thioureido-S-ethyl)-amine, Immunopharmacology 47, 25–33, 2000; Galvani, M., Rovatti, L., Hamdan, M. et al., Protein alkylation in the presence/absence of thiourea in proteome analysis: a matrix-assisted laser desorption/ionization-time-of-flight-mass spectrometry investigation, Electrophoresis 22, 2066–2074, 2001; Castellanos-Serra, L. and Paz-Lago, D., Inhibition of unwanted proteolysis during sample preparation: evaluation of its efficiency in challenge experiments, Electrophoresis 23, 1745–1753, 2002; Tyagarajan, K., Pretzer, E., and Wiktorowicz, J.E., Thiol-reactive dyes for fluorescence labeling of proteomic samples, Electrophoresis 24, 2348–2358, 2003; Fuerst, D.E., and Jacosen, E.N., Thiourea-catalyzed enantioselective cyanosilylation of ketones, J. Am. Chem. Soc. 127, 8964–8965, 2005; Gomez, D.E., Fabbrizzi, L., Licchelli, M., and Monzani, E., Urea vs. thiourea in anion recognition, Org. Biomol. Chem. 3, 1495–1500, 2005; George, M., Tan, G., John, V.T., and Weiss, R.G., Urea and thiourea derivatives as low molecular-mass organochelators, Chemistry 11, 3243–3254, 2005; Limbut, W., Kanatharana, P., Mattiasson, B. et al., A comparative study of capacitive immunosensors based on self-assembled monolayers formed from thiourea, thioctic acid, and 3-mercaptopropionic acid, Biosens. Bioelectron. 22, 233–240, 2006. TNBS
Trinitrobenzenesulfonic Acid
293.2
Reagent for the determination of amino groups in proteins; also reacts with sulfydryl groups and hydrazides; used to induce animal model of colitis.
NO2
O2N
NO2 SO3–
Habeeb, A.F., Determination of free amino groups in proteins by trinitrobenzenesulfonic acid, Anal. Biochem. 14, 328–336, 1966; Goldfarb, A.R., A kinetic study of the reactions of amino acids and peptides with trinitrobenzenesulfonic acid, Biochemistry 5, 2570–2574, 1966; Scheele, R.B. and Lauffer, M.A., Restricted reactivity of the epsilon-amino groups of tobacco mosaic virus protein toward trinitrobenzenesulfonic acid, Biochemistry 8, 3597–3603, 1969; Godin, D.V. and Ng, T.W., Trinitrobenzenesulfonic acid: a possible chemical probe to investigate lipid–protein interactions in biological membranes, Mol. Pharmacol. 8, 426–437, 1972; Bubnis, W.A. and Ofner, C.M., III, The determination of epsilon-amino groups in soluble and poorly soluble proteinaceous materials by a spectrophotometric method using trinitrobenzenesulfonic acid, Anal. Biochem. 207, 129–133, 1992; Cayot, P. and Tainturier, G., The quantification of protein amino groups by the trinitrobenzenesulfonic acid method: a reexamination, Anal. Biochem. 249, 184–200, 1997; Neurath, M., Fuss, I., and Strober, W., TNBS-colitis, Int. Rev. Immunol. 19, 51–62, 2000; Lindsay, J., Van Montfrans, C., Brennen, F. et al., IL-10 gene therapy prevents TNBS-induced colitis, Gene Ther. 9, 1715–1721, 2002; Whittle, B.J., Cavicchi, M., and Lamarque, D., Assessment of anticolitic drugs in the trinitrobenzenesulfonic acid (TNBS) rat model of inflammatory bowel disease, Methods Mol. Biol. 225, 209–222, 2003; Necefli, A., Tulumoglu, B., Giris, M. et al., The effects of melatonin on TNBS-induced colitis, Dig. Dis. Sci. 51, 1538–1545, 2006. TNM
Tetranitromethane
196.03
Modification of tyrosine residues in proteins; crosslinking a side reaction as a reaction with cysteine; antibacterial and antiviral agent.
NO2 O2N
C
NO2
NO2
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806
Chemicals Commonly Used in Biochemistry and Molecular Biology and Their Properties (Continued) Common Name
Chemical Name
M.W.
Properties and Comment
Sokolovsky, M., Riordan, J.F., and Vallee, B.L., Tetranitromethane. A reagent for the nitration of tyrosyl residues in proteins, Biochemistry 5, 3582–3589, 1966; Nishikimi, M. and Yagi, K., Reaction of reduced flavins with tetranitromethane, Biochem. Biophys. Res. Commun. 45, 1042–1048, 1971; Kunkel, G.R., Mehrabian, M., and Martinson, H.G., Contact-site crosslinking agents, Mol. Cell. Biochem. 34, 3–13, 1981; Rial, E. and Nicholls, D.G., Chemical modification of the brown-fat-mitochondrial uncoupling protein with tetranitromethane and N-ethylmaleimide. A cysteine residue is implicated in the nucleotide regulation of anion permeability, Eur. J. Biochem. 161, 689–694, 1986; Prozorovski, V., Krook, M., Atrian, S. et al., Identification of reactive tyrosine residues in cysteine-reactive dehydrogenases. Differences between liver sorbitol, liver alcohol, and Drosophila alcohol dehydrogenase, FEBS Lett. 304, 46–50, 1992; Gadda, G., Banerjee, A., and Fitzpatrick, P.F., Identification of an essential tyrosine residue in nitroalkane oxidase by modification with tetranitromethane, Biochemistry 39, 1162–1168, 2000; Hodges, G.R. and Ingold, K.U., Superoxide, amine buffers, and tetranitro-methane: a novel free radical chain reaction, Free Radic. Res. 33, 547–550, 2000; Capeillere-Blandin, C., Gausson, V., Descamps-Latscha, B., and Witko-Sarsat, V., Biochemical and spectrophotometric significance of advanced oxidation protein products, Biochim. Biophys. Acta 1689, 91–102, 2004; Lundblad, R.L., Chemical Reagents for Protein Modification, CRC Press, Boca Raton, FL, 2004; Negrerie, M., Martin, J.L., and Nghiem, H.O., Functionality of nitrated acetylcholine receptor: the two-step formation of nitrotyrosines reveals their differential role in effectors binding, FEBS Lett. 579, 2643–2647, 2005; Carven, G.J. and Stern, L.J., Probing the ligand-induced conformational change in HLA-DR1 by selective chemical modification and mass spectrometry mapping, Biochemistry 44, 13625–13637, 2005. Trehalose
α-D-glucopyranoglucopyranosyl-1,1-α-Dglucopyranoside; Mycose
342.3
A nonreducing sugar that is found in a variety of organisms where it is thought to protect against stress such as dehydration; there is considerable interest in the use of trehalose as a stabilizer in biopharmaceutical proteins.
OH
CH2OH O HO
CH2OH
OH O
OH
O
OH
OH Elbein, A.D., The metabolism of alpha, alpha-trehalose, Adv. Carbohydr. Chem. Biochem. 30, 227–256, 1974; Wiemken, A., Trehalose in yeast, stress protectant rather than reserve carbohydrate, Antonie Van Leeuwenhoek, 58, 209–217, 1990; Newman, Y.M., Ring, S.G., and Colaco, C., The role of trehalose and other carbohydrates in biopreservation, Biotechnol. Genet. Eng. Rev. 11, 263–294, 1993; Panek, A.D., Trehalose metabolism — new horizons in technological applications, Braz. J. Med. Biol. Res. 28, 169–181, 1995; Schiraldi, C., Di Lernia, I., and De Rosa, M., Trehalose production: exploiting novel approaches, Trends Biotechnol. 20, 420–425, 2002; Elbein, A.D., Pan, Y.T., Pastuszak, I., and Carroll, D., New insights on trehalose: a multifunctional molecule, Glycobiology 13, 17R–27R, 2003; Gancedo, C. and Flores, C.L., The importance of a functional trehalose biosynthetic pathway for the life of yeasts and fungi, FEMS Yeast Res. 4, 351–359, 2004; Cordone, L., Cottone, G., Giuffrida, S. et al., Internal dynamics and protein-matrix coupling in trehalose-coated proteins, Biochim. Biophys. Acta 1749, 252–281, 2005. Trichloroacetic Acid
163.4
Protein precipitant.
O Cl OH Cl
9168_Book.indb 806
Cl
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807
Chemicals Commonly Used in Biochemistry and Molecular Biology and Their Properties (Continued) Common Name
Chemical Name
M.W.
Properties and Comment
Chang, Y.C., Efficient precipitation and accurate quantitation of detergent-solubilized membrane proteins, Anal. Biochem. 205, 22–26, 1992; Sivaraman, T., Kumar, T.K., Jayaraman, G., and Yu. C., The mechanism of 2,2,2-trichloroacetic acid-induced protein precipitation, J. Protein Chem. 16, 291–297, 1997; Arnold, U. and Ulbrich-Hoffman, R., Quantitative protein precipation form guandine hydrochloride-containing solutions by sodium deoxycholate/trichloroacetic acid, Anal. Biochem. 271, 197–199, 1999; Jacobs, D.I., van Rijssen, M.S., van der Heijden, R., and Verpoorte, R., Sequential solubilization of proteins precipitated with trichloroacetic acid in acetone from cultured Catharanthus roseus cells yields 52% more spots after two-dimensional electrophoresis, Proteomics 1, 1345–1350, 2001; Garcia-Rodriguez, S., Castilla, S.A., Machado, A., and Ayala, A., Comparison of methods for sample preparation of individual rat cerebrospinal fluid samples prior to two-dimensional polyacrylamide gel electrophoresis, Biotechnol. Lett. 25, 1899–1903, 2003; Chen, Y.Y., Lin, S.Y., Yeh, Y.Y. et al., A modified protein precipitation procedure for efficient removal of albumin from serum, Electrophoresis 26, 2117–2127, 2005; Zellner, M., Winkler, W., Hayden, H. et al., Quantitative validation of different protein precipitation methods in proteome analysis of blood platelets, Electrophoresis 26, 2481–2489, 2005; Carpentier, S.C., Witters, E., Laukens, K. et al., Preparation of protein extracts from recalcitrant plant tissues: an evaluation of different methods for two-dimensional gel electrophoresis analysis, Proteomics 5, 2497–2507, 2005; Manadas, B.J., Vougas, K., Fountoulakis, M., and Duarte, C.B., Sample sonication after trichloroacetic acid precipitation increases protein recovery from cultured hippocampal neurons, and improves resolution and reproducibility in two-dimensional gel electrophoresis, Electrophoresis 27, 1825–1831, 2006; Wang, A., Wu, C.J., and Chen, S.H., Gold nanoparticle-assisted protein enrichment and electroelution for biological samples containing low protein concentration — a prelude of gel electrophoresis, J. Proteome Res. 5, 1488–1492, 2006. Triethanolamine Tris(2-hydroxyethyl)amine 149.2 Buffer; transdermal transfer reagent. H2 C
H2 C
HO C H2
OH C H2
N H2C CH2
H2 C
HO C H2 pKa approx. 9.5
H N+
OH C H2
–
Cl
H2C CH2 OH
OH Triethanolamine
H2 C
Triethanolamine hydrochloride
Fitzgerald, J.W., The Tris-catalyzed isomerization of potassium D-glucose 6-O-sulfate, Can. J. Biochem. 53, 906–910, 1975; Buhl, S.N., Jackson, K.Y., and Graffunder, B., Optimal reaction conditions for assaying human lactate dehydrogenase pyruvate-to-lactate at 25, 30, and 37 degrees C, Clin. Chem. 24, 261–266, 1978; Myohanen, T.A., Bouriotas, V., and Dean, P.D., Affinity chromatography of yeast alpha-glucosidase using ligandmediated chromatography on immobilized phenylboronic acids, Biochem. J. 197, 683–688, 1981; Shinomiya, Y., Kato, N., Imazawa, M., and Miyamoto, K., Enzyme immunoassay of the myelin basic protein, J. Neurochem. 39, 1291–1296, 1982; Arita, M., Iwamori, M., Higuchi, T., and Nagai, Y., 1,1,3,3-tetramethylurea and triethanolaminme as a new useful matrix for fast atom bombardment mass spectrometry of gangliosides and neutral glycosphingolipids, J. Biochem. 93, 319–322, 1983; Cao, H. and Preiss, J., Evidence for essential arginine residues at the active site of maize branching enzymes, J. Protein Chem. 15, 291–304, 1996; Knaak, J.B., Leung, H.W., Stott, W.T. et al., Toxicology of mono-, di-, and triethanolamine, Rev. Environ. Contim. Toxicol. 149, 1–86, 1997; Liu, Q., Li, X., and Sommer, S.S., pK-matched running buffers for gel electrophoresis, Anal. Biochem. 270, 112–122, 1999; Sanger-van de Griend, C.E., Enantiomeric separation of glycyl dipeptides by capillary electrophoresis with cyclodextrins as chiral selectors, Electrophoresis 20, 3417–3424, 1999; Fang, L., Kobayashi, Y., Numajiri, S. et al., The enhancing effect of a triethanolamine-ethanol-isopropyl myristate mixed system on the skin permeation of acidic drugs, Biol. Pharm. Bull. 25, 1339–1344, 2002; Musial, W. and Kubis, A., Effect of some anionic polymers of pH of triethanolamine aqueous solutions, Polim. Med. 34, 21–29, 2004. Triethylamine CH3
N,N-diethylethanamine
101.2
Ion-pair reagent; buffer.
CH3
N
CH3
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808
Chemicals Commonly Used in Biochemistry and Molecular Biology and Their Properties (Continued) Common Name
Chemical Name
M.W.
Properties and Comment
Brind, J.L., Kuo, S.W., Chervinsky, K., and Orentreich, N., A new reversed-phase, paired-ion thin-layer chromatographic method for steroid sulfate separations, Steroids 52, 561–570, 1988; Koves, E.M., Use of high-performance liquid chromatography-diode array detection in forensic toxicology, J. Chromatog. A 692, 103–119, 1995; Cole, S.R. and Dorsey, J.G., Cyclohexylamine additives for enhanced peptide separations in reversed-phase liquid chromatography, Biomed. Chromatog. 11, 167–171, 1997; Gilar, M. and Bouvier, E.S.P., Purification of crude DNA oligonucleotides by solid-phase extraction and reversed-phase high-performance liquid chromatography, J. Chromatog. A 890, 167–177, 2000; Loos, R. and Barcelo, D., Determination of haloacetic acids in aqueous environments by solid-phase extraction followed by ion-pair liquid chromatography-electrospray ionization mass spectrometric detection, J. Chromatog. A 938, 45–55, 2001; Gilar, M., Fountain, K.J., Budman, Y. et al., Ion-pair reversed-phase high-performance liquid chromatography analysis of oligonucleotides: retention prediction, J. Chromatog. A 958, 167–182, 2002; El-dawy, M.A., Mabrouk, M.M., and El-Barbary, F.A., Liquid chromatographic determination of fluoxetine, J. Pharm. Biomed. Anal. 30, 561–571, 2002; Yang, X., Zhang, X., Li, A. et al., Comprehensive two-dimensional separations based on capillary high-performance liquid chromatography and microchip electrophoresis, Electrophoresis 24, 1451–1457, 2003; Murphey, A.T., Brown-Augsburger, P., Yu, R.Z. et al., Development of an ion-pair reverse-phase liquid chromatographic/tandem mass spectrometry method for the determination of an 18-mer phosphorothioate oligonucleotide in mouse liver tissue, Eur. J. Mass Spectrom. 11, 209–215, 2005; Xie, G., Sueishi, Y., and Yamamoto, S., Analysis of the effects of protic, aprotic, and multi-component solvents on the fluorescence emission of naphthalene and its exciplex with triethylamine, J. Fluoresc. 15, 475–483, 2005. Trifluoroacetic Acid
114.0
Ion-pair reagent; HLPC; peptide synthesis.
O F OH F
F
Rosbash, D.O. and Leavitt, D., Decalcification of bone with trifluoroacetic acid, Am. J. Clin. Pathol. 22, 914–915, 1952; Katz, J.J., Anhydrous trifluoroacetic acid as a solvent for proteins, Nature 174, 509, 1954; Uphaus, R.A., Grossweiner, L.I., Katz, J.J., and Kopple, K.D., Fluorescence of tryptophan derivatives in trifluoroacetic acid, Science 129, 641–643, 1959; Acharya, A.S., di Donato, A., Manjula, B.N. et al., Influence of trifluoroacetic acid on retention times of histidine-containing tryptic peptides in reverse phase HPLC, Int. J. Pept. Protein Res. 22, 78–82, 1983; Tsugita, A., Uchida, T., Mewes, H.W., and Ataka, T., A rapid vapor-phase acid (hydrochloric and trifluoroacetic acid) hydrolysis of peptide and protein, J. Biochem. 102, 1593–1597, 1987; Hulmes, J.D. and Pan, Y.C., Selective cleavage of polypeptides with trifluoroacetic acid: applications for microsequencing, Anal. Biochem. 197, 368–376, 1991; Eshragi, J. and Chowdhury, S.K., Factors affecting electrospray ionization of effluents containing trifluoroacetic acid for high-performance liquid chromatography/mass spectrometry, Anal. Chem. 65, 3528–3533, 1993; Apffel, A., Fischer, S., Goldberg, G. et al., Enhanced sensitivity for peptide mapping with electrospray liquid chromatography-mass spectrometry in the presence of signal suppression due to trifluoroacetic acid- containing mobiles phases, J. Chromatog. A 712, 177–190, 1995; Guy, C.A. and Fields, G.B., Trifluoroacetic acid cleavage and deprotection of resin-bound peptides following synthesis by Fmoc chemistry, Methods Enzymol. 289, 67–83, 1997; Morrison, I.M. and Stewart, D., Plant cell wall fragments released on solubilization in trifluoroacetic acid, Phytochemistry 49, 1555–1563, 1998; Yan, B., Nguyen, N., Liu, L. et al., Kinetic comparison of trifluoroacetic acid cleavage reactions of resin-bound carbamates, ureas, secondary amides, and sulfonamides from benzyl-, benzhyderyl-, and indole-based linkers, J. Comb. Chem. 2, 66–74, 2000; Ahmad, A., Madhusudanan, K.P., and Bhakuni, V., Trichloroacetic acid- and trifluoroacetic acid-induced unfolding of cytochrome C: stabilization of a nativelike fold intermediate(1), Biochim. Biophys. Acta 1480, 201–210, 2000; Chen, Y., Mehok, A.R., Mant, C.T. et al., Optimum concentration of trifluoroacetic acid for reversed-phase liquid chromatography of peptide revisited, J. Chromatog. A 1043, 9–18, 2004. Tris(2-carboxyethyl) phosphine H3C
P
TCEP
250.2
Reducing agent.
CH3
H3C
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Chemicals Commonly Used in Biochemistry and Molecular Biology and their Properties
809
Chemicals Commonly Used in Biochemistry and Molecular Biology and Their Properties (Continued) Common Name
Chemical Name
M.W.
Properties and Comment
Gray, W.R., Disulfide structures of highly bridged peptides: a new strategy for analysis, Protein Sci. 2, 1732–1748, 1993; Gray, W.R., Echistatin disulfide bridges: selective reduction and linkage assignment, Protein Sci. 2, 1749–1755, 1993; Han, J.C. and Han, G.Y., A procedure for quantitative determination of Tris(2-carboxyethyl)phosphine, an odorless reducing agent more stable and effective than dithiothreitol, Anal. Biochem. 220, 5–10, 1994; Wu, J., Gage, D.A., and Watson, J.T., A strategy to locate cysteine residues in proteins by specific chemical cleavage followed by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry, Anal. Biochem. 235, 161–174, 1996; Han, J., Yen. S., Han, G., and Han, F., Quantitation of hydrogen peroxide using Tris(2-carboxyethyl) phosphine, Anal. Biochem. 234, 107–109, 1996; Han, J., Clark, C., Han, G. et al., Preparation of 2-nitro-5-thiobenzoic acid using immobilized Tris(2-carboxyethyl) phosphine, Anal. Biochem. 268, 404–407, 1999; Anderson, M.T., Trudell, J.R., Voehringer, D.W. et al., An improved monobromobimane assay for glutathione utilizing Tris-(2-carboxyethyl)phosphine as the reductant, Anal. Biochem. 272, 107–109, 1999; Shafer, D.E., Inman, J.K. and Lees, A. Reaction of Tris(2-carboxyethyl)phosphine (TCEP) with maleimide and alpha-haloacyl groups: anomalous elution of TCEP by gel filtration, Anal. Biochem. 282, 161–164, 2000; Rhee, S.S. and Burke, D.H., Tris(2-carboxyethyl)phosphine stabilization of RNA: comparison with dithiothreitol for use with nucleic acid and thiophosphoryl chemistry, Anal. Biochem. 325, 137–143, 2004; Legros, C., Celerier, M.L., and Guette, C., An unusual cleavage reaction of a peptide observed during dithiothreitol and Tris(2-carboxyethyl)phosphine reduction: application to sequencing of HpTx2 spider toxin using nanospray tandem mass spectrometry, Rapid Commun. Mass Spectrom. 19, 1317–1323, 2004; Xu, G., Kiselar, J., He, Q., and Chance, M.R., Secondary reactions and strategies to improve quantitative protein footprinting, Anal. Chem. 77, 3029–3037, 2005; Valcu, C.M. and Schlink, K., Reduction of proteins during sample preparation and two-dimensional gel electrophoresis of woody plant samples, Proteomics 6, 1599–1605, 2006; Scales, C.W., Convertine, A.J., and McCormick, C.L., Fluorescent labeling of RAFT-generated poly(N-isopropylacrylamide) via a facile maleimide-thiol coupling reaction, Biomacromolecules 7, 1389–1392, 2006. Urea Carbamide 60.1 Chaotropic agent. O
H2N
NH2
Edelhoch, H., The effect of urea analogues and metals on the rate of pepsin denaturation, Biochim. Biophys. Acta 22, 401–402, 1956; Steven, F.S. and Tristram, G.R., The denaturation of ovalbumin. Changes in optical rotation, extinction, and viscosity during serial denaturation in solution of urea, Biochem. J. 73, 86–90, 1959; Nelson, C.A. and Hummel, J.P., Reversible denaturation of pancreatic ribonuclease by urea, J. Biol. Chem. 237, 1567–1574, 1962; Herskovits, T.T., Nonaqueous solutions of DNA; denaturation by urea and its methyl derivatives, Biochemistry 2, 335–340, 1963; Subramanian, S., Sarma, T.S., Balasubramanian, D., and Ahluwalia, J.C., Effects of the urea–guanidinium class of protein denaturation on water structure: heats of solution and proton chemical shift studies, J. Phys. Chem. 75, 815–820, 1971; Strachan, A.F., Shephard, E.G., Bellstedt, D.U. et al., Human serum amyloid A protein. Behavior in aqueous and urea-containing solutions and antibody production, Biochem. J. 263, 365–370, 1989; Gervais, V., Guy, A., Teoule, R., and Fazakerley, G.V., Solution conformation of an oligonucleotide containing a urea deoxyribose residue in front of a thymine, Nucleic Acids Res. 20, 6455–6460, 1992; Smith, B.J., Acetic acid-urea polyacrylamide gel electrophoresis of proteins, Methods Mol. Biol. 32, 39–47, 1994; Buck, M., Radford, S.E., and Dobson, C.M., Amide hydrogen exchange in a highly denatured state. Hen egg-white lysozyme in urea, J. Mol. Biol. 237, 247–254, 1994; Shirley, B.A., Urea and guanidine hydrochloride denaturation curve, Methods Mol. Biol. 40, 177–190, 1995; Bennion, B.J. and Daggett, V., The molecular basis for the chemical denaturation of proteins by urea, Proc. Natl. Acad. Sci. USA 100, 5142–5147, 2003; Soper, A.K., Castner, E.W., and Luzar, A., Impact of urea on water structure: a clue to its properties as a denaturant? Biophys.Chem.105, 649–666, 2003; Smith, L.J., Jones, R.M., and van Gunsteren, W.F., Characterization of the denaturation of human alpha-1-lactalbumin in urea by molecule dynamics simulation, Proteins 58, 439–449, 2005; Idrissi, A., Molecular structure and dynamics of liquids: aqueous urea solutions, Spectrochim. Acta A Mol. Biomol. Spectrosc. 61, 1–17, 2005; Chow, C., Kurt, N., Murphey, R.M., and Cavagnero, S., Structural characterization of apomyoglobin self-associated species in aqueous buffer and urea solution, Biophys. J. 90, 298–309, 2006. Vinyl Pyridine
4-vinylpyridine
105.1
Modification of cysteine residues in protein.
CH2 CH
N
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Chemicals Commonly Used in Biochemistry and Molecular Biology and Their Properties (Continued) Common Name Water
Chemical Name Hydrogen Oxide
M.W. 18.0
Properties and Comment Solvent.
Lumry, R. and Rajender, S., Enthalpy-entropy compensation phenomena in water solutions of proteins and small molecules: a ubiquitous property of water, Biopolymers 9, 1125–1227, 1970; Cooke, R. and Kuntz, I.D., The properties of water in biological systems, Annu. Rev. Biophys. Bioeng. 3, 95–126, 1974; Fettiplace, R. and Haydon, D.A., Water permeability of lipid membranes, Physiol. Rev. 60, 510–550, 1980; Lewis, C.A. and Wolfenden, R., Antiproteolytic aldehydes and ketones: substituent and secondary deuterium isotope effects on equilibrium addition of water and other nucleophiles, Biochemistry 16, 4886–4890, 1977; Wolfenden, R.V., Cullis, P.M., and Southgate, C.C., Water, protein folding, and the genetic code, Science 206, 575–577, 1979; Wolfenden, R., Andersson, L., Cullis, P.M., and Southgate, C.C., Affinities of amino acid side chains for solvent water, Biochemistry 20, 849–855, 1981; Cullis, P.M. and Wolfenden, R., Affinity of nucleic acid bases for solvent water, Biochemistry 20, 3024–3028, 1981; Radzicka, A., Pedersen, L., and Wolfenden, R., Influences of solvent water on protein folding: free energies of salvation of cis and trans peptides are nearly identical, Biochemistry 27, 4538–4541, 1988; Dzingeleski, G.D. and Wolfenden, R., Hypersensitivity of an enzyme reaction to solvent water, Biochemistry 32, 9143–9147, 1993; Timasheff, S.N., The control of protein stability and association by weak interactions with water: how do solvents affect these processes? Annu. Rev. Biophys. Biomol. Struct. 22, 67–97, 1993; Wolfenden, R. and Radzcika, A., On the probability of finding a water molecule in a nonpolar cavity, Science 265, 936–937, 1994; Jayaram, B. and Jain, T., The role of water in protein–DNA recognition, Annu. Rev. Biophys. Biomol. Struct. 33, 343–361, 2004; Pace, C.N., Trevino, S., Prabhakaran, E., and Scholtz, J.M., Protein structure, stability, and solubility in water and other solvents, Philos. Trans. R. Soc. Lond. B Biol. Sci. 359, 1225–1234, 2004; Rand, R.P., Probing the role of water in protein conformation and function, Philos. Trans. R. Soc. Lond. B Biol. Sci. 359, 1277–1284, 2004; Bagchi, B., Water dynamics in the hydration layer around proteins and micelles, Chem. Rev. 105, 3179–3219, 2005; Raschke, T.M., Water structure and interactions with protein surfaces, Curr. Opin. Struct. Biol. 16, 152–159, 2006; Levy, Y. and Onuchic, J.N., Water mediation in protein folding and molecular recognition, Annu. Rev. Biophys. Biomol. Struct. 35, 389–415, 2006; Wolfenden, R., Degrees of difficulty of water-consuming reactions in the absence of enzymes, Chem. Rev. 106, 3379–3396, 2006.
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9168_Book.indb 811
Type
Composition
Form
Brij 35
Nonionic
Tergitol TMN
Nonionic
Triton X-45
Nonionic
Solid white wax; density 1.18–1.22 (6) Pale yellow liquid, 90%; density, 1.024 (6) Amber liquid, 100% (6)
Triton X-100
Nonionic
Amber liquid (6)
0.16 g/l (4)
64°C (5)
Tween 20 (Polysorbate 20) Tween 80 (Polysorbate 80) CTAB
Nonionic
Polyoxyethylene lauryl ether (E23) Polyoxyethylene trimethyl nonanol (E6) Polyoxyethylene octylphenol (E5) Polyoxyethylene tertoctylphenol (E10) Polyoxyethylene sorbitan monolaurate (E22) Polyoxyethylene sorbitan monooleate (E25) Cetyltrimethyl-ammonium bromide
Yellow oily liquid (6)
0.14 g/l
95°C (4)
93°C (4)
0.33 g/l (10)
SDS
Anionic
2.3 g/l (10)
Cholate
Anionic
Amber liquid; density 1.05–1.10 (6) Creamy-white voluminous powder (13) White crystals, flakes, or powder (13) White Powder
Deoxycholate
Anionic
Lubrol W
Nonionic
Atlas G
Nonionic
Span 20
Nonionic
Nonionic Cationic
Sodium dodecyl (lauryl) sulfate 3, 7, 12-Trihydroxy-5 cholanate 3, 12-Dihydroxy-5 cholanate A fatty alcohol ethylene oxide condensate Polyoxyethylene (sorbitol) hexanolate Sorbitan mono-laurate
Compiled by A. Fulmer. * Critical micelle concentration in H2O, 25°C.
CMC*
Cloud Point
Solubility
0.058 g/1 (4)
100°C (4)
—
36°C (4)
Soluble in H2O and (6) most organic solvents insoluble in oils Completely soluble in H2O (6) Soluble in most organic solvents; insoluble in H2O (6) Soluble in H2O and (6) alcohols; slightly soluble in aromatic solvents Soluble in H2O and organic solvents (6) Soluble in H2O and most organic solvents; insoluble in oils (6) Soluble in H2O and organic solvents (13)
Applications Column chromatography (7) Membrane solubilization (2) Membrane solubilization (2) Membrane solubilization (2) gel electrophoresis (9) Solubilizer (1, 4) Solubilizer (1, 4) Membrane solubilization (2)
Fawn colored waxy solids (6)
Protein solubilization (11), gel electrophoresis (12) Partially soluble in H2O; soluble in most Membrane solubilization (2) organic solvents (13) Partially soluble in H2O; soluble in most Membrane solubilization (2) organic solvents (13) Soluble in H2O, vegetable oils and fatty Membrane solubilization (14) acids (6) Membrane solubilization (14)
White Powder
H2O, 25°C
Soluble in H2O
Membrane solubilization (14)
Common Detergents Used in Biochemical Research
Name
811
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Handbook of Biochemistry and Molecular Biology
812
References 1. Elworthy, Florence, and Macfarlane, Solubilization by Surface-Active Agents, Chapman and Hall Ltd., London, 1968. 2. Foxx and Keith, Membrane Molecular Biology, Sinaur Associates, Stamford, Conn., 1972. 3. Swanson, Bradford, and McIlwain, Biochem. J., 92, 235 (1964). 4. Schönfeldt, Surface-Active Ethylene Oxide Adducts, Pergamon Press, New York, 1969. 5. Shinoda, Nakagawa, Tamamuchi, and Isemura, Colloidal Surfactants, Academic Press, New York, 1963. 6. Sisley and Wood, Encyclopedia of Surface-Active Agents, Chemical Publishing, New York, 1964. 7. Morris and Morris, Separation Methods in Biochemistry, Pitman Publishing, New York, 1963. 8. Roodyn, Biochem J., 85,177 (1962). 9. Alfageme, Zweider, Mahowald, and Cohen, J. Biol. Chem., 249, 3729 (1974). 10. Mukerjee and Mysels, Critical Micelle Concentrations of Aqueous Surfactants Systems, Natl. Stand. Ref. Data Ser., Natl. Bureau Stand., 1971. 11. Reynolds and Tanford, Proc. Natl. Acad. Sci. U.S.A., 66, 1002, (1970). 12. Dunder and Reukert, J. Biol. Chem., 244, 5074 (1969). 13. Stecher, Ed., The Merck Index of Chemicals and Drugs, 7th ed., Merck & Co., Rahway, N.J., 1960. 14. Umbreit and Strominger, Proc. Natl. Acad. Sci. U.S.A., 70, 2997 (1973).
General references for surfactants and detergents Siskey, J.P.(trans. P.J. Wood), Encyclopedia of Surface-Active Agents, Chemical Publishing Company, New York, NY, USA, 1952 Ferguson, L.N., On the water solubilities of ethers, J.Amer.Chem.Soc. 77, 5288-5289, 1955 Nonionic Surfactants, ed. M.J. Schick, Marcel Dekker, New York, NY, USA, 1966 Jungermann, E., Cationic Surfactants, Marcel Dekker, New York, NY, USA, 1970 Cabane, B., Structure of some polymer-detergent aggregates in water, J.Phys.Chem. 81, 1639-1645, 1977 Helenius, A., McCaslin, D.R., Fries, E., and Tanford, C., Properties of detergents, Methods Enzymol. 56, 734-749, 1979 Membranes Detergents, and Receptor Solubilization, ed. J.C. Venter and L.C. Harrison, A.R.Liss, New York, NY, USA, 1984 Industrial Applications of Surfactants II, ed. D.R. Karsa, Royal Society of Chemistry, Cambridge, UK, 1990 Neugebauer, J.M., Detergents: an overview, Methods Enzymol. 182, 239253, 1990 Industrial Applications of Surfactants III, ed. D.R. Karsa, Royal Society of Chemistry, Cambridge, UK, 1992 Surfactants in Lipid Chemistry: Recent Synthetic, Physical and Biodegradation Studies, ed. J.H.P. Tyman, Royal Society of Chemistry, Cambridge, UK, 1992
9168_Book.indb 812
Lawrence, M.J., Surfactant systems: Their use in drug delivery, Chem.Soc. Rev. 23, 417-223, 1994 Porter, M.R., Handbook of Surfactants, 2nd edn., Blackie Academic & Professional, Glasgow, UK, 1994 Structure and Flow in Surfactant Solutions, ed. C.A. Herb and R.K. Prud’homme, American Chemical Society, Washington, DC, 1994 – Chapter 23 (pps 320-336), Li, Y. and Dubin, P.L., Polymersurfactant complexes – Chapter 26 (pps. 370-379), Smith, B.C., Chou, L.-C., Lu, B., and Zakin, J.L., Effect of counterion structure in flow birefringence and dray reduction behavior of quaternary ammonium salt cationic surfactants – Chapter 27 (pps. 380-393), Burgess, D.J. and Sahin, N.O., Interfacial rheology of b-casein solutions Stevens, L., Solutions used in enzymology, in Enzymology LabFax, ed. P.C. Engel, Bios Scientific Publishers, Oxford, UK, Chaper 9, pps. 269289, 1996 Cogdell, R.J. and Lindsay, J.G., Integral Membrane Proteins, in Protein LabFax, ed. N.C. Price, Bios Scientific Publishers, Oxford, UK, Chapter 10, pps. 101-107, 1996 Jönsson, B., Lindman, B., Holmberg, K., and Kronberg, B., Surfactants and Polymers in Aqueous Solution, John Wiley & Sons, Chichester, UK, 1998 Tsujii, K., Surface Activity Principles, Phenomena, and Applications, Academic Press, San Diego, CA, USA, 1998 Holmberg, K., Novel Surfactants Preparations, Applications and Biodegradability, Marcel Dekker, New York, NY, USA, 1998 Hill, R.M., Silicone Surfactants, Marcel Dekker, New York, NY, 1999 Industrial Applications of Surfactants IV, ed. D.R. Karsa, Royal Society of Chemistry, Cambridge, UK, 1999 Specialist Surfactants, ed. I.D. Robb, Blackie Academic & Professional, London, UK, 1997 Liposomes Rational Design, ed. A.S. Janoff, Marcel Dekker, New York, NY, USA, 1999 Hummel, D.O., Handbook of Surfactant Analysis. Chemical, PhysicoChemical and Physical Methods, John Wiley & Sons, Ltd., Chichester, UK, 2000 Corrigan, O.I. and Healy, A.M., Surfactants in pharmaceutical products and systems, in Encyclopedia of Pharmaceutical Technology, ed. J. Swarbrick and J.C. Boylan, Marcel Dekker, New York, NY, Volume 3, pp 2639-2653, 2002 Encyclopedia of Surface and Colloid Science, ed. A.T. Hubbard, Marcel Dekker, Inc, New York, NY, USA, 2002 – Hirata, H. Surfactant molecular complexes, Volume 4, pps 5178-5204 – Imamura, T., Surfactant-protein interactions, Volume 4, pps 5230-5243 Rosen, M.J., Surfactants and Interfacial Phenomena, Wiley-Interscience, Hoboken, NJ, USA, 2004 Goodwin, J.W., Colloids and Interfaces with Surfactants and Polymers: An Introduction, John Wiley, Hoboken, NJ, USA, 2004 Tadros, T.F., Applied Surfactants. Principles and Applications, Wiley-VCH, Weinheim, Germany, 2005 Handbook of Functional Lipids, ed. C.C. Akoh, CRC/Taylor & Francis, Boca Raton, FL, USA, 2006
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Some Properties of Detergents and Surfactants Used in Biochemistry and Molecular Biology Structures for these compounds may be found on p. 816–818. Detergent/Surfactant
Molecular Weight
Tween 20a Tween 80b Triton X-100c Nonidet P-40d Brij Deterentse (polyoxyethylene derivatives) Lubrolf Sodium dodecyl dulfate (SDS)g Sodium deoxycholateh Cetylpyridinium chloridei Cetyltrimethylammonium bromidej Tetradecyltrimethyl-ammonium bromidek Betainesulfonate,l sulfobetaine, alkylsulfobetaine derivatives CHAPSm CHAPSOn
Footnotes Tween 20 is a polysorbate surfactant (polyoxyethylene sorbitan fatty acid ester) useful in immunoassays and pharmaceutical development. Tween 20 reduced protein interaction and protein binding to surfaces. It is related to Tween 80. The Tween surfactants are condensation products of mono-substituted fatty acyl derivatives of sorbitan (SPAN®; a registered trademark of Atlas Chemical) and ethylene oxide. Span® 20 is sorbitan monolaurate and Tween® 20 (polyethoxyethylene (20) sorbitan monolaurate) is derived from Span® 20. Tween®20 has a viscosity of 200-400 cp at 25°C.
a
Tween® 80 (polysorbate 80) is a polysorbate surfactant (polyoxyethylene sorbitan mono-oleate) is used in pharmaceutical formulation where it stabilizes proteins and in various diagnostics tests. Tween® 80 was added early to bacterial culture media and was also demonstrated to enhance the anti-bacterial activity of certain antibiotics. Tween® 80 has also been used for protein membrane studies and were originally manufactured by Atlas Power Company. Tween 80 has a viscosity of 600-800 cp at 25°C. Triton X-100 is a nonionic detergent known as octoxynol, octylphenoxy polyethoxyethanol. The Triton family of surfactants which is manufactured by the condensation of ethylene oxide with alkylphenols and contain between 5 and 15 ethylene oxide units; with Triton X-100, there is 9 or 10 ethylene oxide units while Triton X-30 contains three ethyloxide groups. Triton X-100 is also known as Igepal CA or Polydetergent G. These alkylaryl polyether alcohols were first manufacture by I.G. Farbenindustrie as Igepal products and subsequently manufactured and marketed in the United States as Triton. Triton X-100 is used for the lysis of cells and preparation of subcellular fractions and in the study of membrane proteins.
d Nonidet® is a registered trademark of the Shell Oil Company; however, it is not clear that Shell Oil Company is still associated with the manufacture and/or distribution of this material. A search of the Shell Oil website did not a result for Nonidet. Nonidet® P-40 [octylphenolpoly(ethyleneglycolether)] is a popular alkylphenyl ethoxylate nonionic detergent; also referred to as nonylphenylpolyethylene glycol; polyethyleneglycol-p-isooctylphenyl ether; octylphenoxy polyethoxy ethanol; Igepal CO 630. There is question as to the relation of nonidet P-40 to NP-40. Related to the Triton surfactants. It is useful to assure the provenance of a product labeled Nonidet P40 or NP-40.
nonionic nonionic nonionic nonionic nonionic nonionic anionic anionic cationic cationic cationic zwitterionic zwitterionic zwitterionic
1,300 650 650 582 288.4 432 340.0 364.5 336 614.9 630.9
Key References 1-5 6-10 11-15 16-20 21-25 26-30 31-35 36-40 41-45 46-50 51-55 56-60 61-65 66-70
e Brij® detergents are a series of polyoxyethylene ethers such as lauryl polyoxyethylene (dodecyl polyoxyethylate). This class of surfactants is referred to as poloxamers and includes polyoxypropylene. The number of ethylene oxide units determines the solubility of the dodecyl alcohol derivative:
Moles Ethylene Oxide/ Dodecyl Alcohol 0 2 4 6 7
b
c
Classification
Approximate Solubility of Product Insoluble Insoluble Somewhat miscible Slightly soluble Soluble
Lubrol® is a trademark of ICI referring to a series of nonionic surfactants similar to the Brij® surfactants in being a series of polyoxyethylene alkyl ethers. Lubrol® surfactants are used for membrane solubilization and more recently in the study of lipid rafts. While there are similarities between the various nonionic surfactants, there are also distinct differences.
f
Sodium Dodecyl Sulfate; also known as sodium lauryl sulfate. An anionic detergent used for membrane solubilization and protein denaturation. Used in a popular electrophoretic procedure, SDS-gel electrophoresis where separation is presumed to occur on the basis of molecular weight; there are some exceptions to this assumption (Noel, D., Nikaido, K., and Ames, G.F., A single amino acid substitution in a histidine-transport protein drastically alters its mobility in sodium dodecyl sulfatepolyacrylamide gel electrophoresis, Biochemistry 18, 4159-4165, 1979; Briggs, M.M., Klevit, R.E., and Schachat, F.H., Heterogeneity of contractile proteins-purification and characterization of 2 species of troponin-T from rabbit fast skeletal-muscle, J.Biol.Chem. 259, 10369-10375, 1984; Sakakura, Y., Hirabayashi, J.,. Oda, Y. et al., Structure of chicken 16-kDa b-galactoside-binding lectincomplete amino-acid-sequence, cloning of cDNA, and production of recombinant lectin, J.Biol.Chem. 265, 21573-21579, 1990; Okumura, N., Terasawa, F., Fujita, K. et al., Difference in electrophoretic mobility and plasmic digestion profile between four recombinant fibrinogens, gamma 308K, gamma 3081, gamma 308A, and wild type (gamma 308N), Electrophoresis 21, 2309-2315, 2000). The
g
813
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Handbook of Biochemistry and Molecular Biology
814 counterion to the lauryl sulfate moiety can also influence the interaction with proteins (Kubo, K. and Takagi, T., Modulation of the behavior of a protein in polyacrylamide gel electrophoresis in the presence of dodecyl sulfate by varying the cations, Anal.Biochem. 224, 572-579, 1995; Kubo, K., Effect of incubation of solutions of proteins containing sodium dodecyl sulfate on the cleavage of peptide bonds by boiling, Anal.Biochem. 225, 351-353, 1995). h Deoxycholic acid (3,12-dihydroxycholan-24-oic acid (3a, 5b, 12a), MW 392.6) is relatively insoluble, the sodium salt is more soluble. Deoxychlolate (desoxycholate) is used for membrane solubilization. A natural constituent of bile secretions in man and other mammals.
Cetylpyridinium chloride (1-hexadecylpyridinium chloride) is a cationic detergents which has pharmaceutical use as a preservative and a topical disinfectant. There has been significant use of this material as an active ingredient in mouthwashes. The interaction of cetylpyridinium chloride with glycosaminoglycans such as heparin is useful for characterization. Cetylpyridinium chloride has been used for the isolation of glycosaminoglycans and for the histochemical staining of glycosaminoglycans.
i
Cetyltrimethylammonium bromide (N,N,N-trimethylhexadecaaminium bromide; CTAB; cetrimonium bromide) was developed as a disinfectant and antiseptic. It has been used as detergent in a manner similar to SDS in the determination of protein molecular weight. CTAB is also used for the isolation and assay of nucleic acids. j
k Tetradecyltrimethyl-ammonium bromide (TTABr). TTABr is one of the components of Cetrimide®, a disinfectant. TTABr has seen occasional use for membrane protein solubilization and in the preparation of mixed micelles.
The “parent” compound for this class of surfactants is betaine sulfonate (sulfobetaine). There are non-detergent sulfobetaines (non detergent sulfobetaines such as 3-(1-pyridino)-1-propanesulfonate; NDSB 201)) which have been useful in preventing unwanted protein-protein aggregation ( Vuillard, L., Braun-Breton, C., and Rabilloud, T., Non-detergent sulfobetaines: a new class of mild solubilization agents for protein purification, Biochem.J. 305, 337-343, 1995; Collins, T., D’Amico, S., Georlette, D., et al., A nondetergent sulfobetaine prevents protein aggregation in microcalorimetric studies, Anal.Biochem. 352, 299-301,2006). Detergent sulfobetaines such Zwittergent® 3-12 (n-Dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate) contain a long chain alkyl function (Wollstadt, K.H., Karkhanis, Y.D., Gnozzio, M.J. et al., Potential of the sulfobetaine detergent Zwittergent 3-12 as a desorbing agent in biospecific and bioselective affinity chromatography, J.Chromatog. 497, 87-100, 1989). The CHAPS class of detergents are derivatives of sulfobetaine which contain a cholamide function. l
m
3-[(3-cholamidopropyl)dimethylamino]propanesulfonic acid
n 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate
References for table 1. Liljas, L., Lundahl, P., and Hjerten, S., Selective solubilization with Tween 20 of proteins from water-extracted human erythrocyte membranes. Analysis by gel electrophoresis in dodecylsulfate and in Tween 20, Biochim.Biophys.Acta. 352, 327-337, 1974. 2. Hoffman, W.L., and Jump, A.A., Tween 20 removes antibodies and other proteins from nitrocellulose, J.Immunol.Methods 94, 191-196, 1986.
9168_Book.indb 814
3. Vandenberg, E.T. and Krull, U.J., The prevention of adsorption of interferents to radiolabeled protein by Tween 20, J.Biochem.Biophys. Methods 22, 269-277, 1991 4. Feng, M., Morales, A.B., Poot, A., et al., Effects of Tween 20 on the desorption of protein from polymer surfaces, J.Biomater.Sci.Polym. Ed. 7, 415-424, 1995 5. Kreilgaard, L., Jones, L.S., Randolph, T.W., et al., Effect of Tween 20 on freeze-thawing- and agitation-induced aggregation of recombinant human factor XIII, J.Pharm.Sci. 87, 1597-1603, 1998. 6. Youmans, A.S. and Youmans, G.P., The effect of “tween 80” in vitro on the bacteriostatic activity of twenty compounds for Mycobacterium tuberculosis, J.Bacteriol. 56, 245-252, 1948. 7. Young, M., Dinda, M., and Singer, M., Effect of Tween 80 on lipid vesicle permeability, Biochim. Biophys.Acta. 735, 429-432, 1983. 8. Kerwin, B.A., Heller, M.C., Levin, S.H., and Randolph, T.W., Effects of Tween 80 and sucrose on acute short-term stability and longterm storage at -20°C of a recombinant hemoglobin, J.Pharm.Sci. 87, 1062-1068, 1998. 9. Arakawa, T. and Kita, Y., Protection of bovine serum albumin from aggregation by Tween 80, J.Pharm.Sci. 89, 646-651, 2000. 10. Hillgren, A., Lindgren, J., and Alden, M., Protection mechanism of Tween 80 during freeze-thawing of a model protein, LDH, Int.J.Pharm. 237, 57-69, 2002. 11. De Duve, C. and Wattiaux, R., Tissue fractionation studies. VII. Release of bound hydrolases by means of triton X-100, Biochem.J. 63, 606-608, 1956. 12. Ashani, Y. and Catravas, G.N., Highly reactive impurities in Triton X-100 and Brij 35: partial characterization and removal, Anal. Biochem. 109, 55-62, 1980. 13. Labeta, M.O., Fernandez, N., and Festenstein, H., Solubilization effect of Nonidet P-40, Triton X-100 and CHAPS in the detection of MHC-like glycoproteins, J.Immunol.Methods 112, 133-138, 1988. 14. Partearroyo, M.A., Urbaneja, M.A., and Goni, F.M., Effective detergent/lipid ratios in the solubilization of phosphatidylcholine vesicles by Triton X-100, FEBS Lett. 302, 138-140, 1992. 15. Blonder, J., Yu, L.R., Radeva, G., et al., Combined chemical and enzymatic stable isotope labeling for quantitative profiling of detergentinsoluble membrane proteins isolated membrane proteins isolated using Triton X-100 and Brij-96, J.Proteome Res. 5, 349-360, 2006. 16. Schwartz, B.D. and Nathenson, S.G., Isolation of H-2 alloantigens solubilized by the detergent NP-40, J.Immunol. 107, 1363-1367, 1971. 17. Hosaka, Y. and Shimizu, Y.K., Artificial assembly of envelope particles of HVJ (Sendai virus). I. Asssembly of hemolytic and fusion factors from envelopes solubilized by Nonidet P40, Virology 49, 627-639, 1972. 18. Hart, D.A., Studies on nonidet P40 lysis of murine lymphoid cells. I. Use of cholera toxin and cell surface Ig to determine degree of dissociation of the plasma membrane, J.Immunol. 115, 871-875, 1975. 19. Soloski, M.J., Cabrera, C.V., Esteban, M., and Holowczak, J.A., Studies concerning the structure of and organization of the vaccinia virus nucleod. I. Isolation and characterization of subviral particles prepared by treating virions with guanidine-HCl, nonidet-P40, and 2-mercaptoethanol, Virology 99, 209-217, 1979. 20. Lanuti, P., Marchisio, M., Cantilena, S., et al., A flow cytometry procedure for simultaneous characterization of cell DNA content and expression of intracellular protein kinase C-zeta, J.Immunol.Methods 315, 37-48, 2006. 21. Godson, G.N. and Sinsheimer, R.L., Use of Brij as a general method to prepare polyribosomes from Escherichia coli., Biochim.Biophys. Acta 149, 489-495, 1967. 22. Ashani, Y. and Catravas, G.N., Highly reactive impurities in Triton X-100 and Brij 35: partial characterization and removal, Anal. Biochem. 109, 55-62, 1980 23. Krause, M., Rudolph, R., and Schwartz, E., The non-ionic detergent Brij 58P mimics chaperone effects, FEBS Lett. 532, 253-255, 2002. 24. Lee, Y.C., Simamora, P., and Yalkowsky, S.H., Effect of Brij-78 on systemic delivery of insulin from an ocular device, J.Pharm.Sci. 86, 430-433, 1997. 25. Chakraborty, T., Ghosh, S., and Moulik, S.P., Micellization and related behavior of binary and ternary surfactant mixtures in aqueous medium: cetyl pyridinium chloride (CPC) cetyl trimethyl ammonium bromide (CTAB), and polyoxythelene (10) cetyl ether (Brij-56) derived system,
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Some Properties of Detergents and Surfactants Used in Biochemistry and Molecular Biology 26. Flawia, M.M. and Torres, H.N., Adenylate cyclase activity in lubroltreated membranes from Neurospora crassa, Biochim.Biophys.Acta 289, 428-432, 1972 27. Young, J.L. and Stansfield, D.A., Solubilization of bovine corpusluteum adenylate cyclase in Lubrol-PX, triton X-100 or digitonin and the stabilizing effect of sodium fluoride present in the solubilization medium, Biochem.J. 173, 919-924, 1978. 28. Hommes, F.A., Eller, A.G., Evans, B.A., and Carter, A.L., Reconstitution of ornithine transport in liposomes with Lubrol extracts of mitochondria, FEBS Lett. 170, 131-134, 1984. 29. Chamberlain, L.H., Detergents as tools for the purification and classification of lipid rafts, FEBS Lett. 559, 1-5, 2004. 30. Gil, C., Cubi, R., Blasi, J., and Aguilera, J., Synaptic proteins associate with a sub-set of lipid rafts when isolated from nerve endings at physiological temperature, Biochem.Biophys.Res.Commun. 348, 1334-1342, 2006 31. Emerson, M.F. and Holtzer, A., The hydrophobic bond in micellar systems. Effects of various additives on the stability of micelles of sodium dodecyl sulfate and of n-dodecyltrimethylammonium bromide, J.Phys.Chem. 71, 3320-3330, 1967. 32. Fish, W.W., Reynolds, J.A., and Tanford, C., Gel chromatography of proteins in denaturing solvents. Comparison between sodium dodecyl sulfate and guanidine hydrochloride as denaturants, J.Biol. Chem. 245, 5166-5168, 1970 33. Weber, K. and Kuter, D.J., Reversible denaturation of enzymes by sodium dodecyl sulfate, J.Biol.Chem. 246, 4504-4509, 1971. 34. Dai, S. and Tam, K.C., Effects of cosolvents on the binding interaction between poly(ethylene oxide) and sodium dodecyl sulfate, J.Phys. Chem.B Condens Mater Surf Interfaces Biophys. 110, 20794-20800, 2006. 35. Keller, S., Heerklotz, H., Jahnke, N., and Blume, A., Thermodyanamics of lipid membrane solubilization by sodium dodecyl sulfate, Biophys.J. 90, 4509-4521, 2006. 36. Benzonana, G., Study of bile salts micelles: properties of mixed oleate-deoxycholate solutions at pH 9.0, Biochim.Biophys.Acta. 176, 836-848, 1969 37. Olsenes, S., Removal of structural proteins from ribosomes by treatment with sodium dexoycholate I the presence of EDTA, FEBS Lett. 7, 211-213, 1970. 38. Ehrhart, J.C., and Chaveau, J., Differential solubilization of proteins, phospholipids, free and esterified cholesterol of rat liver cellular membranes by sodium deoxycholate, Biochim.Biophys.Acta 375, 434-445, 1975. 39. Robinson, N.C. and Tanford, C., The binding of deoxycholate, Triton X-100, sodium dodecyl sulfate, and phosphatidylcholine vesicles to cytochrome b5, Biochemistry 14, 369-378, 1975 40. Ranganathan, R., Tcacenco, C.M., Rossetto, R., and Hajdu, J., Characterization of the kinetics of Phospholipase C activity toward mixed micelles of sodium deoxycholate and dimyristoylphosphatidylcholine, Biophys.Chem. 122 79-89, 2006 41. Malchiodi Albedi, F., Cassano, A.M., Ciaralli, F., et al., Influence of cetylpyridinium chloride on the ultrastructural appearance of sulphated glycosaminoglycans in human colonic mucosa, Histochemistry 89, 397-401, 1988 42. Chardin, H., Septier, D., and Goldberg, M., Visualization of glycosaminoglycans in rat incisor predentin and dentin with cetylpyridinium chloride-glutaraldehyde as fixative, J.Histochem.Cytochem. 38, 885-894, 1900 43. Savolainen, H., Isolation and separation of proteoglycans, J.Chromatogr.B Biomed. Sci.Appl. 722, 255-262, 1999 44. Benamor, M., Aguersif, N., and Draa, M.T., Spectrophotometric determination of cetylpyridinium chloride in pharmaceutical products, J.Pharm.Biomed.Anal. 26, 151-154, 2001 45. Arrigler, V., Kogej, K., Majhenc, J., and Svetina, S., Interaction of cetylpyridinium chloride with giant vesicles, Langmuir 21, 76537661, 2005 46. Davies, G.E., Quaternary ammonium compounds: a new technique for the study of their bactericidal action and the results obtained with cetavlon (cetyltrimethylammonium bromide), J.Hyg.(Lond), 47, 271-277, 1949 47. Akin, D.T., Shapira, R., and Kinkade, J.M., Jr., The determination of molecular weights of biologically active proteins by cetyltrimethylammonium bromide-polyacrylamide gel electrophoresis, Anal. Biochem. 145, 170-176, 1985
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48. Jost, J.P., Jiricny, J. and Saluz, H., Quantitative precipitation of short oligonucleotides with low concentrations of cetyltrimethylammonium bromide, Nucleic Acid Res. 17, 2143, 1989 49. Li, Y.F., Shu, W.Q., Feng, P., et al., Determination of DNA with cetyltrimethylammonium bromide by the measurement of resonance light scattering, Anal.Sci. 17, 693-696, 2001 50. Carra, A., Gambino, G., and Schubert, A., A cetyltrimethylammonium bromide-based method to extract low-molecular-weight RNA from polysaccharide-rich plant tissues, Anal.Biochem. 360, 318-320, 2007. 51. Hayakawa, K., Santerre, J.P., and Kwak, J.C, The binding of cationic surfactants by DNA, Biophys.Chem. 17, 175-181, 1983 52. Castedo, A., Castillo, J.L.D., Suarez-Filloys, M.J., and Rodriguez, J.R., Effect of temperature on the mixed micellar tetradecyltrimethylammonium bromide-butanol system, J.Colloid Interface Sci. 196, 148156, 1997 53. Medrzycka, K. and Zwierzykowski, W., Adsorption of alkyltrimethylammonium bromides at the various interfaces, J.Colloid Interface Sci. 230, 67-72, 2000 54. Stodghill, S.P., Smith, A.E., and O’haver, J.H., Thermodynamics of micellization and adsorption of three alkyltrimethylammonium bromides using isothermal titration calorimetry, Langmuir 20, 1138711392, 2004 55. Rasmussen, C.D., Nielsen, H.B., and Andersen, J.E., Analysis of the purity of cetrimide by titrations, PDA J. Pharm.Sci.Technol. 60, 104110, 2006. 56. Sims, N.R, Horvath, L.B., and Carnegie, P.R., Detergent solubilization and solubilization of 2’:3’-cyclic nucleotide 3’-phosphodiesterase from isolated myelin and c6 cells, Biochem.J. 181, 367-375, 1979 57. Wong, R.K., Nichol, C.P., Sekar, M.C., and Roufogalis, B.D., The efficiency of various detergents for extraction and stabilization of acetylcholinesterase from bovine erythrocytes, Biochem.Cell Biol. 65, 8-18, 1987 58. Wydro, P. and Paluch, M., A study of the interaction of dodecyl sulfobetaine with cationic and anionic surfactant in mixed micelles and monolayers at the air/water interface, J.Colloid Interface Sci. 286, 387-391, 2005 59. Nyuta, K., Yoshimura, T., and Esumi, K., Surface tension and micellization of heterogemini surfactants containing quaternary ammonium salt and sulfobetaine moiety, J.Colloid Interface Sci. 301, 267-273, 2006. 60. Zanna, L. and Haeuw, J.F., Separation and quantitative analysis of alkyl sulfobetaine-type detergents by high-performance liquid chromatography and light-scattering detection, J.Chromatog.B Analyt. Technol.Biomed.Life Sci., in press, 2007. 61. Hjelmeland, L.M., A nondenaturing zwitterionic detergent for membrane biochemistry: Design and synthesis, Proc.Nat.Acad.Sci.USA 77, 6368-6370, 1980 62. Ray, J.P., Mernoff, S.T., Sangameswaran, L., and de Blas, A.L., The Stokes Radius of the CHAPS-solubilized benzodiazepine receptor complex, Neurochem.Res. 10, 1221-1229, 1985 63. Labeta, M.O., Fernandez, N., and Festenstein, H., Solubilisation effect of Nonidet P-40, Triton X-100 and CHAPS in the detection of MHC-like glycoprotein, J.Immunol.Methods 112, 133-138, 1988 64. Banerjee, P., Buse, J.T., and Dawson, G., Asymmetric extraction of membrane lipids by CHAPS, Biochim.Biophys.Acta 1044, 305-314, 1990 65. Rouvinski, A., Gahali-Sass, I., Stav, I., et al., Both raft- and non-raft proteins associate with CHAPS-insoluble complexes: some APP in large complexes, Biochem.Biophys.Res.Commun. 308, 750-758, 2003 66. Womack, M.D., Kendall, D.A., and MacDonald, R.C., Detergent effects on enzyme activity and solubilization of lipid bilayer membranes, Biochim.Biophys.Acta. 733, 210-215, 1983. 67. Saunders, C.R., 2nd and Prestegard, J.H., Magnetically orientable phospholipid bilayers containing small amounts of a bile salt analogue CHAPSO, Biophys.J. 58, 447-460, 1990 68. Gartner, W., Ullrich, D. and Vogt, K., Quantum yield of CHAPSOsolubilized rhodopsin and 3-hydroxy retinal containing bovine opsin, Photochem.Photobiol. 54, 1047-1055, 1991 69. Banerjee, P., Joo, J.B., Buse, J.T., and Dawson, G., Differential solubilization of lipids along with membrane proteins by different classes of detergents, Chem.Phys.Lipids 77, 65-78, 1995 70. Gehrig-Burger, K., Kohout, L., and Gimpl, G., CHAPSETEROL. A novel cholesterol-based detergent, FEBS J. 272, 800-812, 2005
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Handbook of Biochemistry and Molecular Biology
816 Tween
R O
C O O
Where R = fatty acid
HO
HO Sorbitan Fatty Acid Ester, SPAN®
OH
O R
C
O
H2 C
H2 C
O
H2 C
H2 C
CH2
O m
H
O
O n
H
H2 C
H2 C
O
n
O
O
H2 C
H2 C
O
H n
Polysorbate (Tween) Triton Detergents O
CH3
H2 C
H2 C
O
H3C
H n = 5–15, usually 9
CH3 H3C
CH3 Octoxynol, Triton X®, Igepal® CA
Alkylphenoxy ethoxylate nonionic detergent O
OH n
H3C
nonoxynol; non-ionic detergent Polyoxyethyleneglycol Ethers (Polyoxyethylene ethers) H2 C
R O
H C H2
n
R=alkyl such as cetyl, dodecyl
Sodium dodecylsulfate, SDS, lauryl sulfate, sodium salt
H3C
9168_Book.indb 816
O O
S
O–
O
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Some Properties of Detergents and Surfactants Used in Biochemistry and Molecular Biology
817
H3C OH CH3
COO– Na+ H
CH 3
H
H
H HO H
Cetylpyridinium Chloride Cl– N+
Betaine –O
O
O
CH3
–O
N+
S O
CH3
CH3 N+
CH3
CH3
CH3 Betaine
Sulfobetaine
Sulfobetaine Derivatives Cl NaHSO3 +
Cl
O Epichlorohydrin
S OH
O
O
O–
Na+
3-chloro-2-hydroxypropanesulfonate, sodium salt O CH3 R
alkylamidopropyldimethylamine
O CH3 R
N H
N+
N H H3C
OH
O
N CH3
O– S O
alkylsulfobetaine derivative
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Handbook of Biochemistry and Molecular Biology
818 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) CH3 OH
O
H3C CH3
H
CH3
CH3
H3C
H
O
S
+ N
HO
O–
O
N H
OH H HO
BigChap N,N-bis(3-gluconamidopropyl)cholamide
HO OH HO HO
OH HO
O NH
HO OH HN
OH O
N H3C OH
O
CH3
CH3
HO
OH
CHAPSO CH3 OH
H3C
O
CH3 H
N H
CH3
S
+ N H
O–
O
H3C
O
CH3 HO
HO
OH H 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate (CHAPSO)
9168_Book.indb 818
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Some Biological Stains and Dyes Name
Description
Acridine Orange MW 320 as the chloride hydrate
CH3
Acridine dyes are strongly yellow fluorescent dyes; stains for nucleic acids and is used for identification of the malaria parasite. Acridine orange is weakly basic, is permeable to membranes and tends to accumulate in intracellular acidic regions. Some use in photodynamic therapy for tumors. The binding of acridine orange to nucleic acids has been extensively studied. H N+
CH3 Cl–
N
H3C
CH3
Steiner, R.F. and Beers, R.F., Jr., Spectral changes accompanying binding of acridine orange by polyadenylic acid, Science 127, 335-336, 1958; Mayor, H.D. and Hill, N.O., Acridine orange staining of a single-stranded DNA bacteriophage, Virology 14, 264-266, 1961; Boyle, R.E., Nelson, S.S., Dollish, F.R., and Olsen, M.J., The interaction of deoxyribonucleic acid and acridine orange, Arch.Biochem.Biophys. 96, 47-50, 1962; Leith, J.D., Jr., Acridine orange and acriflavine inhibit deoxyribonuclease action, Biochim.Biophys.Acta. 72, 643-644, 1963; Morgan, R.S. and Rhoads, D.G., Binding of acridine orange to yeast ribosomes, Biochim.Biophys.Acta 102, 311-313, 1965; Yamabe, S., A spectrophotometric study on binding of acridine orange with DNA, Mol.Pharmacol. 3, 556-560, 1967; Stewart, C.R., Broadening by acridine orange of the thermal transition of DNA, Biopolymers 6, 1737-1743, 1968; Clerc, S. and Barenholz, Y., A quantitative model for using acridine orange as a transmembrane pH gradient probe, Anal.Biochem. 259, 104-111, 1998; Zoccarto, F., Cavallini, L., and Alexandre, A., The pH-sensitive dye acridine orange as a tool to monitor exocytosis/endocytosis, J.Neurochem. 72, 625-633, 1999; Lyles, M.B., Cameron, I.L., and Rawls, H.R., Structural basis for the binding efficiency of xanthines with the DNA intercalator acridine orange, J.Med.Chem. 44, 4650-4660, 2001; Lyles, M.B. and Cameron, I.L., Interactions of the DNA intercalator acridine orange, with itself, with caffeine, and the double stranded DNA, Biophys.Chem. 96, 53-76, 2002; Keiser, J., Utzinger, J., Premji, Z. et al., Acridine orange for malaria diagnosis: its diagnostic performance, its promotion and implementation in Tanzania, and the implications for malaria control, Ann.Trop.Med.Paristol. 96, 643-654, 2002; Lauretti, F., Lucas de Mel, F., Benati, F.J., et al., Use of acridine orange staining for the detection of rotavirus RNA in polyacrylamide gels, J.Virol.Methods 114, 29-35, 2003; Ueda, H.., Murata, H., Takeshita, H., et al., Unfiltered xenon light is useful for photodynamic therapy with acridine orange, Anticancer Res. 25, 3979-3983, 2005; Wang, F., Yang, J., Wu, X., et al., Improvement of the acridine orange-protein-surfactant system for protein estimation based on aromatic ring stacking effect of sodium dodecyl benzene sulphonate, Luminescence 21, 186-194, 2006; Hiruma, H., Katakura, T., Takenami, T., et al., Vesicle disruption, plasma membrane bleb formation, and acute cell death caused by illumination with blue light in acridine orange-loaded malignant melanoma cells, J.Photochem.Photobiol.B 86, 1-8, 2007. Alizarin Blue MW 292
a carbonyl dye; used as a pH indicator and a stain for copper
Meloan, S.N. and Puchtler, H., Iron alizarin blue S stain for nuclei, Stain Technol. 49, 301-304, 1974; Rosenthal, A.R. and Appleton, B., Histochemical localization of intraocular copper foreign bodies, Am.J.Ophthalmol. 79, 613-625, 1975; Rao, N.A., Tso, M.O., and Rosenthal, A.R., Chalcosis in the human eye. A clinicopathologic study, Arch Ophthalmol. 94, 1379-1384, 1976; Amin, A.S. and Dessouki, H.A., Facile colorimetric methods for the quantitative determination of tetramisole hydrochloride, Spectrochim.Acta A. Mol.Biomol.Spectros. 58, 2541-2546, 2002
O N
OH O
OH
819
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Handbook of Biochemistry and Molecular Biology
820
SOME BIOLOGICAL STAINS AND DYES (Continued) Name
Description
Amido Black 10B(Naphthal Blue Black; Amido Schwarz) MW 617 as sodium salt
Protein staining; originally developed as stain for collagen; protein determination.
NO2
N N
O
OH S
H2N
O HO
N N O
S
O
OH Mundkar, B. and Brauer, B., Selective localization of nucleolar protein with amido black 10B, J.Histochem.Cytochem. 14, 94-103, 1966; Mundkar, B. and Greenwood, H., Amido black 10B as a nucleolar stain for lymph nodes in Hodgkin’s disease, Acta Cytol. 12, 218-226, 1968; Schaffner, W. and Weissmann, C., A rapid, sensitive, and specific method for the determination of protein in dilute solution, Anal.Biochem. 56, 502-514, 1973; Kolakowski, E., Determination of peptides in fish and fish products. Part 1. Application of amido black 10B for determination of peptides in trichloroacetic acid extracts of fish meat, Nahrung 18, 371-383, 1974; Kruski, A.W. and Narayan, K.A., Some quantitative aspects of the disc electrophoresis of ovalbumin using amido black 10B stain, Anal.Biochem. 60, 431-440, 1974; Wilson, C.M., Studies and critique of Amido Black 10B, Coomassie Blue R, and Fast Green FCT as stains for protein after polyacrylamide gel electrophoresis, Anal.Biochem. 96, 263-278, 1979; Kaplan, R.S. and Pedersen, P.L., Determination of microgram quantities of protein in the presence of milligram levels of lipid with amido black 10B, Anal.Biochem. 150, 97-104, 1985; Nettleton, G.S., Johnson, L.R., and Sehlinger, T.E., Thin layer chromatography of commercial samples of amido black 10B, Stain Technol. 61, 329-336, 1986; Tumakov, S.A., Elanskaia, L.N., Esin, M.S., and Drozdova, N.I., Quantitative determination of protein in small volumes of biological substances using amido black 10B(article in Russian), Lab.Delo. (5), 54-56, 1988; Schulz, J., Dettlaff, S., Fritzsche, U., et al., The amido black assay: a simple and quantitative multipurpose test of adhesion, proliferation, and cytotoxicity in microplate cultures of keratinocytes (HaCaT) and other cell types growing adherently or in suspension, J.Immunol.Methods 167, 1-13, 1994; Gentile, F., Bali, E., and Pignalosa, G., Sensitivity and applications of the nondenaturing staining of proteins on polyvinylidene difluoride membranes with Amido Black 10B in water followed by destaining in water, Anal.Biochem. 245, 260-262, 1997; Plekhanov, A.Y., Rapid staining of lipids on thin-layer chromatograms with amido black 10B and other water-soluble stains, Anal.Biochem. 271, 186-187, 1999; Butler, P.J.G., Ubarretxene-Belandia, I., Warne, T., and Tate, C.G., The Escherichia coli multidrug transporter EmrE is a dimer in the detergent-solubilised state, J.Mol.Biol. 340, 797-808,. 2004 Azure Dyes (Azure A, Azure B or azure blue)
Cationic dyes which are used to stain nucleic acid and sulfated glycosaminoglycans such as heparin. The sensitivity for staining sulfated glycosaminoglycans is increased with the presence of silver.
N
CH3 H2N
S
N+
Cl–
CH3
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821
SOME BIOLOGICAL STAINS AND DYES (Continued) Name
Description
Klein, F. and Szirmai, J.A., Quantitative studies on the interaction of azure A with deoxyribonucleic acid and deoxyribonucleoprotein, Biochim.Biophys. Acta 72, 48-61, 1963; Goldstein, D.J., A further note on the measurement of the affinity of a dye (Azure A) for histological substrates, Q.J.Microsc.Sci. 106, 299-306, 1965; Wollin, A. and Jaques, L.B., Analysis of heparin—azure A metachromasy in agarose gel, Can.J.Physiol.Pharmacol. 50, 65-71, 1972; Lohr, W., Sohmer, I., and Wittekind, D., The azure dyes: their purification and physicochemical properties. I. Purification of azure A, Stain Technol. 49, 359-366, 1974; Bennion, P.J., Horobin, R.W., and Murgatroyd, L.B., The use of a basic dye (azure A or toluidine blue) plus a cationic surfactant for selective staining of RNA: a technical and mechanistic study, Stain Technol. 50, 307-313, 1975; Dutt, M.K., Staining of DNA-phosphate groups with a mixture of azure A and acridine orange, Microsc.Acta 82, 285-289, 1979; Tadano-Aritomi, K., and Ishizuka, I., Determination of peracetylated sulfoglycolipids using the azure A method, J.Lipid Res. 24, 1368-1375, 1983; Gundry, S.R., Klein, M.D,. Drongowski, R.A., and Kirsh, M.M., Clinical evaluation of a new rapid heparin assay using the dye azure A, Am.J.Surg. 148, 191-194, 1984; Lyon, M. and Gallagher, J.T., A general method for the detection and mapping of submicrogram quantities of glycosaminoglycan oligosaccharides on polyacrylamide gels by sequential staining with azure A and ammoniacal silver, Anal.Biochem. 185, 63-70, 1990; van de Lest, C.H., Versteeg, E.M., Veerkamp, J.H. and van Kuppevelt, T.H., Quantification and characterization of glycosaminoglycans at the nanogram level by a combined azure A-silver staining in agarose gels, Anal.Biochem. 221, 356-361, 1994; Wang, L., Malsch, P. and Harenberg, J., Heparins, low-molecular weight heparins, and other glycosaminoglycans analyzed by agarose gel electrophoresis and azure A-silver staining, Semin.Throm.Hemost. 23, 11-16, 1997. Biebrich Scarlet (Ponceau B), MW 556 as disodium salt
Anionic diazo dye used as cytoplasmic stain and a stain for basic proteins. Biebrich scarlet also binds specifically to lysozyme and chymotrypsin in a specific manner and inhibits enzyme activity HO
N NaO3S
N N
N SO3Na
Douglas, S.D., Spicer, S.S., and Bartels, P.H., Microspectrophotometric analysis of basic protein rich sites stained with Biebrich scarlet, J.Histochem. Cytochem. 14, 352-360, 1966; Winkelman, J.W. and Bradley, D.F., Binding of dyes to polycations. I. Biebrich scarlet and histone interaction parameters, Biochim.Biophys.Acta 126, 536-539, 1966; Saint-Blancard, J., Allary, M., and Jolles, P., Influence of Biebrich scarlet on the lysis kinetics of Micrococcus lysodeikticus by several lysozymes, Biochemie 54, 1375-1376, 1972; Holler, E., Rupley, J.A., and Hess, G.P., Productive and unproductive lysozymechitosaccharide complexes. Equilibrium measurements, Biochemistry 14, 1088-1094, 1975; Giannini, I. and Grasselli, P., Proton transfer to a charged dye bound to the alpha-chymotrypsin active site studied by laser photolysis, Biochim.Biophys.Acta 445, 420-425, 1976; Clark, G. and Spicer, S.S., The assessing of acidophilia with Biebrich scarlet, ponceau de zylidine and woodstain scarlet, Stain Techol. 54, 13-16, 1979; Smith-Gill, S.J., Wilson, A.C., Potter, M., et al., Mapping the antigenic epitope for a monoclonal antibody against lysozyme, J.Immunol. 128, 314-322, 1982; Mlynek, M.L., Comparative investigations on the specificity of Adams’ reaction and the Biebrich scarlet stain for the demonstration of eosinophilic granules, Klin. Wochenschr. 63, 646-647, 1985; Garvey, W., Fathi, A., Bigelow, F., et al., A combined elastic, fibrin and collagen stain, Stain Technol. 62, 365-368, 1987; Allcock, H.R. and Ambrosio, A.M., Synthesis and characterization of pH-sensitive poly(organophosphazene)hydrogels, Biomaterials 17, 2295-2302, 1996; Ma, F., Koike, K., Higuchi, T., et al., Establishment of a GM-CSF-dependent megakaryoblastic cell line with the potential to differentiate into an eosinophilic linage in response to retinoic acids, Br.J.Haematol. 100, 427-435, 1998; Tan, K., Li, Y., and Huang, C., Flow-injection resonance light scattering detection of proteins at the nanogram level, Luminescence 20, 176-180, 2005.
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822
SOME BIOLOGICAL STAINS AND DYES (Continued) Name
Description
Blue tetrazolium; (Tetrazolium blue, bimethoxyneotetrazolium), MW 728 as the dichloride salt. Also nitro blue tetrazolium and tetranitro blue tetrazolium
N
A relatively large hydrophobic cation; histochemical stain, used for oxidoreductases. Forms a blue color in the presence of reducing agents which provided the basis for the early use of sulfydryl groups and other reducing compounds. Also used to measure free radicals, superoxide, and Amadori glycation products.
N
N
N Cl–
N
N+
N+
Cl– O
N
O H3C
CH3 Tetrazolium Blue
NO2
NO2
N
N
N
N Cl–
N
N+
N+
CI– O
N
O CH3
H3C Nitro Blue Tetrazolium
Sulkowitch, H., Rutenburg, A.M., Lesses, M.F., et al., Estimation of urinary reducing corticosteroids with blue tetrazolium, N.Engl.J.Med. 252, 1070-1075, 1955; Litteria, M. and Recknagel, R.O., A simplified blue tetrazolium reaction, J.Lab.Clin.Med. 48, 463-468, 1956; Leene, W. and van Iterson, W., Tetranitro—blue tetrazolium reduction in Bacillus subtilis, J.Cell Biol. 27, 237-241, 1965; Sedar, A.W. and Burde, R.M., The demonstration of the succinic dehydrogenase system in Bacillus subtilis using tetranitro—blue tetrazolium combined with techniques of electron microscopy, J.Cell Biol. 27, 53-66, 1965; Bhatnagar, R.S. and Liu, T.Z., Evidence for free radical involvement in the hydroxylation of proline: inhibition by nitro blue tetrazolium, FEBS Lett. 26, 32-34, 1972; Graham, R.E., Biehl, E.R., Kenner, C.T., et al., Reduction of blue tetrazolium by corticosteroids, J.Pharm.Sci. 64, 226-230, 1975; DeChatelet, L.R. and Shirley, P.S., Effect of nitro blue tetrazolium dye on the hexose monophosphate shunt activity of human polymorphonuclear leukocytes, Biochem.Med. 14, 391-398, 1975; Oteiza, R.M., Wooten, R.S., Kenner, C.T., et al., Kinetics and mechanism of blue tetrazolium reaction with corticosteroids, J.Pharm.Sci. 66, 1385-1388, 1977; DeBari, V.A. and Needle, M.A., Mechanism for transport of nitro-blue tetrazolium into viable and non-viable leukocytes, Histochemistry 56, 155-163, 1978; Biehl, E.R., Wooten, R., Kenner, C.T., and Graham, R.E., Kinetic and mechanistic studies of blue tetrazolium reaction with phenylhydrazines, J.Pharm.Sci. 67, 927-930, 1978; Schopf, R.E., Mattar, J., Meyenburg, W., et al., Measurement of the respiratory burst in human monocytes and polymorphonuclear leukocytes by nitro blue tetrazolium reduction and chemiluminescence, J.Immunol.Methods 67, 109-117, 1984; Jue, C.K. and Lipke, P.N., Determination of reducing sugars in the nanomole range with tetrazolium blue, J.Biochem.Biophys.Methods 11, 109-115, 1985; Walker, S.M., Howie, A.F., and Smith, A.F., The measurement of glycosylated albumin
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823
SOME BIOLOGICAL STAINS AND DYES (Continued) Name
Description
by reduction of alkaline nitro-blue tetrazolium, Clin.Chim.Acta 156, 197-206, 1986; Ghiggeri, G.M., Candiano, G., Ginevri, F., et al., Spectrophotometric determination of browning products of glycation of protein amino groups based on their reactivity with nitro blue tetrazolium salts, Analyst 113, 1101-1104, 1988; Brenan, M. and Bath, M.L., Indoxyl-tetranitro blue tetrazolium method for detection of alkaline phosphatase in immunohistochemistry, J.Histochem.Cytochem. 37, 1299-1301, 1989; Issopoulos, P.B., Sensitive colorimetric assay of cardidopa and methyldopa using tetrazolium blue chloride in pharmaceutical products, Pharm.Weekbl.Sci. 11, 213-217, 1989; Fattorossi, A., Nisini, R., Le Moli, S., et al., Flow cytometric evaluation of nitro blue tetrazolium (NBT) reduction in human polymorphonuclear leukocytes, Cytometry 11, 907-912, 1990; Albiach, M.R., Guerri, J., and Moreno, P., Multiple use of blotted polyvinylidene difluoride membranes immunostained with nitro blue tetrazolium, Anal. Biochem. 221, 25-28, 1994; Chanine, R., Huet, M.P., Oliva, L., and Nadeau, R., Free radicals generated by electrolysis reduces nitro blue tetrazolium in isolated rat heart, Exp.Toxicol.Pathol. 49, 91-95, 1997. Bromophenol Blue (bromphenol blue); tetrabromophenolsulfonphthalein, MW sultone, 670; sodium salt of sulfonic acid, 692
A vital stain; used for determination of protein, pH indicator. There are reports of specific binding to sites on proteins.
OH
Br
OH
Br
Br
Br Br
Br HO
HO O
O S
Br
OH
NaOH Br
SO2–
Na+
O
Bromophenol Blue (sultone) Bjerrum, O.J., Interaction of bromophenol blue and bilirubin with bovine and human serum albumin determined by gel filtration, Scand.J.Clin.Invest. 22, 41-48, 1968; Ramalingam, K. and Ravidranath, M.H., An evaluation of the metachromasia of bromophenol blue, Stain Technol. 47, 179-184, 1972; Harruff, R.C. and Jenkins, W.T., The binding of bromophenol blue to aspartate aminotransferase, Arch.Biochem.Biophys. 176, 206-213,1976; Krishnamoorthy, G. and Prabhananda, B.S., Binding site of the dye in bromophenol blue-lysozyme complex. Protein magnetic resonance study in aqueous solutions, Biochim.Biophys.Acta 709, 53-57, 1982; Ahmad, H. and Saleemuddin, M., Bromophenol blue protein assay: improvement in buffer tolerance and adaptation for the measurement of proteolytic activity, J.Biochem.Biophys.Methods 7, 335-343, 1983; Subrahanian, M., Sheshadri, B.S., and Venkatappa, M.P., Interaction of lysozyme with dyes. II. Binding of bromophenol blue, J.Biochem. 96, 245-252, 1984; Ma, C.Q., Li, K.A., and Tong, S.Y., Microdetermination of proteins by resonance light scattering spectroscopy with bromophenol blue, Anal.Biochem. 239, 86-91, 1996; Cathey, J.C., Schmidt, C.A., and DeWoody, J.A., Incorporation of bromophenol blue enhances visibility of polyacrylamide gels, BioTechniques 22, 222, 1997; Trivedi, V.D., On the role of lysine residues in the bromophenol blue-albumin interaction, Ital.J.Biochem. 46, 67-73, 1997; Bertsch, M., Mayburd, A.L., and Kassner, R.J., The identification of hydrophobic sites on the surface of proteins using absorption difference spectroscopy of bromophenol blue, Anal.Biochem. 313, 187-195, 2003; Li, J., Chatterjee, K., Medek, A., et al., Acid-base characteristics of bromophenol blue-citrate buffer systems in the amorphous state, J.Pharm.Sci. 93, 697-712, 2004; Sarma, S. and Dutta, R.K., Electronic spectral behavior of bromophenol blue in oil in water microemulsions stabilized by sodium dodecyl sulfate and n-butanol, Spectrochim.Acta A Mol.Biomol. Spectrosc. 64, 623-627, 2006; You, L., Wu, Z., Kim, T., and Lee, K., Kinetics and thermodynamics of bromophenol blue adsorption by a mesoporous hybrid gel derived from tetraethoxysilane and bis(trimethoxysilyl)hexane, J.Colloid Interface Sci. 300, 526-535, 2006; Zeroual, Y., Kim, B.S., Kim, C.S., et al., A comparative study on biosorption characteristics of certain fungi for bromophenol blue dye, Appl.Biochem.Biotechnol. 134, 51-60, 2006
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824
SOME BIOLOGICAL STAINS AND DYES (Continued) Name
Description
Bromothymol Blue or Bromthymol Blue; dibromothymolsulfonphthalein, MW as sultone, 624; as sodium salt, 646
A lipophilic dye; serves as a vital stain to trace fluid movements; used as an ion pair reagent for the measurement of certain drugs; extensive use as pH indicator as there is a change from yellow to blue as pH changes from 6 to 8.0
CH3
CH3 H 3C
OH
CH
OH
CH
H3C
Br
Br CH3
CH3 Br
Br CH3
O SO2
HO
H3C
CH
Base (NaOH)
OH
CH3
SHO2
HO
H3C
CH3
CH CH3
Azzone, G.F., Piemonte, G., and Massari, S., Intramembrane pH changes and bromthymol blue translocation in liver mitochondria, Eur.J.Biochem. 6, 207-212, 1968; Mitchell, P., Moyle, J., and Smith, L., Bromthymol blue as a pH indicator in mitochondrial suspensions, Eur.J.Biochem. 4, 9-19, 1968; Das Gupta, V. and Cadwallader, D.E., Determination of first pKa’ value and partition coefficients of bromthymol blue, J.Pharm.Sci. 57, 2140-2142, 1968; Jackson, J.B. and Crofts, A.R., Bromothymol blue and bromocresol purple as indicators of pH changes in chromatophores of Rhodospirillum rubrum, Eur.J.Biochem. 10,226-237, 1969; Gromet-Elhanan, Z. and Briller, S., On the use of bromthymol blue as an indicator of internal pH changes in chromatophores from Rhodospirillum rubrum, Biochem.Biophys.Res.Commun. 37, 261-265, 1969; Smith, L., Bromthymol blue as a pH indicator in mitochondrial suspensions, Ann.N.Y.Acad.Sci 147, 856, 1969; Lowry, J.B., Direct spectrophotometric assay of quaternary ammonium compounds using bromthymol blue, J.Pharm.Sci. 68, 110-111, 1979; Mashimo, T., Ueda, I., Shieh, D.D., et al., Hydrophilic region of lecithin membranes studied by bromothymol blue and effects of an inhalation anesthetic, enflurane, Proc.Natl.Acad.Sci.USA 76, 5114-5118, 1979; Yamamoto, A., Utsumi, E., Sakane, T. et al., Immunological control of drug absorption from the gastrointestinal tract: the mechanism whereby intestinal anaphylaxis interferes with the intestinal absorption of bromthymol blue in the rat, J.Pharm.Pharmacol. 38, 357-362, 1986; Dean, V.S., Dingley, J., and Vaughan, R.S., The use of bromothymol blue and sodium thiopentone to confirm tracheal intubation, Anaesthesia 51, 29-32, 1996; Gorbenko, G.P., Bromothymol blue as a probe for structural changes of model membranes induced by hemoglobin, Biochim.Biophys.Acta 1370, 107-118, 1998; Ramesh, K.C., Gowda, B.G., Melwanki, M.B., et al., Extractive spectrophotometric determination of antiallergic drugs in pharmaceutical formulations using bromopyrogallol and bromothymol blue, Anal.Sci. 17, 1101-1103, 2001; Rahman, N., Ahmed Khan, N. and Hejaz Azmi, S.N., Extractive spectrophotometric methods for the determination of nifedipine in pharmaceutical formulations using bromocresol green, bromophenol blue, bromothymol blue and eriochrome black T, Farmaco 59, 47-54, 2004; Erk, N., Spectrophotometric determination of indinavir in bulk and pharmaceutical formulations using bromocresol purple and bromothymol blue, Pharmazie 59, 183-186, 2004; HO O
HO
O
S S
O N N
NH2
9168_Book.indb 824
N
O N
NH2
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Some Biological Stains and Dyes
825
SOME BIOLOGICAL STAINS AND DYES (Continued) Name
Description
Congo Red (C.I. direct red 28); 696.7 as the disodium salt
Developed as acid-base indicator (Congo Red paper, Riegel’s paper); more recent use to detect amyloid peptide aggregates and other fibril structures (crossed b structures); also cellulose surfaces. See also thioflavin T
HO
HO
O S O
O S
O
N
N
N
O
O
N
NH2
NH2
Glenner, G.G., The basis of the staining of amyloid fibers: their physico-chemical nature and the mechanism of their dye-substrate interaction, Prog. Histochem.Cytochem. 13, 1-37, 1981; Elghetany, M.T. and Saleem, A., Methods for staining amyloid in tissues: a review, Stain Technol. 63, 201-212, 1988; Lorenzo, A. and Yankner, B.A., Amyloid fibril toxicity in Alzheimer’s disease and diabetes, Ann.N.Y.Acad.Sci. 777, 89-95, 1996; Sipe, J.D. and Cohen, A.S., Review: history of the amyloid fibril, J.Struct.Biol. 130, 88098, 2000; Piekarska, B., Konieczny, L, Rybarska, J., et al., Intramolecular signaling in immunoglobulins—new evidence emerging from the use of supramolecular protein ligands, J.Physiol.Pharmacol. 55, 487-501, 2004; Nilsson, M.R., Techniques to study amyloid fibril formation in vitro, Methods 34, 151-160, 2004; Ho, M.R., Lou, Y.C., Lin, W.C., et al., Human pancreatitis-associated protein forms fibrillar aggregates with a native-like conformation, J.Biol.Chem. 281, 33566-33576, 2006; Hatters, D.M., Zhong, N., Rutenber, E., and Weisgraber, K.H., Amino-terminal domain stability mediates apolipoprotein E aggregation into neurotoxic fibrils, J.Mol.Biol. 361, 932-944, 2006; McLaughlin, R.W., De Stigter, J.K., Sikkink, L.A., et al., The effects of sodium sulfate, glycosaminoglycans, and Congo red on the structure, stability, and amyloid formation of an immunoglobulin light-chain protein, Protein Sci. 15, 1710-1722, 2006; Sladewski, T.E., Shafer, A.M., and Hoag, C.M., The effect of ionic strength on the UV-vis spectrum of congo red in aqueous solution, Spectrochim.Acta A Mol.Biomol.Spectrosc. 65, 985-987, 2006; Eisert, R., Felau, L., and Brown, L.R., Methods for enhancing the accuracy and reproducibility of Congo Red and thioflavin T assays, Anal.Biochem. 353, 144-146, 2006; Sereikaite, J. and Bumelis, V.A., Congo red interaction with a-proteins, Acta Biochim.Pol. 53, 87-92, 2006; Frid, P., Anisimov, S.V. and Popovic, N., Congo red and protein aggregation in neurodegenerative diseases, Brain Res.Brain Res.Rev. 53, 135-160, 2007; Goodrich, J.D. and Winter, W.T., Alpha-chitin nanocrystals prepared from shrimp shells and their specific surface area measurement, Biomacromolecules 8, 252-257, 2007; Lencki, R.W., Evidence for fibril-like structure in bovine casein micelles, J.Dairy Sci. 90, 75-89, 2007; Diaminobenzidine (DAB); 3,3’-dimethyl-aminobenzidine; 3,3’,4,4’-tetraaminobiphenyl; MW 214; 360 as the tetrahydrochloride H2N
H2N
Histochemical demonstration of peroxidases, oxidases, catalases where DAB serves as electron acceptor forming a polymeric brown product; also used for Western blotting.
NH2
NH2
Seligman, A.M., Karnovsky, M.J., Wasserkrug, H.L., and Hanker, J.S., Nondroplet ultrastructural demonstration of cytochrome oxidase activity with a polymerizing osmiophilic reagent, diaminobenzidine (DAB), J.Cell Biol. 38, 1-14, 1968; Novikoff, A.B., and Goldfischer, S., Visualization of peroxisomes (microbodies) and mitochondria with diaminobenzidine, J.Histochem.Cytochem. 17, 675-680, 1969; Ekes, M., The use of diaminobenzidine (DAB) for the histochemical demonstration of cytochrome oxidase activity in unfixed plant tissues, Histochemie 27, 103-108, 1971; Herzog, V. and Fahimi, H.D., A new sensitive colorimetric assay for peroxidase using 3,3’-diaminobenzidine as hydrogen donor, Anal.Biochem. 55, 554-562, 1973; Nishimura, E.T. and Cooper, C., Peroxidatic reaction of catalase-antibody complex of leukocyte demonstrated by diaminobenzidine, Cancer Res. 34, 2386-2392, 1974; Pelliniemi, L.J., Dym, M., and Karnovsky, M.J., Peroxidase histochemistry using diaminobenzidine tetrahydrochloride stored as a frozen solution, J.Histochem.Cytochem. 28, 191-192, 1980; van Bogaert, L.J., Quinones, J.A., and van Craynest, M.P., Difficulties involved in diaminobenzidine histochemistry of endogenous peroxidase, Acta Histochem. 67, 180-194, 1980; Perotti, M.E., Anderson, W.A., and Swift, H., Quantitative cytochemistry of the diaminobenzidine cytochrome oxidase reaction product in mitochondria of cardiac muscle and pancreas, J.Histochem.Cytochem. 31, 351-365, 1983; Bosman, F.T., Some recent developments in immunocytochemistry, Histochem.J. 15, 189-200, 1983; Deimann, W., Endogenous peroxidase activity in mononuclear phagocytes, Prog. Histochem.Cytochem. 15, 1-58, 1984; Kugler, P., Enzyme histochemical applied in the brain, Eur.J.Morphol. 28, 109-120, 1990; Deitch, J.S., Smith, K.L., Swann, J.W., and Turner, J.N., Parameters affecting imaging of the horseradish-peroxidase-diaminobenzidine reaction product in the confocal scanning laser microscope, J.Microsc. 160, 265-278, 1990; Ludany, A.,Gallyas, F., Gaszner, B. et al., Skimmed-milk blocking improves silver staining post-intensification of peroxidase-diaminobenzidine staining on nitrocellulose membrane in immunoblotting, Electrophoresis 14, 78-80, 1993; Fritz, P., Wu, X., Tuczek, H., et al., Quantitation in immunohistochemistry. A research method or a diagnostic tool in surgical pathology?, Pathologica 87, 300-309, 1995; Werner, M., Von Wasielewski, R. and Komminoth, P., Antigen retrieval, signal amplification and intensification in immunohistochemistry, Histochem.Cell Biol. 105, 253-260, 1996; Horn, H., Safe diaminobenzidine (DAB) disposal, Biotech.Histochem. 77, 229, 2002; Kiernan, J.A., Stability and solubility of 3,3’-diaminobenzidine (DAB), Biotech. Histochem. 78, 135, 2003; Rimm, D.L., What brown cannot do for you, Nat.Biotechnol. 24, 914-916, 2006
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826
SOME BIOLOGICAL STAINS AND DYES (Continued) Name
Description
Dichlorofluorescin diacetate (2’,7’-dichlorofluorescin diacetate) MW 487 H3C
Histochemical demonstration of peroxidases and esterases; detection of reactive oxygen species (ROS). Not be confused with the fluorescein derivatives
O
O
O
O
CH3
O
Cl
Cl O
OH Hassan, N.F., Campbell, D.E., and Douglas, S.D., Phorbol myristate acetate induced oxidation of 2’,7’-dichlorofluorescin by neutrophils from patients with chronic granulomatous disease, J.Leukoc.Biol. 43, 317-322, 1988; Rosenkranz, A.R., Schmaldienst, S., Stuhlmeier, K.M., et al., A microplate assay for the detection of oxidative products using 2’,7’-dichlorofluorescin diacetate, J.Immunol.Methods 156, 39-45, 1992; Royall, J.A. and Ischiropoulos, H., Evaluation of 2’,7’-dichlorofluorescin and dihydrorhodamine 123 as fluorescent probes for intracellular H2O2 in cultured endothelial cells, Arch.Biochem.Biophys. 302, 348-355, 1993; Kooy, N.W., Royall, J.A., and Ishiropoulos, H., Oxidation of 2’,7’-dichlorofluorescin by peroxynitrite, Free Radic.Res. 27, 245-254, 1997; van Reyk, D.M., King, N.J., Dinauer, M.C., and Hunt, N.M., The intracellular oxidation of 2’.7’-dichlorofluorescin in murine T lymphocytes, Free Rad.Biol.Med. 30, 82-88, 2001; Burkitt, M.J. and Wardman, P., Cytochrome C is a potent catalyst of dichlorofluorescin oxidation: implications for the role of reactive oxygen species in apoptosis, Biochem.Biophys.Res.Commun. 282, 329-333, 2001; Chignell, C.F. and Sik, R.H., A photochemical study of cells loaded with 2’,7’-dichlorofluorescin: implications for the detection of reactive oxygen species generated during UVA irradiation, Free Rad.Biol.Med. 34, 1029-1034, 2003; Afzal, M. Matsugo, S., Sesai, M. et al., Method to overcome photoreaction , a serious drawback to the use of dichlorofluorescin in evaluation of reactive oxygen species, Biochem. Biophys.Res. Commun. 304, 619-624, 2003; Lawrence, A., Jones, C.M., Wardman, P., and Burkitt, M.J., Evidence for the role of a peroxidase compound I-type intermediate in the oxidation of glutathione, NADH, ascorbate, and dichlorofluorescein by cytochrome c/H2O2. Implications for oxidative stress during apoptosis, J.Biol.Chem. 278, 29410-29419, 2003; Myhre, O., Andersen, J.M., Aarnes, H., and Fonnum, F., Evaluation of the probes 2’,7’-dichlorofluorescin diacetate, luminol, and lucigenin as indicators of reactive species formation, Biochem.Pharmacol. 65, 15751582, 2003; Laggner, H., Hermann, M., Gmeiner, B.M., and Kapiotis, S., Cu2+ and Cu+ bathocuproine disulfonate complexes promote he oxidation of the ROS-detecting compound dichlorofluorescin (DCFH), Anal.Bioanal.Chem. 385, 959-961, 2006; Matsugo, S., Sasai, M., Shinmori, H. et al., Generation of a novel fluorescent product, monochlorofluorescein from dichlorofluorescin by photo-irradiation, Free Radic.Res. 40, 959-965, 2006 3,3’-Dimethyl-9-methyl-4,5,4’,5’dibenzothiacarbocyanine; DBTC; “Stains all”; MW 560
A cationic dye with a broad specificity for interaction including glycosaminoglycans and nucleic acids. DBTC has been used to stain for calmodulin and other calcium-binding proteins.
H3C C
S
CH
CH N
S N+ Br–
H3C H3C
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Some Biological Stains and Dyes
827
SOME BIOLOGICAL STAINS AND DYES (Continued) Name
Description
Scheres, J.M., Production of C and T bands in human chromosomes after heat treatment at high pH and staining with “stains-all”, Humangenetik, 23, 311-314, 1974; Green, M.R. and Pastewka, J.V., The cationic carbocyanine dyes Stains-all, DBTC, and Ethyl-stains-all, DBTC-3,3’9-triethyl, J.Histochem.Cytochem. 27, 797-799, 1979; Caday, C.G. and Steiner, R.F., The interaction of calmodulin with the carbocyanine dye (Stains all), J.Biol. Chem. 260, 5985-5990, 1985; Caday, C.G., Lambooy, P.K., and Steiner, R.F., The interaction of Ca2+-binding proteins with the carbocyanine dye stains-all, Biopolymers 25, 1579-1595, 1986; Sharma, Y., Rao, C.M. Rao, S.C., et al., Binding site conformation dictates the color of the dye stains-all. A study of the binding of this dye to the eye lens proteins crystallins, J.Biol.Chem. 264, 20923-20927, 1989; Lu, M., Guo, Q., Seeman, N.C. and Kallenbach, N.R., Drug binding by branched DNA: selective interaction of the dye stains-all with an immobile junction, Biochemistry 29, 3407-3412, 1990; Nakamura, K., Masuyama, E., Wada, S., and Okuno, M., Applications of stains’ all staining to the analysis of axonemal tubulins: identification of beta-tubulin and beta-isotubulins, J.Biochem.Biophys.Methods 21, 237-245, 1990; Gruber, H.E. and Mekikian, P., Application of stains-all for demarcation of cement lines in methacrylate embedded bone, Biotech.Histochem. 66, 181-184, 1991; Sharma, Y., Gapalakrishna, A., Balasubramanian, D., et al., Studies on the interaction of the dye, stains-all, with individual calcium-binding domains of calmodulin, FEBS Lett. 326, 59-64, 1993; Lee, H.G. and Cowman, K., An agarose gel electrophoretic method of analysis of hyaluronan molecular weight distribution, Anal.Biochem. 219, 278-287, 1994; Myers, J.M., Veis, A., Sabsay, B., and Wheeler, A.P., A method for enhancing the sensitivity and stability of stains-all for Phosphoproteins separated in sodium dodecyl sulfate-polyacrylamide gels, Anal.Biochem. 240, 300-302, 1996; Goldberg, H.A. and Warner, K.J., The staining of acidic proteins on polyacrylamide gels: enhanced sensitivity and stability of “Stains All” staining in combination with silver nitrate, Anal.Biochem. 251, 227-233, 1997; Volpi, N., and Maccari, F., Detection of submicrogram quantities of glycosaminoglycans on agarose gels by sequential staining with toluidine blue and Stains All, Electrophoresis 23, 4060-4066, 2002; Volpi, N., Macari, F., and Titze, J., Simultaneous detection of submicrogram quantities of hyaluronic acid and dermatan sulfate on agarose-gel by sequential staining with toluidine blue and Stains All, J.Chromatog.B. Analyt. Technol. Biomed.Life Sci. 820, 131-135, 2005 Evans Blue (C.I. Direct Blue 53); MW 961 as tetrasodium salt.
Early use as a method for determining blood volume; histochemical use as a protein stain, extensive use as a vital stain; more recent use to demonstrate vascular leakage and surgical dye. H3C
NaO3S N
NaO3S
N
N N
OH CH3
NH2
SO3Na
OH SO3Na
NH2
Gibson, J.G., and Evans, W.A., Clinical studies of the blood volume. I. Clinical application of a method employing the azo dye “Evans Blue” and the spectrophotometer, J.Clin.Invest. 16, 301-316, 1937; Morris, C.J., The determination of plasma volume by the Evans blue method: the analysis of haemolyzed plasma, J.Physiol. 102, 441-445, 1944; Morris, C.J., Chromatographic determination of Evans blue in plasma and serum, Biochem.J. 38, 203-204, 1944; McCord, W.M. and Ezell, H.K., Cell volume determinations with Evans blue, Proc.Soc.Exptl.Biol.Med. 76, 727-728, 1951; Caster, W.O., Simon, A.B., and Armstrong, W.D., Evans blue space in tissues of the rat, Am.J.Physiol. 183, 317-321, 1955; Clausen, D.F. and Lifson, N., Determination of Evans blue dye in blood in tissues, Proc.Soc.Exp.Biol.Med. 91, 11-14, 1956; Larsen, O.A. and Jarnum, S., The Evans Blue test in amyloidosis, Scand.J.Clin.Lab.Invest. 17, 287-294, 1965; Rabinovitz, MK. and Schen, R.J., The characteristics of certain alpha globulins in immunoelectrophoresis of human serum, using Evans blue dye, Clin.Chim.Acta 17, 499-503, 1967; Crippen, R.W. and Perrier, J.L., The use of neutral red and Evans blue for live-dead determination of marine plankton (with comments on the use of rotenone for inhibition of grazing), Stain Technol. 49, 97-104, 1974; Fry, D.L., Mahley, R.W., Weisgraber, K.H. and Oh, S.Y., Simultaneous accumulation of Evans blue dye and albumin in the canine aortic wall, Am.J.Physiol. 233, H66-H79, 1977; Shoemaker, K., Rubin, J., Zumbro, G.L., and Tackett, R., Evans blue and gentian violet: alternatives to methylene blue as a surgical marker dye, J.Thorac.Cardiovasc.Surg. 112, 542-544, 1996; Skowronek, M., Roterman, Konieczny, L., et al., The conformational characteristics of Congo Red, Evans blue and Trypan blue, Comput.Chem. 24, 429-450, 2000; Hamer, P.W., McGeachie, J.M., Davies, M.J., and Grounds, M.D., Evans Blue dye as an in vivo marker of myofibre damage: optimizing parameters for detecting initial myofibre membrane permeability, J.Anat. 200, 69-79, 2002; Kaptanoglu, E., Okutan, O., Akbiyik, F., et al., Correlation of injury severity and tissue Evans glue content, lipid peroxidation and clinical evaluation in acute spinal cord injury in rats, J.Clin.Neurosci. 11, 879-885, 2004; Green, M., Frashid, G., Kollias, J. et al., The tissue distribution of Evans blue dye in a sheep model of sentinel node biopsy, Nucl.Med.Commun. 27, 695-700, 2006;
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Handbook of Biochemistry and Molecular Biology
828
SOME BIOLOGICAL STAINS AND DYES (Continued) Name
Description
Fast Green (Fast Green FCF; C.I. food green 3) M.W. 809 as the trisodium salt
A histochemical cytoplasmic counterstain; a stain for protein; used to demonstrate histones; marker dye; a food dye FD & C fast green 3).
SO3H
SO3H H3C
CH3
N
+
N
SO3H
CH3 Bryan, J.H., Differential staining with a mixture of safranin and fast green FCF, Stain Technol. 30, 153-157, 1955; Garcia, A.M., Studies on deoxyribonucleoprotein in leukocytes and related cells of mammals. VII. The fast green histone content of rabbit leukocytes after hypertonic treatment, Stain Technol. 30, 153-157, 1955; Hunt, D.E. and Caldwell, R.C., Use of fast green in agar-diffusion microbiological assays, Appl.Microbiol. 18, 1098-1099, 1969; Gorovsky, M.A., Carlson, K., and Rosenbaum, J.L., Simple method for quantitative densitometry of polyacrylamide gels using fast green, Anal. Biochem. 35, 359-370, 1970; Entwhistle, K.W., Congo red-fast green fcf as a supra-vital stain for ram and bull spermatozoa, Aust.Vet.J. 48, 515-519, 1972; Noeske, K., Discrepancies between cytophotometric alkaline Fast Green measurements and nuclear histone protein content, Histochem.J. 5, 303-311, 1973; McMaster-Kaye, R. and Kaye, J.S., Staining of histones on polyacrylamide gels with amido blank and fast green, Anal. Biochem. 61, 120-132, 1974; Medugorac, I., Quantitative determination of cardiac myosin subunits stained with fast green in SDS-electrophoretic gels, Basic Res. Cardiol. 74, 406-416, 1979; Glimore, L.B. and Hook, G.E., Quantitation of specific proteins in polyacrylamide gels by the elution of Fast Green FCF, J.Biochem.Biophys.Methods 5, 57-66, 1981; Smit, E.F., de Vries, E.G., Meijer, C., et al., Limitations of the fast green assay for chemosensitivity testing in human lung cancer, Chest 100, 1358-1363, 1991; Li, Y.F., Huang, C.Z., and Li, M., A resonance light-scattering determination of proteins with fast green FCF, Anal.Sci.18, 177-181, 2002; Tsuji, S., Yoshii, K., and Tonogai, Y. Identification of isomers and subsidiary colors in commercial Fast Green FCF (FD & C Green No. 3, Food Green No. 3) by liquid chromatography-mass spectrometry and comparison between amounts of the subsidiary colors by high-performance liquid chromatography and thin-layer chromatography-spectrometry, J.Chromatog.A. 1101, 214-221, 2006; Luo, S., Wehr, N.B., and Levine, R.L., Quantitation of protein on gels and blots by infrared fluorescence of Coomassie glue and Fast Green, Anal.Biochem. 350, 233-238, 2006; Ali, M.A. and Bashier, S.A., Effect of fast green dye on some biophysical properties of thymocytes and splenocytes of albino mice, Food Addit.Contam. 23, 452-561, 2006
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Some Biological Stains and Dyes
829
SOME BIOLOGICAL STAINS AND DYES (Continued) Name
Description
Fluorescein (C.I. solvent yellow 94) MW as free acid is 332 while the disodium salt is 376.
Fluorescein is an acidic hydroxyxanthene which exists in a variety of forms. Depending on pH, fluorescein is in equilibrium the free acid and a dianion form. When the dianion is present as the sodium salt, the term uranin has been used to describe the molecule. Fluorescein has a broad range of use including use in large quantities to trace environment water flow. Fluorescein has been used to vascular flow with particular interest in ophthalmology. Fluorescein has been modified to include reactive functional groups (isothiocyanate; for covalent insertion of fluorescent probes into protein and other biological macromolecules. Care must be taken to avoid confusion with fluorescin.
Fluorescein
Uranin O
HO
OH
HO
O
NaOH
O
HO
COO–Na+
HCl
O
O
O
O
HO
O
O
COOH
COOH
Thiophosgene
S
N
NH2
C Fluorescein Isothiocyanate
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Handbook of Biochemistry and Molecular Biology
830
SOME BIOLOGICAL STAINS AND DYES (Continued) Name
Description
Ray, R.R. and Binkhorst, R.D., The diagnosis of papillary block by intravenous injection of fluorescein, Am.J.Ophthamol. 61, 480-483, 1966; Hill, D.W., Fluorescein angiography in fundus diagnosis, Br.Med.Bull. 26, 161-165, 1970; Gass, J.D., Fluorescein angiography. An aid in the differential diagnosis of intraocular tumors, Int.Ophthalomol.Clin. 12, 85-120, 1972; Ohkuma, S., Use of fluorescein isothiocynate-dextran to measure proton pumping in lysosomes and related organelles, Methods Enzymol. 174, 131-154, 1989; Klose, A.D. and Gericke, K.R., Fluorescein as a circulation determinant, Ann. Pharmacother. 28, 891-893, 1994; Wischke, C. and Borchert, H.H., Fluorescein isothiocyanate labelled bovine serum albumin (FITC-BSA) as a model protein drug: opportunities and drawbacks, Pharmazie 61, 770-774, 2006; Berginc, K., Zakelj, S., Levstik, L., et al., Fluorescein transport properties across artificial lipid membranes, Caco-2 cell monolayers and rat jejuna, Eur.J.Pharm.Biopharm., in press, 2006. Malachite Green (Victoria Green, C.I. basic green 4); MW leukobase, 330; carbinol free base as the hydrochloride, 383)
A cationic diaminotriphenylmethane which is used to stain a variety of cells including bacterial spores, pH indicators, stain for phospholipid; measurement of inorganic phosphate
Malachite Green Victoria Green B
H3C N
N+ H3C
CH3
Cl– CH3
H3C N
CH3
OH
CH3 N CH3
Cl– N H3C
CH3
Leukomalachite green Leuko form
9168_Book.indb 830
N+ H3C
CH3
Malachite Green Carbinol Chloride
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Some Biological Stains and Dyes
831
SOME BIOLOGICAL STAINS AND DYES (Continued) Name
Description
Norris, D., Reconstitution of virus X-saturated potato varieties with malachite green, Nature 172, 816, 1953; Kanetsuna, F., A study of malachite green staining of leprosy bacilli, Int.J.Lepr. 32, 185-194, 1964; Solari, A.A., Herrero, M.M., and Painceira, M.T., Use of malachite green for staining flagella in bacteria, Appl.Microbiol. 16, 792, 1968; Teichman, R.J., Takei, G.H., and Cummins, J.M., Detection of fatty acids, fatty aldehydes, phospholipids, glycolipids and cholesterol on thin-layer chromatograms stained with malachite green, J.Chromatog. 88, 425-427, 1974; Singh, E.F., Moawad, A., and Zuspan, F.P., Malachite green—a new staining reagent for prostaglandins, J.Chromatog 105, 194-196, 1975; Nefussi, J.R., Septier, D., Sautier, J.M. et al., Localization of malachite green positive lipids in the matrix of bone nodule formed in vitro, Calif.Tissue Int. 50, 273-282, 1992; Henderson, A.L., Schmitt, T.C., Heinze, T.M., and Cerniglia, C.E., Reduction of malachite green to leucomalachite green by intestinal bacteria, Appl.Environ.Microbiol 63, 4099-4101, 1997; Nguyen, D.H., DeFina, S.C., Fink, W.H., and Dieckmann, T., Binding to an RNA aptamers changes the charge distribution and conformation of malachite green, J.Am.Chem.Soc. 124, 15081-15084, 2002; Dutta, K., Bhattacharjee, S., Chauduri, B., and Mukhopadhyay, S., Oxidative degradation of malachite green by Fenton generated hydroxyl radicals in aqueous acidic media, J.Environ.Sci.Health A. Tox. Hazard Subst. Environ.Eng. 38, 1311-1326, 2003; Jadhav, J.P. and Govindwar, S.P., Biotransformation of malachite green by Saccharomyces cerevisiae MTCC 463, Yeast 23, 315-323, 2006;. Methylene Blue, MW 374 as the chloride monohydrate
CH3 N
A weakly hydrophilic cationic diaminothiazine. Methylene blue is used for a variety of purposes including development of the Romanowsky stain, bacterial staining, assay of redox reactions, vital staining, photooxidation of proteins, surgical marker (this use appears to undergoing serious reconsideration), and determination of cell wall permeability. Methylene blue with UV irradiation is used for the inactivation of pathogens in blood plasma.
Cl– S+
CH3 N
H3C
CH3
N Methylene Blue Reduction
CH3 N
CH3 S
H3C
N CH3
N H Methylene Blue Leuko Form
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Handbook of Biochemistry and Molecular Biology
832
SOME BIOLOGICAL STAINS AND DYES (Continued) Name
Description
Wishart, G.M., On the reduction of methylene blue by tissue extracts, Biochem.J. 17, 103-114, 1923; Whitehead, H.R., The reduction of methylene blue in milk: The influence of light, Biochem.J. 24, 579-584, 1930; Worley, L.G., The relation between the Golgi apparatus and “Droplets” in the cell stainable vitally with methylene blue, Proc.Natl.Acad.Sci.USA 29, 228-231, 1943; Weil, L., Gordon, W.G., and Buchert, A.R., Photooxidation of amino acids in the presence of methylene blue, Arch.Biochem. 33, 90-109, 1951; Moore, T., Sharman, I.M., Ward, R.J., The vitamin E activity of substances related to methylene blue, Biochem.J. 54, xvi-xvii, 1953; Yamazaki, I., Fujinaga, K., and Takehara, I., The reduction of methylene blue catalyzed by the turnip peroxidase, Arch.Biochem.Biophys. 72, 42-48, 1957; Borzani, W. and Vairo, M.L., Adsorption of methylene blue as a means of determining cell concentration of dead bacteria in suspensions, Stain Technol. 35, 77-81, 1960; Tinne, J.E., A methylene blue medium for distinguishing virulent mycobacteria, Scott.Med.J. 4, 130-132, 1959; Barbosa, P. and Peters, T.M., The effects of vital dyes on living organisms with special reference to methylene blue and neutral red, Histochem.J. 3, 71-93, 1971; Bentley, S.A., Marshall, P.N., and Trobaugh, F.E., Jr., Standardization of the Romanowksy staining procedure: an overview, Anal.Quant.Cytol. 2, 15-18, 1980; Wittekind, D.H. and Gehring, T., On the nature of Romanowsky-Giemsa staining and the Romanowsky-Giemsa effect. I. Model experiments on the specificity of azure B-eosin Y stain as compared with other thiazine dye – eosin Y combinations, Histochem.J. 17, 263-289, 1985; Schulte, E. and Wittekind, D., The influence of Romanowsky-Giemsa type stains on nuclear and cytoplasmic features of cytological specimens, Anal.Cell Pathol. 1, 83-86, 1989; Tuite, E.M. and Kelly, J.M., Photochemical interactions of methylene blue and analogues with DNA and other biological substrates, J.Photochem.Photobiol.B. 21, 103-124, 1993; Bradbeer, J.N., Riminucci, M., and Bianco, P., Giemsa as a fluorescent stain for mineralized bone, J.Histochem.Cytochem. 42, 677-680, 1994; Wainwright, M. and Crossley, K.B., Methylene blue – a therapeutic dye for all seasons?, J.Chemother. 14, 431-443, 2002; Inamura, K., Ikeda, E., Nagayasu, T., et al., Adsorption behavior of methylene blue and its congeners on a stainless steel surface, J.Colloid Interface Sci. 245, 50-57, 2002; Floyd, R.A., Schneider, J.E., Jr., and Dittmern, D.P., Methylene blue photoinactivation of RNA viruses, Antiviral Res. 61, 141-151, 2004; Rider, K.A. and Flick, L.M., Differentiation of bone and soft tissues in formalin-fixed, paraffin-embedded tissue by using methylene blue/acid fuchsin stain, Anal.Quant.Cytol.Histol. 26, 246-248, 2004; Rider, K.A. and Flick, L.M., Differentiating of bone and soft tissues in formalin-fixed, paraffin-embedded tissue by using methylene blue/acid fuchsin stain, Anal. Quant.Cytol. Histol. 26, 246-248, 2004; Papin, J.F., Floyd, R.A., and Dittmer, D.P., Methylene blue photoinactivation abolishes West Nile virus infectivity in vivo, Antiviral Res. 68, 84-87, 2005; Dilgin, Y. and Nisli, G., Fluorometric determination of ascorbic acid in vitamin C tablets using methylene blue, Chem.Pharm.Bull. 53, 1251-1254, 2005; Itoh, K., Decolorization and degradation of methylene blue by Arthrobacter globiformis, Bull. Environ.Contam.Toxicol. 75, 1131-1136, 2005; D’Amico, F., A polychromatic staining method for epoxy embedded tissues: a new combination of methylene blue and basic fuchsine for light microscopy, Biotech.Histochem. 80, 207-210, 2005; Cheng, Y., Liu, W.F., Yan, Y.B., and Zhou, H.M., A nonradiometric assay for poly(a)-specific ribonuclease activity by methylene blue colorimetry, Protein Pept.Lett. 13, 125-128, 2006; Jurado, E., Fernandez-Serrano, M., Nunez-Olea, J., et al., Simplified spectrophotometric method using methylene blue for determining anionic surfactants: applications to the study of primary biodegradation in aerobic screening tests, Chemosphere 65, 278-285, 2006; Dinc, S., Ozaslan, C., Kuru, B., et al., Methylene blue prevents surgery-induced peritoneal adhesions but impairs the early phase of anastomotic wound healing, Can.J.Surg. 49, 321-328, 2006; Appadurai, I.R. and Scott-Coombes, D., Methylene blue for parathyroid localization, Anaesthesia 62, 94, 2007; Mihai, R., Mitchell, E.W., and Warwick, J., Dose-response and postoperative confusion following methylene blue infusion during parathyroidectomy, Can.J.Anaesth. 54, 79-81, 2007; McCullagh, C. and Robertson, P., Effect of polyethyleneimine, a cell permeabiliser, on the photo-sensitised destruction of algae by methylene blue and nuclear fast red, Photochem.Photobiol., in press, 2007; Methyl Orange, Orange III, Gold Orange, C.I. Orange 52; MW 327 as sodium salt
An azo dye with limited use in histology; use for the assay of redox reactions. Used for the study of protein conformation; early use for assay of albumin; binding to cationic proteins
O
OH S O
N N
H2N Wetlaufer, D.B. and Stahmann, M.A., The interaction of methyl orange anions with lysine polypeptides, J.Biol.Chem. 203, 117-126, 1953; Colvein, J.R., The adsorption of methyl orange by lysozyme, Can.J.Biochem.Physiol. 32, 109-118, 1954; Lundh, B., Serum albumin as determined by the methyl orange method and by electrophoresis, Scand.J.Clin.Lab.Invest. 17, 503-504, 1965; Barrett, J.F., Pitt, P.A., Ryan, A.J., and Wright, S.E., The demethylation of m-methyl orange and methyl orange in vivo and in vitro, Biochem.Pharmacol. 15, 675-680, 1966; Shikama, K., Denaturation and renaturation of binding sites of bovine serum albumin for methyl orange, J.Biochem. 64, 55-63, 1968; Lang, J., Auborn, J.J., and Eyring, E.M., Kinetic studies of the interaction of methyl orange with beta-lactoglobulin between pH 3.7 and 2.0, J.Biol.Chem. 246, 5380-5383, 1971; Browner, C.J. and Lindup, W.E., Decreased plasma protein binding of o-methyl red methyl orange and phenytoin (diphenylhydantoin) in rats with acute renal failure,
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Some Biological Stains and Dyes
833
SOME BIOLOGICAL STAINS AND DYES (Continued) Name
Description
Br.J.Pharmacol. 63, 367P, 1978; Ford, C.L. and Winzor, D.J., A recycling gel partition technique for the study of protein-ligand interactions: the binding of methyl orange to bovine serum albumin, Anal.Biochem. 114, 146-152, 1981; Chung, K.T., Stevens, S.E., Jr., and Cerniglia, C.E., The reduction of azo dyes by the intestinal microflora, Crit.Rev.Microbiol. 18, 175-190, 1992; Yang Y. Jung, D.W., Bai, D.G., et al., Counterion-dye staining method for DNA in agarose gels using crystal violet and methyl orange, Electrophoresis 22 855-859, 2001; Nam, W., Kim, J., and Han, G., Photocatalytic oxidation of methyl orange in a three-phase fluidized bed reactor, Chemosphere 47, 1019-1024, 2002; Marci, G., Augugliaro, V., Bianco Prevot, A., et al., Photocatalytic oxidation of methyl-orange in aqueous suspension: comparison of the performance of different polycrystalline titanium dioxide, Ann.Chim. 93, 639-648, 2003; Del Nero, J., de Araujo, R.E., Gomes, A.S., and de Melo, C.P., Theoretical and experimental investigation or the second hyperpolarizabilities of methyl orange, J.Chem. Phys. 122, 104506, 2005; de Oliveira, H.P., Oliveira, E.G., and de Melo, C.P., J.Colloid Interface Sci. 303, 444-449, 2006; Bejarano-Perez, N.J. and SuarezHerrera, M.F., Sonophotocatalytic degradation of congo red and methyl orange in the presence of TiO2 as a catalyst, Ultrason.Sonochem. in press, 2006; Methyl Red (2-[[4-(dimethylamino)phenyl]azo] benzoic acid; C.I. acid red 2) MW 270; 306 as the hydrochloride
pH indicator; use as signal in redox reactions; rare use in histochemistry; some use for staining protozoa and other bacteria; standard for spectroscopy
O OH CH3 N
N
N
CH3 Cowan, S.T., Micromethod for the methyl red test, J.Gen.Microbiol. 9, 101-109, 1953; Ljutov, V., Technique of methyl red test, Acta Pathol.Microbiol. Scand. 51, 369-380, 1961; Barry, A.L., Berhsohn, K.L., Adams, A.P., and Thrupp, L.D., Improved 18-hour methyl red test, Appl.Microbiol. 20, 866-870, 1970; Korzun, W.J. and Miller, W.G., Monitoring the stability of wavelength calibration of spectrophotometers, Clin.Chem. 32, 162-165, 1986; Chung, K.T., Stevens, S.E., Jr., and Cerniglia, C.E., The reduction of azo dyes by the intestinal microflora, Crit.Rev.Microbiol. 18, 175-190, 1992; Miyajima, M., Sagami, I., Daff, S., et al., Azo reduction of methyl red by neuronal nitric oxide synthase: the important role of FMN in catalysis, Biochem.Biophys.Res. Commun. 275, 752-758, 2000; Kashida, H., Tanaka, M., Baba, S., et al., Covalent incorporation of methyl red dyes into double-stranded DNA for their ordered clustering, Chemistry 12, 777-784, 2006; Kalyuzhnyi, S., Yemashova, N., and Fedorovich, V., Kinetics of anaerobic biodecolourisation of azo dyes, Water Sct.Technol. 54, 73-79, 2006; Katsuda, T. Ooshima, H., Azuma, M., and Kato, J., New detection method for hydrogen gas for screening hydrogen-producing microorganisms using water-soluble Wilkinson’s catalyst derivative, J.Biosci.Bioeng. 102, 220-226, 2006; Hsueh, C.C. and Chen, B.Y., Comparative study on reaction selectivity of azo dye decolorization by Pseudomonas luteola,, J.Hazard.Mater., in press, 2006 Methyl Violet (C.I. Basic violet 1); Molecular weight depends on the degree of methylation
Methyl violet is a mixture of N-methylated pararoanilines which is used a pH indicator and a biological stain. The term gentian violet is used for this mixture in Europe and for crystal violet (methyl violet 10B, the hexamethyl derivative) in the United States. There is some use of methyl violet for staining DNA. The industrial use is for dyeing fabrics blue and violet. The depth of color is dependent on the degree of methylation.
Methyl Violet CH3
CH3
N
N+
Cl– CH3
H3C
HN CH3
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834
SOME BIOLOGICAL STAINS AND DYES (Continued) Name
Description
Bancroft, J.D., Methyl green as a differentiator and counterstain in the methyl violet technique for demonstration of amyloid in fresh cryostat sections, Stain Technol. 38, 336-337, 1963; Campbell, L.M. and Roth, I.L., Methyl violet: a selective agent for differentiation of Klebsiella pneumonia from Enterobacter aerogenes and other gram-negative organisms, Appl.Microbiol. 30, 258-261, 1975; Dutt, M.K., Staining of depolymerized DNA in mammalian tissues with methyl violet 6B and crystal violet, Folia Histochem.Cytochem. 18, 79-83, 1980; Liu, Y., Ma, C.Q., Li, K.A., et al., Rayleigh light scattering study on the reaction of nucleic acids and methyl violet, Anal.Biochem. 268, 187-192, 1999; Dogan, M. and Aikan, M., Removal of methyl violet from aqueous solution by perlite, J.Colloid Interface Sci. 267, 32-41, 2003; Jin, L.T. and Choi, J.K., Usefulness of visible dyes for the staining of protein of DNA in electrophoresis, Electrophoresis 25, 2429-2438, 2004; Neutral Red (C.I. Basic Red 5; nuclear fast red; 3-amino-7-dimethylamino2-methylphenazine hydrochloride; toluylene red as the unprotonated form), MW 289 as the hydrochloride H3C
pH indicator, histological stain for Golgi, nuclear. Also a vital stain for intracellular organelles and cytotoxicity testing.
N Cl–
H2N
N
N+
H
CH3
CH3
Lepper, E.H. and Martin, C.J., The protein error in estimating pH with neutral red and phenol red, Biochem.J. 21, 356-361, 1927; Morse, W.C., Dail, M.C., and Olitzky, I., A study of the neutral red reaction for determining virulence of Mycobacteria, Am.J.Public Health 43, 36-39, 1953; Vivian, D.L. and Belkin, M., Unexpected anomalies in the behavior of neutral red and related dyes, Nature 178, 154, 1956; Darnell, J.E., Jr., Lockart, R.Z., Jr., and Sawyer, T.K., The effect of neutral red on plaque formation in two virus-cell systems, Virology 6, 567-568, 1958; Crowther, D. and Melnick, J.L., The incorporation of neutral red and acridine orange into developing poliovirus particles making them photosensitive, Virology 14, 11-21, 1961; Boyer, M.G., The role of tannins in neutral red staining of pine needle vacuoles, Stain Technol. 38, 117-120, 1967; Sawicki, W., Kieler, J., and Briand, P., Vital staining with neutral red and trypan blue of 3H-thymidine-labeled cells prior to autoradiography, Stain Technol. 42, 143-146, 1967; Barbosa, P. and Peters, T.M., The effects of vital dyes on living organisms with special reference to methylene blue and neutral red, Histochem.J. 3, 71-93, 1971; Gutter, B., Speck, W.T., and Rosenkranz, H.S., Light-induced mutagenicity of neutral red (3-amino-7-dimethylamino-2-methylphenazine hydrochloride), Cancer Res. 37, 1112-1114, 1977; Nemes, Z., Dietz, R., Luth, J.B., et al., The pharmacological relevance of vital staining with neutral red, Experientia 35, 1475-1476, 1979; Gray, D.W., Millard, P.R., McShane, P., and Morris, P.J., The use of the dye neutral red as a specific, non-toxic, intra-vital stain of islets of Langerhans, Br.J.Exp.Pathol. 64, 553-558, 1983; LaManna, J.C., Intracellular pH determination by absorption spectrophotometry of neutral red, Metab.Brain Dis 2, 167-182, 1987; Elliott, W.M. and Auersperg, N., Comparison of the neutral red and methylene blue assays to study cell growth in culture, Biotech.Histochem. 68, 29-35, 1993; Fautz, R., Husein, B., and Hechenberger, C., Application of the neutral red assay (NR assay) to monolayer cultures of primary hepatocytes: rapid colorimetric viability determination for the unscheduled DNA synthesis test (UDS), Mutat.Res. 253, 173-179, 1991; Kado, R.T., Neutral red: a specific fluorescent dye in the cerebellum, Jpn.J.Physiol. 43(Suppl 1), S161-S169, 1993; Ishiyama, M., Tominaga, H. Shiga, M., A combined assay of cell viability and in vitro cytotoxicity with a highly water-soluble tetrazolium salt, neutral red and crystal violet, Biol.Pharm.Bull. 19, 1518-1520, 1996; Ciapetti, G., Granchi, D., Verri, E., et al., Application of a combination of neutral red and amido black staining for rapid, reliable cytotoxicity testing of biomaterials, Biomaterials 17, 1259-1264, 1996; Sousa, C., Sá e Melo, T., Geze, M., et al., Solvent polarity and pH effects on the spectroscopic properties of neutral red: application to lysosomal microenvironment probing in living cells, Photochem.Photobiol. 63, 601-607, 1996; Baker, C.S., Crystallization of neutral red vital stain from minimum essential medium due to pH instability, In Vitro Cell.Dev.Biol.Anim. 34, 607-608, 1998; Okada, D., Neutral red as a hydrophobic probe for monitoring neuronal activity, J.Neurosci.Methods 101, 85-92, 2000; Zuang, V., The neutral red release assay: a review, Altern.Lab.Anim. 29, 575-599, 2001; Svendsen, C., Spurgeon, D.J., Hankard, P.K., and Weeks, J.M., A review of lysosomal membrane stability measured by neutral red retention: is a workable earthworm biomarker?, Ecotoxicol.Environ. Saf. 57, 20-29, 2004.
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835
SOME BIOLOGICAL STAINS AND DYES (Continued) Name
Description
Nile Red (Nile pink); MW 318
A lipophilic benzooxazone used for lipid staining and as a hydrophobic probe for proteins; Nile red has been used as a stain for protein on acrylamide gel. The fluorescence of Nile red is very dependent on solvent. Nile red is poorly soluble in aqueous systems but the recent development of water-soluble derivatives has improved utility.
H3C
H3C
N
O
O
N
Greenspan, P., Mayer, E.P., and Fowler, S.D., Nile red; a selective fluorescent stain for intracellular lipid droplets, J.Cell Biol. 100, 965-973, 1985; Fowler, S.D. and Greenspan, P., Application of Nile red, a fluorescent hydrophobic probe, for the detection of neutral lipid deposits in tissue sections: comparison with oil red O, J.Histochem.Cytochem. 33, 833-836, 1985; Sackett, D.L. and Wolff, J., Nile red as a polarity-sensitive fluorescent probe of hydrophobic protein surfaces, Anal.Biochem. 167, 229-234, 1987; Brown, W.J., Warfel, J., and Greenspan, P., Use of Nile red stain in the detection of cholesteryl ester accumulation in acid lipase-deficient fibroblasts, Arch.Pathol.Lab.Med. 112, 295-297, 1988; Brown, W.J., Sullivan, T.R., and Greenspan, P, Nile red staining of lysosomal phospholipid inclusions, Histochemistry 97, 349-354, 1992; Brown, M.B., Miller, J.N., and Seare, N.J., An investigation of the use of Nile red as a long-wavelength fluorescent probe for the study of alpha 1-acid glycoprotein-drug interactions, J.Pharm. Biomed.Anal. 13 1011-1017, 1995; Alba, F.J., Bermudez, A., Bartolome, S. and Daban, J.R,. Detection of five nanograms of protein by two-minute nile red staining of unfixed SDS gels, BioTechniques 21, 625-626, 1996; Daban, J.R., Fluorescent labeling of proteins with Nile red and 2-methoxy-2,4diphenyl-3(2H)-furanone: physicochemical basis and application to the rapid staining of sodium dodecyl sulfate polyacrylamide gels and western blots, Electrophoresis 22, 874-880, 2001; Hendriks, J., Gensch, T., Hviid, L., et al., Transient exposure of hydrophobic surface in the photoactive yellow protein monitored with Nile red, Biophys.J. 82, 1632-1643, 2002; Prokhorenko, I.A., Dioubankova, N.N., and Korshun, V.A., Oligonucleotide conjugates of Nile Red, Nucleosides Nucleotides Nucleic Acids 23, 509-520, 2004; Yablon, D.G. and Schilowitz, A.M., Solvatochromism of Nile Red in nonpolar solvents, Appl.Spectros. 58, 843-847, 2004; Genicot, G., Leroy, J.L., Soom, A.V., and Donnay, I., The use of a fluorescent dye, Nile red, to evaluate the lipid content of single mammalian oocytes, THeriogeneology 63, 1181-1194, 2005; Sebok-Nagy, K., Miskoczy, Z. and Biczok, L., Interaction of 2-hydroxy-substituted Nile red fluorescent probe with organic nitrogen compounds, Photochem.Photobiol. 81, 1212-1218, 2005; Thomas, K.J., Sherman, D.B., Amiss, T.J., et al., A long-wavelength fluorescent glucose biosensor based on bioconjugates on galactose/glucose binding protein and Nile red derivatives, Diabetes Technol.Ther. 8, 261-268, 2006; Jose, J. and Burgess, K., Syntheses and properties of water-soluble Nile red derivatives, J.Org.Chem. 71, 7835-7839, 2006; Mukherjee, S., Raghuraman, H., and Chattopadhyay, A., Membrane localization and dynamics of Nile red: effect of cholesterol, Biochim.Biophys.Acta 1768, 59-66, 2007 Oil Red O (Sudan Red 5B; C.I. Solvent Red 27)
An extremely lipophilic dye; used for the demonstration of lipid depositions.
CH3
HO N
N
N
N H 3C
H3C
Moran, P. and Heyden, G., Enzyme histochemical studies on the formation of hyaline bodies in the epithelium of odontogenic cysts, J.Oral Pathol. 4, 120-127, 1975; Merrick, J.M., Schifferle, R., Zadarlik, K., et al., Isolation and partial characterization of the heterophile antigen of infectious mononucleosis from bovine erythrocytes, J.Supramol.Struct. 6, 275-290, 1977; Anderson, L.C. and Garrett, J.R., Lipid accumulation in the major salivary glands of streptozotocin-diabetic rats, Arch.Oral Biol. 31, 469-475, 1986; Chastre, J., Fagon, J.Y., Soler, P., et al., Bronchoalveolar lavage for rapid diagnosis of the fat embolism syndrome in trauma patients, Ann.Intern.Med. 113, 583-588, 1990; Aleksic, I., Ren, M., Popov, A., et al., In vivo liposome-mediated transfection of HLA-DR alpha-chain gene into pig hearts, Eur.J.Cardiothorac.Surg. 12, 792-797, 1997.
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836
SOME BIOLOGICAL STAINS AND DYES (Continued) Name
Description
Rhodamine B (C.I. Basic Violet 10) MW 479
An aminoxanthene which is a lipophilic cation in acid solution and a lipophilic anion in basic solution. Metal binding reagent, a neutral stain; a fluorescent lipid stain; stain for Phosphoproteins for gel electrophoresis. Isothiocyanate derivative used for labeling proteins.
CH3
H3C H2C H3C
N
N+
O
Cl–
CH3
COOH
Martin, G., Colorimetric determination of zinc by thiocyanate derivatives; a new method using rhodamine B, Bull.Soc.Chim.Biol. 34, 1174-1177, 1952; Webb, J.M., Hansen, W.H., Desmond, A., and Fitzhugh, O.G., Biochemical and toxicological studies of rhodamine B and 3,6-diaminofluroan, Toxicol.Appl.Pharmacol. 3, 696-706, 1961; Miketukova, V., Detection of metals on paper chromatograms with Rhodamine B., J.Chromatog. 24, 302-304, 1966; Liisberg, M.F., Rhodamine B as an extremely specific stain for cornification, Acta Anat. 69, 52-57, 1968; Shelley, W.B., Fluorescent staining of elastic tissue with Rhodamine B and related xanthene dyes, Histochemie 20, 244-249, 1969; Oshima, G.T. and Nagasawa, K., Fluorometric method for determination of mercury(II) with Rhodamine B, Chem.Pharm.Bull. 18, 687-692, 1970, Zahradnicek, L., Kratochvila, J., and Garcis, A., Determination of inorganic phosphorus using Rhodamine B by the continuous-flow technique, Clin.Chim.Acta 80, 431-433, 1977; Wessely, Z., Shapiro, S.H., Klavins, J.V., and Tinberg, H.M., Identification of Mallory bodies with rhodamine B fluorescence and other strains for keratin, Stain Technol. 56, 169-176, 1981; Debruyne, I., Inorganic phosphate determination: colorimetric assay based on the formation of a rhodamine B-phosphomolybdate complex, Anal.Biochem. 130, 454-460, 1983; Debruyne, I., Staining of alkali-labile Phosphoproteins and alkaline phosphatases on polyacrylamide gels, Anal.Biochem. 133, 110-115, 1983; Balcerzak, M., Sensitive spectrophotometric determination of osmium with tin(II) chloride and rhodamine B after flotation using cyclohexane, Analyst 113, 129-132, 1988; Glimcher, M.J. and Lefteriou, B., Soluble glycosylated phosphoproteins of cementum, Calcif.Tissue Int. 45, 165-172, 1989; Fernandez-Busquets, X. and Burger, M.M., Use of rhodamine B isothiocyanate to detect proteoglycan core proteins in polyacrylamide gels, Anal.Biochem. 227, 394-396, 1995; Jung, D.W., Yoo, G.S. and Choi, J.K., Mixed-dye staining method for protein detection in polyacrylamide gel electrophoresis using calconcarboxylic acid and rhodamine B, Electrophoresis 19, 2412-2415, 1998; Pal, J.K., Godbole, D., and Sharma, K., Staining of proteins on SDS polyacrylamide gels and on nitrocellulose membranes by Alta, a colour used as a cosmetic, J.Biochem.Biophys.Methods 61, 339-347, 2004; dos Santos Silva, A.L, Joekes, I., Rhodamine B diffusion in hair as a probe for structural integrity, Colloids Surf.B.Biointerfaces 40, 19-24, 2005; Moreno-Villoslada, I., Jofre, M., Miranda, V., et al., pH dependence of the interaction between rhodamine B and the water-soluble poly(sodium 4-styrenesulfonate), J.Phys.Chem.B Condens. Mater. Surf.Interfaces Biophys. 110, 11809-11812, 2006. Rose Bengal (Rose Bengal B; rose bengale; C.I. acid red 94; the disodium salt is referred to as rose Bengal extra); MW 1049 as dipotassium salt and 1018 as disodium salt I
An acidic hydroxyxanthene soluble in water used as a cellular stain; diagnostic aid in ophthalmology; source of singlet oxygen in photooxidation; use as a probe for surface protein structure; early use of the radioactive derivative as a tracer for liver function.
I
NaO
O
I
I Cl
COOH
Cl
Cl Cl
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837
SOME BIOLOGICAL STAINS AND DYES (Continued) Name
Description
Conn, H.J., Rose Bengal as a general bacterial stain, J.Bacteriol. 6, 253-254, 1921; Forster, H.W., Jr., Rose Bengal test in diagnosis of deficient tear formation, AMA Arch.Ophthalmol. 45, 419-424, 1951; Rippa, M. and Picco, C., Rose Bengal as a reporter of the polarity and acidity of the TPN binding site in 6-phosphoglucoonate dehydrogenase, Ital.J.Biochem. 19, 178-192, 1970; Lyons, A.B. Ashman, L.K., The Rose Bengal assay for monoclonal antibodies to cell surface antigens: comparisons with common hybridoma screening methods, J.Immunoassay 6, 325-345, 1985; Hederstedt, L. and Hatefi, Y., Modification of bovine heart succinate dehydrogenase with ethoxyformic anhydride and rose Bengal: evidence for essential histidyl residues protectable by substrates, Arch.Biochem.Biophys. 247, 346-354, 1986; Allen, M.T., Lynch, M., Lagos, A. et al., A wavelength dependent mechanism for rose bengalsensitized photoinhibition of red cell acetylcholinesterase, Biochim.Biophys.Acta. 1075, 42-49, 1991; Feenstra, R.P. and Tseng, S.C., What is actually stained by rose bengal?, Arch.Ophthalmol. 110, 984-993, 1992; Shan, M.A. and Ali, R., Modification of pig kidney diamine oxidase with ethoxyformic anhydride and rose bengal: evidence for essential histidine residue at the active site, Biochem.Mol.Biol.Int. 33, 9-19, 1994; Tseng, S.C. and Zhang, S.H., Interaction between rose bengal and different protein components, Cornea 14, 427-435, 1995; Singh, R.J., Hogg, N. and Kalyanaraman, B., Interaction of nitric oxide with photoexcited rose bengal: evidence for one-electron reduction of nitric oxide to nitroxyl anion, Arch.Biochem.Biophys. 324, 367-373, 1995; Bottiroli, G., Croce, A.C., Balzarini, P., et al., Enzyme-assisted cell photosensitization: a proposal for an efficient approach to tumor therapy and diagnosis. The rose bengal fluorogenic substrate, Photochem.Photobiol. 66, 374-383, 1997; Perez-Ruiz, T., Martinez-Lozano, C., Tomas, V. and Fenoll, J., Determination of proteins in serum by fluorescence quenching of rose bengal using the stopped-blow mixing technique, Analyst 125, 507-510, 2000; Lin, W., Garnett, M.C., Davis, S.S., et al., Preparation and characterization of rose Bengal-loaded surface-modified albumin nanoparticles, J.Control. Res. 71, 117-126, 2001; Posadez, A., Biasutti, A., Casale, C., et al., Rose Bengal-sensitized photooxidation of the dipeptides l-tryptophyl-l-phenylalanine, l-trytophyl-l-tyrosine and l-tryptophyl-l-tryptophan: kinetics, mechanism and photoproducts, Photochem.Photobiol. 80, 132-138, 2004; Luiz, M., Biasutti, M.A., and Garcia, N.A., Effect of reverse micelles on the Rose Bengal-sensitized photo-oxidation of 1- and 2-hydroxynaphthalene, Redox Rep. 9, 199-205, 2004; Khan-Lim, D. and Berry, M., Still confused about rose bengal?, Curr.Eye Res. 29, 311-317, 2004; Soldani, C., Bottone, M.G., Croce, A.C., et al., The Golgi apparatus is a primary site of intracellular damage after photosensitization with Rose Bengal acetate, Eur.J.Histochem. 48, 443-449, 2004; de Lima Santos, H., Forest Rigos. C., Claudio Tedesco, A., and Ciancaglini, P., Rose Bengal located within liposome do not affect the activity of inside-out oriented Na,K-ATPase, Biochim.Biophys.Acta 1715 96-103, 2005; Miller, J.S., Rose bengal-stimulated photooxidation of 2-chlorophenol in water using solar simulated light, Water Res. 39, 412-422, 2005; Shimizu, O., Watanabe, J., Naito, S. and Shibata, Y., Quenching mechanism of Rose Bengal triplet state involved in photosensitization of oxygen in ethylene glycol, J.Phys.Chem.A Mol.Spectrosc.Kinet.Environ.Gen.Theory 110, 1735-1739, 2006; Seitzman, G.D., Cevallos, V. and Margolis, T.P., Rose bengal and lissamine green inhibit detection of herpes simplex virus by PCR, Am.J.Ophthalmol. 141,756-758, 2006; Fini, P., Loseto, R., Catucci, L., et al., Study on the aggregation and electrochemical properties of Rose Bengal in aqueous solution of cyclodextrins, Bioelectrochemistry 70, 44-49, 2007. SITS (stilbene isothiocyanate sulfonic acid; 4-Acetamido-4’isothiocyanostilbene-,2’-disulfonic acid); MW 498 as disodium salt
O
Fluorescent stain; use on fixed tissues. Reactive groups permits use as a label for antibodies. Also used as a vital stain and as general cytoplasmic stain; used to characterize membrane anion-transport processes
SO3H
H3C
N
N HN
C
S
N HO3S
Benjaminson, M.A. and Katz, I.J., Properties of SITS (4-acetamido-4’-isothiocyanostilbene-2,2’-disulfonic acid): fluorescence and biological staining, Stain Technol. 45, 57-62, 1970; Rothbarth, P.H., Tanke, H.J., Mul, N.A., et al., Immunofluorescence studies with 4-acetamido-4’-isothiocyanato stilbene -2-2′-disulphonic acid (SITS), J.Immunol.Methods 19,101-109, 1978; Schmued, L.C. and Swanson, L.W., SITS: a covalently bound fluorescent retrograde tracer that does not appear to taken up by fibers-of-passage, Brain Res. 249, 137-141, 1982; Gilbert, P., Kettenmann, H., Orkland, R.K., and Schachner, M., Immunocytochemical cell identification in nervous system culture combined with intracellular injection of a blue fluorescing dye (SITS), Neurosci.Lett. 34, 123-128, 1982; Ploem, J.S., van Driel-Kulker, A.M., Goyarts-Veldstra, L., et al., Image analysis combined with quantitative cytochemistry. Results and instrumental developments for cancer diagnosis, Histochemistry 84, 549-555, 1986; Pedini, V., Ceccarelli, P., and Gargiulo, A.M., A lectin histochemical study of the zygomatic salivary gland of adult dogs, Vet.Res.Commun. 19, 363-375, 1995; Papageorgiou, P.. ,Shmukler, B.E., Stuant-Tilley, A.K., et al., AE anion exchangers in atrial tumor cells, Am.J.Physiol.Heart Circ.Physiol. 280, H937-H945, 2001; Quilty, J.A., Cordat, E., and Reithmeier, R.A., Impaired trafficking of human kidney anion exchanger (kAE1) caused by hetero-oligomer formation with a truncated mutant associated with distal renal tubular acidosis, Biochem.J. 368, 895-903, 2002. For SITS-sensitive anion transport see Villereal, M.L. and Levinson, C., Chloride-stimulated sulfate efflux in Ehrlich ascites tumor cells: evidence for 1:1 coupling, J.Cell Physiol. 90, 553-563, 1977; Kimelberg, H.K., Bowman, C.L., and Hirata, H., Anion transport in astrocytes, Ann.N.Y.Acad.Sci. 481, 334-353, 1986; Montrose, M., Randles, J., and Kimmich, G.A., SITS-sensitive Cl– conductance pathway in chick intestinal cells, Am.J.Physiol. 253, C693-C699, 1987; Bassnett, S., Stewart, S., Duncan, G., and Crogha, P.C., Efflux of chloride from the rat lens: influence of membrane potential and intracellular acidification, Q.J.Exp.Physiol. 73, 941-949, 1988; Ishibashi, K., Rector, F.C., Jr., and Berry, C.A., Chloride transport across the basolateral membrane of rabbit proximal convoluted tubules, Am.J.Physiol. 258, F1569-F1578, 1990; Wilson, J.M., Laurent, P., Tufts, B.L. et al., NaCl uptake by the branchial epithelium I freshwater teleost fish: an immunological approach to ion-transport protein localization, J.Exp.Biol. 203, 2279-2296, 2000; Small, D.L. and Tauskela, J., and Xia, Z., Role for chloride but not potassium channels in apoptosis in primary rat cortical cultures, Neurosci.Lett. 334, 95-98, 2002.
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838
SOME BIOLOGICAL STAINS AND DYES (Continued) Name
Description
Texas Red (TR, sulphorhodamine 101 acid chloride; MW 625
N
The presence of a reactive sulfonyl chloride function allows the labeling of protein amino groups. General probe for proteins and other molecules including DNA and carbohydrate to follow cellular transit.
N+
O
SO3–
SO2Cl Titus, J.A., Haugland, R., Sharrow, S.O., and Segal, D.M., Texas Red, a hydrophilic, red-emitting fluorophore for use with fluorescein in dual parameter flow microfluorimetric and fluorescence microscopic studies, J.Immunol.Methods 50, 193-204, 1982; Schneider, H., Differential intracellular staining of identified neurones in Locusta with texas red and lucifer yellow, J.Neurosci.Methods 30, 107-115, 1989; Leonce, S. and Cudennec, C.A., Modification of membrane permeability measured by Texas-Red during cell cycle progression and differentiation, Anticancer Res. 10, 369-374, 1990; Srour, E.F., Lemmhuis, T., Brandt, J.E, et al., Simultaneous use of rhodamine 123, phycoerythrin, Texas red, and allophycocyanin for the isolation of human hematopoietic progenitor cells, Cytometry 12, 179-183, 1991; Wessendorf, M.W. and Brelje, T.C., Which fluorophore is brightest? A comparison of the staining obtained using fluorescein, tetramethylrhodamine, lissamine rhodamine, Texas red, and cyanine 3.18, Histochemistry 98, 81-85, 1992; Belichenko, P.V. and Dahlstrom, A., Dual channel confocal laser scanning microscopy of Lucifer yellow-microinjected human brain cells combined with Texas red immunofluorescence, J.Neurosci.Methods 52, 111-118, 1994; Brismar, H., Trepte, O. and Ulfhake, B., Spectra and fluorescence lifetimes of lissamine rhodamine, tetramethylrhodamine isothiocyanate, texas red, and cyanine 3.18 fluorophores: influences of some environmental factors recorded with a confocal laser scanning microscope, J.Histochem.Cytochem. 43, 699-707, 1995; Anees, M., Location of tumour cells in colon tissue by Texas red labelled pentosan polysulphate, an inhibitor of a cell surface protease, J.Enzyme Inhib. 10, 203-214, 1996; Simon, S., Reipert, B., Eibl, M.M., Steinkasserer, A., Detection of phosphatidylinositol glycan class A gene transcripts by RT in situ PCR hybridization. A comparative study using fluorescein, Texas Red, and digoxigenin-11 dUTP for color detection, J.Histochem.Cytochem. 45, 1659-1664, 1997; Larramendy, M.L., El-Rifai, W., and Knutila, S., Comparison of fluorescein isothiocyanate- and Texas red-conjugated nucleotides for direct labeling in comparative genomic hybridization, Cytometry 31,174-179, 1998; Hembry, R.M., Detection of focal proteolysis using Texas-red-gelatin, Methods Mol.Biol. 151, 417-424, 2001; Kahn, E., Lizard, G., Frouin, F., et al., Confocal analysis of phosphatidylserine externalization with the use of biotinylated annexin V revealed with streptavidinFITC, -europium, -physcoerythrin or –Texas Red in oxysterol-treated apoptotic cells, Anal.Quant.Cytol.Histol. 23, 47-55, 2001; Alba, F.J. and Daban, J.R., Detection of Texas red-labelled double-stranded DNA by non-enzymatic peroxyoxalate chemiluminescence, Luminescence 16, 247-249, 2001; Watanabe, K. and Hattori, S., Real-time dual zymographic analysis of matrix metalloproteinases using fluorescein-isothiocyanate-labeled gelatin and Texas-red-labeled casein, Anal.Biochem. 307, 390-392, 2002; Tan, H.H., Thornhill, J.A., Al-Adhami, B.H., et al.,, A study of the effect of surface damage on the uptake of Texas Red-BSA by schistosomula of Shistosoma mansoni, Paristology 126, 235-240, 2003; Wippersteg, V., Ribeiro, F., Liedtke, S., et al., The uptake of Texas Red-BSA in excretory system of schistosomes and its colocalisation with ER60 promoter-induced GFP in transiently transformed adult males, Int.J.Parasitol. 33, 1139-1143, 2003; Unruh, J.R., Gokulrangan, G., Wilson, G.S., and Johnson, C.K., Fluorescence properties of fluorescein, tetramethylrhodamine and Texas Red linked to a DNA aptamer, Photochem.Photobiol. 81, 682-690, 2005. Thioflavin T (Basic Yellow 1; 318.9) H3C
Fluorescent dye use to detect b-sheet structures such as amyloid protein; also used to detect conformational changes in therapeutic proteins on drying. CH3
S N Cl–
N+
CH3
CH3
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Some Biological Stains and Dyes
839
SOME BIOLOGICAL STAINS AND DYES (Continued) Name
Description
Rogers, D.R., Screening for amyloid with the thioflavin-T fluorescent method, Am.J.Clin.Pathol. 44, 59-61, 1965; Saeed, S.M. and Fine, G., Thioflavin-T for amyloid detection, Am.J.Clin.Pathol. 47, 588-593, 1967; De Ferrari, G.V., Mallender, W.D., Inestrose, N.C. and Rosenberry, T.L., Thioflavin T is a fluorescent probe of the acetylcholinesterase peripheral site that reveals conformational interactions between the peripheral and acylation sites, J.Biol.Chem. 276, 23282-23287, 2001; Mathis, C.A, Bacskai, B.J., Kajdasz, S.T., et al., A lipophilic thioflavin-T derivative for positron emission tomography (PET) imaging of amyloid in brain, Bioorg. Med.Chem.Lett. 12, 295-298, 2002; Kramenburg, O., Bouma, B., Kroon-Batenberg, M.J., et al., Tissue-type plasminogen activator is a multiligand cross-b structure receptor, Curr.Biol.12, 1833-1839, 2002: Bouma, B., Loes, M., Kroon-Batenberg, M.J., et al., Glycation induced formation of amyloid cross-b-structure, J.Biol. Chem. 278, 41810-41819, 2003; Krebs, M.R., Bromley, E.H., and Donald, A.M., The binding of thioflavin-T to amyloid fibrils: localization and implications, J.Struct.Biol. 149, 30-37, 2005; Khurana, R., Coleman, C., Ionescu-Zanetti, C., et al., Mechanism of thioflavin T binding to amyloid fibrils, J.Struct.Biol. 151, 229-238, 2005; Inbar, P., Li, C.Q., Takayama, S.A., et al., Oligo (ethylene glycol) derivatives of Thioflavin T as inhibitors of protein-amyloid interactions, Chem.Bio. Chem 7, 1563-1566, 2006; Okuno, A., Kato, M., and Taniguchi, Y., The secondary structure of pressure- and temperature-induced aggregation of equine serum albumin studied by FT-IR spectroscopy, Biochim.Biophys.Acta 1764, 1407-1412, 2006; Groenning, M., Olsen, L,. van de Weert, M., et al., Study on the binding of Thioflavin T to b-sheet-rich and non-b-sheet cavities, J.Struct.Biol., in press, 2006; Maskevich, A.A., Stsiapura, V.I., Kuzmitsky, V.A., et al., Spectral properties of Thioflavin-T in solvents with different dielectric properties and in a fibril-incorporated form, J.Proteome Res., in press, 2007; Maas, C., Hermeling, S., Bouma, B., et al., A role for protein misfolding in immunogenicity of biopharmaceuticals, J.Biol.Chem. 282, 2229-2236, 2007 Toluidine Blue (C.I. Basic Blue 17; Methylene Blue T5O, TBO, toluidine blue O, tolonium chloride) MW 306
A diaminothiazine which can exist in a cation form which is hydrophilic. Used for metachromatic staining of biological molecules such as mucins and macromolecular structures. Used for demarcation of tumor tissue for surgery. Very early use for heparin neutralization in blood coagulation
N
H3C
Cl N+
S
NH2
CH3 Haley, T.J. and Rhodes, B., Effect of toluidine blue on the coagulation of fibrinogen by thrombin, Science 117, 604-606, 1953; Ball, J. and Jackson, D.S., Histological, chromatographic and spectrophotometric studies of toluidine blue, Stain Technol. 28, 33-40, 1953; Gustafsson, B.E. and Cronberg, S., The effect of hyaluronidase and toluidine blue on the mast cells in rats and hamsters, Acta Rheumatol.Scand. 5, 179-189, 1959; Schueller, E., Peutsch, M., Bohacek, L.G., and Gupta, R.K., A simplified toluidine blue stain for mast cells, Can.J.Med.Technol. 29, 137-138, 1967; Itzhaki, R.F., Binding of polylysine and Toluidine Blue to deoxyribonucleoprotein, Biochem.J. 121, 25P-26P, 1971; Sakai, W.S., Simple method for differential staining of paraffin embedded plant material using toluidine blue O, Stain Technol. 48, 247-249, 1973; Koski, J.P. and McGarvey, K.E., Toluidine blue as a capricious dye, Am.J.Clin.Pathol. 73, 457, 1980; Busing, C.M. and Pfiester, P., Permanent staining of rapid frozen section with toluidine blue, Pathol.Res.Pract. 172, 211-215, 1981; Drzymala, R.E., Liebman, P.A. and Romhanyi, G., Acid polysaccharide content of frog rod outer segments determined by metachromatic toluidine blue staining, Histochemistry 76, 363-379. 1982; O’Toole, D.K., The toluidine blue-membrane filter method: absorption spectra of toluidine blue stained bacterial cells and the relationship between absorbance and dry mass of bacteria, Stain Technol. 58, 357-364, 1983; Waller, J.R., Hodel, S.L., and Nuti, R.N., Improvement of two toluidine blue O-mediated techniques for DNase detection, J.Clin.Microbiol. 21, 195-199, 1985; Lior, H. and Patel, A., Improved toluidine blue-DNA agar for detection of DNA hydrolysis by campylobacters, J.Clin.Microbiol. 25, 2030-2031, 1987; Paardekooper, M., De Bruijne, A.W., Van Steveninck, J., and Van den Broek, P.J., Inhibition of transport systems in yeast by photodynamic treatment with toluidine blue, Biochim.Biophys.Acta 1151, 143-148, 1993; Passmore, L.J., and Killeen, A.A., Toluidine blue dye-binding method for measurement of genomic DNA extracted from peripheral blood leukocytes, Mol.Diagn. 1, 329-334, 1996; Korn, K., Greiner-Stoffele, T. and Hahn, U., Ribonuclease assays utilizing toluidine blue indicator plates, methylene blue, or fluorescence correlation spectroscopy, Methods Enzymol. 341, 142-153, 2001; Sanchez, A., Guzman, A., Ortiz, A.. et al., Toluidine blue-O of prion protein deposits, Histochem.Cell Biol. 116, 519-524, 2001; Prat, E., Camps, J., del Rey, J., et al., Combination of toluidine blue staining and in situ hybridization to evaluate paraffin tissue sections, Cancer Genet. Cytogenet. 155, 89-91, 2004; Kaji, Y., Hiraoka, T., and Oshika, T., Vital staining of squamous cell carcinoma of the conjunctive using toluidine blue, Acta Ophthalmol.Scand. 84, 825-826, 2006; Missmann, M., Jank, S., Laimer, K., and Gassner, R., A reason for the use of toluidine blue staining in the presurgical management of patients with oral squamous cell carcinomas, Oral Surg.Oral Med. Oral Pathol. Oral Radiol.Endod. 102, 741-743, 2006.
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Handbook of Biochemistry and Molecular Biology
840
SOME BIOLOGICAL STAINS AND DYES (Continued) Name
Description
Trypan Blue (diamine blue; C.I. Direct Blue 14) MW 961 as tetrasodium salt
A tetrasulfated anionic dye composed of a large planar aromatic system. Moderately soluble in water, more soluble in ethylene glycol but essentially insoluble in ethanol. Used a biological stain with particular interest as vital stain. Used in the early polychrome stains. Current use also as surgical stain for cataract surgery. SO3Na
NaO3S
N N
N
NaO3S
N
OH H3C
NH2
SO3Na
CH3 HO H2N
Menkin, V., Effects of ACTH on the mechanism of increased capillary permeability to trypan blue in inflammation, Am.J.Physiol. 166, 518-523, 1951; Wislocki, G.B. and Leduc, E.H., Vital staining of the hematoencephalic barrier by silver nitrate and trypan blue, and cytological comparisons of the neurohypophysis, pineal body, area postrema, intercolumnar tubercule and supraoptic crest, J.Comp.Neurol. 96, 371-413, 1952; Auskaps, A.M. and Shaw, J.H., Vital staining of calcifying bone and dentin with trypan blue, J.Dent.Res. 34, 452-459, 1955; Ferm, V.H., Permeability of the rabbit blastocyst to trypan blue, Anat.Rec. 125, 745-759, 1956; Kelly, J.W., Staining reactions of some anionic disazo dyes and histochemical properties of the red impurity in trypan blue, Stain Technol. 33, 89-94, 1958; Tennant, J.R., Evaluation of the trypan blue technique for determination of cell viability, Transplantation 2, 685-694, 1964; Holl, A., Vital staining by trypan blue: its selectivity for olfactory receptor cells of the brown bullhead, Ictalurus natalis, Stain Technol. 40, 269-273, 1965; Estupinan, J. and Hanson, R.P., Congo red and trypan blue as stains for plaque assay of Newcastle disease virus, Avian Dis. 13, 330-339. 1969; Lloyd, J.B. and Field, F.E., The red impurity in trypan blue, Experientia 26, 868-869, 1970; Dickinson, J.P. and Apricio, S.G., Trypan blue: reaction with myelin, Biochem.J. 122, 65P-66P, 1971; Davies, M., The effect of triton WR-1339 on the subcellular distribution of trypan blue and 125I-labelled albumin in rat liver, Biochem.J. 136, 57-65, 1973; Davis, H.W. and Sauter, R.W., Fluorescence of Trypan Blue in frozen-dried embryos of the rat, Histochemistry 54, 177-189, 1977; Loike, J.D. and Silverstein, S.C., A fluorescence quenching technique using trypan blue to differentiate between attached and injested glutaraldehyde-fixed red blood cells in phagocytosing murine macrophages, J.Immunol. Methods. 57, 373-379, 1983; Boiadjieva, S., Hallberg, C., Hogstrom, M., and Busch, C., Methods in laboratory investigation. Exclusion of trypan blue from microcarriers by endothelial cells: an in vitro barrier function test, Lab.Invest. 50, 239-246, 1984; Lee, R.M., Chambers, C., O’Brodovich, H., and Forrest, J.B., Trypan blue method for the identification of damage to airway epithelium due to mechanical trauma, Scan.Electron Microsc. (Pt. 3), 1267-1271, 1984; Shen, W.C., Yang, D., and Ryser, H.J., Colorimetric determination of microgram quantities of polylysine by trypan blue precipitation, Anal.Biochem. 142, 521-524, 1984; Perry, S.W., Epstein, L.G., and Gelbard, H.A., In situ trypan blue staining of monolayer cell cultures for permanent fixation and mounting, Biotechniques 22, 1020-1024, 1997; Mascotti, K., McCullough, J., and Burger, S.R., HPC viability measurement: trypan blue versus acridine orange and propidium iodide, Transfusion 40, 693-696, 2000; Igarashi, H. Nagura, K., and Sugimura, H., Trypan blue as a slow migrating dye for SSCP detection in polyacrylamide gel electrophoresis, Biotechniques 29, 42-44, 2000. Zincon; 2-[1-(2-hydroxy-5sulfonatophenyl)-3-phenyl-5formazano] benzoic acid, sodium salt MW 462
A metal complexing formazan dye which is used in histochemistry to demonstrate the presence of zinc; zincon is also used to stain for zinc proteins on solid support electrophoresis. Zincon is also used to demonstrate the presence of zinc in solution using spectrophotometric estimation of the zinc-dye complex
OH H N
N N
N
COOH SO3H
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Some Biological Stains and Dyes
841
SOME BIOLOGICAL STAINS AND DYES (Continued) Name
Description
Corns, C.M., A new colorimetric method for the measurement of serum calcium using a zinc-zincon indicator, Ann.Clin.Biochem. 24, 591-597, 1987; Richter, P., Toral, M.I., Tapia, A.E., and Fuenzalida, E., Flow injection photometric determination of zinc and copper with zincon based on the variation of the stability of the complexes with pH, Analyst. 122, 1045-1048, 1997; Choi, J.K., Tak, K.H., Jin, L.T., et al., Background-free, fast protein staining in sodium dodecyl sulfate polyacrylamide gel using counterion dyes, zincon and ethyl violet, Electrophoresis 23, 4053-4059, 2002; Smejkal, G.B. and Hoff, H.F., Use of the formazan dye zincon for staining proteins in polyacrylamide gels, Biotechniques 34, 486-468, 2003; Choi, J.K., Chae, H.Z., Hwang, S.Y., et al., Fast visible dye staining of proteins in one- and two-dimensional sodium dodecyl sulfate-polyacrylamide gels compatible with matrix assisted laser desorption/ionization-mass spectrometry, Electrophoresis 25, 1136-1141, 2004; Morais, I.P., Souto, M.P., and Rangel, A.O., A double-line sequential injection system for the spectrophotometric determination of copper, iron, manganese, and zinc in waters, J.AOAC Int. 88, 639-644, 2005; Ribiero, M.F., Dias, A.C., Santos, J.L., et al., Fluidized beds in flow analysis: use with ion-exchange separation for spectrophotometric determination of zinc in plant digests, Anal.Bioanal.Chem. 384, 1019-1024, 2006.
Biological stains and dyes – general references
Conn’s Biological Stains a Handbook of Dyes, Stains, and Fluorochromes for use in Biology and Medicine, ed. R.W. Horobin and J.A. Kiernan, Bios, Oxford University Press, Oxford, UK, 2002.
The Chemistry of Synthetic Dyes and Pigments, ed. H.A. Lubs, American Chemical Society, Reinhold Publishing, New York, NY, USA, 1955 Kiernan, J.A., Classification and naming of dyes, stains and fluorochromes, Biotech.Histochem. 76, 261-278, 2001
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Mordant Dyes Mordant dye is a dye that interacts with a tissue, cell, or subcellular organelle via an interaction with a substance, a mordant, which interacts with the substrate and the dye. The interaction may involve covalent interaction. A mordant can be defined as a substance which interacts with both the substrate (for example tissue, cell, subcellular organelle) and the dye1. Most mordants are metal salts such ferric chloride and those of chromium and vanadium. Other compounds such as tannic acid1 and galloylglucoses2 can serve as mordants. Hematoxylin used in Hematoxylin and eosin (H and E) staining which contains a mixture of chelates formed between hematein and aluminum ions3,4. Mordant blue 3 (Chromoxane cyanine R) uses iron5. There has been a significant increase in our understanding of mordant dyes4,6.
2. Simionescu, N. and Simionescu, M., Galloylgluose of low molecular weight as mordant in electron microscopy. I. Procedure, and evidence mordanting effect, J.Cell Biol. 70, 608-621, 1976. 3. Bettinger, C. and Zimmermann, H.W., New investigations on hematoloxylin, hematein and hematein-aluminum complexes. 2. Hemateinaluminum complexes and hemalum staining, Histochemistry 96, 215-228, 1991. 4. Horobin, R.W., Biological staining: mechanisms and theory, Biotechnic & Histochemistry 77, 3-13, 2002. 5. Kiernan, J.A., Chromoxane cyanine R. I. Physical and chemical properties of the dye and of some of its iron complexes, J.Microsc. 134, 13-23, 1984. 6. Dapson, R.W., Dye-tissue interactions: mechanisms, quantification and bonding parameters for dyes used in biological staining, Biotech. Histochem. 80, 49-72, 2005.
References 1. Afzelius, B.A., Section staining for electron microscopy using tannic acid as a mordant: a simple method for visualization of glycogen or collagen, Microsc.Res.Tech. 21, 65-72, 1992.
General references for mordant dyes Conn’s Biological Stains, 10th edn., ed. R.W. Horobin and J.A. Kiernan, Bios Scientific Publishers, Oxford, UK, 2002
Mordant Dyes Dye Name Mordant Blue 1; chrome azurol A; chromoxane pure blue B Mordant Blue 3; eriochrome cyanine R; chromoxane cyanine R Mordant Blue 10; Gallocyanine; Mordant Blue 14; Celestine blue; Mordant Blue 29; chromoxane pure blue BLD Mordant Blue 45; Gallamine blue Mordant Blue 80; Chromotrope 2R Mordant Red 3; Alizarin red S Mordant Violet 39; aurin tricarboxylic acid (ATA); aluminon; chrome violet TG a
Metal Ion Specificitya Al, B, Ca, Al, Cr, Fe Cr, Fe, Pb Fe Be Al, Ca Cr Al, Ca Al, Be, Cr, Fe
Comments Calcium staining in histochemistry Iron staining in histochemistry; also Al Chromium complex for staining DNA and RNA Complex with iron use as nuclear stain Be staining in histochemistry Al complex for staining nuclei Cytoplasmic counterstain Calcified tissue stain Aluminum staining in histochemistry
Metal ion specificity is broad with most mordant stains so other metal ions can be complexed by the respective mordant stain.
843
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Handbook of Biochemistry and Molecular Biology
844 HO
O
HO
O
H3C
NH2
O
C
C
HO
H3C
O
O
N
O
CH3
H3C Cl
O OH
Cl Mordant blue 14; Celestine blue
COOH
Mordant blue 1; Chrome azurol B
COOH
HO COOH
O
COOH
HO
O
H3C
CH3 Cl
H3C
Cl
CH3 SO3H
SO3H Mordant blue 29; Chromoxane pure blue
Mordant blue 3; Eriochrome cyanine R
CH3 N
N
O
H3C
O
O
H3C
OH
CH3
OH
O
N CONH2
N
Mordant blue 45; Gallamine blue
COOH Mordant blue 10; Gallocyanine
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Mordant Dyes
845 HO
COOH
HO
HO
O
SO3H N HOOC N HO3S Mordant blue 80; Chromotrope 2R COOH O
OH
OH OH
Mordant Violet 39; Aurin Tricarboxylic Acid
SO3H O Mordant red 3; Alizarin red S
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Metal Chelating Agentsa Name
M.W.
BAPTA [1,2-bis(o-aminophenoxy) ethane, N,N,N’,N’-tetraacetic acid
476.4
Chelating agent with higher affinity for zinc than calcium can be used as with EGTA for chelation of intracellular metal ions. BAPTA-AM, the acetoxymethyl ester, is a useful derivative.
COOH
HOOC
HOOC
Description
N
N COOH O
O
Harrison, S.M. and Bers, D.M., The effect of temperature and ionic strength on the apparent Ca-affinity of EGTA and the analogous Ca-chelators BAPTA and dibromo-BAPTA, Biochim.Biophys.Acta. 925, 133-143, 1987; Minta, A., Kao, J.P., and Tsien, R.Y., Fluorescent indicators for cytosolic calcium based on rhodamine and fluorescein chromophores, J.Biol.Chem. 264, 8171-8178, 1989; Csermely, P., Sandor, P., Radics, L., and Somogyi, J., Zinc forms complexes with higher kinetic stability than calcium, 5-F-BAPTA as a good example, Biochem.Biophys.Res.Commun. 165, 838-844, 1989; Marks, P.W. and Maxfield, F.R., Preparation of solutions with free calcium concentration is the nanomolar range using 1,2-bis(o-aminophenoxy)ethane-N,N,N’,N’-tetraacetic acid, Anal. Biochem. 193, 61-71, 1991; Brooks, S.P. and Storey, K.B., Bound and determined: a computer program for making buffers of defined ion concentrations, Anal. Biochem. 201, 119-126, 1992; Dieter, P., Fitzke, E., and Duyster, J., BAPTA induces a decrease of intracellular free calcium and a translocation and inactivation of protein kinase C in macrophages, Biol.Chem.Hoppe Seyler 374, 171-174, 1993; Natarajan, V., Scribner, V.M., and Taher, M.M., 4-Hydroxynonenal, a metabolite of lipid peroxidation, activates phospholipase D in vascular endothelial cells, Free Radic.Biol.Med. 15, 365-375, 1993; Bers, D.M., Patton, C.W., and Nuccitelli, R., A practical guide to the preparation of Ca2+ buffers, Methods Cell Biol. 40, 3-29, 1994; Oiki, S., Yamamoto, T., and Okada, Y., A simultaneous evaluation method of purity and apparent stability constant of Ca-chelating agents and selectivity coefficient of Ca-selective electrodes, Cell Calcium 15, 199-208, 1994; Aballay, A., Sarrouf, M.N., Colombo, M.I., et al., Zn2+ depletion blocks endosome fusion, Biochem.J. 312, 919-923, 1995; Britigan, B.E., Rasmussen, G.T., and Cox, C.D., Binding of iron and inhibition of iron-dependent oxidative cell injury by the “calcium chelator” 1,2-bis(2-aminophenoxy) ethane, N,N,N’,N,’-tetraacetic acid, Biochem.Pharmacol. 55, 287-295, 1998; Kim-Park, W.K., Moore, M.A., Hakki, Z.W., and Kowolik, M.J., Activation of the neutrophil respiratory burst requires both intracellular and extracellular calcium, Ann.N.Y.Acad.Sci. 832, 394-404, 1997; Barbouti, A., Doulias, P.T., Zhu, B., et al., Intracellular iron, but not copper, plays a critical role in hydrogen peroxide-induced DNA damage, Free Radic.Biol.Med. 31, 490-498, 2001; Nielsen, A.D., Fuglsang, C.C., and Westh, P., Isothermal titration calorimetric procedure to determine protein-metal ion binding parameters in the presence of excess metal ion or chelator, Anal.Biochem. 314, 227-234, 2003; Gee, K.R., Rukavishnikov, A. and Rothe, A., New Ca2+ fluoroionophores based on the BODIPY fluorophore, Comb.Chem.High Throughput Screen. 6, 363-366, 2003; Swystum, V., Chen, L., Factor, P., et al., Apical trypsin increases ion transport and resistance by a phospholipase C-dependent rise of Ca2+, Am.J.Physiol.Lung Cell.Mol.Physiol. 288, L820-L830, 2005; Lazzaro, M.D., Cardenas, L., Bhatt, A.P., et al., Calcium gradients in conifer pollen tube; dynamic properties differ from those seen in angiosperms, J.Exp.Bot. 56, 2619-2628, 2005 α-Benzoin oxime (cupron); 2-hydroxy-1,2-diphenylethanone oxime
227.3
Complexes cupric ions, molybdenum, chromium, lead, or tungsten as well as other metal ions. Used for the analysis of these metal ions. More recently, a-Benzoin oxime has been immobilized and used as a chelating resin for concentration and separation of metal ions. Copper complexes have been used as biocidal agent.
OH
N OH alpha-benzoin oxime
OH
N HO beta-benzoin oxime
OH
O benzoin
847
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Handbook of Biochemistry and Molecular Biology
848
METAL CHELATING AGENTS (Continued) Name
M.W.
Description
Borkow, G. and Gabbay, J., Putting copper into action-impregnated products with potent biocidal activities, FASEB J. 18, 1728-1730, 2004; Borkow, G. and Gabbay, J., Copper as a biocidal tool, Curr.Med.Chem. 12, 2163-2175, 2005; Ghaedi, M., Asadpour, E., and Vafaie, A., Sensitized spectrophotometric determination of Cr(III) ion for speciation of chromium ion in surfactant media using a-benzoin oxime, Spectrochim.Acta A Mol. Biomol.Spectrosc. 63, 182-188, 2006; Soylak, M. and Tuzen, M., Dianion SP-850 resin as a new solid phase extractor for preconcentration-separation of trace metal ions in environmental samples, J.Hazard.Mater. 137, 1496-1501, 2006 Chromotropic acid; 4,5-dihydroxy-2,7naphthalenedisulfonic acid OH
320.3
Chelation and fluorometric determination of aluminum and other metal ion. Also used for the analysis formaldehyde.
OH
O
O S HO
S O
O
OH
Metal Ions: Durham, A.C. and Walton, J.M., A survey of the available colorimetric indicators for Ca2+ and Mg2+ ions in biological experiments, Cell Calcium 4, 47-55, 1983; Prestel, H., Gahr, A., and Niessner, R., Detection of heavy metals in water by fluorescence spectroscopy: on the way to a suitable sensor system, Fresenius J.Anal.Chem. 368, 182-191, 2000; Destandau, E., Alain, V., and Bardez, E., Chromotropic acid, a fluorogenic chelating agent for aluminum(III), Anal.Bioanal.Chem. 378, 402-410, 2004; Themelis, D.G. and Kika, F.S., Flow and sequential injection methods for the spectrofluorometric determination of aluminum in pharmaceutical products using chromotropic acid as chromogenic reagent, J.Pharm.Biomed.Anal. 41, 1179-1185, 2006; Lemos, V.A., Santos, L.N., Alves, A.P., and David, G.T., Chromotropic acid-functionalized polyurethane foam: A new sorbent for on-line preconcentration and determination of cobalt and nickel in lettuce samples, J.Sep.Sci. 29, 1197-1204, 2006; Formaldehyde: Manius, G.J., Wen, L.F., and Palling, D., Three approaches to the analysis of trace formaldehyde in bulk and dosage from pharmaceuticals, Pharm.Res.10, 449-453, 1993; Flyvholm, M.A., Tiedmann, E., and Menne, T., Comparison of 2 tests for clinical assessment of formaldehyde exposure, Contact Dermatitis 34, 35-38, 1996; Pretto, A., Milani, M.R., and Cardoso, A.A., Colorimetric determination of formaldehyde in air using a hanging drop of chromotropic acid, J.Environ.Monit. 2, 566-570, 2000 Citric acid (2-hydroxy-1,2,3propanetricarboxylic acid
HO
H2C
COOH
C
COOH
H2C
COOH
192.1
Moderately strong chelating agent. Used frequently for calcium and iron
Hopkins, E.W. and Herbst, E.J., An explanation for the apparent chelation of calcium by tetrodotoxin, Biochem.Biophys.Res.Commun. 30, 528-533, 1968; Steinmetz, W.L., Glick, M.R., and Oei, T.O., Modified aca method for determination of iron chelated by deferoxoamine and other chelators, Clin. Chem. 26, 1593-1597, 1980; Ford, W.C. and Harrison, A., The role of citrate in determining the activity of calcium ions in human semen, Int.J.Androl. 7, 198-202, 1984; Lund, A.J. and Aust, A.E., Iron mobilization from asbestos by chelators and ascorbic acid, Arch.Biochem.Biophys. 278, 61-64, 1990; Francis, B., Seebart, C., and Kaiser, I.I., Citrate is an endogenous inhibitor of snake venom enzymes by metal-ion chelation, Toxicon 30, 1239-1246, 1992; Morgan, J.M., Navabi, H., Schmid, K.W., and Jasani, B., Possible role of tissue-bound calcium ions in citrate-mediated high temperature antigen retrieval, J.Pathol. 174, 301-307, 1994; Rhee, S.H. and Tanaka, J., Effect of citric acid on the nucleation of hydroxyapatite in a simulated body fluid, Biomaterials 20, 2155-2160, 1999; Engelmann, M.D., Bobier, R.T., Hiatt, T., and Cheng, I.F., Variability of the Fenton reaction characteristics of the EDTA, DTPA, and citrate complexes of iron, Biometals 16, 519-527, 2003; Fernandez, V. and Winkelmann, G., The determination of ferric iron in plants by HPLC using the microbial iron chelator desferrioxamine E, Biometals 18, 53-62, 2005; Matinaho, S., Karhumäki, P., and Parkkinen, J., Bicarbonate inhibits the growth of Staphylococcus epidermidis in platelet concentrates by lowering the level of non-transferrin-bound iron, Transfusion 45, 1768-1773, 2005; Reynolds, A.J., Haines, A.H., and Russell, D.A., Gold glyconanoparticles for mimics and measurement of metal ion-mediated carbohydrate-carbohydrate interactions, Langmuir 22, 1156-1163, 2006
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Metal Chelating Agentsa
849 METAL CHELATING AGENTS (Continued)
Name
M.W.
Cupferron; N-hydroxy-Nnitrosobenzene, ammonium salt
155.2
Description Chelation of and precipitation of iron, copper, zinc, vanadium
O NH4+ –O
N N
Kolthoff, I.M. and Liberti, A., Amperometric titration of copper and ferric iron with cupferron, Analyst 74, 635-641, 1949; Ahuja, B.S., Kiran, U., and Sudershan, In vivo & in vitro inhibition of mung bean superoxide dismutase by cupferron, Indian J. Biochem.Biophys. 18, 86-87, 1981; Walsh, K.A., Daniel, R.M., and Morgan, H.W., A soluble NADH dehydrogenase (NADH: ferricyanide oxidoreductase) from Thermus aquaticus strain T351, Biochem.J. 209, 427-433, 1983; Danzaki, Y., Use of cupferron as a precipitant for the determination of impurities in high-purity iron by ICP-AES, Anal. Bioanal.Chem. 356, 143-145, 1996; Heinemann, G. and Vogt, W., Quantification of vanadium in serum by electrothermal atomic absorption spectrometry, Clin.Chem. 42, 1275-1282, 1996; Oztekin, N. and Erim, F.B., Separation and direct UV detection of lanthanides complexed with cupferron by capillary electrophoresis, J.Chromatog.A. 895, 263-268, 2000; Hou, Y., Xie, W., Janczuk, A.J., and Wang, P.G. O-Alkylation of cupferron: aiming at the design and synthesis of controlled nitric oxide releasing agents, J.Org.Chem. 65, 4333-4337, 2000; Bourque, J.R., Burley, R.K., and Bearne, S.L., Intermediate analogue inhibitors of mandelate racemase: N-hydroxyformanilide and cupferron, Bioorg.Med.Chem.Lett. 17, 105-108, 2007 Sodium diethyldithiocarbamate; Diethyldithiocarbamate, sodium salt (Dithio carb sodium); diethylcarbamothioic acid, sodium salt; diethyldithiocarbamic acid sodium salt
171.2
Chelation of zinc, copper, mercury, and nickel
S
Na+
–S
N
CH3
CH3 Rigas, D.A., Eginitis-Rigas, C. and Head, C., Biphasic toxicity of diethyldithiocarbamate, a metal chelation, to T lymphocytes and polymorphonuclear granulocytes: reversal by zinc and copper, Biochem.Biophys.Res.Commun. 88, 373-379, 1979; Khandelwal, S., Kachru, D.N., and Tandon, S.K., Chelation in metal intoxication. IX. Influence of amino and thiol chelators on excretion of manganes in poisoned rabbits, Toxicol.Lett. 6, 131-135, 1980; Marciani, D.J. Wilkie, S.D., and Schwartz, C.L., Colorimetric determination of agarose-immobilized proteins by formation of copper-protein complexes, Anal.Biochem. 128, 130-137, 1983; O’Callaghan, J.P. and Miller, D.B., Diethyldithiocarbamate increases distribution of cadmium to brain but prevents cadmium-induced neurotoxicity, Brain Res. 170, 354-358, 1986; Khandelwal, S., Kachru, D.N., and Tandon, S.K., Influence of metal chelators on metalloenzymes, Toxicol.Lett. 37, 213-219, 1987; Tandon, S.K., Hashmi, N.S., and Kashru, D.N., The lead-chelating effects of substituted dithiocarbamates, Biomed.Environ.Sci. 3, 299-305, 1990; Borrello, S., De Leo, M.E., Landricina, M. et al., Diethyldithiocarbamate treatment up regulates manganese superoxide dismutase gene expression in rat liver, Biochem.Biophys.Res.Commun. 220, 546-552, 1996 Dimethylglyoxime; 2,3-butanedionedioxime; diacetyldioxime H3C
116.1
Primary for chelation and detection of nickel; also for separation of lead and chelation of copper
N OH OH
H3C
9168_Book.indb 849
N
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850
METAL CHELATING AGENTS (Continued) Name
M.W.
Description
Lee, D.W. and Halmann, M., Selective separation of nickel (II) by dimethylglyoxime-treated polyurethane foam, Anal.Chem. 48, 2214-2218, 1976; Dixon, N.E., Gazzola, C., Asher, C.J. et al., Jack Bean urease (EC 3.5.1.5)-II. The relationship between nickel, enzymatic activity, and the “abnormal” ultraviolet spectrum. The nickel content of jack beans, Can.J.Biochem. 58, 474-480, 1980; Huber, K.R., Sridhar, R., Griffith, E.H., et al., Superoxide dismutase-like activities of copper(II) complexes tested in serum, Biochim.Biophys.Acta 915, 267-276, 1987; Heo, J., Staples, C.R., Halbleib, C.M., and Ludden, P.W., Evidence for a ligand CO that is required for catalytic activity of CO dehydrogenase from Rhodospirilum rubrum, Biochemistry 39, 7956-7963, 2000; Celo, V., Murimboh, J., Salam, M.S., and Chakrabarti, C.L., A kinetic study of nickel complexation in model systems by adsorptive catholic stripping voltammetry, Environ.Sci.Technol. 35, 1084-1089, 2001; Ponnuswamy, T. and Chyan, O., Detection of Ni2+ by a dimethylglyoxime probe using attenuated total-reflection infrared spectrometry, Anal.Sci. 18, 449-453, 2002 Dithizone; diphenylthiocarbazone
256.3
Chelating and measurement of mercury, zinc, cobalt, copper, and lead. Extensive use for the histochemical detection of zinc.
S H N
N N H
N
Landry, A.S., Optimum range for maximum accuracy in biological lead analyses by dithizone, Ind.Health Mon. 11, 103, 1951; Mager, M., McNary, W.F., Jr., and Lionetti, F., The histochemical detection of zinc, J.Histochem.Cytochem. 1, 493-504, 1953; McNary, W.F., Jr., Dithizone staining of myeloid granules, Blood 12, 644-648, 1957; Butler, E.J., and Newman, G.E., An absorptiometric method for the determination of traces of copper in biological materials with dithizone, Clin.Chim.Acta 11, 452-460, 1965; Shendriker, A.D. and West, P.W., Microdetermination of lead with dithizone and the ring-oven technique, Anal.Chim.Acta 61, 43-48, 1972; Nabrzyski, M., Spectrophotometric method for copper and mercury determination in the same food sample using dithizone and lead diethyldithiocarbamate, Anal.Chem. 47, 552-553, 1975; Song, M.K., Adham, N.F., and Rinderknecht, R., A simple, highly sensitive colorimetric method for the determination of zinc in serum, Am.J.Clin.Pathol. 65, 229-233, 1976; Holmquist, B., Elimination of adventitious metals, Methods Enzymol. 158, 6-12, 1988; Goldberg, E.D., Eschenko, V.A., and Bovt, V.D., Diabetogenic activity of chelators in some mammalian species, Endocrinologie 28, 51-55, 1990; Kawamura, C., Kizaki, M., Fukuchi, Y., and Ikeda, Y., A metal chelator, diphenylthiocarbazone, induces apoptosis, in acute promyelocytic leukemia (APL): cells mediated by a caspase-dependent pathway with a modulation of retinoic acid signaling pathways, Leuk.Res. 26, 661-668, 2002; Shaw, M.J., Jones, P., and Haddad, P.R., Dithizone derivatives as sensitive water soluble chromogenic reagents of the ion chromatographic determination of inorganic and organo-mercury in aqueous matrices, Analyst 128, 1209-1212, 2003; Santos, I.G., Hagenbach, A., and Abram, U., Stable gold(III) complexes with thiosemicarbazone derivatives, Dalton Trans. (4), 677-682, 2004; Khan, R., Ahmed, M.J., and Bhanger, M.I., A rapid spectrophotometric method for the determination of trace level lead using 1,5-diphenylthiocarbazone in aqueous micellar solutions, Anal.Sci. 23, 193-199, 2007 α,α-Dipyridyl; 2,2’-dipyridyl; bipyridyl; BIPY
156.2
Used for chelation of ferrous iron and other divalent metal cations
N
N Fredens, K. and Danscher, G., The effect of intravital chelation with dimercaprol, calcium disodium edentate, 1-10-phenanthroline and 2,2’-dipyridyl on the sulfide silver strainability of the rat brain, Histochemie 37, 321-331, 1973; Evans, S.A. and Shore, J.D., The role of zinc-bound water in liver alcohol dehydrogenase catalysis, J.Biol.Chem. 255, 1509-1514, 1980; Rao, G.H., Cox, A.C., Gerrard, J.M., and White, J.G., Effects of 2,2’-dipyridyl and related compounds on platelet prostaglandin synthesis and platelet function, Biochim.Biophys.Acta 628, 468-479, 1980; Ikeda, H., Wu, G.Y., and Wu, C.H., Evidence that an iron chelator regulates collagen synthesis by decreasing the stability of procollagen mRNA, Hepatology 15, 282-287, 1992; Hales, N.J. and Beattie, J.F., Novel inhibitors of prolyl 4-hydroxylase. 5. The intriguing structure-activity relationships seen with 2,2’-bipyridine and its 5,5’-dicarboxy acid derivatives, J.Med.Chem. 36, 3853-3858, 1993; Henley, R. and Worwood, M., The enhancement of iron-dependent luminal peroxidation by 2,2’-dipyridyl and nitrilotriacetate, J.Biolumin.Chemilumin. 9, 245-250,1994; Nocentini, G. and Barzi, A., The 2,2’-bipyridyl-6-carbothioamide copper (II) complex differs from the iron(II) complex in its biochemical effects in tumor cells, suggesting possible differences in the mechanism leading to cytotoxicity, Biochem.Pharmacol. 52, 65-71, 1996; Romeo, A.M., Christen, L, Niles, E.G., and Kosman, D.J., Intracellular chelation of iron by bipyridyl inhibits DNA virus replication: ribonucleotide reductase maturation as a probe of intracellular iron pools, J.Biol.Chem. 276, 24301-24308, 2001; Slingsby, R.W., Bordunov, A, and Grimes, M., Removal of metallic impurities from mobile phases in reversed-phase high-performance liquid chromatography by the use of an in-line chelation column, J.Chromatog. A. 913, 159-163, 2001; Huang, K., Dai, J., Fournier, J., et al., Ferrous ion autooxidation and its chelation in iron-loaded human liver HepG2 cells, Free Radic.Biol.Med. 32, 84-92, 2002; Pfister, A. and Fraser, C.L., Synthesis and unexpected reactivity of iron tris(bipyridine) complexes with poly(ethylene glycol) macroligands, Biomacromolecules 7, 459-468, 2006
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Metal Chelating Agentsa
851 METAL CHELATING AGENTS (Continued)
Name EDTA ( ethylenediaminetetraacetic acid)
M.W.
Description
292
General chelation agentb Structure serves as the basis for therapeutic agents which deliver radioactive materials to tumor cells. EDTA is used to detach adherent cells from tissue culture surfaces. Frequently included in protease inhibitor “cocktails.” The affinity for metal ions decrease as pH decreases and there may be a change in the relative affinity for specific metal ionsb.
HOOC N HOOC
COOH N COOH
Sakabe, I., Paul, S., Mitsumoto, T., et al., A factor that prevents EDTA-induced cell growth inhibition: purification of transethyretin from chick embryo brain, Endocr.J. 44, 375-391, 1999; Rocken, C. and Roessner, A., An evaluation of antigen retrieval procedures for immunoelectron microscopic classification of amyloid deposits, J.Histochem.Cytochem. 47, 1385-1394, 1999; Sciaudone, M.P., Chattopadhyay, S., and Freake, H.C., Chelation of zinc amplifies induction of growth hormone mRNA levels in cultured rat pituitary tumor cells, J.Nutr. 130, 158-163, 2000; Welch, K.D., Davis, T.Z., and Aust, S.D., Iron autoxidation and free radical generation: effects of buffers, ligands, and chelators, Arch.Biochem.Biophys. 397, 360-369, 2002; Breccia, J.D., Andersson, M.M., and Hatti-Kaul, R., The role of poly(ethyleneimine) in stabilization against metal-catalyzed oxidation of proteins: a case study with lactate dehydrogenase, Biochim.Biophys.Acta 1570, 165-173, 2002; Cowart, R.E, Reduction of iron by extracellular iron reductases: implications for microbial iron acquisition, Arch.Biochem.Biophys. 430, 273-281, 2002; Geebelen, W., Vangronaveld, J., Adriano, D.C., et al., Effects of Pb-EDTA and EDTA on oxidative stress reactions and mineral uptake in Phaseolus vulgaris, Physiol.Plant 115, 377-382, 2002; Powis, D.A. and Zerbes, M., In situ chelation of Ca(2+) in intracellular stores induces capacitative Ca(2+) entry in bovine adrenal chromaffin cells, Ann.N.Y.Acad.Sci. 971, 150-152, 2002; Tarasov, K.A., O’Hare, D., and Isupov, V.P., Solid-state chelation of metal ions by ethylenediaminetetraacetate intercalated in a layered double hydroxide, Inorg.Chem. 42, 1919-1927, 2003; Nichols, N.M., Benner, J.S., Martin, D.D., and Evans, T.C., Jr., Zinc ion effects on individual Ssp DnaE intein-splicing steps: regulating pathway progression, Biochemistry 42, 5301-5311, 2003; Conzone, S.D., Hall, M.M., Day, D.E., and Brown, R.F., Biodegradable radiation delivery system utilizing glass microspheres and ethylenediaminetetraacetate chelation therapy, J.Biomed.Mater.Res.A. 70, 256-264, 2004; Reynolds, A.J., Haines, A.H., and Russell, D.A., Gold glyconanoparticles for mimics and measurement of metal ion-mediated carbohydrate-carbohydrate interactions, Langmuir 22, 1156-1163, 2006; Fernandes, C.M., Zamuner, S.R., Zuliani, J.P., et al., Inflammatory effects of BaP1: a metalloproteinase isolated from Bothrops asper snake venom: leukocyte recruitment and release of cytokines, Toxicon 47, 549-559, 2006; Pajak, B. and Orzechowski, A., Ethylenediaminetetraacetic acid affects subcellular expression of clusterin protein in human colon adenocarcinoma COLO 205 cell line, Anticancer Drugs 18, 55-63, 2007 EGTA (ethyleneglycoltetraacetic acid)
380.4
COOH
HOOC
HOOC
Chelating agent with much greater affinity for calcium ions than for magnesium.
O
O
COOH
Schor, S.L., The effects of EGTA and trypsin on the serum requirements for cell attachment to collagens, J.Cell Sci. 40, 271-279, 1979; Bers, D.M., A simple method for the accurate determination of free [Ca] in Ca-EGTA solutions, Am.J.Physiol. 242, C404-408, 1982; Miller, D.J. and Smith, G.L., EGTA purity and the buffering of calcium ions in physiological solutions, Am.J.Physiol. 246, C160-C166, 1984; Sulakhe, P.V. and Hoehn, E.K., Interaction of EGTA with a hydrophobic region inhibits particular adenylate cyclase from rat cerebral cortex: a study of an EGTA-inhibitable enzyme by using alamethicin, Int.J.Biochem. 16, 1029-1035, 1984; Bryant, D.T. and Andrews, P., High-affinity binding of Ca2+ to bovine a-lactalbumin in the absence and presence of EGTA, Biochem.J. 220, 617-620, 1984; Smith, G.L. and Miller, D.J., Potentiometric measurements of stoichiometric and apparent affinity constants of EGTA for protons and divalent cations including calcium, Biochim.Biophys.Acta 839, 287-299, 1985; Marini, M.A., Evans, W.J., and Berger, R.L., The determination of binding constants with a differential thermal and potentiometric titration apparatus. II. EDTA, EGTA and calcium, J.Biochem.Biophys.Methods 12, 135-146, 1986; Harrison, S.M. and Bers, D.M., The effect of temperature and ionic strength on the apparent Ca-affinity of EGTA and the analogous Ca-chelators BAPTA and dibromo-BAPTA, Biochim.Biophys.Acta 925, 133-143, 1987; Guan, Y.Y., Kwan, C.Y., and Daniel, E.E., The effects of EGTA on vascular smooth muscle contractility in calcium-free medium, Can.J.Physiol.Pharmacol. 66, 1053-1056, 1988Youatt, J., Calcium and microorganisms, Crit.Rev.Microbiol. 19, 83-97, 1993; Yingst, D.R., and Barrett, V.E., Binding and elution of EGTA to anion exchange columns: implications for study of (Ca+Mg)-ATPase inhibitors, Biochim.Biophys.Acta 1189, 113-118, 1994; Lee, Y.C., Fluorometric determination of EDTA and EGTA using terbium-salicylate complex, Anal.Biochem. 293, 120-123, 2001; Rothen-Rutishauser, B., Riesen, F.K., Braun, A., et al., Dynamics of tight and adherens junctions under EGTA treatment, J.Membr.Biol. 188, 151-162, 2002; Chen, J.L., Ahluwalia, J.P., and Stamnes, M., Selective effects of calcium chelators on anterograde and retrograde protein transport in the cell, J.Biol.Chem. 277, 35682-35687, 2002; Fisher, A.E., Hague, T.A., Clarke, C.L., and Naughton, D.P., Catalytic superoxide scavenging by metal complexes of the calcium chelator EGTA and contrast agent EHPG, Biochem.Biophys.Res. Commun. 323, 163-167, 2004; Dweck, D., Reyes-Alfonso, A., Jr., and Potter, J.D., Expanding the range of free calcium regulation in biological solutions, Anal.Biochem. 347, 303-315, 2005; Ellis-Davies, G.C. and Barsotti, R.J., Tuning caged calcium: photolabile analogues of EGTA with improved optical and chelation properties, Cell Calcium 39, 75-83, 2006; Zhou, J.L., Li, X.C., Garvin, J.L., et al., Intracellular ANG II induces cytosolic Ca2+ mobilization by stimulating intracellular AT1 receptors in proximal tubule cells, Am.J.Physiol.Renal Physiol. 290, F1382-1390, 2006
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852
METAL CHELATING AGENTS (Continued) Name
M.W.
Oxalic Acid
126.1 as hydrate
Description Modest chelator; history of use in blood and in dentistry.
O HO OH O Lu, H., Mou, S., Yan, Y., et al., On-line pretreatment and determination of Pb, Cu and Cd at the µ l-1 level in drinking water by chelation ion chromatography, J.Chromatog.A. 800, 247-255, 1998; Bruer, W, Ronson, A., Slotki, I.N., et al., The assessment of serum nontransferrin-bound iron in chelation therapy and iron supplementation, Blood 95, 2975-2982, 2000; Salovaara, S., Sandberg, A.S., and Andlid, T., Combined impact of pH and organic acids on iron uptake by Caco-2 cells, J.Agric.Food Chem. 51, 7820-7824, 2003; Gerken, B.M., Wattenbach, C., Linke, D., Tweezing-absorptive bubble separation. Analytical method for the selective and high enrichment of metalloenzymes, Anal.Chem. 77, 6113-6117, 2005 o-Phenanthroline; 1,10-phenanthroline
198.2 as hydrate
Moderately strong chelating agent. Historical use for zinc. More recent studies have copper and iron complexes as specific nuclease activity and there is some evidence to indicate protease activity. Possible role of metal complexes as oxidizing agentsd
N N
o-phenanthroline; 1,10-phenanthroline
N Cu2+
N
N
N
Sytkowski, A.J. and Vallee, B.L., Chemical reactivities of catalytic and noncatalytic zinc or cobalt atoms of horse liver alcohol dehydrogenase: differentiation by their thermodynamic and kinetic properties, Proc.Nat.Acad.Sci.USA. 73, 344-348, 1976; Kidani, Y. and Hirose, J., Coordination chemical studies on metalloenzyme. II. Kinetic behavior of various types of chelating agents towards bovine carbonic anhydrase, J.Biochem. 81, 1383-1391, 1977; Evans, C.W., The spectrophotometric determination of micromolar concentrations of Co2+ using o-phenanthroline, Anal.Biochem. 135, 335-339, 1983; Wu, H.B. and Tsou, C.L., A comparison of Zn(II)_ and Co(II) in the kinetics of inactivation of aminoacylase by 1,10-phenanthroline and reconstitution of the apoenzyme, Biochem.J. 296, 435-441, 1993; Auld, D.S., Removal and replacement of metal ions in metallopeptidases, Methods Enzymol. 248, 228-242, 1995; Leopold, I. and Fricke, B., Inhibition, reactivation and determination of metal ions in membrane metalloproteases of bacterial origin using high-performance liquid chromatography coupled on-line with inductively coupled plasma mass spectrometry, Anal.Biochem. 252, 277-285, 1997; Ciancaglini, P., Pizauro, J.M., and Leone, F.A., Dependence of divalent metal ions on phosphotransferase activity of osseous plate alkaline phosphatase, J.Inorg.Biochem. 66, 51-55, 1997
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Metal Chelating Agentsa
853 METAL CHELATING AGENTS (Continued)
Name
M.W.
8-Quinolinol; 8-hydroxyquinoline
145.2
Description Metal chelating agent with higher affinity for Fe, Cu, and Zn. Lower affinity for Ca and Mg. Therapeutic use as antiseptic/bacteriostatic agent.
OH N
Fayez, M. and El-Tarras, M., Potentiometric titration of 8-hydroxyquinoline with Cu(II) using Cu(II)sulphide-ion selective electrode, Pharmazie 30, 799, 1975; Eskeland, T., The effect of various metal ions and chelating agents on the formation of noncovalently and covalently linked IgM polymers, Scand.J.Immunol. 6, 87-95, 1977; Albro, P.W., Corbett, J.T. and Schroeder, J.L., Generation of hydrogen peroxide by incidental metal ion-catalyzed autoxidation of glutathione, J.Inorg.Biochem. 27, 191-203, 1986; Yasui, T., Yuchi, A., Wade, H., and Nagagawa, S., Reversed-phase high-performance liquid chromatography of several metal-S-quinolinethiol complexes, J.Chromatog. 596, 73-78, 1992; Vieira, N.E., Yergey, A.L., and Abrams, S.A., Extraction of magnesium from biological fluids using 8-hydroxyquinoline and cation-exchange chromatography for isotopic enrichment using thermal ionization mass spectrometry, Anal.Biochem. 218, 92-97, 1994; Zachariouis, M., and Hearn, M.T., Adsorption and selectivity characteristics of several human serum proteins with immobilized hard Lewis metal ion-chelate adsorbents, J.Chromatog.A. 890, 95-116, 2000; Anfossi, L., Giraudi, G., Grassi, G., et al., Binding properties of a polyclonal antibody directed against toward lead complexes, Ann.Chim. 93, 499-512, 2003; Yamada, H., Hayashi, H., and Yasui, T., Utility of 1-octanol/octane mixed solvents for the solvent extraction of aluminum(III), gallium(III), and indium(III) with 8-quinolinol, Anal.Sci. 22, 371-376, 2006; Song, K.C., Kim, J.S., Park, S.M., et al., Fluorogenic Hg2+-selective chemodosimeter derived from 8-hydroxyquinoline, Org. Lett. 8, 3413-3416, 2006; Mittal, S.K., Kumar, A., Gupta, N., et al., 8-Hydroxyquinoline based neutral tripodal ionophore as a Cu(II) selective electrode and the effect of remote substituents on electrode properties, Anal.Chim.Acta 585, 161-170, 2007 a
Metal chelating systems can be viewed as metal ion buffer systems • MY = metal chelate complex; M is metal and Y is chelating agent. Metal ion are electron acceptors can be viewed as Lewis acids while chelating agents donate electrons and are Lewis bases. Also, chelating agents are polydentate • MYn–m = M+n + M–m • K MY = [MYn+m]/[M+n][Y–m] • pM = log K MY + log [Y–m]/[MYn–m]
The values for stability(association constants presented below are dependent of solvent conditions such as pH as well as relative amounts of chelating agent and metal ion. b Stability of some metal ion complexes with EDTA Metal Ion
Log K (MY) 10.96 12.70 14.04 14.33 25.1 16.31 18.62 18.80 16.50
Ca V+2 Mn+2 Fe2+ Fe3+ Co2+ Ni2+ Cu2+ Zn2+ 2+
Adapted from Mingos, D.M.P., Essential Trends in Inorganic Chemistry, Oxford University Press, Oxford, United Kingdom, 1998. c
Influence of pH on pM for EDTA where pK1, 2.0; pK2, 2.67; pK3, 6.16; pK4 11.26. pH
Cu(II)
Zn(II)
Mg(II)
Ca(II)
Mn(II)
Fe(III)
4 6 8 10
8.4 12.6 15.0 16.8
6.7 10.5 12.8 14.6
2.0 3.8 5.4 7.2
2.3 5.5 7.3 9.2
4.5 7.3 10.1 12.0
15.7 19.5 21.8 23.7
Adapted from Chaberek, S. and Martell, A.F., Organic Sequestering Agents. A Discussion of the Chemical Behavior and Applications of Metal Chelate Compounds in Aqueous Systems, John Wiley & Sons, London, UK, 1959
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854 d
Nuclease activity: Que, B.G., Downey, K.M., and So, A.G., Degradation of deoxyribonucleic acid by a 1,10-phenanthroline-copper complex: the role of hydroxyl radicals, Biochemistry 19, 5987-5891, 1980; Goldstein, S. and Czapski, G., The role and mechanism of metal ions and their complexes in enhancing damage in biological systems or in protecting these systems from the toxicity of O2-, J.Free Radic.Biol.Med. 2, 3-11, 1986; Sigman, D.S. and Chen, C.H, Chemical nucleases: new reagents in molecular biology, Annu.Rev.Biochem. 59, 207-236, 1990; Sigman, D.S., Chemical nucleases, Biochemistry 29, 9097-9105, 1990; Montenay-Garestier, T., Helene, C., and Thuong, N.T., Design of sequence-specific bifunctional nucleic acid ligands, Ciba. Found.Sym. 158, 147-157, 1991; Pan, C.Q., Landgraf, R., and Sigman, D.S., DNA-binding proteins as site-specific nucleases, Mol.Microbiol. 12, 335-342, 1994. Protease activity: Kito, M. and Urade, R., Protease activity of 1,10-phenanthroline-copper systems, Met.Ions Biol.Syst. 38, 187196, 2001. Oxidation: McArdle, J.V., Gray, H.B., Creutz, C., and Sutin, N., Kinetic studies of the oxidation of ferrocytochrome c from horse heart and Candida krusei by tris(1,10-phenanthroline)cobalt(3), J.Amer.Chem.Soc. 96, 5737-5741, 1974; McArdle, J.V., Coyle, C.L., Gray, H.B., Yoneda, G.S., and Holwerda, R.A., Kinetics studies of the oxidation of blue copper proteins by tris(1-,10-phenanthroline)cobalt(III) ions, J.Amer.Chem.Soc. 99, 2483-2389, 1977; Lau, O.W. and Luk, S.F., Spectrophotometric determination of ascorbic acid in canned fruit juices, cordials, and soft drink with iron(III) and 1,10-phenanthroline as reagents, J.Assoc.Off.Anal.Chem. 70, 518-520, 1987; Mandal, S., Kazmi, N.H., and Sayre, L.M., Ligand dependence in the copper-catalyzed oxidation of hydroquinones, Arch.Biochem.Biophys. 435, 21-31, 2005; Hung, M. and Stanbury, D.W., Oxidation of thioglycolate by [Os(phen)3]3+: an unusual example of redox-mediated aromatic substitution, Inorg.Chem. 44, 9952-9960, 2005; Ishrat, Q.U. and Iftikhar, A., Kinetics of copper(II) catalyzed oxidation of iodide by iron(III)orthophenanthroline complex in aqueous solution, Pak.J.Pharm.Sci. 18, 20-24, 2005; Ozyurek, M., Guglu, K., Bektasoglu, B., and Apak, R., Spectrophotometric determination of ascorbic acid by the modified CUPRAC method with extractive separation of flavonoids-La(III) complexes, Anal.Chim.Acta 588, 88-95, 2007
Some Stability Constants for Divalent Metal Ion Chelate Complexes (log k) Chelating Agent
Ca
Mg
Zn
Fe
Cu
BAPTA EDTA EGTA 8-Hydroxyquinoline 1,10-Phenanthroline Citrate Oxalate
6.8 10.6 11.0 3.3 0.5 3.6 3.0
8.7 5.2 4.7 1.5 3.6 2.6
16.4 12.9 8.5 6.4 5.0 4.9
6 14.2 11.8 8.0 5.8 4.4 4.7
18.8 12.9 8.5 6.3 5.9 4.4
General references for metal chelating agents Analytical Uses of Ethylene Diamine Tetraacetic Acid, D. Van Nostrand, Princeton, New Jersey, USA, 1950 Martell, A.E. and Calvin, M., Chemistry of the Metal Chelate Compounds, Prentice-Hall, Englewood Cliffs, NJ, USA, 1952 Chelating Agents and Metal Chelates, ed. F.P. Dwyer and D.P. Mellon, Academic Press, New York, NY, USA, 1964
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Data for Biochemical Research, ed. R.M.C. Dawson, Clarendon Press, Oxford, UK, 1986 Handbook on Metals in Clinical and Analytical Chemistry, ed. H.G. Seiler, A. Sigel, and H. Sigel, Marcel Dekker, New York, NY, USA, 1994 Bertini, I., Gray, H.B., Strefel, E.I., and Valentine, J.S., Biological Inorganic Chemistry. Structure and Reactivity, University Science Books, Sausalito, CA, USA, 2007
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Water Water Purity and Water Purity Classification Water Type Deionized Purified Apyrogenic High Purity Ultrapure a b c d
Resistancea Megohm @25°C
Bioburden (cfu)b
Dissolved Solids (mg/L)
— 100 0.1 1 1
10 1 1 0.5 0.005
0.05 0.2 0.8 10 18
British Pharmacopoeia 2007, TSO Norwich, Norwich, UK, 2007; http://www.pharmacopoeia.org.uk Process water is defined as that water which is used during the pharmaceutical manufacturing process. A higher purity water may be required for formulation of the final drug product and/or reconstitution of final product prior to use. A high purity water is required for a product which is to be injected as opposed to an oral administered product. http://www.fda.gov/ora/inspect_ref/ itg/itg46.html
Resistance (R), determined by conductivity(A/Vc ), measured in siemans (S) , R is measured in ohms (V/Ad) (Ω) Colony-forming units SI unit for conductivity – m–2 . kg–1 . s–3 . A2; one S equal to the conductance of one ohm–1 SI unit for resistance – m–2 . kg–1 . s–3 . A–2; one R is equal to one ohm
Some Definitions of Pharmaceutical Water (FDA/ORA) • • • • • • •
Meltzer, T.H., High Purity Water Preparation, Tall Oaks Publications, Littleton, CO, 1993 Fischbacher, C., Quality assurance in analytical chemistry, in Encyclopedia of Analytical Chemistry, ed. R.A. Meyers, Wiley, New York, NY, USA, pps 13563-13587, 2000 Environmental water quality issues are a separate issue with different classification issues
Non-potable Potable USP purified USP water for injection (WFI) USP sterile water for injection USP bacteriostatic water for injection USP sterile water for irrigation
See 2007 USP/NF. The Official Compendia of Standards, US Pharmacopeia, Rockville, MD, USA, 2007, http://www.usp.org;
• Kannel, P.R., Lee, S., Lee, Y.S., et al., Application of water quality indices and dissolved oxygen as indicators for river water classification and urban impact assessment, Environ. Monit.Assess., in press, 2007 • Kowalkowski, T., Zhytniewski, R., Szpejna, J., and Buszewski, B., Application of chemometrics in river water classification, Water Res. 40, 7544-752, 2006
WATER PURIFICATION The Following is a Representation of Estimates of Effectiveness. Effectiveness of Removal of Contaminant/Impurity Techniquea Activated Carbon Ion Exchange Distillation Reverse Osmosis Ultrafiltration
Inorganic or Ionized Organic
Organic
Pyrogens
Particulates
Partial
Partial
No
No
Yes Yes Partial
No Yesb Partial
No Yes Yes
No Yes Yes
Partial
Partial
Good
Yes
In general, a combination of technologies is required–For example, 1. Input water (outside line) 2. Filtration/settling (depends on quality of input water) 3. Distillation 4. Reverse osmosis 5. Terminal filtration (e.g. 0.2 µ filter) Other techniques such as ultraviolet irradiation may be used b Effectiveness of separation depends on vapor pressure (boiling point) differences between water and specific contaminant impurity and lack of formation of a azeotropic mixture. http://www.fda.gov/ora/inspect_ref/igs/high.html a
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856
PROPERTIES OF WATER Absorption of Light (adapted from Morton, R.A., Biochemical Spectroscopy, Wiley, New York, NY, USA, 1975) Wavelength (nm)
Absorbancea
Wavelength(nm)
Absorbancea
220 250 280 300 320 340 400
1.1 0.8 0.4 0.3 0.2 0.2 0.1
500 600 700 800 1000 1500 1800
0.02 0.1 0.8 2.4 40.0 194.0 170.0
Water quality and Water analysis references Water general references
Dorsey, N.E., Properties of Ordinary Water-Substance, Reinhold Publishing Company, New York, NY, USA, 1940 Eisenberg, D. and Kauzmann, W., The Structure and Properties of Water, Oxford University Press, New York, NY, USA, 1969 Water: A Comprehensive Treatise, ed. F. Frank, Plenum Press, London, 1972 Water and Aqueous Solutions, ed, G.W. Nelson and J.E. Enderby, Adam Hilger, Bristol, UK, 1985 Robison, G.W., Zhu, S.B., Singh, S., and Evans, M.W., Water in Biology, Chemistry, and Physics Experimental Overviews and Computational Methodologies, World Scientific Press, Singapore, 1996 Frank, F., Water, 2nd edition, A Matrix of Life, Royal Society of Chemistry, Cambridge, UK, 2000
Water purity
Swaddle, T.W., Applied Inorganic Chemistry, University of Calgary Press, Calgary, Alberta, Canada, Chapter 12 (Water conditioning), 1990 Afshar, A., Zhao, X., Heckert, R.A., and Trotter, H.C., Suitability of autoclaved tap water for preparation of ELISA reagents and washing buffer, J.Virol.Methods 46, 275-278, 1994 Stewart, K.K. and Ebel, R.E., Chemical Measurements in Biological Systems, John Wiley & Sons, New York, NY, USA, Chapter 2 (Water, pH, and Buffers), 2000
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Mabic, S. and Kano, I., Impact of purified water quality on molecular biology experiments, Clin.Chem.Lab.Med. 41, 486-491, 2003. Regnault, C., Kano, I., Darbouret, D., and Mabic, D., Ultrapure water for liquid chromatography-mass spectrometry studies, J.Chromatog.A. 1030, 289-295, 2004. Bennett, A., Process Water: Analyzing the lifecycle cost of pure water, Filtration & Separation, March, 2006.
Water analysis
Water analysis, in Encyclopedia of Analytical Sciences, ed A. Townshend, Academic Press, London, UK, Volume 9, pps. 5445-5559, 1995 Fischbacher, C., Quality assurance in analytical chemistry, in Encyclopedia of Analytical Chemistry, ed. R.A. Meyers, Wiley, New York, NY, USA, pps 13563-13587, 2000 Reid, D., Water determination in food, in Encyclopedia of analytical chemistry, ed. R.H. Meyers, Wiley, New York, NY, USA, pps. 4318-4332, 2000
Spectroscopy of water
Morton, R.A., Biochemical Spectroscopy, Wiley, New York, NY, USA, 1975 Symans, M.C.R., Spectroscopic studies of water and aqueous solutions, in Water and Aqueous Solutions, ed G.W. Nelson and J.E. Enderby, Adam Hilger, Bristol, UK, pps. 41-55, 1985 Googin, P.L. and Carr, C., Far infrared spectroscopy and aqueous solutions, in Water and Aqueous Solutions, ed. G.W. Nelson and J.E., Enderby, Adam Hilger, Bristol, UK, pps. 149-161, 1985 Mehrotra, R., Infrared spectroscopy, gas chromatography/infrared in food analysis, in Encyclopedia of Analytical Chemistry, ed. R.H. Meyers, pps. 4007-4024, 2000
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Stability of Solutions for GLP and cGMP Use
A. There are three factors which influence the stability of solutions 1. Purity of solvent used for the preparation of the solution. This is usually water and should be of USP or higher grade. There is an assumption of sterility of water obtained in a GLP or cGMP environment. The presence of trace amounts of metal ions in solution can contribute to stability issues for certain solutes. 2. Storage conditions – plastic container, glass container, under nitrogen, temperature of storage 3. Lability of the solute in solutions. Solutes such as sodium chloride are indefinitely stable while solutions of carbohydrates such as glucose or glycerol can undergo oxidation and mutarotation B. There is little information of the storage stability of solutions while there is considerable information on the storage stability of reference standards and specific laboratory reagents1-6. The several pharmacopeia (US, UK) discuss the stability of certain reagents and standards but do not contain information about solution stability. Compendia of chemical data7,8 will state if a given chemical decomposes in solution. C. It is possible to make the following recommendations with the caveat that there will be exceptions. The preferable approach to stability would involve (1) defining the critical attributes of the solvent for the specific process or analysis and (2) measurement of such property or properties over a period of one year. Storage of a laboratory solvent for more than a period of one year is not acceptable. It is assumed that the solutions are prepared with USP quality or higher water under clean conditions.
No solution should be retained for a period of time longer than 3 months unless it has been documented that effectiveness is retained beyond that time period. There may be some solutions such as dilute phosphate buffers at neutrality that are stable for much shorter periods of time. Solutions should be stored in the cold (2-4°C) unless it has been documented that storage at 23°C (room temperature) is effective. Any dating of an solvent used in a GLP or cGMP process shall be consistent with information in the SOP for the preparation of such solvent and information in any run sheet/analytical SOP which uses such solvent.
References 1. Urone, P.F., Stability of colorimetric reagent for chromium, S-diphenylcarbazide, in various solvents, Analyt. Chem. 27, 13541355, 1955. 2. Grant, D.R., Reagent stability in Rosen’s ninhydrin method of analysis for amino acids, Analyt.Biochem. 6, 109, 1963. 3. Peterson, R.C., Stablity of Folin phenol reagent, J.Pharm.Sci. 55, 523, 1966. 4. Durham, B.W., Reagent stability, Analyt.Chem. 51, A922, 1979 5. Beck, J., Coleman, P., and Grzesiak, J., Cholesterol rate reagent with extended stability, Clin.Chem. 31, 949, 1985. 6. Georghiou, P.E., Winsor, L., Shirtliff, C.J., and Svec, J., Storage stability of formaldehyde containing paraosaniline reagent, Analyt.Chem. 59, 2432-2435, 1987 7. Reagent Chemicals. Specifications and Procedures, 10th edn., ed. P. A. Bovis, American Chem.Soc, Washington, DC, 2006 8. CRC Handbook of Chemistry and Physics, 86th edn., ed. D.Lide, CRC Press, Boca Raton, FL, 2006
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General Information on Spectroscopy UV-Vis Spectroscopy Absorbance Data for Common Solvents
Spectroscopy – the study of the interaction of electromagnetic radiation with matter – excluding chemical effects. Spectrophotometer – an instrument which measures the relationship between the absorption of light by a substance and the wavelength of the incident light Spectrometer – an instrument which measures the distribution of wavelengths in electromagnetic radiation; also an instrument which measures the energies and masses in a distribution of particles as in a mass spectrometer Beer’s Law (Beer-Lambert Law or Beer-Lambert-Bouguer Law) – states that while the relationship between transmittance and concentration is non-linear, the relationship between absorbance and concentration is linear. The practical consequence is that with high absorbance values, one is measuring small differences in large numbers with attendant inaccuracies. I/Io (Transmittance)
%Transmittance (I/Io × 100)
1.0 0.1 0.01 0.001 0.0001 0.00001 0.000001
Absorbance (A)
100 10 1 0.1 0.01 0.001 0.0001
0 1.0 2.0 3.0 4.0 5.0 6.0
log10 (I/I0) = εcl = A; where I is the intensity of transmitted light; Io is the intensity of incident light; ε is the molar extinction coefficient (L mol–1 cm–1), c is concentration (mol L–1), l is pathlength (cm); and A is absorbance. Electromagnetic Radiation Ranges Frequently Used in Biochemistry and Molecular Biology Definition Ultraviolet (UV) Visible (Vis) Near Infrared (NIR) Infrared (IR) Far Infrared (FIR) a
Range (Wavelength)
Range (Wavenumber)
190–360 nm 360–780 nm 780–2500 nm
12,800 – 4000 cm–1
Comments Qualitative and Quantitative Qualitative and Quantitative Qualitative and Quantitative
Solvent CHCl3 Hexanes MeOH/EtOH H2O Dioxane Acetonitrile a
4000 – 250 cm–1
4 × 104–106 nma
250 – 10–cm
Mostly Qualitative Mostly Qualitative
Usually used reciprocal centimeters ( cm-1) for wavelength description. This quantity is the wave number (reciprocal of the wavelength in cm-1)
Hyperchromic – increase in absorbance Hypochromic – decrease in absorbance Hypsochromic – decrease in wavelength; also known as a “blue” shift Bathochromic – increase in wavelength; also known as a “red” shift
240 nm 200 nm 205 nm 190 nm 205 nm 190 nm
In this context, “cut-off ” is the lowest wavelength at which the solvent can be used; solvent absorbance is sufficiently high below this wavelength to marginalize results.
Water transmits light satisfactorily between 400 nm and 800 nm; from 600 nm to 900 nm, above 900 nm, transmission decreases by a factor 50 and above 1.3 μm, transmission decreases more rapidly; transmission increases from 400 to 220 while a further decrease in wavelength results in markedly decreased transmission. • Opticalx Properties of Water (Morton, R.A., Biochemical Spectroscopy, John Wiley & Sons (A Halsted Press Book), New York, New York, 1975.) Optical Properties of Plastics Used For Microplate Assaysa,b Material Quartz Polystyrened Polypropylenee Polyvinyl chloride (PVC) a
b
c
d
2500–40,000 nma
UV-VIS “Cut-Off” Wavelengtha
e
UV “Cut-off”c 180 nm 300 nm 300 nm 300 nm
These are values for the “common” microplate thickness; with thin-thickness (10 μ), the “cut-off ” value of polyethylene is 180 nm (Andrady, A.L., Ultraviolet radiation and polymers, in Physical Properties of Polymers Handbook, ed. J.E. Mark, American Institute of Physics, AIP Press, Woodberry, NY, USA, 1996). There are a number of UV-transparent microplates available on the market. These are made of proprietary plastics; most likely unique blends. The microplates will permit use at 260 nm and 280 nm making them useful for biochemical analyses. However, since the composition of these microplates is proprietary, the microplates must be evaluated for any unique binding properties. In this context, “cut-off ” is the lowest wavelength at which the microplate can be used; the zero absorbance of the microplate is sufficiently high such to marginalize results. Tends to be hydrophilic so an aqueous sample tends to “film” and stick the sides of well (surface tension effect) Tends to be hydrophobic so aqueous sample tends to “bead.”
Near Infrared Spectroscopy • The spectra of water • Symons, M.C.R., Spectroscopy of aqueous solutions: protein and DNA interactions with water, Cell.Mol. Life Sci.57, 999-1007, 2000. • Gregory, R.B., Protein hydration and glass transition behavior, in Protein-Solvent Interactions, ed. R.B. Gregory, Marcel Dekker, Inc., New York, New York, USA, Chapter 3, pps 191-264, 1995.
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860 • Application to human tissue • Chance, B., Nioka, S., Warren, W., and Yurtsever, G., Mitochondrial NADH as the bellwether of tissue O2 delivery, Adv.Exp.Med. Biol. 566, 231-242, 2005. • Cerussi, A.E., Berger, A.J., Bevilacqua, F., Sources of absorption and scattering contrast for near-infrared optical mammography, Academic Radiology 8, 211-218, 2001. • Eikje, N.S., Ozaki, Y., Aizawa, K., and Arase, S., Fiber optic near-infrared Raman spectroscopy for clinical noninvasive determination of water content in diseased skin and assessment of cutaneous edema, J.Biomed.Opt. 10, 14013, 2005. • Pickup, J.C., Hussain, F., Evans, N.D., and Sachedina, N., In vivo glucose monitoring: the clinical reality and the promise, Biosens.Bioelectron. 20, 1897-1902, 2005. • Hielscher, A.H., Bluestone, A.Y., Abdoulaev, G.S., et al., Near-infrared diffuse optical tomography, Dis. Markers 18, 313-337, 2002. • Christian, N.A., Milone, M.C., Ranka, S.S., et al., Tatfunctionalized near-infrared emissive polymerosomes for dendritic cell labeling, Bioconjug.Chem. 18, 31-40, 2007. • Process Monitoring • Liu, J., Physical characterization of pharmaceutical formulations in frozen and freeze-dried solid states: techniques and applications in freeze-drying development, Pharm.Dev.Technol. 11, 3-28, 2006. • Scaftt, M., Arnold, S.A., Harvey, L.M., and McNeil, B., Near infrared spectroscopy for bioprocess monitoring and control: current status and future trends, Crit.Rev. Biotechnol. 26, 17-39, 2006. • Reich, G., Near-infrared spectroscopy and imaging: basic principles and pharmaceutical applications, Adv. Drug Deliv.Rev. 57, 1109-1143, 2005. • Suehara, K., and Yano, T., Bioprocess Monitoring using near-infrared spectroscopy, Adv.Biochem.Eng. Biotechnol. 90, 173-198, 2004. • General • Ferrari, M., Mottola, L., and Quaresima, V., Principles, techniques, and limitations of near infrared spectroscopy, Can.J.Appl.Physiol. 29, 463-487. 2004. • McWhorter, S. and Soper, S.A., Near-infrared laserinduced fluorescence detection in capillary electrophoresis, Electrophoresis 21, 1267-1280, 2000. • Symons, M.C., Spectroscopy of aqueous solutions: proteins and DNA interactions with water, Cell Mol.Life Sci. 57, 999-1007, 2000. • Nir, S., Nicol, F., and Szoka, F.C., Jr., Surface aggregation and membrane penetration by peptides: relation to pore formation and fusion, Mol.Membr.Biol. 16, 95-101, 1999.
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Some Spectrometer Window Materials for Infrared Spectroscopy Wavelength Range (cm–1)
Refractive Index
NaCl KBr BaF2
40,000 – 600 43,500 – 400 66,666 – 800
1.52 1.54 1.45
ZnSe Si
20,000 – 500 8,333 – 33
2.43 3.42
Material
Characteristics Soluble in H2O, Etoh Soluble in H2O, EtOH Insoluble in H2O, soluble in acid Insoluble in H2O Insoluble in H2O
General references for spectrometry Twyman, F. and Allsop, C.B., The Practice of Absorption Spectrophotometry, Adam Hilger, London, 1934. Brode, W.R., Chemical Spectroscopy, John Wiley & Sons, New York, New York, 1943. Analytical Absorption Spectroscopy, ed. M.G.Mellon, John Wiley & Sons, New York, New York, 1950. Morton, R.A. Biochemical Spectroscopy, John Wiley & Sons (A Halsted Press Book), New York, New York, 1975. Campbell, L.D. and Dwek, R.A., Biological Spectroscopy, Benjamin/ Cummings, Menlo Park, California, 1984. Practical Absorption Spectroscopy, ed A. Knowles and C. Burgess (UV Spectrometry Group), Chapman & Hall, London, UK, 1984. Osborne, B.G., Fearn, T., and Hindle, P.H., Practical NIR Spectroscopy with Applications in Food and Beverage Analysis, Longman Scientific and Technical, Harrow, Essex, UK, 1993. UV Spectroscopy Techniques, Instrumentation, Data Handling, UV Spectrometry Group, ed. B.J. Clark, T. Frost, and M.A. Russell, Chapman & Hall, London, UK, 1993. Stuart, B., Biological Applications of Infrared Spectroscopy, ACOL Series, Wiley, Chichester, UK, 1997. Standards and Best Practices in Absorption Spectrometry, ed. C. Burgess and T. Frost (UVSG), Blackwell Science, Oxford, UK, 1999. Stewart, K.K. and Ebel, R.E., Chemical Measurements in Biological Systems, John Wiley & Sons, New York, NY, USA, 2000. Workman, J., Jr., Handbook of Organic Compounds. NIR, IR, Raman, and UV-Vis Featuring Polymers and Surfactants, Academic Press, San Diego, California, 2001. Volume 1 Methods and Interpretation, Volume 2 UV-Vis and NIR Spectra, Volume 3 IR and Raman Spectra. Near-Infrared Spectroscopy, ed. H.W. Siesler, Ozaki, Y., Kawate, S., and Heise, H.M., Wiley-VCH, Weinheim, Germany, 2002. Stuart, B.H., Infrared Spectroscopy: Fundamentals and Applications, John Wiley & Sons, Ltd., Chichester, UK, 2004. Burns, D.A. and Ciurczak, E.W., Handbook of Near-Infrared Analysis, Third Edition, CRC Press, Boca Raton, USA, 2007. Workman, J. and Weyer, L., Practical Guide to Interpretive Near-Infrared Spectroscopy, CRC Press, Boca Raton, USA, 2007.
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Microplates Attributes for Microplates Used for Assay • • • •
Absorbance of light (see Spectroscopy) Adsorption of materials Intraplate Variation (well-to-well reproducibility) Vendor Reproducibility
Attributes for Microplates Used for Reactions and Cell Culture • Adsorption of materials • Intraplate Variation (well-to-well reproducibility) • Vendor Reproducibility
Selected references on the use of microplates Adsorption of materials/factors influencing adsorption to microplates • Bulter, J.E., Ni., L., Nessler, R., et al., The physical and functional behavior of capture antibodies adsorbed on polystyrene, J.Immunol.Methods 150, 77-90, 1992 • Davies, J., Roberts, C.J., Dawkes, A.C., et al., Use of scanning probe microscopy and surface-plasmon resonance as analytical tools in the study of antibody-coated microtiter wells, Langmuir 10, 2654-2661, 1994 • Douglas, A.S. and Monteith, C.A., Improvements to immunoassays by use of covalent binding assay plates, Clin. Chem. 40, 1833-1837, 1994 • Elsner, H.I. and Mouritsen, S., Use of psoralens for covalent immobilization of biomolecules in solid-phase assays, Bioconjugate Chem. 5, 463-467, 1994 • Stevens, P.W. and Kelso, D.M., Estimation of the proteinbinding capacity of microplate wells using sequential ELISAs, J.Immunol.Methods 178, 59-70, 1995 • Page, J.D., Derango, R., and Huang, A.E., Chemical modification of polystyrene’s surface and its effect on immobilized antibodies, Colloids and Surfaces A – Physicochem. Eng.Aspects 132, 193-201, 1998 • Baumann, S., Grob, P., Stuart, F., et al., Indirect immobilization of recombinant proteins to a solid phase using the albumin binding domain of streptococcal protein G and immobilized albumin, J.Immunol.Methods, 221, 95-106, 1998 • Ricoux, R., Chazaud, B., Tresca, J.P., and Pontet, M., Quality control of coated antibodies: New, rapid determination of binding affinity, Clin.Chem.Lab.Med. 38, 239-243, 2000 • Qian, W.P., Yao, D.F., Yu, F., et al., Immobilization of antibodies on ultraflat polystyrene surfaces, Clin.Chem. 46, 1456-1463, 2000
• Bulter, J.E., Solid supports in enzyme-linked immunosorbent assay and other solid-phase immunoassays, Methods 22, 4-23, 2000 • Sugihara, T., Seong, G.H., Kobatake, E., and Aziawa, M., Genetically synthesized antibody-binding protein selfassembled on hydrophobic matrix, Bioconjugate Chem. 11, 789-794, 2000 • Julián, E., Cama, M., Martinez, P., and Luquin, M., An ELISA for five glycolipids from the cell wall of Mycobacterium tuberculosis: Tween 20 interference in the assay, J.Immunol.Methods 251, 21-30, 2001 • Peluso, P., Wilson, D.S., Do, D., et al., Optimizing antibody immobilization strategies for the construction of protein microarrays, Analyt.Biochem. 312, 113-124, 2003 • Johnson, J.C., Nettikadan, S.R., Vengasandra, S.G., and Henderson, E., Analysis of solid-phase immobilized antibodies by atomic force microscopy, J.Biochem.Biophys. Meth. 59, 167-180, 2004 • Clinchy, B., Youssefi, M.R., and Håjensson, L., Differences in adsorption of serum proteins and production of IL-1ra by human monocytes incubated in different tissue culture plates, J.Immunol.Methods 282, 53-61, 2003 • Shrivastav, T.G., Basu, A., and Karlya, K.P., Substitution of carbonate buffer by water for IgG immobilization in enzyme linked immunosorbent assay, J.Immunoassay Immunchem. 23, 191-203, 2003 • Clinchy, B., Gunnerås, M., Håkensson, L., and Håkensson, L., Production of IL-1Ra by human mononuclear blood cells in vitro: Influence of serum factors, Cytokine 34, 320330, 2006
Issues of well-to-well variation
• Faessel, H.M., Levasseur, L.M., Slocum, H.K., and Greco, W.R., Parabolic growth patterns in 96-well cell growth experiments, In Vitro Cell Dev.Biol.-Animal 35, 270-278, 1999 • Pitts, B., Hamilton, M.A., Zelver, N., and Stewart, P.S., A microtiter-plate screening method for biofilm disinfection and removal, J.Microbiol.Methods 54, 269-276, 2003 • Patel, M.I., Tuckerman, R., and Dong, Q., A pitfall of the 3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethonylphenol)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay due to evaporation in wells on the edge of a 96 well plate, Biotechnol.Lett. 27, 805-808, 2005 • Heaver, M., Kopun, M., Rittgen, W., and Granzow, C., Cytotoxicity determination with photochemical artifacts, Cancer Lett. 223, 57-66, 2005 • Straetemanns, R., O’Brien, T., Wouters, L., et al., Design and analysis of drug combination experiments, Biometrical J. 47, 299-308, 2005
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Plastics Plastics are a group of materials which are used extensively in biochemistry and molecular biology. Plastics can be defined as nonmetallic polymeric materials which can be molded or extruded into a shape. A plastic can be a single polymeric component such as polypropylene, a blend of several polymers or a block copolymer consisting of joined segments of two or more individual polymers such as a block copolymer of polybutadiene and poly(ethylene oxide). The physical properties of a plastic are a combination of the polymeric composition and additives such as plasticizers. Plasticizers are high molecular weight liquids or solids melting a low temperature which are blended with thermoplastic resins such as polyvinyl chloride to change physical properties. Plasticizers including phthalate derivatives such as di(2-ethyl)hexylphthalate, derivatives of organic acids such as di-2-ethylhexyladipate, and polyglycols (polyethylene glycol). Other materials included in the manufacture of plastics include antioxidants, lubricants, stabilizers, and colorants. All of these components can influence the property of the final plastic product. It must be emphasized that the biomedical market for plastics is quite small in volume compared to the overall market. Thus, unless a vendor makes their own raw material, most suppliers to biochemistry and molecular biology purchase bulk product from a large chemical company. As a result there can be batch-to-batch and vendor-tovendor variation in product. Another issue which confounds the use of plastics is the addition of stabilizers. Stabilizers are chemicals such as hydroxybenzophenones and hydroxyphenylbensotriazoles which are added to prevent damage from ultraviolet irradiation. These compounds do absorb ultraviolet light in the 200-400 nm range and do present problems in biochemical analyses. The careful investigator will assure the source of the plastics used in products such as microplates and incubation flasks (Clinchy, B., Youssefi, M.R., and Håkansson, L., Differences in adsorption of serum proteins and production of IL-1ra by human monocytes incubated in different tissue culture microtiter plates, J.Immunol.Methods 282, 53-61, 2003).
General references for plastics Dubois, J.H., and John, F.W., Plastics, 5th Edn, Van Nostrand Reinhold, New York, NY, USA, 1974 Billmeyer, F.W., Textbook of Polymer Science, Wiley, New York, NY, 1984 Griffin, G.J.L., Chemistry and Technology of Biodegradable Polymers, Blackie Academic & Professional, London, UK, 1994 Physical Properties of Polymers Handbook, ed. J.E. Mark, American Institute of Physics, AIP Press, Woodberry, NY, USA, 1996 Araki, T. and Qui, T-C., Structure and Properties of Multiphase Polymeric Materials, Marcel Dekker, New York, NY, 1998 Polymer Data Book., ed. J.E. Mark, Oxford University Press, Oxford, UK, 1999 Plastics Additives Handbook, 5th edn., ed. H. Zweifel, Hanser Publications, Munich, Germany, 2001 Bart, J.C.J., Plastics Additives. Advanced Industrial Analysis, IOS Press, Amsterdam, Netherlands, 2006 Chiellini, E., Biomedical Polymers and Polymer Therapeutics, Kluwer Academic, New York, NY, USA, 2002 Ramakrishna, S., An Introduction to Biocomposites, Imperial College Press, London, UK, 2004 Carraher, C.A., Jr., Introduction to Polymer Chemistry, CRC Press, Boca Raton, FL, USA, 2006 Polymeric Nanofibers, ed. D.H. Reneker and H. Fong, American Chemical Society, Washington, DC, USA, 2006. American Chemical Council; http://www.plasticsresource.com.
Plasticizers Crawford, R.R. and Esmerian, O.K., Effect of plasticizers on some physical properties of cellulose acetate phthalate films, J.Pharm.Sci. 60, 312-314, 1971 Ekwall, B., Nordensten, C., and Albanus, L., Toxicity of 29 plasticizers to HeLa cells in the MIT-24 system, Toxicology 24, 199-210, 1982 Goldstein, D.B., Feistner, G.J., Faull, K.F., and Tomer, K.B., Plasticizers as contaminants in commercial ethanol, Alcohol Clin.Exp.Res. 11, 521524, 1987 Sager, G. and Little, C., The effect of tris-(2-butoxyethyl)-phosphate (TBEP) and di-(2-ethylhexyl)-phthalate (DEHP) and the b-adrenergic receptor-blockers [3H]-(-)-dihydroalprenolol ([3H]-(-)-DHA) and [3H-(-)-CGP 12177 were tested for their ability to interact with b-adrenergic binding to α 1-acid glycoprotein and mononuclear leukocytes, Biochem.Pharmacol. 38,2551-2557, 1989 Baker, J.K., Characterization of phthalate plasticizers by HPLC/thermospray mass spectrometry, J.Pharm.Biomed.Anal. 15, 145-148, 1996 Wahl, H.G., Hoffman, A., Haring, H.U., and Liebich, H.M., Identification of plasticizers in medical products by a combined direct thermodesorption—cooled injection system and gas chromatography—mass spectrometry, J.Chromatog.A 847, 1-7, 1999 Cano, J.M., Marin, M.L., Sanchez, A., and Hernandis, V., Determination of adipate plasticizers in poly(vinyl chloride) by microwave-assisted extraction, J.Chromatog.A 963, 401-409, 2002 Siepmann, F., le Brun, V., and Siepmann, J., Drugs acting as plasticizers in polymeric systems: a quantitative treatment, J.Control.Release 115, 298-306, 2006
Some Properties of Plastics Used in Biochemistry and Molecular Biology Structures for these plastics may be found on p. 866–868 Plastic AcrylonitrileButadiene elastomers (Nitrile rubber) Nylon (aliphatic polyamides) Polyacrylamide
Polyacrylate (PA; polyacrylic acid)1 Polybutadiene Polycarbonate
Polyethylene Polyethylene oxide (PEO) Poly(ethylene terephthalate) (PET)
Uses Soft rubber applications such as gloves and pharmaceutical stoppers Matrix for tissue engineering, suture material, matrix for adsorptive technologies Primary use as a matrix for protein electrophoresis (PAGE, polyacrylamide gel electrophoresis). Early use as gel filtration matrix; more recent use as a hydrogel and implant material Dental cement, use for manufacture of microparticles and nanoparticles Chromatographic matrix such as polybutadiene-coated zirconia, microarray plates Matrix for biological assays; material for tissue culture flasks; implants; filters; use as “solid” component of copolymers; tyrosine-derived polycarbonate used in tissue engineering Implants, tubing Component of hydrogels; some use in implant biology; use in copolymers Matrix for tissue engineering; cell culture matrix; used in block copolymers
References 1-4 6-10 11-18
19-24 25-31 32-42
43-45 46-51 52-57
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Handbook of Biochemistry and Molecular Biology
864 Some Properties of Plastics Used in Biochemistry and Molecular Biology (Continued) Plastic Poly(methacrylate)1 Polypropylene Polypropylene oxide Polystyrene
Poly(vinyl chloride)
1
Uses Used in monolithic chromatographic columns fiber used in surgery Hydrogels, block copolymers, Microplates, can be modified by irradiation for covalent binding of probes; chromatographic matrices; microbeads for assays; frequently a copolymer with divinylbenzene Use in biosensors; use as flexible tubing when phthalate stabilizers are used; use as copolymer for encapsulation
References 58 59-61 62-64 65-82
83-88
The nomenclature for acrylic acid and derivatives can be confusing. The following definitions are used in this text. Acrolein (2-propenal) Acrylamide (2-propenamide) Acrylic acid (propenoic acid) Acrylonitrile (2-propenenitrile) Methacrylic acid (2-methylpropenoic acid) Methyl methacrylic acid (2-methylpropenoic acid methyl ester)
References 1. Shanker, J., Gibaldi, M., Kanig, J.L. et al., Evaluation of the suitability of butadiene-acrylonitrile rubbers as closures for parenteral solutions, J.Pharm.Sci. 56, 100-108, 1967 2. Williams, J.R., Permeation of glove materials by physiologically harmful chemicals, Am.Ind.Hyg.Assoc.J. 40, 877-882, 1979 3. Parker, S. and Braden, M., Soft prosthesis materials based on powdered elastomers, Biomaterials 11, 482-490, 1990 4. Walsh, D.L., Schwerin, M.R., Kisielewski, R.W., et al., Abrasion resistance of medical glove materials, J.Biomed.Mater.Res.B Appl. Biomater. 68, 81-87, 2004. 5. McConway, M.G. and Chapman, R.S., Application of solid-phase antibodies to radioimmunoassay. Evaluation of two polymeric microparticles, Dynospheres and nylon, activated by carbonyldiimidazole or tresyl chloride, J.Immunol.Methods 95, 259-266, 1986 6. Absolom, D.R., Zingg, W., and Neumann, A.W., Protein adsorption to polymer particle: role of surface properties, J.Biomed.Mater.Res. 21, 161-171, 1987 7. Alicata, R., Mantaudo, G., Puglisi, C., and Samperi, F. Influence of chain end groups on the matrix-assisted laser desorption/ionization spectra of polymer blends, Rapid Commun.Mass.Spectrom. 16, 248260, 2002 8. Zhu, X., Cai, J., Yang, J., et al., Films coated with molecular imprinted polymers for the selective stir bar sorption extraction of monocrotophos, J.Chromatog.A. 1131, 37-44, 2006. 9. Dennes, T.J., Hunt, G.C., Schwarzbauer, J.E., and Schwartz, J., Highyield activation of scaffold polymer surfaces to attach cell adhesion molecules, J.Am.Chem.Soc. 129, 93-97, 2007 10. Friedrich, J., Zalar, P., Mororcic, M., et al., Ability of fungi to degrade synthetic polymer nylon-6, Chemosphere, in press, 2007 11. Hjerten, S. and Mosbach, R., “Molecular-sieve” chromatography of proteins on columns of cross-linked polyacrylamide, Anal.Biochem. 3, 109-118, 1962 12. Goodfriend, T., Ball, D., and Updike, S., Antibody in polyacrylamide gel, a solid phase reagent for radioimmunoassay, Immunochemistry 6, 481-484, 1969 13. John, M., Skrabei, H., and Dellweg, H., Use of polyacrylamide gel columns for the separation of nucleotides, FEBS Lett. 5, 185-186, 1969 14. Bovin, M.V., Polyacrylamide-based glycoconjugates as tools in glycobiology, Glycoconj. J. 15, 431-446, 1998
9168_Book.indb 864
15. Patrick, T., Polyacrylamide gel in cosmetic procedures: experience with Aquamid®, Semin.Cutan.Med.Surg. 23, 233-235, 2004. 16. Plieva, F., Bober, B., Dainiak, M., et al., Macroporous polyacrylamide monolithic gels with immobilized metal affinity ligands: the effect of porous structure and ligand coupling chemistry on protein binding, J.Mol.Recognit. 19, 305-312, 2006. 17. Sairam, M., Babu, V.R., Vijaya, B.. et al., Encapsulation efficiency and controlled release characteristics of crosslinked polyacrylamide particles, Int.J.Pharm. 320, 131-136, 2006. 18. Sefton, M.V. and Broughton, R.L., Microencapsulation of erythrocytes, Biochim.Biophys.Acta 717, 473-477, 1982 19. Stevenson, W.T. and Sefton, M.V., Graft copolymer emulsions of sodium alginate with hydroxylalkyl methacrylates for microencapsulation, Biomaterials 8, 449-457, 1987 20. Laemmli, U.K., Characterization of DNA condensates induced by poly(ethylene oxide) and polylysine, Proc.Nat.Acad.Sci.USA 72, 4288-4292, 1975 21. Sefton, M.V. and Nishimura, E., Insulin permeability of hydrophilic polyacrylate membranes, J.Pharm.Sci. 69, 208-209, 1980 22. Svensson, A., Norrman, J., and Piculell, L., Phase behavior of polyion-surfactant ion complex salts: effects of surfactant chain length and polyion length, J.Phys.Chem.B Condens.Matter Mater. Surf. Interface Biophys. 110, 10332-10340, 2006 23. Turos, E., Shim, J.Y., Wang, Y., et al., Antibiotic-conjugated polyacrylate nanoparticles: new opportunities for development of antiMRSA agents, Bioorg.Med. Chem Lett. 17, 53-56, 2007 24. Herdt, A.R., Kim, B.S., and Taton, T.A., Encapsulated magnetic nanoparticles as supports for proteins and recyclable biocatalysts, Bioconjug.Chem. 18, 183-189, 2007 25. Sun, L. and Carr, P.W., Chromatography of proteins using polybutadiene-coated zirconia, Anal.Chem. 67, 3717-3721, 1995 26. Alvarez, C., Strumia, M. and Bertorello, H., Synthesis and characterization of a biospecific adsorbent containing bovine serum albumin as a ligand and its use for bilirubin retention, J.Biochem.Biophys. Methods 49, 649-656, 2001 27. Davoras, E.M. and Coutsolelos, A.G., Efficient biomimetic catalytic epoxidation of polyene polymers by manganese porphyrins, J.Inorg. Biochem. 94, 161-170, 2003 28. Erhardt, R., Zhang, M., Boker, A., et al., Amphiphilic Janus particles with polystyrene and poly(methacrylic acid) hemispheres, J.Am. Chem.Soc. 125, 3260-3267, 2003 29. Xu, J. and Zubarev, E.R., Supramolecular assemblies of starlike and V-shaped PB-PEO amphiphiles, Angew.Chem.Int.Ed.Engl. 43, 54915496, 2004 30. Geng, Y., Discher, D.E., Justynska, J., and Schlaad, H., Grafting short peptides onto polybutadiene-block-poly(ethylene oxide): a platform for self-assembling hybrid amphiphiles, Angew.Chem.Int.Ed.Engl. 45, 7578-7581, 2006 31. Kassu, A., Taguenang, J.M., and Sharma, A., Photopatterning of butadiene substrates by interferometric ultraviolet lithography: fabrication of phospholipid microarrays, Appl.Opt. 46, 489-494, 2007 32. Chandy, T. and Sharma, C.P., Changes in protein adsorption on polycarbonate due to L–ascorbic acid, Biomaterials 6, 416-420, 1985 33. Hough, T., Singh, M.B., Smart, I.J., and Knox, R.B., Immunofluorescent screening of monoclonal antibodies to surface antigens of animal and plant cells bound to polycarbonate membranes, J.Immunol. Methods 92, 103-107, 1986 34. Thelu, J., and Ambroise-Thomas, P., A septate polycarbonate cell culture unit used for Plasmodium falciparum and hybridomas, Trans.R.Soc.Trop.Med.Hyg. 82, 360-362, 1988 35. Bignold, L.P., Rogers, S.D., and Harkin, D.G., Effects of plasma proteins on the adhesion, spreading, polarization in suspension, random motility and chemotaxis of neutrophil leukocytes on polycarbonate (Nucleopore) filtration membranes, Eur.J.Cell Biol. 53, 27-34, 1990 36. Lee, J.H., Lee, S.J., Khang, G., and Lee, H.B., Interaction of fibroblasts on polycarbonate membrane surfaces with different micropore sizes and hydrophilicity, J.Biomater.Sci.Poly.Ed. 10, 283-294, 1999
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Plastics 37. Liu, Y., Ganser, D., Schneider, A., et al., Microfabricated polycarbonate CE devices for DNA analysis, Anal.Chem. 73, 4196-4201, 2001 38. Liu, J., Zeng, F., and Allen, C., Influence of serum protein on polycarbonate-based copolymer micelles as a delivery system for a hydrophobic anti-cancer agents, J.Control Release 103, 481-487, 2005 39. Meechaisue, C., Dubin, R., Supaphol, P., et al., Electrospun mat of tyrosine-derived polycarbonate fibers for potential use as tissue scaffolding material, J.Biomater.Sci. Polym.Ed. 17, 1039-1056, 2006 40. Li, Y., Wang, Z., Ou, L.M., and Yu, H.Z., DNA detection on plastic: surface activation protocol to convert polycarbonate substrates to biochip platforms, Anal.Chem. 79, 426-433, 2007 41. Carion, O., Souplet, V., Olivier, C., et al., Chemical micropatterning of polycarbonate for site-specific peptide immobilization and biomolecular interactions, ChemBioChem 8, 315-322, 2007 42. Tripathi, A., Wang, J., Luck, J.A., and Suni, L.L., Nanobiosensor design utilizing a periplasmic E.coli receptor protein immobilized within Au/polycarbonate nanopores, Anal.Chem. 79, 1266-1270, 2007 43. Raff, R.A.V. and Allison, J.B., Polyethylene, Interscience Publishers, New York, NY, USA, 1956 44. Bhat, S.V., Biomaterials, Kluwer Academic Publishers, Boston, MA, USA, 2002 45. Shanbhag, A. and Rubash, H.E., Joint Replacement and Bone Resorption: Pathology, Biomaterials, and Clinical Practice, Taylor & Francis, New York, NY, USA, 2006 46. Desai, N.P. and Hubbell, J.A., Biological responses to polyethylene oxide modified polyethylene terephthalate surfaces, J.Biomed.Mater. Res. 25, 829-843, 1991 47. Lopina, S.T., Wu, G., Merrill, E.W., and Griffith-Cima, L., Heptocyte culture on carbohydrate-modified star polyethylene oxide hydrogels, Biomaterials 17, 559-569, 1996 48. Vereschagin, E.I., Han, D.H., Troitsky, A.W., et al., Radiation technology in the preparation of polyethylene oxide hydrophilic gels and immobilization of proteases for use in medical practice, Arch.Pharm. Res. 24, 229-233, 2001 49. Liu, L.S. and Berg, R.A., Adhesion barriers of carboxymethylcellulose and polyethylene oxide composite gels, J.Biomed.Mat.Res. 63, 326-332, 2002 50. Wu, N., Wang, L.S., Tan, D.C., et al., Mathematical modeling and in vitro study of controlled drug release via a highly swellable and dissoluble polymer matrix: polyethylene oxide with high molecular weights, J.Control.Release 102, 569-581, 2005 51. Unsworth, L.D., Sheardown, H., and Brash, J.L., Polyethylene oxide surfaces of variable chain density by chemisorption of PEO-thiol on gold: adsorption of proteins from plasma studied by radiolabeling and immunoblotting, Biomaterials 26, 5927-5933, 2005 52. Nair, P.D. and Sreenivasan, K., Effect of steam sterilization on polyethylene terephthalate, Biomaterials 5, 305-306, 1984 53. Dadsetan, M., Mirzadeh, H., Sharifi-Sanjani, N., and Daliri, M., Cell behavior on laser surface-modified polyethylene terephthalate in vitro, J.Biomed.Mater.Res. 57, 183-189, 2001 54. Cenni, E., Granchi, D., Ciapetti, G., et al., Interleukin-6 expression by cultured human endothelial cells in contact with carbon coated polyethylene terephthalate, J.Mater.Sci.Mater.Med. 12, 365-369, 2001 55. Neves, A.A., Medcalf, N., and Brindle, K.M., Influence of stirringinduced mixing on cell proliferation and extracellular matrix deposition in meniscal cartilage constructs based on polyethylene terephthalate scaffolds, Biomaterials 26, 4828-4836, 2005 56. Basu, S. and Yang, S.T., Astrocyte growth and glial cell line-derived neurotrophic factor secretion in three-dimensional polyethylene terephthalate fibrous matrices, Tissue Eng. 11, 940-952, 2005 57. Alisch-Mark, M., Herrmann, A., and Zimmermann, W., Increase of the hydrophilicity of polyethylene terephthalate fibres by hydrolases from Thermomospora fusca and Fusarium solani f.sp.pisi, Biotechnol. Lett. 28, 681-685, 2006
9168_Book.indb 865
865 58. Jungbauer, A. and Hahn, R., Polymethacrylate monoliths for preparative and industrial separation of biomolecular assemblies, J.Chromatog.A. 1184, 62-79, 2008 59. Peter, F.H., Polypropylene, Gordon and Breach Science Publishers, New York, NY, USA, 1968 60. Karger-Kocsis, J., Polypropylene: Structure, Blends and Composites, Chapman & Hall, London, UK, 1995 61. Karger-Kocsis, J., Polypropylene an A-Z Reference, Dordrecht, Netherlands, 1998 62. Topchieva, I.N. and Efremova, N.V., Conjugates of proteins with block co-polymers of ethylene and polypropylene oxides, Biotechnol. Genet.Eng.Rev. 12, 357-382, 1994 63. Newman, M.J., Actor, J.K., Balusubramanian, M., and Jagannath, C., Use of nonionic block copolymers in vaccines and therapeutics, Crit. Rev.Ther.Drug Carrier Syst. 15, 89-142, 1998 64. Gutowska, A., Jeong, B., and Jasionowski, M., Injectable gels for tissue engineering, Anat.Rec. 263, 342-349, 2001 65. Catarero, L.A., Butler, J.E., and Osborne, J.W., The adsorptive characteristics of proteins for polystyrene and their significance in solidphase immunoassays, Anal.Biochem. 105, 375-382, 1980 66. Zouali, M. and Stollar, B.D., A rapid ELISA for measurement of antibodies to nucleic acid antigens using UV-treated polystyrene microplates, J.Immunol.Methods 90, 105-110, 1986 67. Piskin, E., Tuncel, A., Denizli, A., and Ayhan, H., Monosize microbeads based on polystyrene and their modified forms for some selected medical and biological applications, J.Biomater.Sci.Polym. Ed. 5, 451-471, 1994 68. Kochanowska, I.E., Rapak, A., and Szewczuk, A., Effect of pretreatment of wells in polystyrene plates on adsorption of some human serum proteins, Arch.Immunol.Ther.Exp.(Warsz.) 42, 135139, 1994 69. Staak, C., Salchow, R., Clausen, P.H., and Luge, E., Polystyrene as an affinity chromatography matrix for the purification of antibodies, J.Immunol.Methods 194, 141-146, 1996 70. Davankov, V., Tsyurupa, M., Ilyin, M., and Pavlova, L., Hypercrosslinked polystyrene and its potential for liquid chromatography: a mini-review, J.Chromatog.A. 965, 65-73, 2002 71. Gessner, A., Lieske, A., Paulke, B.R., and Muller, R.H., Functional groups on polystyrene model nanoparticles: influence on protein adsorption, J.Biomed.Mater.Res.A 65, 319-326, 2003 72. Saitoh, T., Hattori, N., and Hiraide, M., Protein separation with surfactant-coated polystyrene involving Cibacron Blue 3GA-conjugated Triton X-100, J.Chromatog.A. 1028, 149-153, 2004 73. van Kooten, T.G., Spijker, H.T., and Busscher, H.J., Plasma-treated polystyrene surfaces: model surfaces for studying cell-biomaterial interactions, Biomaterials 25, 1735-1747, 2004 74. Recknor, J.B., Recknor, J.C., Sakaguchi, D.S., and Mallapragada, S.K., Oriented astroglial cell growth on micropatterned polystyrene substrates, Biomaterials 25, 2753-2767, 2004 75. Turner, S.F., Clarke, S.M., Rennie, A.R., et al., Adsorption of gelatin to a polystyrene /water interface as a function of concentration, pH, and ionic strength, Langmuir 21, 10082-10088, 2005 76. Rosado, E., Caroll, H., Sanchez, O., and Peniche, C., Passive adsorption of human antirrabic immunoglobulin onto a polystyrene surface, J.Biomater.Sci.Polym.Ed. 16, 435-448, 2005 77. Carvalho, R.S., Ianzer, D.A., Malavolta, L., et al., Polystyrene-type resin used for peptide synthesis: application for anion-exchange and affinity chromatography, J.Chromatog.B.Analyt.Technol.Biomed.Life Sci. 817, 231-238, 2005 78. Jodar-Reyes, A.B., Ortega-Vinuesa, J.L., and Martin-Rodriguez, A., Adsorption of different amphiphilic molecules onto polystyrene latices, J.Colloid Interface Sci. 282, 439-447, 2005 79. Mitchell, S.A., Davidson, M.R., and Bradley, R.H., Improved cellular adhesion to acetone plasma modified polystyrene surfaces, J.Colloid Interface Sci. 281, 122-129, 2005 80. Cao, Y.C., Hua, X.F., Zhu, X.X., et al., Preparation of Au coated polystyrene beads and their application in an immunoassay, J.Immunol. Methods 317, 163-170, 2006
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Handbook of Biochemistry and Molecular Biology
866 81. Tirri, M.E., Wahlroos, R., Meltola, N.J., et al., Effect of polystyrene microsphere surface to fluorescence lifetime under two-photon excitation, J.Fluoresc. 16, 809-816, 2006 82. Baker, S.C., Atkin, N., Gunning, P.A., et al., Characterization of electrospun polystyrene scaffolds for three-dimensional in vitro biological studies, Biomaterials 27, 3136-3146, 2006 83. Titow, W.V., PVC Technology, Elsevier Applied Science, London, UK, 1984 84. Encyclopedia of PVC, ed. L.I. Nass and C.A. Heiberger, Marcel Dekker, New York, NY, USA, 1986
85. Cha, G.S., Liu, D., Meyerhoff, M.E., et al., Electrochemical performance, biocompatibility, and adhesion of new polymer matrices for solid-state ion sensors, Anal.Chem. 63, 1666-1672, 1991 86. Zielinski, B.A. and Aebischer, P., Chitosan as a matrix for mammalian cell encapsulation, Biomaterials 15, 1049-1056, 1994 87. Immobilization of Enzymes and Cells, ed. G.F. Bickerstaff, Humana Press, Totowa, NJ, USA, 1997 88. Karakus, E., Pekyardimci, S., and Esma, K., Urea biosynthesis based on PVC membrane containing palmitic acid, Artif.Cells Blood Substit.Immobil.Biotechnol. 33, 329-341, 2005 N
*
* x
y
Acrylonitrile-butadiene (Nitrile) O * *
N H
n
Nylon 3 O H N *
* n Nylon 6 *
*
O
NH2
n Polyacrylamide
CH2 OH H
COOO Acrylic Acid
9168_Book.indb 866
n Polyacrylic acid (usually as sodium salt
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Plastics
867
1,3-butadiene
n Polybutadiene (one of several structures shown)
CH3 HO
OH CH3 bisphenol A
Phosgene
CH3 *
O
O C
CH3
O
*
n
polycarbonate
9168_Book.indb 867
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Handbook of Biochemistry and Molecular Biology
868
O CH3
CH3 H3C
Propylene Oxide
n Polyethylene
OH
KOH
HO CH3 O
O *
HO
* n
CH3
Polyethylene oxide
n Polypropylene Oxide (Polypropylene Glycol)
O
*
O
C
C
*
O O
Ph
n
*
poly(ethylene)terephthalate (PET)
* n Polystyrene
* Cl
*
O
n
O
Polyvinyl chloride
CH3
H
Cl
Cl
H
H
H
n Poly(methyl acrylate)
Isotactic *
CH3
Cl
H
Cl
*
H
Cl
H
Syndiotactic H
H
Cl
H
Cl
Cl
n Polypropylene
9168_Book.indb 868
Atactic (an irregular)
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9168_Book.indb 869
Polyethylene High Polypropylene Density Copolymer Polypropylene
PolymethylPentene
Teflon FEP
Polyphenylene Polycarbonate Oxide
Polystyrene General StyrenePolyvinyl Purpose Acrylonitrile Chloride
PHYSICAL PROPERTIES Temperature Limit, °C Specific Gravity Tensile Strength, psi Brittleness Temperature, °C Water Absorption, % Flexibility Transparency Relative O2 Permeability Autoclavable
80 0.92 2,000 –100 0.01 Excellent Translucent 0.40 No
120 0.95 4,000 –100 0.01 Rigid Opaque 0.08 With caution
130 0.90 2,900 –40 0.02 Slight Translucent 0.20 Yes
135 0.90 5,000 0 0.02 Rigid Translucent 0.11 Yes
175 0.83 4,000 — 0.01 Rigid Clear 2.0 Yes
205 2.15 3,000 –270 0.01 Excellent Translucent 0.59 Yes
135 1.20 8,000 –135 0.35 Rigid Clear 0.15 Yes
135 1.06 9,600 — 0.07 Rigid Opaque — Yes
70 1.07 6,000 Brittlea 0.05 Rigid Clear 0.11 No
95 1.07 11,000 –25 0.23 Rigid Clear 0.03 No
70 1.34 6,500 –30 0.06 Rigid Clear 0.01 No
CHEMICAL RESISTANCEb Acids, inorganic Acids, organic Alcohols Aldehydes Amines Bases Dimethyl sulfoxide (DMSO) Esters Ethers Foods Glycols Hydrocarbons, aliphatic Hydrocarbons, aromatic Hydrocarbons, halogenated Ketones Mineral oil Oils, essential Oils, lubricating Oils, vegetable Proteins, unhydrolyzed Salts Silicones Water a b
E E E G G E E
E E E G G E E
E E E G G E E
E E E G G E E
E E E G G E E
E E E E E E E
E G G F N N N
G E E G F E E
N G G N G G N
E E G F G E N
G G G F N E N
E G E E G G G
E G E E G G G
E G E E G G G
E G E E G G G
E G E E G F F
E E E E E E E
N F E G F N N
F N G E G N N
N F E G N N N
N N G G E N N
F F G F F N N
G E G G E E E G E
G E G E E E E E E
G E G E E E E E E
G E G E E E E E E
G E G E E E E E E
E E E E E E E E E
N E G G E E E E E
N E F E E G E E E
N G N G G G E G E
N G F G E E E G E
N E N E E G E G E
Normally somewhat brittle at room temperatures. E, Excellent. Long exposures (up to one year) at room temperatures have no effect. G, Good. Short exposures (less than 24 hours) at room temperature cause no damage. F, Fair. Short exposures at room temperature cause little or no damage under unstressed conditions. N, Not recommended. Short exposures may cause permanent damage.
869
By permission of Thermo Fisher Scientific.
Chemical and Physical Properties of Various Commercial Plastics
Polyethylene Low Density
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Generic Source-Based Nomenclature for Polymers (IUPAC Recommendations 2001) Prepared by a Working Group consisting of
R. E. Bareiss (Germany), R. B. Fox (Usa), K. Hatada (Japan), K. Horie (UK), A. D. Jenkins (UK), J. Kahovec (Czech Republic), R Kubisa (Poland), E. Maréchal (France), I. Meisel (Germany), W. V. Metanomski (USA), I. Mita (Japan), R. F. T. Stepto (UK), and E. S. Wilks (USA) Prepared for publication by
E. Maréchal1 and E. S. Wilks2,† Université Pierre et Marie Curie (Paris VI), Laboratoire de Synthèse Macromoléculaire, Boîte 184, 4 Place Jussieu F-75252, Paris Cédex 05, France; 2113 Meriden Drive, Canterbury Hills, Hockessin, DE 19707 USA
1
*Membership of the Commission during the preparation of this report (1993–1999) was as follows: Titular Members: R. E. Bareiss (Germany, 1983–1993); M. Barón (Argentina, from 1996, Secretary from 1998); K. Hatada (Japan, 1989–1997); M. Hess (Germany, from 1998); K. Horie (Japan, from 1997); J. Kahovec (Czech Republic, to 1999); P. Kubisa (Poland, from 1999); E. Maréchal (France, from 1994); I. Meisel (Germany, from 2000); W. V. Metanomski (USA, 1994–1999); C. Noël (France, to 1997); V. P. Shibaev (Russia, to 1995); R. F. T. Stepto (UK, 1989–1999, Chairman to 1999); E. S. Wilks (USA, from 2000); W. J. Work (USA, 1987–1999, Secretary, 1987–1997); Associate Members: M. Barón (Argentina, 1991–1995); K. Hatada (Japan, 1998–1999); J.-I. Jin (Korea, from 1993); M. Hess (Germany, 1996–1997); K. Horie (Japan, 1996–1997); O. Kramer (Denmark, from 1996); P. Kubisa (Poland, 1996–1998); E. Maréchal (France, 1991–1993); I. Meisel (Germany, 1997–1999); S. Penczek (Poland, from 1994); L. Shi (China, 1987–1995); V. P. Shibaev (Russia, 1996–1999); E. S. Wilks (USA, 1998–1999). †
Corresponding author
Abstract: The commission has already published two documents on the source-based names of linear copolymers and nonlinear polymers; however, in some cases this nomenclature leads to ambiguous names. The present document proposes a generic source-based nomenclature that solves these problems and yields clearer source-based names. A generic source-based name comprises two parts:
1) polymer class (generic) name followed by a colon 2) the actual or hypothetical monomer name(s), always parenthesized in the case of a copolymer
The formula, the structure-based name, the source-based name, and the generic source-based name of the polymer are given for each example in the document. In some cases, only generic source-based give unambiguous names, for example, when a polymer has more than one name or when it is obtained through a series of intermediate structures. The rules concern mostly polymers with one or more types of functional group or heterocyclic system in the main chain, but to some extent they are also applicable to polymers with sidegroups, carbon-chain polymers such as vinyl or diene polymers, spiro and cyclic polymers, and networks.
Contents 1. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 871 2. Source-based nomenclature for homopolymers. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 872 3. Generic nomenclature. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 872 3.1 Fundamental principles. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 872 3.2 General rules. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 872 4. Further applications of generic names. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 875 5. References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 876
1. Introduction The IUPAC Commission on Macromolecular Nomenclature has published three documents [1–3] on the structure-based nomenclature for polymers that enable most polymers, except networks, to be named. The Commission has also produced two documents Reproduced from:
Pure Appl. Chem., Vol. 73, No. 9, pp. 1511–1519, 2001. © 2001 IUPAC
[4,5] on the source-based nomenclature of linear copolymers and nonlinear polymers. In general, source-based names are simpler and less rigorous than structure-based names. However, there are cases in which the simplicity of the source-based nomenclature leads to ambiguous names for polymers. For example, the condensation of a dianhydride (A) with a diamine (B) gives first a polyamide-acid, which can be cyclized to a polyimide; however, both products have the same name poly(A-alt-B) according to current source-based nomenclature. If the class name of the polymer “amide-acid” or “imide” is incorporated in the name, 871
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872 differentiation is easily accomplished. Even in cases where only a single product is formed, use of the class name (generic name) may help to clarify the structure of the polymer, especially if it is very complex. Examples of ambiguous names exist also for homopolymers. The source-based name “polybutadiene” does not indicate whether the structure is 1,2-, 1,4-cis-, or 1,4-trans-; supplementary information is needed to distinguish between the possibilities. It is the objective of the present document to introduce a generic nomenclature system to solve these problems, and to yield better source-based names. Most trivial names, such as polystyrene, are source-based names. Hitherto, the Commission has not systematically recommended source-based names for homopolymers because it considered that the more rigorous structure-based names were more appropriate for scientific communications. However, since the publication of “Nomenclature of Regular Single-Strand Organic Polymers” in 1976, scientists, in both industry and academia, have continued to use trivial names. Even the Commission itself adopted (1985) a source-based nomenclature for copolymers owing to its simplicity and practicality. Based on these facts, the Commission has now decided to recommend source-based nomenclature as an alternative official nomenclature for homopolymers. In this document, the rules for generating source-based names for homopolymers are described. Consequently, source-based and structure-based names are available for most polymers. Names of the monomers in the source-based names of polymers should preferably be systematic but they may be trivial if well established by usage. Names of the organic groups, as parts of constitutional repeating units (CRU) in structure-based names, are those based on the principles of organic nomenclature and recommended by the 1993 A Guide to IUPAC Nomenclature of Organic Compounds [6].
2. Source-based nomenclature for homopolymers RULE 1
The source-based name of a homopolymer is made by combining the prefix “poly” with the name of the monomer. When the latter consists of more than one word, or any ambiguity is anticipated, the name of the monomer is parenthesized. Example 1.1 CH
CH2
n
3. Generic nomenclature 3.1 Fundamental principles
The basic concept for generic source-based nomenclature is very simple; just add the polymer class name to the source-based name of the polymer. Addition of the polymer class name is frequently OPTIONAL; in some cases, the addition is necessary to avoid ambiguity or to clarify. However, the addition is undesirable if it fails to add clarification. The system presented here can be applied to almost all homopolymers, copolymers, and others, such as networks. However, generic source-based nomenclature should not be considered as a third nomenclature system to be added to the other two systems of nomenclature; it must be considered as an auxiliary system and a simple extension of current source-based nomenclature. When the generic part of the name is eliminated from the name of a polymer, the well-established source-based name remains.
3.2 General rules RULE 2
A generic source-based name of a polymer has two components in the following sequence: (1) a polymer class (generic) name (polyG) followed by a colon and (2) the actual or hypothetical monomer name(s) (A, B, etc.), always parenthesized in the case of a copolymer. In the case of a homopolymer, parentheses are introduced when it is necessary to improve clarity. polyG: A polyG: (B) polyG: (A-co-B) polyG: (A-alt-B) Note 1 The polymer class name (generic name) describes the most appropriate type of functional group or heterocyclic ring system. Note 2 All the rules given in the two prior documents on source-based nomenclature [4,5] can be applied to the present nomenclature system, with the addition of the generic part of the name. Note 3 A polymer may have more than one name; this usually occurs when it can be prepared in more than one way. Note 4 If a monomer or a pair of complementary monomers can give rise to more than one polymer, or if the polymer is obtained through a series of intermediate structures, the use of generic nomenclature is essential (see examples 2.1, 2.3, and 2.4). Example 2.1 O n
CH
CH2
CH
O n
CH
CH2
Source-based name: poly(vinyl chloride) Structure-based name: poly(1-chloroethylene)
9168_Book.indb 872
CH
CH2
n
CI
n
O I
Source-based name: Polystyrene Structure-based name: poly(1-phenylethylene) Example 1.2
CH2
O
CH
CH2
CH II
CH2
n
Generic source-based name: I. polyalkylene:vinyloxirane II. polyether:vinyloxirane
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873
Source-based names: I and II have the same source-based name:poly(vinyloxirane). Structure-based names: I. poly(1-oxiranylethylene) II. poly[(oxy(1-vinylethylene)]
2
C
Structure-based names: I. poly[imino (2,5-diamino-1,4-phenylene)iminoterephthaloyl] II. poly[(1,5-dihydrobenzo[1,2-d:4,5-d′]diimidazole-2,6diyl)-1,4-phenylene] Example 2.4
Example 2.2
N
poly[(terephthaloyl dichloride)-alt-benzene-1,2,4,5-tetramine]
N+ O
C
N O 3
1
N
H2N—NH2 5
+
NH
C
C
C
NH
OH
O
n
O
O
HO
C
I
4
O NH
Generic source-based name: polyoxadiazole:(4-cyanobenzonitrile N-oxide)
I
O C
+ CI
NH2
H2N
CI
C O
Source-based name: I and II have the same source-based name: poly[hydrazinealt-(terephthalic acid)] Structure-based names: I. poly(hydrazine-1,2-diylterephthaloyl) II. poly(1,3,4-oxadiazole-2,5-diyl-1,4-phenylene)
O
C NH2 O
C n
I
C NH2 O
O C n
O
C
r
O (CH2)4 C NH (CH2)6
n
+
H N N
O (CH2)2 C
Example 2.6
NH C
N H
O
Generic source-based names: poly urethane:[butane-1,4 -diol-alt-(hexane-1,6 -diyl diisocyanate)]-block-polyester:[(ethylene glycol)-alt(terephthalic acid)]
I
N
p
O
Structure-based name: poly(oxybutane-1,4-diyloxycarbonyliminohexane-1,6diyliminocarbonyl)-block-poly(oxyethyleneoxyterephthaloyl)
H2N NH
O
O (CH2)4 O C NH (CH2)6 NH C
O
NH
NH
II
O
Example 2.5
H2N NH
n
N N
n
Generic source-based names: I. polyhydrazide:[hydrazine-alt-(terephthalic acid)] II. polyoxadiazole:[hydrazine-alt-(terephthalic acid)]
Example 2.3 NH2
O C
Structure-based name: poly(1,2,4-oxadiazole-3,5-diyl-1,4-phenylene)
H2N
C
NH
n
O
O
n
II
Generic source-based name: I. polyamide:[(terephthaloyl dichloride)-alt-benzene-1,2,4, 5-tetramine] II. polybenzimidazole:[(terephthaloyl dichloride)-alt-benzene1,2,4,5-tetramine]
O NH C
CH2
O
CH2 CH2
O (CH2)4 C NH (CH2)6
p
O CH3
O
/
N
C
O (CH2)4 C NH (CH2)6
n
CH2 CH CH2
O
CH2 CH2
p
O CH3
OH
Source-based name: I and II have the same source-based name:
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874 Generic source-based name: polyamide:[hexane-1,6-diamine-alt-(adipic acid)]-graftpolyether: (ethylene oxide) Note 5 I t is assumed that this reaction is limited to only one graft for each CRU.
RULE 3
Example 4.1 O
O O
H2N
O
O O
O
When more than one type of functional group or heterocyclic system is present in the polymer structure, names should be alphabetized; for example, poly(GG′):(A-alt-B). Note 6 I t is preferable, but not mandatory, to cite all generic classes.
O C NH HO C
O
O
O C NH C OH
O
Example 3.1 O
O
(CH2)2 O C
(CH2)4 C
CH3
O p
O (CH2)2 O
C
NH
O
n
Structure-based name: poly{[oligo(oxyethyleneoxyadipoyl)]oxyethyleneoxycar bonylimino(x-methyl-1,4-phenylene)iminocarbonyl)} Example 3.2 F
+
HO
OH
C O
O O
I
n
C O
N
N O
O
O
Polymer class names relevant only to the main chain are specified in the name; names of side-chain functional groups may also be included after a hyphen if they are formed during the polymerization reaction.
n
II
Generic source-based names: I. poly(amide-acid):[(pyromellitic dianhydride)-alt-(4,4′oxydianiline)] (Both carboxy groups result from the polymerization reaction.) II. polyimide: [(pyromellitic dianhydride)-alt-(4,4′-oxydianiline)] Structure-based names: I. poly[oxy-1,4-phenyleneiminocarbonyl(4,6-dicarboxy-1, 3-henylene)carbonylimino-1,4-phenylene] II. poly[(5,7-dihydro-1,3,5,7-tetraoxobenzo[1,2-c:4,5-c′] dipyrrole-2,6(1H,3H)-diyl)-1,4-phenyleneoxy-1,4-phenylene] Example 4.2 CI
O
CH2
+
HO
OH
H3C
Structure-based name: poly(oxy-1,4-phenyleneoxy-1,4-phenylenecarbonyl-1,4phenylene)
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O
O
Generic source-based name: polyetherketone:(4,4′-difluorobenzophenone-att-hydroquinone)
RULE 4
n
O NH C
Generic source-based name: polyesterurethane:(α,ω-dihydroxyoligo[(ethylene glycol)alt-(adipic acid)]-alt-(2,5-tolylene diisocyanate)}
F
NH2
+
CH3
OH O
CH2
CH
O n
CH2 H3C
CH3
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Generic Source-Based Nomenclature for Polymers Generic source-based names: poly(ether-alcohol):(epichlorohydrin-alt-bisphenol A) Structure-based name: poly[oxy(2-hydroxypropane-1,3-diyl)oxy-1,4-phenylene(1methylethane-1,1-diyl)-1,4-phenylene]
RULE 5
875
4. Further applications of generic names Generic source-based nomenclature can be extended to more complicated polymers such as spiro and cyclic polymers and networks. Example 6.1 HO
In the case of carbon-chain polymers such as vinyl polymers or diene polymers, the generic name is to be used only when different polymer structures may arise from a given monomeric system.
CH2 OH
CH2
+
C HO
O
O
CH2 OH
CH2
Example 5.1 CH
CH2
CH
CH2
n
Generic source-based name: polyalkylene:(buta-1,3-diene) Source-based name: poly(buta-1,3-diene) Structure-based name: poly(1-vinylethylene)
CH
CH2 CH2
O
O
O
n
Generic source-based name: polyspiroketal:{[2,2-bis(hydroxymethyl)-propane-1,3diol]-alt-cyclohexane-1,4-dione} or polyspiroketal: (pentaerythritol-alt-cyclohexane-1,4-dione) Structure-based name: poly[2,4,8,10-tetraoxaspiro[5.5]undecane-3,3,9,9-tetrayl9,9-bis(ethylene)]
Example 5.2 CH
O
n
Example 6.2
Generic source-based name: polyalkenylene:buta-1,3-diene
O
CH2 CH2
O
O
C
C
n
Source-based name:poly(buta-1,3-diene) Structure-based name: poly(but-1-ene-1,4-diyl) Example 5.3 CH
CH2
Note 8 There is no IUPAC nomenclature for cyclic polymers.
n
C
Example 6.3
NH2
O
Generic source-based name: cyclo-polyester: [(ethylene glycol)-alt-(terephthalic acid)]
O
Generic source-based name: polyalkylene: acrylamide
O
C
O CH
CH
O
C O (CH2)4
O
C
O C O (CH2)4
n
Structure-based name: poly[1-(aminocarbonyl)ethylene]
+
Example 5.4
CH
O NH
C
(CH2)2
n
Generic source-based name: polyamide:acrylamide Structure-based name: poly[imino(1-oxopropane-1,3-diyl)] Note 7 The terms polyalkylene and polyalkenylene have been defined in ref. 7, p. 149.
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CH2
network
Generic source-based name: polyester: {butane-1,4-diol-alt-[(maleic anhydride);(phthalic anhydride)]}-net-polyalkylene: (maleic anhydride)-costyrene]
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876
5. References 1. “Nomenclature of regular single-strand organic polymers, 1975”, Pure Appl. Chem. 48, 373–385 (1976). Reprinted as chapter 5 in Ref. 7. 2. “Nomenclature of regular double-strand (ladder and spiro) organic polymers 1993”, Pure Appl. Chem. 65, 1561–1580 (1993). 3. “Structure-based nomenclature for irregular single-strand organic polymers 1994”, Pure Appl. Chem. 66, 873–889 (1994).
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4. “Source-Based Nomenclature for Copolymers 1985”, Pure Appl. Chem. 57, 1427–1440 (1985). Reprinted as chapter 7 in Ref. 7. 5. “Source-based nomenclature for non-linear macromolecules and macromolecular assemblies”, Pure Appl. Chem. 69, 2511–2521 (1997). 6. A Guide to IUPAC Nomenclature of Organic Compounds, R. Panico, W. H. Powell, J-C. Richer (Eds.), Blackwell Scientific Publications, Oxford (1993). 7. Compendium of Macromolecular Nomenclature, W. V. Metanomski (Ed.), Blackwell Scientific Publications, Oxford (1991).
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Definitions of Terms Relating to Reactions of Polymers and to Functional Polymeric Materials (IUPAC Recommendations 2003) Prepared by a Working Group consisting of
K. Horie1,‡, M. Barón2, R. B. Fox3, J. He4, M. Hess5, J. Kahovec6, T. Kitayama7, P. Kubisa8, E. Maréchal9, W. Mormann10, R. F. T. Stepto11, D. Tabak12, J. Vohlídal13, E. S. Wilks14, and W. J. Work15 *Members of the Commission on Macromolecular Nomenclature (1997–2001) and the Subcommittee on Macromolecular Nomenclature (2002–2003) contributing to this report were: G. Allegra (Italy); M. Barón (Argentina, Commission and Subcommittee Secretary); A. Fradet (France); K. Hatada (Japan); J. He (China); M. Hess (Germany, Commission and Subcommittee Chairman); K. Horie (Japan); A. D. Jenkins (UK); J.-I. Jin (Korea); R. G. Jones (UK, Subcommittee Secretary); J. Kahovec (Czech Republic); T. Kitayama (Japan); P. Kratochvíl (Czech Republic); P. Kubisa (Poland); E. Maréchal (France); I. Meisel (Germany); W. V. Metanomski (USA); G. Moad (Australia); W. Mormann (Germany); S. Penczek (Poland); L. P. Rebelo (Portugal); M. Rinaudo (France); I. Schopov (Bulgaria); M. Schubert (USA); V. P. Shibaev (Russia); S. Slomkowski (Poland); R. F. T. Stepto (UK, Commission Chairman); D. Tabak (Brazil); J. Vohlídal (Czech Republic); E. S. Wilks (USA); W. J. Work (USA, Commission Secretary); Other Contributors: K. Dorfner (Germany); J. M. J. Fréchet (USA); W. I. Harris (USA); P. Hodge (UK); T. Nishikubo (Japan); C. K. Ober (USA); E. Reichmanis (USA); D. C. Sherrington (UK); M. Tomoi (Japan); D. Wöhrle (Germany). 1Department of Organic and Polymer Materials Chemistry, Tokyo University of Agriculture and Technology, 2-24-16 Nakacho, Koganei-shi, Tokyo 184-8588, Japan; 2Facultad de Ciências Exactas y Naturales, Universidad de Belgrano, Villanueva 1324, Buenos Aires 1426, Argentina; 36115 Wiscasset Road, Bethesda, MD 20816, USA; 4State Key Laboratory of Engineering Plastics, The Chinese Academy of Sciences, Institute of Chemistry, Beijing 100080, China; 5Fachbereich 6: Physikalische Chemie, Universitat Duisburg-Essen, D-47048 Duisburg, Germany; 6Ústav Makromolekulární Chemie, Akademie ved Ceské Republiky, Heyrovského námestí 2, CZ-162 06 Praha 6, Czech Republic; 7Department of Chemistry, Osaka University, Toyonaka, Osaka 560-8531, Japan; 8Centrum Badan Molek. i Makromolek., Polska Akademia Nauk, Sienkiewicza 112, PL-90 363 Lódz, Poland; 9Laboratoire de Synthèse Macromoléculaire, Université Pierre et Marie Curie (Paris VI), Boîte 184 - Tour 54, 4e étage, 4 place Jussieu, F-75252 Paris Cédex 05, France; 10Makromolekulare Chemie, Universität - Gesamthochschule Siegen, Adolf Reichwein Strasse 2, D-57068 Siegen, Germany; 11Polymer Science and Technology Group (MMSC), University of Manchester and UMIST, Grosvenor Street, Manchester M1 7HS, UK; 12Praca Pio X, 78 Sala 1213 - Candelaria, Rio de Janerio - RJ 20091-040, Brazil; 13Katedra Fyzikalni a Makromolekularni Chemie, Universita Karlova, Albertov 2030, CZ-128 40 Praha 2, Czech Republic; 14113 Meriden Drive, Canterbury Hills, Hockessin, DE 19707, USA; 151288 Burnett Road, Huntingdon Valley, PA 19006, USA ‡
Corresponding author: E-mail: [email protected]
Abstract: The document defines the terms most commonly encountered in the field of polymer reactions and functional polymers. The scope has been limited to terms that are specific to polymer systems. The document is organized into three sections. The first defines the terms relating to reactions of polymers. Names of individual chemical reactions are omitted from the document, even in cases where the reactions are important in the field of polymer reactions. The second section defines the terms relating to polymer reactants and reactive polymeric materials. The third section defines the terms describing functional polymeric materials.
Contents Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 877 1. Reactions involving polymers. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 878 2. Polymer reactants and reactive polymeric materials. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 880 3. Functional polymeric materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 881 References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 884
Introduction Chemical reactions of polymers have received much attention during the last two decades. Many fundamentally and industrially important reactive polymers and functional polymers are prepared by the reactions of linear or cross-linked polymers and by Reproduced from: Pure Appl. Chem., Vol. 76, No. 4, pp. 889–906, 2004.
© 2004 IUPAC
the introduction of reactive, catalytically active, or other groups onto polymer chains. Characteristics of polymer reactions may be appreciably different from both reactions of low-molar-mass compounds and polymerization reactions. Basic definitions of polymerization reactions have been included in the original [1] and revised [2] documents on basic terms in polymer science published by the IUPAC Commission on Macromolecular Nomenclature. Furthermore, the basic classification and definitions of polymerization reactions [3] and some polymer reactions such as degradation, aging, and related chemical transformations 877
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878 of polymers have been defined [4]. However, in spite of the growing importance of the field, a clear and uniform terminology covering the field of reactions and the functionalization of polymers has not been presented until now. For example, combinatorial chemistry using reactive polymer beads has become a new field in recent years. The development of a uniform terminology for such multidisciplinary areas can greatly aid communication and avoid confusion. This document presents clear concepts and definitions of general and specific terms relating to reactions of polymers and functional polymers. The document is divided into three sections. In Section 1 terms relating to reactions of polymers are defined. Names of individual chemical reactions (e.g., chloromethylation) are omitted from this document, even in cases where the reactions are important in the field of polymer reactions, because such names are usually already in widespread use and are well defined in organic chemistry and other areas of chemistry [5]. Sections 2 and 3 deal with the terminology of reactive and functional polymers. The term “functional polymer” has two meanings: (a) a polymer bearing functional groups (such as hydroxy, carboxy, or amino groups) that make the polymer reactive and (b) a polymer performing a specific function for which it is produced and used. The function in the latter case may be either a chemical function such as a specific reactivity or a physical function like electric conductivity. Polymers bearing reactive functional groups are usually regarded as polymers capable of undergoing chemical reactions. Thus, Section 2 deals with polymers and polymeric materials that undergo various kinds of chemical reactions (i.e., show chemical functions). Section 3 deals with terms relating to polymers and polymeric materials exhibiting some specific physical functions. For definitions of some physical functions, see also Compendium of Chemical Terminology (“Gold Book”) [6]. A functional polymer according to Definition 3.6 of the present document is a polymer that exhibits specified chemical reactivity or has specified physical, biological, pharmacological, or other uses that depend on specific chemical groups. Thus, several terms concerned with properties or the structure of polymers are included in Section 3 whenever they are closely related to specific functions. Terms that are defined implicitly in the notes and related to the main terms are given in bold type.
1. Reactions involving polymers 1.1 Chemical amplification
Process consisting of a chemical reaction that generates a species that catalyzes another reaction and also the succeeding catalyzed reaction. Note 1: Chemical amplification can lead to a change in structure and by consequence to a change in the physical properties of a polymeric material. Note 2: The term “chemical amplification” is commonly used in photoresist lithography employing a photo-acid generator or photo-base generator.
1.2 Chemical modification
Process by which at least one feature of the chemical constitution of a polymer is changed by chemical reaction(s). Note: A configurational change (e.g., cis–trans isomerization) is not usually referred to as a chemical modification.
1.3 Cross-linking
Reaction involving sites or groups on existing macromolecules or an interaction between existing macromolecules that results in the formation of a small region in a macromolecule from which at least four chains emanate. Note 1: See [6], p. 94 and Definition 1.59 in [2] for cross-link. Note 2 : The small region may be an atom, a group of atoms, or a number of branch points connected by bonds, groups of atoms, or oligomeric chains. Note 3: A reaction of a reactive chain end of a linear macromolecule with an internal reactive site of another linear macromolecule results in the formation of a branch point, but is not regarded as a cross-linking reaction.
1.4 Curing
Chemical process of converting a prepolymer or a polymer into a polymer of higher molar mass and connectivity and finally into a network. Note 1: Curing is typically accomplished by chemical reactions induced by heating (thermal curing), photoirradiation (photo-curing), or electron-beam irradiation (EB curing), or by mixing with a chemical curing agent. Note 2: Physical aging, crystallization, physical crosslinking, and postpolymerization reactions are sometimes referred to as “curing”. Use of the term “curing” in these cases is discouraged. Note 3: See also Definition 1.22.
1.5 Depolymerization
Process of converting a polymer into its monomer or a mixture of monomers (see [6], p. 106 and Definition 3.25 in [2]).
1.6 Grafting
Reaction in which one or more species of block are connected to the main chain of a macromolecule as side chains having constitutional or configurational features that differ from those in the main chain. Note: See [6], p. 175 and Definition 1.28 in [2] for graft macromolecule.
1.7 Interchange reaction
Reaction that results in an exchange of atoms or groups between a polymer and low-molar-mass molecules, between polymer molecules, or between sites within the same macromolecule.
Note 3: An example of chemical amplification is the transformation of [(tert-butoxycarbonyl)oxy]phenyl groups in polymer chains to hydroxyphenyl groups catalyzed by a photo-generated acid.
Note: An interchange reaction that occurs with polyesters is called transesterification.
Note 4: The term “amplification reaction” as used in analytical chemistry is defined in [6], p. 21.
Chemical reaction that results in the breaking of main-chain bonds of a polymer molecule.
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1.8 Main-chain scission
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Note 1: See [6], p. 64 and Definition 3.24 in [2] for chain scission.
main chain), degradation means changes in chemical structure. It can also be accompanied by cross-linking.
Note 2: Some main-chain scissions are classified according to the mechanism of the scission process: hydrolytic, mechanochemical, thermal, photochemical, or oxidative scission. Others are classified according to their location in the backbone relative to a specific structural feature, for example, a-scission (a scission of the C-C bond alpha to the carbon atom of a photo-excited carbonyl group) and b-scission (a scission of the C-C bond beta to the carbon atom bearing a radical), etc.
Note 2: Usually, degradation results in the loss of, or deterioration in useful properties of the material. However, in the case of biodegradation (degradation by biological activity), polymers may change into environmentally acceptable substances with desirable properties (see Definition 3.1).
1.9 Mechanochemical reaction
Chemical reaction that is induced by the direct absorption of mechanical energy. Note: Shearing, stretching, and grinding are typical methods for the mechanochemical generation of reactive sites, usually macroradicals, in polymer chains that undergo mechanochemical reactions.
1.10 Photochemical reaction
Chemical reaction that is caused by the absorption of ultraviolet, visible, or infrared radiation ([6], p. 302). Note 1: Chemical reactions that are induced by a reactive intermediate (e.g., radical, carbene, nitrene, or ionic species) generated from a photo-excited state are sometimes dealt with as a part of photochemistry. Note 2: An example of a photochemical reaction concerned with polymers is photopolymerization. Note 3: See also Definitions 1.1, 1.18, 3.14, and 3.25.
1.11 Polymer complexation polymer complex formation
Process that results in the formation of a polymer–polymer complex or a complex composed of a polymer and a low-molar-mass substance.
1.12 Polymer cyclization
Chemical reaction that leads to the formation of ring structures in or from polymer chains. Note 1: Examples of cyclization along polymer chains are: (a) cyclization of polyacrylonitrile, (b) acetalization of poly(vinyl alcohol) with an aldehyde, (c) cyclization of polymers of conjugated dienes such as polyisoprene or polybutadiene leading to macrocycles. Note 2: Examples of cyclization of polymer molecules are: (a) cyclization of poly(dimethylsiloxane), (b) back-biting reaction during ionic polymerizations of heterocyclic monomers.
1.13 Polymer degradation
Chemical changes in a polymeric material that usually result in undesirable changes in the in-use properties of the material. Note 1: In most cases (e.g., in vinyl polymers, polyamides) degradation is accompanied by a decrease in molar mass. In some cases (e.g., in polymers with aromatic rings in the
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Note 3: See Definition 16 in [4] for degradation.
1.14 Polymer functionalization
Introduction of desired chemical groups into polymer molecules to create specific chemical, physical, biological, pharmacological, or other properties.
1.15 Polymer reaction
Chemical reaction in which at least one of the reactants is a highmolar-mass substance.
1.16 Polymer-supported reaction
Chemical reaction in which at least one reactant or a catalyst is bound through chemical bonds or weaker interactions such as hydrogen bonds or donor-acceptor interactions to a polymer. Note 1: The easy separation of low-molar-mass reactants or products from the polymer-supported species is a great advantage of polymer-supported reactions. Note 2: Typical examples of polymer-supported reactions are: (a) reactions performed by use of polymer-supported catalysts, (b) solid-phase peptide synthesis, in which intermediate peptide molecules are chemically bonded to beads of a suitable polymer support.
1.17 Protection of a reactive group
Temporary chemical transformation of a reactive group into a group that does not react under conditions where the nonprotected group reacts. Note: For example, trimethylsilylation is a typical transformation used to protect reactive functional groups such as hydroxy or amino groups from their reaction with growing anionic species in anionic polymerization.
1.18 Radiation reaction
Chemical reaction that is induced by ionizing radiation with γ-ray, X-ray, electron, or other high-energy beams. Note 1: Radiation reactions involving polymers often lead to chain scission and cross-linking. Note 2: A photochemical reaction (see Definition 1.10) is sometimes regarded as a type of radiation reaction.
1.19 Reactive blending
Mixing process that is accompanied by the chemical reaction(s) of components of a polymer mixture. Note 1: Examples of reactive blending are: (a) blending accompanied by the formation of a polymer-polymer complex, (b) the formation of block or graft copolymers by a combination of radicals formed by the mechanochemical scission of polymers during blending.
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880 Note 2: Reactive blending may also be carried out as reactive extrusion or reaction injection molding (RIM).
1.20 Sol-gel process
Formation of a polymer network by the reaction of monomer(s), liquid or in solution, to form a gel, and in most cases finally to form a dry network. Note: An inorganic polymer (e.g., silica-gel or organic-inorganic hybrid) can be prepared by the sol-gel process.
1.21 Surface grafting
Process in which a polymer surface is chemically modified by grafting or by the generation of active sites that can lead to the initiation of a graft polymerization. Note 1: Peroxidation, ozonolysis, high-energy irradiation, and plasma etching are methods of generating active sites on a polymer surface. Note 2: See also Definition 1.6.
1.22 Vulcanization
Chemical cross-linking of high-molar-mass linear or branched polymer or polymers to give a polymer network. Note 1: The polymer network formed often displays rubberlike elasticity. However, a high concentration of cross-links can lead to rigid materials. Note 2: A classic example of vulcanization is the crosslinking of cis-polyisoprene through sulfide bridges in the thermal treatment of natural rubber with sulfur or a sulfur-containing compound.
Handbook of Biochemistry and Molecular Biology 2.3 Living polymer
Polymer with stable, polymerization-active sites formed by a chain polymerization in which irreversible chain transfer and chain termination are absent. Note 1: See [6], p. 236 and Definition 3.21 in [2] for living polymerization.
2.4 Macromonomer
Polymer or oligomer whose molecules each have one end-group that acts as a monomer molecule, so that each polymer or oligomer molecule contributes only a single monomer unit to a chain of the product polymer. Note 1: The homopolymerization or copolymerization of a macromonomer yields a comb or graft polymer. Note 2: In the present definition, Definition 2.35 in [2] has been combined with Definition 1.9 in [2]. See also [6], p. 241. Note 3: Macromonomers are also sometimes referred to as macromers®. The use of the term “macromer” is strongly discouraged.
2.5 Polymer catalyst
Polymer that exhibits catalytic activity. Note 1: Certain synthetic polymer catalysts can behave like enzymes. Note 2: Poly(4-vinylpyridine) in its basic form and sulfonated polystyrene in its acid form are examples of polymers that can act as catalysts in some base- and acid-catalyzed reactions, respectively.
2. Polymer reactants and reactive polymeric materials
2.6 Polymer-metal complex
2.1 Chelating polymer
2.7 Polymer phase-transfer catalyst
Polymer containing ligand groups capable of forming bonds (or other attractive interactions) between two or more separate binding sites within the same ligand group and a single atom. Note 1: Chelating polymers mostly act as ion-exchange polymers specific to ions that form chelates with chelating ligands of the polymer. Note 2: See [6], p. 68 for chelation.
2.2 Ion-exchange polymer
Polymer that is able to exchange ions (cations or anions) with ionic components in solution. Note 1: See [6], p. 208 for ion exchange. Note 2: An ion-exchange polymer in ionized form may also be referred to as a polyanion or a polycation. Note 3: Synthetic ion-exchange organic polymers are often network polyelectrolytes. Note 4: A membrane having ion-exchange groups is called an ion-exchange membrane. Note 5: Use of the term “ion-exchange resin” for “ionexchange polymer” is strongly discouraged.
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Complex comprising a metal and one or more polymeric ligands. Polymer that acts as a phase-transfer catalyst and thereby causes a significant enhancement of the rate of a reaction between two reactants located in neighboring phases owing to its catalysis of the extraction of one of the reactants across the interface to the other phase where the reaction takes place. Note 1: Polymer phase-transfer catalysts in the form of beads are often referred to as triphase catalysts because such catalysts form the third phase of the reaction system. Note 2: See [6], p. 299 for phase-transfer catalyst.
2.8 Polymer-supported catalyst
Catalyst system comprising a polymer support in which catalytically active species are immobilized through chemical bonds or weaker interactions such as hydrogen bonds or donor-acceptor interactions. Note 1: Polymer-supported catalysts are often based on network polymers in the form of beads. They are easy to separate from reaction media and can be used repeatedly. Note 2: Examples of polymer-supported catalysts are: (a) a polymer-metal complex that can coordinate reactants, (b) colloidal palladium dispersed in a swollen network polymer that can act as a hydrogenation catalyst.
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Definitions of Terms Relating to Reactions of Polymers and to Functional Polymeric Materials Note 3: Polymer-supported enzymes are a type of polymer-supported catalysts.
2.9 Polymer reactant polymer reagent polymer-supported reagent
Reactant (reagent) that is or is attached to a high-molar-mass linear polymer or a polymer network. Note: The attachment may be by chemical bonds, by weaker interactions such as hydrogen bonds, or simply by inclusion.
2.10 Prepolymer
Polymer or oligomer whose molecules are capable of entering, through reactive groups, into further polymerization and thereby contributing more than one structural unit to at least one type of chain of the final polymer. Note: Definition 2.37 in [2] has been combined with Definition 1.11 in [2]. See also [6], p. 318.
2.11 Reactive polymer
Polymer having reactive functional groups that can undergo chemical transformation under the conditions required for a given reaction or application.
2.12 Redox polymer electron-exchange polymer oxidation-reduction polymer
Polymer containing groups that can be reversibly reduced or oxidized. Note 1: Reversible redox reaction can take place in a polymer main-chain, as in the case of polyaniline and quinone/ hydroquinone polymers, or on side-groups, as in the case of a polymer carrying ferrocene side-groups. Note 2: See [7] p. 346. Note 3: Use of the term “redox resin” is strongly discouraged.
2.13 Resin
Soft solid or highly viscous substance, usually containing prepolymers with reactive groups. Note 1: This term was used originally because of its analogy with a natural resin (rosin) and designated, in a broad sense, any polymer that is a basic material for plastics, organic coatings, or lacquers. However, the term is now used in a more narrow sense to refer to prepolymers of thermosets (thermosetting polymers). Note 2: The term is sometimes used not only for prepolymers of thermosets, but also for cured thermosets (e.g., epoxy resins, phenolic resins). Use of the term for cured thermosets is strongly discouraged. Note 3: Use of the term “resin” to describe the polymer beads used in solid-phase synthesis and as polymer supports, catalysts, reagents, and scavengers is also discouraged.
2.14 Telechelic polymer telechelic oligomer
Prepolymer capable of entering into further polymerization or other reactions through its reactive end-groups. Note 1: Reactive end-groups in telechelic polymers come from initiator or termination or chain-transfer agents in
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chain polymerizations, but not from monomer(s) as in polycondensations and polyadditions. Note 2: See [6], p. 414 and the Note to Definition 1.11 in [2] for telechelic molecule.
2.15 Thermosetting polymer
Prepolymer in a soft solid or viscous state that changes irreversibly into an infusible, insoluble polymer network by curing. Note 1: Curing can be by the action of heat or suitable radiation, or both. Note 2: A cured thermosetting polymer is called a thermoset.
3. Functional polymeric materials 3.1 Biodegradable polymer
Polymer susceptible to degradation by biological activity, with the degradation accompanied by a lowering of its molar mass. Note 1: See also Note 2 to Definition 1.13. Note 2: See [6], p. 43 for biodegradation. In the case of a polymer, its biodegradation proceeds not only by catalytic activity of enzymes, but also by a wide variety of biological activities.
3.2 Conducting polymer
Polymeric material that exhibits bulk electric conductivity. Note 1: See [6], p. 84 for conductivity. Note 2: The electric conductivity of a conjugated polymer is markedly increased by doping it with an electron donor or acceptor, as in the case of polyacetylene doped with iodine. Note 3: A polymer showing a substantial increase in electric conductivity upon irradiation with ultraviolet or visible light is called a photoconductive polymer; an example is poly (N-vinyl-carbazole) (see [6], p. 302 for photoconductivity). Note 4: A polymer that shows electric conductivity due to the transport of ionic species is called an ion-conducting polymer; an example is sulfonated polyaniline. When the transported ionic species is a proton as, e.g., in the case of fuel cells, it is called a proton-conducting polymer. Note 5: A polymer that shows electric semiconductivity is called a semiconducting polymer (See [6], p. 372 for semiconductor). Note 6: Electric conductance of a nonconducting polymer can be achieved by dispersing conducting particles (e.g., metal, carbon black) in the polymer. The resulting materials are referred to as conducting polymer composites or solid polymer-electrolyte composites.
3.3 Electroluminescent polymer
Polymeric material that shows luminescence when an electric current passes through it such that charge carriers can combine at luminescent sites to give rise to electronically excited states of luminescent groups or molecules. Note 1: Electroluminescent polymers are often made by incorporating luminescent groups or dyes into conducting polymers.
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882 Note 2: Electrogenerated chemiluminescence (see [6], p. 130) directly connected with electrode reactions may also be called electroluminescence.
3.4 Ferroelectric polymer
Polymer in which spontaneous polarization arises when dipoles become arranged parallel to each other by electric fields. Note 1: See [6], p. 153 for ferroelectric transition. Note 2: Poly(vinylidene fluoride) after being subjected to a corona discharge is an example of a ferroelectric polymer.
3.5 Ferromagnetic polymer
Polymer that exhibits magnetic properties because it has unpaired electron spins aligned parallel to each other or electron spins that can easily be so aligned.
Handbook of Biochemistry and Molecular Biology Note 1: Macroporous polymers are often network polymers produced in bead form. However, linear polymers can also be prepared in the form of macroporous polymer beads. Note 2: Macroporous polymers swell only slightly in solvents. Note 3: Macroporous polymers are used, for example, as precursors for ion-exchange polymers, as adsorbents, as supports for catalysts or reagents, and as stationary phases in size-exclusion-chromatography columns. Note 4: Porous polymers with pore diameters from ca. 2 to 50 nm are called mesoporous polymers.
3.10 Nonlinear optical polymer
Polymer that exhibits an optical effect brought about by electromagnetic radiation such that the magnitude of the effect is not proportional to the irradiance.
3.6 Functional polymer
Note 1: See [6], p. 275 for nonlinear optical effect.
or
Note 2: An example of nonlinear optical effects is the generation of higher harmonics of the incident light wave.
(a) Polymer that bears specified chemical groups
(b) Polymer that has specified physical, chemical, biological, pharmacological, or other uses which depend on specific chemical groups. Note: Examples of functions of functional polymers under definition (b) are catalytic activity, selective binding of particular species, capture and transport of electric charge carriers or energy, conversion of light to charge carriers and vice versa, and transport of drugs to a particular organ in which the drug is released.
3.7 Impact-modified polymer
Polymeric material whose impact resistance and toughness have been increased by the incorporation of phase microdomains of a rubbery material. Note: An example is the incorporation of soft polybutadiene domains into glassy polystyrene to produce highimpact polystyrene.
3.8 Liquid-crystalline polymer
Polymeric material that, under suitable conditions of temperature, pressure, and concentration, exists as a liquid crystalline mesophase (Definition 6.1 in [7]).
Note 3: A polymer that exhibits a nonlinear optical effect due to anisotropic electric susceptibilities when subjected to electric field together with light irradiation is called an electro-optical polymer. A polymer that exhibits electro-optical behavior combined with photoconductivity is called a photorefractive polymer.
3.11 Optically active polymer
Polymer capable of rotating the polarization plane of a transmitted beam of linear-polarized light. Note 1: See [6], p. 282 for optical activity. Note 2: The optical activity originates from the presence of chiral elements in a polymer such as chiral centers or chiral axes due to long-range conformational order in a polymer (helicity) (see [6], p. 182 for helicity).
3.12 Photoelastic polymer
Polymer that under stress exhibits birefringence.
3.13 Photoluminescent polymer
Polymer that exhibits luminescence (i.e., fluorescence or phosphorescence arising from photo-excitation). Note: See [6], p. 304 for photoluminescence.
Note 1: See [4], p. 235 for liquid-crystal. Note 2: A liquid-crystalline polymer can exhibit one or more liquid state(s) with one- or two-dimensional, longrange orientational order over certain ranges of temperatures either in the melt (thermotropic liquid-crystalline polymer) or in solution (lyotropic liquid-crystalline polymer).
3.9 Macroporous polymer
Glass or rubbery polymer that includes a large number of macropores (50 nm–1 μm in diameter) that persist when the polymer is immersed in solvents or in the dry state.
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3.14 Photosensitive polymer
Polymer that responds to ultraviolet or visible light by exhibiting a change in its physical properties or its chemical constitution. Note 1: Examples of the changes in photosensitive polymers are a change in molecular shape (photoresponsive polymer), a change in its constitution (photoreactive polymer), and a reversible change in color (photochromic polymer). Note 2: Photosensitivity in photosensitive polymers means that the polymers are sensitive to the irradiated light
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Definitions of Terms Relating to Reactions of Polymers and to Functional Polymeric Materials leading to some change in properties or structure. It is different from photo-sensitization defined in [6], p. 307. Note 3: See [6], p. 307 for photoreaction and [6], p. 302 for photochromism.
3.15 Piezoelectric polymer
(a) Polymer that exhibits a change in dielectric properties on application of pressure
883
Note 5: See Definition 1.58 in [2] for network. Note 6: The definition for gel in [6], p. 170 does not include a polymer gel.
3.20 Polymer membrane
Thin layer of polymeric material that acts as a barrier permitting mass transport of selected species. Note: See [6], p. 251 for membrane.
or (b) Polymer that shows a change in its dimensions when subjected to an electric field.
3.16 Polyelectrolyte
Polymer composed of molecules in which a portion of the constitutional units has ionizable or ionic groups, or both. Note 1: A polymer bearing both anionic and cationic groups in the same molecule is called an amphoteric polyelectrolyte. Note 2: A polymer bearing acid or basic groups is called a polymer acid or a polymer base, respectively. Note 3: A polymer acid or a polymer base can be used as a matrix for ion-conducting polymers. Note 4: Definition 2.38 in [2] has been combined with Definition 1.65 in [2]. The present definition replaces the one in [6], p. 312.
3.17 Polymer compatibilizer
Polymeric additive that, when added to a blend of immiscible polymers, modifies their interfaces and stabilizes the blend. Note: Typical polymer compatibilizers are block or graft copolymers.
3.18 Polymer drug
Polymer that contains either chemically bound drug molecules or pharmacologically active moieties. Note: A polymer drug is usually used to provide drug delivery targeted to an organ and controlled release of an active drug at the target organ.
3.19 Polymer gel
Gel in which the network component is a polymer network. Note 1: A gel is an elastic colloid or polymer network that is expanded throughout its whole volume by a fluid. Note 2: The polymer network can be a network formed by covalent bonds or by physical aggregation with region of local order acting as network junctions.
3.21 Polymer solvent
Polymer that acts like a solvent for compounds of low molar mass. Note: An example of a polymer solvent is poly(oxyethylene); it can dissolve various inorganic salts by complexation.
3.22 Polymer sorbent
Polymer that adsorbs or absorbs a certain substance or certain substances from a liquid or a gas. Note 1: A polymer sorbent may be a polymer adsorbent or a polymer absorbent. The former acts by surface sorption and the latter by bulk sorption. Note 2: See [6], p. 383 for sorption, [6], p. 11 for adsorption, and [6], p. 3 for absorption.
3.23 Polymer support
Polymer to or in which a reagent or catalyst is chemically bound, immobilized, dispersed, or associated. Note 1: A polymer support is usually a network polymer. Note 2: A polymer support is usually prepared in bead form by suspension polymerization. Note 3: The location of active sites introduced into a polymer support depends on the type of polymer support. In a swollen-gel-bead polymer support the active sites are distributed uniformly throughout the beads, whereas in a macroporous-bead polymer support they are predominantly on the internal surfaces of the macropores.
3.24 Polymer surfactant
Polymer that lowers the surface tension of the medium in which it is dissolved, or the interfacial tension with another phase, or both. Note: See [6], p. 409 for surfactant.
3.25 Resist polymer
Polymeric material that, when irradiated, undergoes a marked change in solubility in a given solvent or is ablated.
Note 3: An example of covalent polymer gels is net-poly(Nisopropylacrylamide) swollen in water, which shows volume phase transition during heating.
Note 1: A resist polymer under irradiation either forms patterns directly or undergoes chemical reactions leading to pattern formation after subsequent processing.
Note 4: Examples of physically aggregated polymer gels are poly(vinyl alcohol) gel and agarose gel, which show reversible sol-gel transitions.
Note 2: A resist material that is optimized for use with ultraviolet or visible light, an electron beam, an ion beam, or X-rays is called a photoresist (see [6], p. 307),
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884 electron-beam resist, ion-beam resist, or X-ray resist, respectively. Note 3: In a positive-tone resist, also called a positive resist, the material in the irradiated area not covered by a mask is removed, which results in an image with a pattern identical with that on the mask. In a negative-tone resist, also called a negative resist, the non-irradiated area is subsequently removed, which results in an image with a pattern that is the complement of that on the mask.
3.26 Shape-memory polymer
Polymer that, after heating and being subjected to a plastic deformation, resumes its original shape when heated above its glasstransition or melting temperature. Note: Crystalline trans-polyisoprene is an example of a shape-memory polymer.
3.27 Superabsorbent polymer
Polymer that can absorb and retain extremely large amounts of a liquid relative to its own mass. Note 1: The liquid absorbed can be water or an organic liquid. Note 2: The swelling ratio of a superabsorbent polymer can reach the order of 1000:1.
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Handbook of Biochemistry and Molecular Biology Note 3: Superabsorbent polymers for water are frequently polyelectrolytes.
References 1. IUPAC. Compendium of Macromolecular Nomenclature, (the IUPAC “Purple Book”), prepared for publication by W. V. Metanomski, Chap. 1, Blackwell, Oxford (1991); IUPAC. “Basic definitions of terms relating to polymers (1974)”, Pure Appl. Chem. 40, 477–491 (1974). 2. A. D. Jenkins, P. Kratochvíl, R. F. T. Stepto, U. W. Suter. “Glossary of basic terms in polymer science (IUPAC Recommendations 1996)”, Pure Appl. Chem. 68, 2287–2311 (1996). 3. I. Mita, R. F. T. Stepto, U. W. Suter. “Basic classification and definitions of polymerization reactions (IUPAC Recommendations 1994)”, Pure Appl. Chem. 66, 2483–2486 (1994). 4. K. Hatada, R. B. Fox, J. Kahovec, E. Maréchal, I. Mita, V. Shibaev. “Definitions of terms relating to degradation, aging, and related chemical transformations of polymers (IUPAC Recommendations 1996)”, Pure Appl. Chem. 68, 2313–2323 (1996). 5. R. A. Y. Jones and J. F. Bunnett. “Nomenclature for organic chemical transformations (IUPAC Recommendations 1989)”, Pure Appl. Chem. 61, 725–768 (1989). 6. IUPAC. Compendium of Chemical Terminology: IUPAC Recommendations, (the IUPAC “Gold Book”), 2nd ed., compiled by A. D. McNaught and A. Wilkinson, Blackwell, Oxford (1997). 7. C. Noël, V. P. Shibaev, M. Baro´n, M. Hess, A. D. Jenkins, Jung-Il Jin, A. Sirigu, R. F. T. Stepto, W. J. Work. “Definitions of basic terms relating to low-molar-mass and polymer liquid-crystals (IUPAC Recommendations 2001)”, Pure Appl. Chem. 73, 845–895 (2001).
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Definitions of Terms Relating to Reactions of Polymers and to Functional Polymeric Materials
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Alphabetical Index of Terms Term aa-scission b-scission amphoteric polyelectrolyte biodegradable polymer biodegradation chelating polymer chemical amplification chemical modification conducting polymer conducting polymer composite cross-linking curing depolymerization EB curing electro-optical polymer electroluminescent polymer electron-beam resist electron-exchange polymer ferroelectric polymer ferromagnetic polymer functional polymer grafting hydrolytic scission impact-modified polymer interchange reaction ion-beam resist ion-conducting polymer ion-exchange membrane ion-exchange polymer liquid-crystalline polymer living polymer lyotropic liquid-crystalline polymer macromonomer macroporous-bead polymer support macroporous polymer main-chain scission mechanochemical reaction mechanochemical scission mesoporous polymer negative resist negative-tone resist nonlinear-optical polymer optically active polymer oxidation-reduction polymer oxidative scission photo-acid generator photo-base generator photo-curing photochemical reaction photochemical scission photochromic polymer photoconductive polymer photoelastic polymer photoluminescent polymer photopolymerization photoreactive polymer photorefractive polymer photoresist photoresponsive polymer photosensitive polymer piezoelectric polymer
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Definition No. 1.8 1.8 3.16 3.1 1.13 2.1 1.1 1.2 3.2 3.2 1.3 1.4 1.5 1.4 3.10 3.3 3.25 2.12 3.4 3.5 3.6 1.6 1.8 3.7 1.7 3.25 3.2 2.2 2.2 3.8 2.3 3.8 2.4 3.23 3.9 1.8 1.9 1.8 3.9 3.25 3.25 3.10 3.11 2.12 1.8 1.1 1.1 1.4 1.10, 1.18 1.8 3.14 3.2 3.12 3.13 1.10 3.14 3.10 3.25 3.14 3.14 3.15
Term polyanion polycation polyelectrolyte polymer absorbent polymer acid polymer adsorbent polymer base polymer catalyst polymer compatibilizer polymer complex formation polymer complexation polymer cyclization polymer degradation polymer drug polymer functionalization polymer gel polymer membrane polymer phase-transfer catalyst polymer reactant polymer reaction polymer reagent polymer solvent polymer sorbent polymer support polymer surfactant polymer-metal complex polymer-supported catalyst polymer-supported enzyme polymer-supported reaction polymer-supported reagent positive resist positive-tone resist prepolymer protection of a reactive group proton-conducting polymer radiation reaction reactive blending reactive polymer redox polymer resin resist polymer semiconducting polymer shape-memory polymer sol-gel process solid polymer-electrolyte composite superabsorbent polymer surface grafting swollen-gel-bead polymer support telechelic oligomer telechelic polymer thermal curing thermal scission thermoset thermosetting polymer thermotropic liquid-crystalline polymer transesterification trimethylsilylation triphase catalyst vulcanization X-ray resist
Definition No. 2.2 2.2 3.16 3.22 3.16 3.22 3.16 2.5 3.17 1.11 1.11 1.12 1.13 3.18 1.14 3.19 3.20 2.7 2.9 1.15 2.9 3.21 3.22 3.23 3.24 2.6 2.8 2.8 1.16 2.9 3.25 3.25 2.10 1.17 3.2 1.18 1.19 2.11 2.12 2.13 3.25 3.2 3.26 1.20 3.2 3.27 1.21 3.23 2.14 2.14 1.4 1.8 2.15 2.15 3.8 1.7 1.17 2.7 1.22 3.25
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Definitions of Terms Related to Polymer Blends, Composites, and Multiphase Polymeric Materials (IUPAC Recommendations 2004) Prepared by a Working Group consisting of
W. J. Work1’‡, K. Horie2, M. Hess3, and R. F. T. Stepto4 11288 Burnett Road, Huntingdon Valley, PA 19006, USA; 2 6-11-21, Kozukayama, Tarumi-ku, Kobe 655-0002, Japan; 3 Universitat Duisburg-Essen, Fachbereich 6: Physikalische Chemie, D-47048 Duisburg, Germany; 4 University of Manchester and UMIST, Polymer Science and Technology Group (MMSC), Grosvenor Street, Manchester, M1 7HS, UK 1
Prepared for publication by
W. J. WORK
*Membership of the Commission on Macromolecular Nomenclature (extant until 2002) during the preparation of this report (1993– 2003) was as follows: Titular Members: R. E. Bareiss (Germany, to 1993); M. Barón (Argentina, Associate Member to 1995, Titular Member from 1996, Secretary from 1998); K. Hatada (Japan, to 1997, Associate Member to 1999); M. Hess (Germany, Associate Member from 1996, Titular Member from 1998, Chairman from 2000); K. Horie (Japan, Associate Member from 1996, Titular Member from 1998); R. G. Jones (UK, Pool Titular Member to 1997, Titular Member from 1998); J Kahovec (Czech Republic, to 1999); P. Kubisa (Poland, Associate Member from 1996, Titular Member from 2000); E. Maréchal (France, Associate Member 1992–1993, 2000–2001, Titular Member 1994–1999); I Meisel (Germany, Associate Member from 1998, Titular Member from 2000); W. V. Metanomski (USA, to 1999); C. Noël (France, to 1997); V. P. Shibaev (Russia, to 1995, Associate Member to 1999); R. F. T. Stepto (UK, Chairman to 1999); E. S. Wilks (USA, Associate Member from 1998, Titular Member from 2000); W. J. Work (USA, Secretary to 1997). Associate Members contributing to this report: J.-I. Jin (Korea, National Representative to 1993, Associate Member from 1994); T. Kitayama (Japan, from 2000); S. Penczek (Poland, from 1994); J. Vohlídal (Czech Republic, from 2000). National Representatives contributing to this report: W. Mormann (Germany, from 2000). **Membership of the Subcommittee on Macromolecular Terminology (extant from 2002) during the preparation of this report (1993– 2003) was as follows: M. Hess (Germany, Chairman); M. Barón (Argentina, Secretary); G. Allegra (Italy); A. Fradet (France); J. He (China); K. Horie (Japan); A. D. Jenkins (UK); J.-II Jin (Korea); R. G. Jones (UK); J. Kahovec (Czech Republic); T. Kitayama (Japan); P. Kratochvíl (Czech Republic); P. Kubisa (Poland); I. Meisel (Germany); W. V. Metanomski (USA); G. Moad (Australia); W. Mormann (Germany); S. Penczek (Poland); L. P. Rebelo (Portugal); M. Rinaudo (France); I. Schopov (Bulgaria); M. Schubert (USA); V. P. Shibaev (Russia); S. Slomkowski (Poland); R. F. T. Stepto (UK); D. Tabak (Brazil); J. Vohlídal (Czech Republic); E. S. Wilks (USA); W. J. Work (USA). Other contributors to this report: S. Akiyama (Japan); P. Avakian (USA); K. Binder (Germany); C. Bucknall (UK); R. Gilbert (Australia); J. He (China); J. S. Higgins (UK); T. Inoue (Japan); B.-J. Jungnickel (Germany); R. Koningsveld (Netherlands); J. Lertola (USA); T. Nishi (Japan); T. Nose (Japan); D. Paul (USA); I. Plotzker (USA); L. A. Utracki (Canada); B. Wood (USA). ‡
Corresponding author
Abstract: The document defines the terms most commonly encountered in the field of polymer blends and composites. The scope has been limited to mixtures in which the components differ in chemical composition or molar mass and in which the continuous phase is polymeric. Incidental thermodynamic descriptions are mainly limited to binary mixtures although, in principle, they could be generalized to multicomponent mixtures. The document is organized into three sections. The first defines terms basic to the description of polymer mixtures. The second defines terms commonly encountered in descriptions of phase domain behavior of polymer mixtures. The third defines terms commonly encountered in the descriptions of the morphologies of phase-separated polymer mixtures.
Reproduced from: Pure Appl. Chem., Vol. 76, No. 11, pp. 1985–2007, 2004. © 2004 IUPAC
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Handbook of Biochemistry and Molecular Biology
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Contents 1. Basic terms in polymer mixtures. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 888 2. Phase domain behavior. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 892 3. Domains and morphologies. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 894 References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 896 Bibliography. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 896 Alphabetical Index of Terms. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 897
Introduction It is the intent of this document to define the terms most commonly encountered in the field of polymer blends and composites. The scope has been limited to mixtures in which the components differ in chemical composition or molar mass or both and in which the continuous phase is polymeric. Many of the materials described by the term “multiphase” are two-phase systems that may show a multitude of finely dispersed phase domains. Hence, incidental thermodynamic descriptions are mainly limited to binary mixtures, although they can be and, in the scientific literature, have been generalized to multi-component mixtures. Crystalline polymers and liquid-crystal polymers have been considered in other documents [1,2] and are not discussed here. This document is organized into three sections. The first defines terms basic to the description of polymer mixtures. The second defines terms commonly encountered in descriptions of phase-domain behavior of polymer mixtures. The third defines terms commonly encountered in the descriptions of the morphologies of phase-separated polymer mixtures. General terms describing the composition of a system as defined in ref. [3] are used without further definition throughout the document. Implicit definitions are identified in boldface type throughout the document.
1. Basic terms in polymer mixtures 1.1 Polymer blend
Macroscopically homogeneous mixture of two or more different species of polymer [3,4].
Notes:
1. Whether or not a single phase exists depends on the chemical structure, molar-mass distribution, and molecular architecture of the components present. 2. The single phase in a mixture may be confirmed by light scattering, X-ray scattering, and neutron scattering. 3. For a two-component mixture, a necessary and sufficient condition for stable or metastable equilibrium of a homogeneous single phase is ∂ 2 ∆ mixG ∂φ 2
>0 T, p
where ΔmixG is the Gibbs energy of mixing and f the composition, where f is usually taken as the volume fraction of one of the components. The system is unstable if the above second derivative is negative. The borderline (spinodal) between (meta)stable and unstable states is defined by the above second derivative equalling zero. If the compositions of two conjugate (coexisting) phases become identical upon a change of temperature or pressure, the third derivative also equals zero (defining a critical state). 4. If a mixture is thermodynamically metastable, it will demix if suitably nucleated (see 2.5). If a mixture is thermodynamically unstable, it will demix by spinodal decomposition (see 2.8) or by nucleation and growth if suitably nucleated, provided there is minimal kinetic hindrance.
Notes:
1. See the Gold Book, p. 312 [3]. 2. In most cases, blends are homogeneous on scales larger than several times the wavelengths of visible light. 3. In principle, the constituents of a blend are separable by physical means. 4. No account is taken of the miscibility or immiscibility of the constituent macromolecules, i.e., no assumption is made regarding the number of phase domains present. 5. The use of the term “polymer alloy” for “polymer blend” is discouraged, as the former term includes multiphase copolymers but excludes incompatible polymer blends (see 1.3). 6. The number of polymeric components which comprises a blend is often designated by an adjective, viz., binary, ternary, quaternary,
1.2 Miscibility
Capability of a mixture to form a single phase over certain ranges of temperature, pressure, and composition.
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1.3 Miscible polymer blend homogeneous polymer blend
Polymer blend that exhibits miscibility (see 1.2). Notes:
1. For a polymer blend to be miscible, it must satisfy the criteria of miscibility (see 1.2). 2. Miscibility is sometimes erroneously assigned on the basis that a blend exhibits a single Tg or optical clarity. 3. A miscible system can be thermodynamically stable or metastable (see note 4 in 1.2). 4. For components of chain structures that would be expected to be miscible, miscibility may not occur if molecular architecture is changed, e.g., by crosslinking.
1.4 Homologous polymer blend
Mixture of two or more fractions of the same polymer, each of which has a different molar-mass distribution.
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Definitions of Terms Related to Polymer Blends, Composites, and Multiphase Polymeric Materials 1.5 Isomorphic polymer blend
Polymer blend of two or more different semi-crystalline polymers that are miscible in the crystalline state as well as in the molten state. Notes:
Notes:
1. Such a blend exhibits a single, composition-dependent glass-transition temperature, Tg, and a single, composition-dependent melting point, Tm. 2. This behavior is extremely rare; very few cases are known.
1.6 Polymer-polymer complex
Complex, at least two components of which are different polymers [3]. Notes:
1. See the Gold Book, p. 313 [3]. 2. A complex is a molecular entity formed from two or more components that can be ionic or uncharged (see the Gold Book, p. 81) [3]. 3. Although the intrinsic binding energy between the individual interacting sites giving rise to the complex is weaker than a covalent bond, the total binding energy for any single molecule may exceed the energy of a single covalent bond. 4. The properties of a complex defined here differ from those given in ref. [3] because, owing to the repeating nature of a polymer molecule, many interacting sites may be present, which together will provide stronger bonding than a single covalent bond.
1.7 Metastable miscibility
Capability of a mixture to exist for an indefinite period of time as a single phase that is separated by a small or zero energy barrier from a thermodynamically more stable multiphase system.
Recommended acronym: SIPN
Polymer comprising one or more polymer network(s) and one or more linear or branched polymer(s) characterized by the penetration on a molecular scale of at least one of the networks by at least some of the linear or branched chains [4]. Notes:
1. See the Gold Book, p. 255 [3]. 2. Mixtures exhibiting metastable miscibility may remain unchanged or they may undergo phase separation, usually by nucleation or spinodal decomposition.
1.8 Metastable miscible polymer blend
Polymer blend that exhibits metastable miscibility. Note: In polymers, because of the low mobility of polymer chains, particularly in a glassy state, metastable mixtures may exist for indefinite periods of time without phase separation. This has frequently led to confusion when metastable miscible polymer blends are erroneously claimed to be miscible.
1.9 Interpenetrating polymer network Recommended acronym: IPN
Polymer comprising two or more polymer networks which are at least partially interlaced on a molecular scale, but not covalently bonded to each other and cannot be separated unless chemical bonds are broken [4].
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1. See the Gold Book, p. 205 [3]. 2. A mixture of two or more preformed polymer networks is not an interpenetrating polymer network. 3. An IPN may be further described by the process by which it is synthesized. When an IPN is prepared by a process in which the second component network is polymerized following the completion of polymerization of the first component network, the IPN may be referred to as a sequential IPN. When an IPN is prepared by a process in which both component networks are polymerized concurrently, the IPN may be referred to as a simultaneous IPN.
1.10 Semi-interpenetrating polymer network
Notes:
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1. See the Gold Book, p. 372 [3]. 2. Semi-interpenetrating polymer networks are different from interpenetrating polymer networks because the constituent linear-chain or branched-chain macromolecule(s) can, in principle, be separated from the constituent polymer network(s) without breaking chemical bonds, and, hence, they are polymer blends. 3. Semi-interpenetrating polymer networks may be further described by the process by which they are synthesized. When an SIPN is prepared by a process in which the second component polymer is polymerized or incorporated following the completion of polymerization of the first component polymer, the SIPN may be referred to as a sequential SIPN. When an SIPN is prepared by a process in which both component polymers are polymerized concurrently, the SIPN may be referred to as a simultaneous SIPN. (This note has been changed from that which appears in ref. [4] to allow for the possibility that a linear or branched polymer may be incorporated into a network by means other than polymerization, e.g., by swelling of the network and subsequent diffusion of the linear or branched chain into the network.).
1.11 Immiscibility
Inability of a mixture to form a single phase. Notes:
1. Immiscibility may be limited to certain ranges of temperature, pressure, and composition. 2. Immiscibility depends on the chemical structures, molarmass distributions, and molecular architectures of the components.
1.12 Immiscible polymer blend heterogeneous polymer blend Polymer blend that exhibits immiscibility.
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Multicomponent material comprising multiple different (nongaseous) phase domains in which at least one type of phase domain is a continuous phase (see 3.12).
1.21 Intercalation
Process by which a substance becomes transferred into pre-existing spaces of molecular dimensions in a second substance. Note: The term as defined here is specific to polymer science. An alternative definition of “intercalation” applies in some other fields of chemistry (see the Gold Book, p. 202 [3]).
Note: Foamed substances, which are multiphased materials that consist of a gas dispersed in a liquid or solid, are not normally considered to be composites.
1.14 Polymer composite
Composite in which at least one component is a polymer.
1.22 Exfoliation
Process by which thin layers individually separate from a multilayered structure.
1.15 Nanocomposite
Note: In the context of a nanocomposite material, the individual layers are of the order of at most a few nanometers in thickness.
Composite in which at least one of the phases has at least one dimension of the order of nanometers.
1.16 Laminate
Material consisting of more than one layer, the layers being distinct in composition, composition profile, or anisotropy of properties. Notes:
1. Laminates may be formed by two or more layers of different polymers. 2. Composite laminates generally consist of one or more layers of a substrate, often fibrous, impregnated with a curable polymer, curable polymers, or liquid reactants. 3. The substrate is usually a sheet-like woven or nonwoven material (e.g., glass fabric, paper, copper foil). 4. A single layer of a laminate is termed a lamina.
1.17 Lamination
Process of forming a laminate.
1.23 Wetting
Process by which an interface between a solid and a gas is replaced by an interface between the same solid and a liquid.
1.24 Adhesion
Holding together of two bodies by interfacial forces or mechanical interlocking on a scale of micrometers or less.
1.25 Chemical adhesion
Adhesion (see 1.25) in which two bodies are held together at an interface by ionic or covalent bonding between molecules on either side of the interface.
1.26 Interfacial adhesion
Adhesion (see 1.25) in which interfaces between phases or components are maintained by intermolecular forces, chain entanglements, or both, across the interfaces.
1.18 Delamination
Process that separates the layers of a laminate by breaking their structure in planes parallel to those layers.
1.19 Impregnation
Notes:
Penetration of monomeric, oligomeric, or polymeric liquids into an assembly of fibers. Notes:
1. The term as defined here is specific to polymer science. An alternative definition of “impregnation” applies in some other fields of chemistry (see the Gold Book, p. 197) [3]. 2. Impregnation is usually carried out on a woven fabric or a yarn.
1.20 Prepreg
Sheets of a substrate that have been impregnated with a curable polymer, curable polymers, or liquid reactants, or a thermoplastic, and are ready for fabrication of laminates. Notes:
1. See 1.16 notes 2 and 3. 2. During the impregnation the curable polymer, curable polymers, or liquid reactants may be allowed to react to a certain extent (sometimes termed degree of ripening).
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1. Interfacial adhesion is also referred to as tack. 2. Adhesive strength (recommended symbol: Fa, unit: N m−2) is the force required to separate one condensed phase domain from another at the interface between the two phase domains divided by the area of the interface. 3. Interfacial tension (recommended symbol: γ, unit: N m−1, J m−2) is the change in Gibbs energy per unit change in interfacial area for substances in physical contact. 4. Use of the term interfacial energy for interfacial tension is not recommended.
1.27 Interfacial bonding
Bonding in which the surfaces of two bodies in contact with one another are held together by inter-molecular forces. Note: Examples of intermolecular forces include covalent, ionic, van der Waals, and hydrogen bonds.
1.28 Interfacial fracture
Brittle fracture that takes place at an interface.
1.29 Craze
Crack-like cavity formed when a polymer is stressed in tension that contains load-bearing fibrils spanning the gap between the surfaces of the cavity.
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Definitions of Terms Related to Polymer Blends, Composites, and Multiphase Polymeric Materials Note: Deformation of continua occurs with only minor changes in volume; hence, a craze consists of both fibrils and voids.
1.30 Additive
Substance added to a polymer.
as incompatibility is related to the weakness of interfacial bonding.
1.35 Compatible polymer blend
Immiscible polymer blend (see 1.12) that exhibits macroscopically uniform physical properties throughout its whole volume. Note: The macroscopically uniform physical properties are usually caused by sufficiently strong interactions between the component polymers.
Notes:
1. The term as defined here is specific to polymer science. An alternative definition of “additive” applies in some other fields of chemistry (see the Gold Book, p. 10) [3]. 2. An additive is usually a minor component of the mixture formed and usually modifies the properties of the polymer. 3. Examples of additives are antioxidants, plasticizers, flame retardants, processing aids, other polymers, colorants, UV absorbers, and extenders.
1.31 Interfacial agent
Additive that reduces the interfacial energy between phase domains.
1.32 Compatibility
Capability of the individual component substances in either an immiscible polymer blend (see 1.12) or a polymer composite (see 1.14) to exhibit interfacial adhesion (see 1.27).
1.36 Compatibilizer
Polymer or copolymer that, when added to an immiscible polymer blend (see 1.12), modifies its inter-facial character and stabilizes its morphology. Note: Compatibilizers usually stabilize morphologies over distances of the order of micrometers or less.
1.37 Coupling agent adhesion promoter
Interfacial agent comprised of molecules possessing two or more functional groups, each of which exhibits preferential interactions with the various types of phase domains in a composite. Notes:
Notes:
1. Use of the term “compatibility” to describe miscible systems is discouraged. 2. Compatibility is often established by the observation of mechanical integrity under the intended conditions of use of a composite or an immiscible polymer blend.
Note: Compatibilization may be achieved by addition of suitable copolymers or by chemical modification of interfaces through physical treatment (i.e., irradiation or thermal) or reactive processing.
Polymeric material, exhibiting macroscopically uniform physical properties throughout its whole volume, that comprises a compatible polymer blend (see 1.35), a miscible polymer blend (see 1.3), or a multiphase copolymer (see 3.3). Note: See note 5 in 1.1.
1.39 Dispersion
Material comprising more than one phase where at least one of the phases consists of finely divided phase domains (see 3.2), often in the colloidal size range, distributed throughout a continuous phase domain. Notes:
1.34 Degree of compatibility
Measure of the strength of the interfacial bonding between the component substances of a composite or immiscible polymer blend (see 1.12). Notes:
1. Estimates of the degree of compatibility are often based upon the mechanical performance of the composite, the interphase thickness (see “Interfacial region interphase”), or the sizes of the phase domains present in the composite, relative to the corresponding properties of composites lacking compatibility. 2. The term degree of incompatibility is sometimes used instead of degree of compatibility. Such use is discouraged
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1. A coupling agent increases adhesion between phase domains. 2. An example of the use of a coupling agent is in a mineralfilled polymer material where one part of the coupling agent molecule can chemically bond to the inorganic mineral while the other part can chemically bond to the polymer.
1.38 Polymer alloy
1.33 Compatibilization
Process of modification of the interfacial properties in an immiscible polymer blend that results in formation of the interphases (see 3.6) and stabilization of the morphology, leading to the creation of a polymer alloy.
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1. The term as defined here is specific to polymer science. An alternative definition of “dispersion” applies in some other fields of chemistry (see the Gold Book, p. 118) [3]. 2. Particles in the colloidal size range have linear dimensions [3] between 1 nm and 1 μm. 3. The finely divided domains are called the dispersed or discontinuous phase domains (see 3.13). 4. For a definition of continuous phase domain, see 3.12. 5. A dispersion is often further characterized on the basis of the size of the phase domain as a macrodispersion or a microdispersion. To avoid ambiguity when using these terms, the size of the domain should also be defined.
1.40 Dispersing agent dispersing aid dispersant
Additive (see 1.30), exhibiting surface activity, that is added to a suspending medium to promote uniform and maximum
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1.41 Agglomeration aggregation
Process in which dispersed molecules or particles form clusters rather than remain as isolated single molecules or particles. Note: See the Gold Book, p. 13 [3].
1.42 Agglomerate aggregate
Clusters of dispersed molecules or particles that results from agglomeration (see 1.41). Note: The term as defined here is specific to polymer science. An alternative definition of “aggregate” is used in some other fields of chemistry (see the Gold Book, p. 13) [3].
2. Phase domain behavior 2.1 Miscibility window
Range of copolymer compositions in a polymer mixture, at least one component substance of which is a copolymer, that gives miscibility (see 1.2) over a range of temperatures and pressures. Notes:
1.43 Extender
Substance, especially a diluent or modifier, added to a polymer to increase its volume without substantially altering the desirable properties of the polymer. Note: An extender may be a liquid or a solid.
1.44 Filler
Area within the coexistence curve of an isobaric phase diagram (temperature vs. composition) or an isothermal phase diagram (pressure vs. composition). Note: A miscibility gap is observed at temperatures below an upper critical solution temperature (UCST) (see 2.15) or above the lower critical solution temperature (LCST) (see 2.14). Its location depends on pressure. In the miscibility gap, there are at least two phases coexisting.
Notes:
1. The term as defined here is specific to polymer science. An alternative definition of “filler” applies in some other fields of chemistry (see the Gold Book, p. 154) [3]. 2. Fillers may be added to modify mechanical, optical, electrical, thermal, flammability properties, or simply to serve as extenders.
1.45 Fill factor Recommended symbol: øfill
Maximum volume fraction of a particulate filler that can be added to a polymer while maintaining the polymer as the continuous phase domain.
1.46 Thermoplastic elastomer
Melt-processable polymer blend or copolymer in which a continuous elastomeric phase domain is reinforced by dispersed hard (glassy or crystalline) phase domains that act as junction points over a limited range of temperature. Notes:
1. The behavior of the hard phase domains as junction points is thermally reversible. 2. The interfacial interaction between hard and soft phase domains in a thermoplastic elastomer is often the result
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1. Outside the miscibility window immiscible mixtures are formed. 2. The compositions of the copolymers within the miscibility window usually exclude the homopolymer compositions of the monomers from which the copolymers are prepared. 3. The miscibility window is affected by the molecular weights of the component substances. 4. The existence of miscibility windows has been attributed to an average force between the monomer units of the copolymer that leads to those units associating preferentially with the monomer units of the other polymers.
2.2 Miscibility gap
Solid extender.
of covalent bonds between the phases and is sufficient to prevent the flow of the elastomeric phase domain under conditions of use. 3. Examples of thermoplastic elastomers include block copolymers and blends of plastics and rubbers.
2.3 Flory–huggins theory flory–huggins–staverman theory
Statistical thermodynamic mean-field theory of polymer solutions, formulated independently by Flory, Huggins, and Staverman, in which the thermodynamic quantities of the solution are derived from a simple concept of combinatorial entropy of mixing and a reduced Gibbs-energy parameter, the “c interaction parameter” (see 2.4). Notes:
1. See the Gold Book, p. 158 [3]. 2. The Flory–Huggins theory has often been found to have utility for polymer blends; however, there are many equation-of-state theories that provide more accurate descriptions of polymer–polymer interactions. 3. The present definition has been modified from that which appears in ref. [5] to acknowledge the contributions of Staverman and to further clarify the statistical basis of the theory.
2.4 c Interaction parameter
Recommended symbol: χ Interaction parameter, employed in the Flory–Huggins theory (see 2.3), to account for the contribution of the noncombinatorial
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Definitions of Terms Related to Polymer Blends, Composites, and Multiphase Polymeric Materials entropy of mixing and the enthalpy of mixing to the Gibbs energy of mixing. Notes:
1. The definition and the name of the term have been modified from that which appears in ref. [5] to reflect its broader use in the context of polymer blends. In its simplest form, the χ parameter is defined according to the Flory–Huggins equation for binary mixtures
2. In the unstable region bounded by the spinodal curve, phase domain separation is spontaneous, i.e., no nucleation step is required to initiate the separation process.
2.8 Spinodal decomposition spinodal phase-demixing
Long-range, diffusion-limited, spontaneous phase domain separation initiated by delocalized concentration fluctuations occurring in an unstable region of a mixture bounded by a spinodal curve. Note: Spinodal decomposition occurs when the magnitude of Gibbs energy fluctuations with respect to composition are zero.
∆ mixG = n1 ln φ1 + n2 ln φ2 + χ x1n1φ2 , RT
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for a mixture of amounts of substance n1 and n2 of components denoted 1 and 2, giving volume fractions f1 and f 2, with the molecules of component 1 each conceptually consisting of x1 segments whose Gibbs energy of interaction with segments of equal volume in the molecules of component 2 is characterized by the interaction parameter χ. 2. The c interaction parameters characterizing a given system vary with composition, molar mass, and temperature. 3. B is an alternative parameter to χ, where B = cRT/Vm, in which Vm is the molar volume of one of the components of the mixture.
2.9 Cloud point
2.5 Nucleation of phase separation
Curve of temperature vs. composition defined by the cloud points (see 2.9) over range of compositions of two substances.
Initiation of phase domain formation through the presence of heterogeneities.
Experimentally measured point in the phase diagram of a mixture at which a loss in transparency is observed due to light scattering caused by a transition from a single- to a two-phase state. Notes:
2.10 Cloud-point curve
Note: Mixtures are observed to undergo a transition from a single- to a two-phase state upon heating or cooling.
Notes:
1. See the Gold Book, p. 277 [3]. 2. In a metastable region of a phase diagram (see 1.2), phase separation is initiated only by nucleation.
2.6 Binodal binodal curve coexistence curve
Curve defining the region of composition and temperature in a phase diagram for a binary mixture across which a transition occurs from miscibility of the components to conditions where single-phase mixtures are metastable or unstable (see note 4 in 1.2). Note: Binodal compositions are defined by pairs of points on the curve of Gibbs energy of mixing vs. composition that have common tangents, corresponding to compositions of equal chemical potentials of each of the two components in two phases.
2.7 Spinodal spinodal curve
Curve defining the region of composition and temperature for a binary mixture across which a transition occurs from conditions where single-phase mixtures are metastable to conditions where single-phase mixtures are unstable and undergo phase separation by spinodal decomposition (see 2.8). Notes:
1. The spinodal curve for a binary mixture is defined as the geometrical locus of all states with
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∂ 2 ∆ mix G ∂φ 2
T, P
= 0 (see 1.2, note 4 )
1. The phenomenon is characterized by the first appearance of turbidity or cloudiness. 2. A cloud point is heating rate- or cooling rate-dependent.
2.11 Cloud-point temperature
Temperature at a cloud point (see 2.9).
2.12 Critical point
Point in the isobaric temperature-composition plane for a binary mixture where the compositions of all coexisting phases become identical. Notes:
1. An alternative definition of “critical solution point” refers strictly to liquid-vapor equilibria (see the Gold Book, p. 93) [3]. 2. Unless specified atmospheric pressure is assumed. 3. In a phase diagram, the slope of the tangent to the spinodal is zero at this point. 4. At a critical point, binodals and spinodals coincide. 5. Although the definition holds strictly for binary mixtures, it is often erroneously applied to multicomponent mixtures. 6. See note 3 in 1.2.
2.13 Lower critical solution temperature Recommended acronym: LCST
Critical temperature below which a mixture is miscible. Notes:
1. See the Gold Book, p. 93 [3]. 2. Below the LCST and above the UCST (see 2.14), if it exists, a single phase exists for all compositions.
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3. The LCST depends upon pressure and the molar-mass distributions of the constituent polymer(s). 4. For a mixture containing or consisting of polymeric components, these may be different polymers or species of different molar mass of the same polymer.
2.14 Upper critical solution temperature Recommended abbreviation: UCST
Critical temperature above which a mixture is miscible.
Notes:
1. See the Gold Book, p. 93 [3]. 2. Above the UCST and below the LCST (see 2.13), if it exists, a single phase exists for all compositions 3. The UCST depends upon the pressure and molar-mass distributions of the constituent polymer(s). 4. For a mixture containing or consisting of polymeric components, these may be different polymers or species of different molar mass of the same polymer.
2.15 Phase inversion
Process by which an initially continuous phase domain becomes the dispersed phase domain and the initially dispersed phase domains become the continuous phase domain. Notes:
1. See the Gold Book, p. 299 [3]. 2. Phase inversion may be observed during the polymerization or melt processing of polymer blend systems. 3. The phenomenon is usually observed during polymerization of a monomer containing a dissolved polymer.
2.16 Interdiffusion
Process by which homogeneity in a mixture is approached by means of spontaneous mutual molecular diffusion.
2.17 Blooming
3. Domains and morphologies Many types of morphologies have been reported in the literature of multiphase polymeric materials. It is the intent of this document to define only the most commonly used terms. In addition, some morphologies have historically been described by very imprecise terms that may not have universal meanings. However, if such terms are widely used they are defined here.
3.1 Morphology
Shape, optical appearance, or form of phase domains in substances, such as high polymers, polymer blends, composites, and crystals. Note: For a polymer blend or composite, the morphology describes the structures and shapes observed, often by microscopy or scattering techniques, of the different phase domains present within the mixture.
3.2 Phase domain
Region of a material that is uniform in chemical composition and physical state.
Process in which one component of a polymer mixture, usually not a polymer, undergoes phase separation and migration to an external surface of the mixture.
2.18 Coalescence
Process in which two phase domains of essentially identical composition in contact with one another form a larger phase domain. Notes:
1. See the Gold Book, p. 75 [3]. 2. Coalescence reduces the total interfacial area. 3. The flocculation of a polymer colloid, through the formation of aggregates, may be followed by coalescence.
2.19 Morphology coarsening phase ripening
Process by which phase domains increase in size during the aging of a multiphase material. Notes:
1. In the coarsening at the late stage of phase separation, volumes and compositions of phase domains are conserved.
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2. Representative mechanisms for coarsening at the late stage of phase separation are: (1) material flow in domains driven by interfacial tension (observed in a co-continuous morphology), (2) the growth of domain size by evaporation from smaller droplets and condensation into larger droplets, and (3) coalescence (fusion) of more than two droplets. The mechanisms are usually called (1) Siggia’s mechanism, (2) Ostwald ripening (or the Lifshitz-Slyozov mechanism), and (3) coalescence. 3. Morphology coarsening can be substantially stopped by, for example, vitrification, crosslinking, and pinning, the slowing down of molecular diffusion across domain interfaces.
Notes:
1. A phase in a multiphase material can form domains differing in size. 2. The term “domain” may be qualified by the adjective microscopic or nanoscopic or the prefix micro- or nano- according to the size of the linear dimensions of the domain. 3. The prefixes micro-, and nano- are frequently incorrectly used to qualify the term “phase” instead of the term “domain”; hence, “microphase domain”, and “nanophase domain” are often used. The correct terminology that should be used is phase microdomain and phase nanodomain.
3.3 Multiphase copolymer
Copolymer comprising phase-separated domains.
3.4 Domain interface domain boundary
Surface forming a boundary between two phase domains. Note: A representation of the domain interface as a twodimensional surface over-simplifies the actual structure. All interfaces have a third dimension, namely, the interphase or interfacial region (see 3.6).
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Definitions of Terms Related to Polymer Blends, Composites, and Multiphase Polymeric Materials 3.5 Domain structure
Morphology of individual phase domains in a multiphase system. Note: Domain structures may be described for phase domains or domains that are themselves multiphased structures.
3.12 Continuous phase domain matrix phase domain
Phase domain (see 3.2) consisting of a single phase in a heterogeneous mixture through which a continuous path to all phase domain boundaries may be drawn without crossing a phase domain boundary.
3.6 Interfacial region interphase
Note: In a polymer blend, the continuous phase domain is sometimes referred to as the host polymer, bulk substance, or matrix.
Region between phase domains in an immiscible polymer blend in which a gradient in composition exists. Note: See the Gold Book, p. 205 [3].
3.7 Phase interaction
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3.13 Discontinuous phase domain discrete phase domain dispersed phase domain
Molecular interaction between the components present in the interphases of a multiphase mixture.
Phase domain in a phase-separated mixture that is surrounded by a continuous phase but isolated from all other similar phase domains within the mixture.
Note: The interphase elasticity is the capability of a deformed interphase to return to its original dimensions after the force causing the deformation has been removed.
Note: The discontinuous phase domain is sometimes referred to as the guest polymer.
3.8 Interfacial-region thickness interphase thickness interfacial width
Linear extent of the composition gradient in an interfacial region. Notes:
1. See the Gold Book, p. 203 [3]. 2. The width at half the maximum of the composition profile across the interfacial region (see 3.6) or the distance between locations where df/dr (with f the composition of a component and r the distance through the interfacial region) has decreased to 1/e are used as measures of the interfacial-region thickness.
3.14 Dual phase domain continuity co-continuous phase domains
Topological condition, in a phase-separated, two-component mixture, in which a continuous path through either phase domain may be drawn to all phase domain boundaries without crossing any phase domain boundary.
3.15 Core-shell morphology
Two-phase domain morphology, of approximately spherical shape, comprising two polymers, each in separate phase domains, in which phase domains of one polymer completely encapsulate the phase domains of the other polymer. Note: This morphology is most commonly observed in copolymers or blends prepared in emulsion polymerization by the sequential addition and polymerization of two different monomer compositions.
3.9 Hard-segment phase domain
Phase domain of microscopic or smaller size, usually in a block, graft, or segmented copolymer (see 3.11), comprising essentially those segments of the polymer that are rigid and capable of forming strong intermolecular interactions. Note: Hard-segment phase domains are typically of 2–15 nm linear size.
3.16 Cylindrical morphology
Phase domain morphology, usually comprising two polymers, each in separate phase domains, in which the phase domains of one polymer are of cylindrical shape. Notes:
3.10 Soft-segment phase domain
Phase domain of microscopic or smaller size, usually in a block, graft, or segmented copolymer (see 3.11), comprising essentially those segments of the polymer that have glass transition temperatures lower than the temperature of use. Note: Soft-segment phase domains are often larger than hard-segment phase domains and are often continuous.
3.11 Segmented copolymer
Copolymer containing phase domains of microscopic or smaller size, with the domains constituted principally of single types of structural unit. Note: The types of domain in a segmented copolymer usually comprise hard- and soft-segment phase domains.
9168_Book.indb 895
1. Phase domains of the constituent polymers may alternate, which results in many cylindrical layers surrounding a central core domain. 2. Cylindrical morphologies can be observed, for example, in triblock copolymers.
3.17 Fibrillar morphology
Morphology in which phase domains have shapes with one dimension much larger than the other two dimensions. Note: Fibrillar phase domains have the appearance of fibers.
3.18 Lamellar domain morphology
Morphology in which phase domains have shapes with two dimensions much larger than the third dimension.
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896 Note: Plate-like phase domains have the appearance of extended planes that are often oriented essentially parallel to one another.
3.19 Microdomain morphology
Morphology consisting of phase microdomains. Notes:
1. See 3.2. 2. Microdomain morphologies are usually observed in block, graft, and segmented copolymers. 3. The type of morphology observed depends upon the relative abundance of the different types of structural units and the conditions for the generation of the morphology. The most commonly observed morphologies are spheres, cylinders, and lamellae.
3.20 Nanodomain morphology
Morphology consisting of phase nanodomains. Note: See 3.2.
3.21 Onion morphology
Multiphase morphology of roughly spherical shape that comprises alternating layers of different polymers arranged concentrically, all layers being of similar thickness.
3.22 Ordered co-continuous double gyroid morphology
Co-continuous morphology in which a set of two gyroid-based phase domains exhibits a highly regular, three-dimensional lattice-like morphology with Ia3d space group symmetry. Notes:
1. The domains are composed of tripoidal units as the fundamental building structures. 2. The two domains are interlaced.
3.23 Multicoat morphology
Morphology observed in a blend of a block copolymer with the homopolymer of one of the blocks and characterized by alternating concentric shells of the copolymer and the homopolymer. Note: The morphology is identical to onion morphology (see 3.21) within a matrix of homopolymer [6].
3.24 Rod-like morphology
Morphology characterized by cylindrical phase domains.
3.25 Multiple inclusion morphology salami-like morphology
Multiphase morphology in which dispersed phase domains of one polymer contain and completely encapsulate many phase
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Handbook of Biochemistry and Molecular Biology domains of a second polymer that may have the same composition as the continuous phase domain (see 3.12).
References 1. IUPAC. “Definitions of terms relating to crystalline polymers (IUPAC Recommendations 1988)” Pure Appl. Chem. 61, 769–785 (1989). 2. IUPAC. “Definitions of basic terms relating to low-molar-mass and polymer liquid crystals (IUPAC Recommendations 2001)”, Pure Appl. Chem. 75, 845–895 (2001). 3. IUPAC. Compendium of Chemical Terminology (the “Gold Book”), compiled by A. D. McNaught and A. Wilkinson, 2nd ed., Blackwell Science, Oxford (1997). 4. IUPAC. “Glossary of basic terms in polymer science (IUPAC Recommendations 1996)”, Pure Appl. Chem. 68, 2287–2311 (1996). 5. D. K. Carpenter. “Solution properties”, in Encyclopedia of Polymer Science and Engineering, Vol. 15, 2nd ed., J. I. Kroschwitz (Ed.), pp. 419–481, Wiley Interscience, New York (1989). 6. J. M. G. Cowie. “Miscibility”, in Encyclopedia of Polymer Science and Engineering, 2nd ed., J. I. Kroschwitz (Ed.), Supplement, pp. 455– 480, Wiley Interscience, New York (1989).
Bibliography 1. IUPAC. “Definitions of terms relating to degradation, aging, and related chemical transformations of polymers (IUPAC Recommendations 1996)”, Pure Appl. Chem. 68, 2313–2323 (1996). 2. ASTM Glossary of ASTM Definitions, 2nd ed., American Society for Testing and Materials, Philadelphia, PA (1973). 3. IUPAC. Compendium of Macromolecular Nomenclature (the “Purple Book”), prepared for publication by W. V. Metanomski, Blackwell Scientific, Oxford (1991). 4. A. N. Gent and G. R. Hamed. “Adhesion”, in Encyclopedia of Polymer Science and Engineering, Vol. 1, 2nd ed., J. I. Kroschwitz (Ed.), pp. 476–517, Wiley Interscience, New York (1985). 5. L. Leibler. “Phase transformations”, in Encyclopedia of Polymer Science and Engineering, Vol. 11, 2nd ed., J. I. Kroschwitz, (Ed.), pp. 30–45, Wiley Interscience, New York (1988). 6. J. Koberstein. “Interfacial properties”, in Encyclopedia of Polymer Science and Engineering, Vol. 8, 2nd ed., J. I. Kroschwitz (Ed.), pp. 237–279, Wiley Interscience, New York (1987). 7. D. W. Fox and R. B. Allen. “Compatibility”, in Encyclopedia of Polymer Science and Engineering, Vol. 3, 2nd ed., J. I. Kroschwitz (Ed.), pp. 758–775, Wiley Interscience, New York (1985). 8. R. A. Orwoll. “Solubility of polymers”, Encyclopedia of Polymer Science and Engineering, Vol. 15, 2nd ed., J. I. Kroschwitz (Ed.), pp. 380–402, Wiley Interscience, New York (1989). 9. L. H. Sperling. “Microphase structure”, in Encyclopedia of Polymer Science and Engineering, Vol. 9, 2nd ed., J. I. Kroschwitz (Ed.), pp. 760–788, Wiley Interscience, New York (1987). 10. D. R. Paul, J. W. Barlow, and H. Keskkula. “Polymer blends”, in Encyclopedia of Polymer Science and Engineering, Vol. 12, 2nd ed., J. I. Kroschwitz (Ed.), pp. 399–461, Wiley Interscience, New York (1988). 11. D. R. Paul and S. Newman. Polymer Blends, Academic Press, New York (1978). 12. D. R. Paul and C. B. Bucknall. Polymer Blends: Formulation and Performance, John Wiley, New York (1999). 13. L. A. Utracki. Polymer Alloys and Blends, Hanser Publishers, New York (1990).
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897
Alphabetical Index of Terms Term additive adhesion adhesion promoter adhesive strength agglomerate agglomeration aggregate aggregation binodal binodal curve blooming bulk substance chemical adhesion cloud point cloud-point curve cloud-point temperature co-continuous phase domains coalescence coexistence curve compatibility compatibilization compatibilizer compatible polymer blend complex composite continuous phase domain core-shell morphology coupling agent craze critical point cylindrical morphology degree of compatibility degree of incompatibility degree of ripening delamination discontinuous phase domain discrete phase domain dispersant dispersed phase domain dispersing agent dispersing aid dispersion domain boundary domain interface domain structure dual phase domain continuity exfoliation extender fibrillar morphology fill factor filler Flory–Huggins theory Flory–Huggins–Staverman theory guest polymer hard-segment phase domain heterogeneous polymer blend homogeneous polymer blend homologous polymer blend host polymer immiscibility immiscible polymer blend impregnation intercalation interdiffusion interfacial adhesion interfacial agent interfacial bonding interfacial energy interfacial fracture interfacial region
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Definition No. 1.30 1.24 1.37 1.26 1.42 1.41 1.42 1.41 2.6 2.6 2.17 3.12 1.25 2.9 2.10 2.11 3.14 2.18 2.6 1.32 1.33 1.36 1.35 1.6 1.13 3.12 3.15 1.37 1.29 2.12 3.16 1.34 1.34 1.20 1.18 3.13 3.13 1.40 3.13 1.40 1.40 1.39 3.4 3.4 3.5 3.14 1.22 1.43 3.17 1.45 1.44 2.3 2.3 3.13 3.9 1.12 1.3 1.4 3.12 1.11 1.12 1.19 1.21 2.16 1.26 1.31 1.27 1.26 1.28 3.6
Term interfacial-region thickness interfacial tension interfacial width interpenetrating polymer network (IPN) interphase interphase elasticity interphase thickness isomorphic polymer blend lamellar domain morphology lamina laminate lamination lower critical solution temperature (LCST) macrodispersion matrix matrix phase domain metastable miscibility metastable miscible polymer blend microdispersion microdomain morphology miscibility miscibility gap miscibility window miscible polymer blend morphology morphology coarsening multicoat morphology multiphase copolymer multiple inclusion morphology nanocomposite nanodomain morphology nucleation of phase separation onion morphology ordered co-continuous double gyroid morphology phase domain phase interaction phase inversion phase microdomain phase nanodomain phase ripening pinning polymer alloy polymer blend polymer composite polymer–polymer complex prepreg rod-like morphology salami-like morphology segmented copolymer sequential IPN sequential SIPN semi-interpenetrating polymer network (SIPN) simultaneous IPN simultaneous SIPN soft-segment phase domain spinodal spinodal curve spinodal decomposition spinodal phase-demixing tack thermoplastic elastomer upper critical solution temperature (UCST) wetting c interaction parameter
Definition No. 3.8 1.26 3.8 1.9 3.6 3.7 3.8 1.5 3.17 1.16 1.16 1.17 2.13 1.39 3.12 3.12 1.7 1.8 1.39 3.19 1.2 2.2 2.1 1.3 3.1 2.19 3.23 3.3 3.25 1.15 3.20 2.5 3.21 3.22 3.2 3.7 2.15 3.2 3.2 2.19 2.19 1.38 1.1 1.14 1.6 1.20 3.24 3.25 3.11 1.9 1.10 1.10 1.9 1.10 3.10 2.7 2.7 2.8 2.8 1.26 1.46 2.14 1.23 2.4
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Organic Name Reactions Useful in Biochemistry and Molecular Biology Akabori Amino Acid Reaction NH2
O
R
O2/Carbohydrate
O
R
C H2
C H2 OH
H +
CO2 + NH3 HCl/EtOH
Na/Hg NH2 R
O C H2 H
Reaction in the presence of hydrazine yields hydrazides which can be coupled to aromatic aldehydes O H2N
CH
O H N
C
CH
C
O H N
CH3
CH3
CH
C
OH
CH3
Hydrazine/Heat
O
O H2N
CH CH3
C
H N
NH2
+
H2N
CH
C
O H N
NH2
+
H2N
CH3
CH
C
OH
CH3
Bose, A.K., et al., Microwave enhanced Akabori reaction for peptide analysis, J.Am.Soc.Mass Spectrom. 13, 839–850, 2002
Originally devised as a method for the conversion of amino acids or amino acid esters to aldehydes. The Akabori reaction has been modified for use in the determination of C-terminal amino acids by performing the reaction in the presence of hydrazine and for the production of derivatives useful for mass spectrometric identification. Ambach, E. and Beck, W., Metal-complexes with biologically important ligands. 35. Nickel, cobalt, palladium, and platinum complexes with Schiff-bases of a-amino acids – A contribution to the mechanism of the Akabori reaction, Chemische Berichte-Recueil 118, 2722-2737, 1985; Bose, A.K., Ing, Y.H., Pramanik, B.N., et al., Microwave enhanced Akabori reaction for peptide analysis, J.Am.Soc.Mass Spectrom. 13, 839-850, 2002; Pramanik, B.N., Ing, Y.H., Bose, A.K., et al., Rapid cyclopeptide analysis by microwave enhanced Akabori reaction, Tetrahedron Lett. 44, 2565-2568, 2003; Puar, M.S., Chan, T.M., Delgarno, D., et al., Sch 486058: A novel cyclic peptide of actinomycete origin, J.Antibiot. 58, 151-154, 2005.
899
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Handbook of Biochemistry and Molecular Biology
900
ORGANIC NAME REACTIONS USEFUL IN BIOCHEMISTRY AND MOLECULAR BIOLOGY (Continued) Aldol Condensation O
OH
O
O
+ H3C
H3C
H
C H2
H3C
H
H
O
O HO
NH2
HO
O
NH2 O
5-Aminolevulinic acid
5-Aminolevulinic acid
H2 C
H2N
H N
H2C H2C O
HO
CH2 OH
O Porphobilinogen O O
OH
OH C
CH2
O
O O CoA H3C
S
HO
C
C
OH
C
+ H2C
OH
H2C
OH
Acetyl-coenzyme A O Oxaloacetic acid
O Citrate
Citrate synthase an aldol-like condensation
OH
O P
OH
O P
HO
H2C
O
C
O
OH
H2C
O
C
O
H2C
OH
Dihydroxyacetone phosphate
CH HC
OH
HC
OH
H2C
Fructose 1,6-bisphosphate aldolase a retro aldol condensation
O CH OH
HC
O
O
H2C
O
P
O P
HO OH
9168_Book.indb 900
OH
HO
OH Glyceraldehyde-3-phosphate
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Organic Name Reactions Useful in Biochemistry and Molecular Biology
901
ORGANIC NAME REACTIONS USEFUL IN BIOCHEMISTRY AND MOLECULAR BIOLOGY (Continued) Condensation of one carbonyl compound with the enol/enolate form of another to form a b-hydroxyaldehyde; the base-catalyzed reaction proceeds via the enolate form while the acid-catalyzed reaction proceeds via the enol form. The basic chemistry of the aldol condensation is observed in several enzymatic reactions including citrate synthase, fructose-1,6-bisphosphate aldolase, and 2-keto-4-hydroxyglutarate aldolase. See Lane, R.S., Hansen, B.A., and Dekker, E.E., Sulfhydryl groups in relation to the structure and catalytic activity of 2-oxo-4-hydroxyglutarate aldolase from bovine liver, Biochim.Biophys.Acta 481, 212-221, 1977; Evans, D.A. and McGee, L.R., Aldol diastereoselection. Zirconium enolates. Product selective, enolate structure independent condensations, Tetrahedron Lett. 21, 3975-3978, 1980; Grady, S.R., Wang, J.K., and Dekker, E.E., Steady-state kinetics and inhibition studies of the aldol condensation reaction catalyzed by bovine liver and Escherichia coli 2-keto-4-hydroxyglutarate aldolase, Biochemistry 20, 2497-2502, 1981; Rokita, S.E., Srere, P.A., and Walsh, C.T., 3-Fluoro-3-deoxycitrate: A probe for mechanistic study of citrate-utilizing enzymes, Biochemistry 21, 3765-3774, 1982; Frere, R., Nentwich, M., Gacond, S., et al., Probing the active site of Pseudomonas aeruginosa porphobilinogen synthase using newly developed inhibitors, Biochemistry, 45, 8243-8253, 2006; Dalsgaard, T.K., Nielsen, J.H., and Larsen, L.B., Characterization of reaction products formed in a model reaction between pentanal and lysine-containing oligopeptides, J.Agric.Food Chem. 54, 6367-6373, 2006. A crossed aldol refers to a condensation reaction with two different aldehydes/ketones; the second aldehyde frequently is formaldehyde as it cannot react with itself although this is not a requirement (Kiehlman, E. and Loo, P.W., Orientation in crossed aldol condensation of chloral with unsymmetrical aliphatic ketones, Canad.J.Chem. 49, 1588, 1971; Findlay, J.A., Desai, D.N., and McCaulay, J.B., Thermally induced crossed aldol condensations, Canad.J.Chem. 59, 3303-3304, 1981; Esmaelli, A.A., Tabas, M.S., Nasseri, M.A., and Kazemi, F., Solvent-free crossed aldol condensation of cyclic ketones with aromatic aldehydes assisted by microwave irradiation, Monatshefte fur Chemie 136, 571-576, 2005). Amadori Rearrangement R
R NH
NH
H
OH
H
OH
CH2 HO
O
Amadori rearrangement A reaction following the formation of the unstable reaction product between an aldehyde (reducing sugar) and an amino group (formation of a Schiff base, an aldimine) which results in a more stable ketoamine. The Amadori rearrangement is part of the Maillard reaction which is also called the Browning reaction and can result in the formation of advanced glycation end products. See Amadori, M. Atti.Accad.Nazl.Lincei 2, 337, 1925; Hodge, J.E., The Amadori rearrangement, Adv.Carbohydrate Chem. 10, 169-205, 1955; Acharya, A.S. and Manning, J.M., Amadori rearrangement of glyceraldehyde-hemoglobin Schiff based adducts. A new procedure for the determination of ketoamine adducts in proteins, J.Biol.Chem. 255, 7218-7224, 1980; Acharya, A.S. and Manning, J.M., Reaction of glycoaldehyde with proteins: latent crosslinking potential of a-hydroxyaldehydes, Proc. Natl.Acad.Sci.USA 80, 3590-3594, 1983; Roper, H., Roper, S., and Meyer, B., Amadori- and N-nitroso-Amadori compounds and their pyrolysis products. Chemical, analytical and biological aspects, IARC Sci.Publ. (57), 101-111, 1984; Baynes, J.W., Watkins, N.G., Fisher, C.I. et al., The Amadori product on protein: structure and reactions, Prog.Clin.Biol.Res. 304, 43-67, 1989; Nacharaju, P. and Acharya, A.S., Amadori rearrangement potential of hemoglobin at its glycation sites is dependent on the three-dimensional structure of protein, Biochemistry 31, 12673-12679, 1992; Zyzak, D.V., Richardson, J.M., Thorpe, S.R., and Baynes, J.W., Formation of reactive intermediates from Amadori compounds under physiological conditions, Arch. Biochem.Biophys. 316, 547-554, 1995; Khalifah, R.G., Baynes, J.W., and Hudson, B.G., Amadorins: novel post-Amadori inhibitors of advanced glycation reactions, Biochem.Biophys.Res.Commun. 257, 251-158,1999; Davidek, T., Clety, N., Aubin, S., and Blank, I., Degradation of the Amadori compound N-(1-deoxy-D-fructos-1-yl)glycine in aqueous model system, J.Agric.Food Chem. 50, 5472-5479, 2002. Baeyer-Villiger Reaction R´
R
R
O R´
Peroxyacid O
O Baeyer−Villiger reaction
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902
ORGANIC NAME REACTIONS USEFUL IN BIOCHEMISTRY AND MOLECULAR BIOLOGY (Continued) The oxidation of a ketone by a peroxy acid to yield an ester. This reaction is catalyzed by bacterial monooxygenases and has proved useful in preparation of optically pure esters and lactones. See Ryerson, C.C., Ballou, D.P. and Walsh, C., Mechanistic studies on cyclohexanone oxygenase, Biochemistry 21, 2644-2655, 1982; Bolm, C., Metal-catalyzed asymmetric oxidations, Med.Res.Rev. 19, 348-356, 1999; Zambianchi, F., Pasta, P., Carrea, G., et al., Use of isolated cyclohexanone monooxygenase from recombinant Escherichia coli as a biocatalyst for Baeyer-Villiger and sulfide oxidations, Biotechnol.Bioeng. 78, 489-496, 2002; Alphand, V., Carrea, G., Wohlgemuth, R., et al., Towards large-scale synthetic application of Baeyer-Villiger monooxygenase, Trends Biotechnol. 21, 318-323, 2003; Walton, A.Z. and Stewart, J.D., Understanding and improving NADPH-dependent reactions by nongrowing Escherichia coli cells, Biotechnol.Prog. 20, 403-411, 2004; Malito, E., Alfieri, A., Fraaije, M.W., and Mattevi, A., Crystal structure of a Baeyer-Villiger monooxygenase, Proc.Nat.Acad.Sci.USA 101, 13157-13162, 2004; ten Brink, G.J., Arends, I.W., And Sheldon, R.A., The Baeyer-Villiger reaction: new developments toward greener procedures, Chem.Rev. 104, 4105-4124, 2004; Boronat, M., Corma. A., Renz, M., et al., A multisite molecular mechanism for Baeyer-Villiger oxidations on solid catalysts using environmentally friendly H2O2 as oxidant, Chemistry 11, 6905-6915, 2005; Mihovilovic, M.D., Rudroff, E., Winninger, A., et al., Microbial Baeyer-Villiger oxidation: stereopreference and substrate acceptance of cyclohexanone monooxygenase mutants prepared by directed evolution, Org.Lett. 8, 1221-1224, 2006; Baldwin, C.V. and Woodley, J.M., On oxygen limitation in a whole cell biocatalytic Baeyer-Villiger oxidation process, Biotechnol.Bioeng. 95, 362-369, 2006. Beckmann Rearrangement R´
R´ HN Acid (protic or Lewis) N
R
R
O
OH Oxime
Amide
Beckmann rearrangement
An acid (protic or Lewis)-catalyzed conversion of an oxime to a substituted carboxylic amide. See Darling, C.M. and Chen, C.P., Rearrangement of N-benzyl-2-cyano(hydroxyimino)acetamide, J.Pharm.Sci. 67, 860-861, 1978; Gayen, A.K., and Knowles, C.O., Penetration and fate of methomyl and its oxime metabolite in insects and two spotted spider mites, Arch.Environ.Contam.Toxicol. 10, 55-67, 1981; Mangold, J.B., Mangold, B.L., and Spina, A., Rat liver aryl sulfotransferase-catalyzed sulfation and rearrangement of 9-fluorenone oxime, Biochim.Biophys.Acta 874, 37-43, 1986; De Luca, L., Giacomelli, G., and Procheddu, A., Beckmann rearrangement of oximes under very mild conditions, J.Org.Chem. 67, 6272-6274, 2002; Torisawa, Y. Nishi, T., and Minamikawa, J., A study on the conversion of indanones into carbostyrils, Bioorg.Med.Chem. 11, 2205-2209, 2003; Furuya, Y., Ishihara, K., and Yamamoto, H., Cyanuric chloride as a mild and active Beckmann rearrangement catalyst, J.Am.Chem.Soc. 127, 11240-11241, 2005; Yamabe, S., Tsuchida, N., and Yamazaki, S., Is the Beckmann rearrangement a concerted or stepwise reaction? A computational study, J.Org.Chem. 70,1063810644, 2005; Ichino, T., Arimoto, H., and Uemura, D., Possibility of a non-amino acid pathway in the biosynthesis of marine-derived oxazoles, Chem. Commun. (16), 1742-1744, 2006. Benzoin Condensation R
O
H HCN
CH
O
OH
R R The conversion of benzaldehyde to benzoin (aromatic a-hydroxyketones) via cyanide-mediated condensation; other aromatic aldehydes can participate in this reaction. See Iding, H., Dunnwald, T., Greiner, L., et al., Benzoylformate decarboxylase from Pseudomonas putida as stable catalyst for the synthesis of chiral 2-hydroxy ketones, Chemistry 6, 1483-1495, 2000; White, M.J. and Leeper, F.J., Kinetics of the thiazolium ion-catalyzed benzoin condensation, J.Org.Chem. 66, 5124-5131, 2001; Dunkelmann, P., Kolter-Jung, D., Nitsche, A. et al., Development of a donor-acceptor concept for enzymatic cross-coupling reactions of aldehydes: the first asymmetric cross-benzoin condensation, J.Am.Chem.Soc. 124, 12084-12085, 2002; Pohl, M., Lingen, B., and Muller, M., Thiamin-diphosphate-dependent enzymes: new aspects of asymmetric C-C bond formation, Chemistry 8, 5288-5295, 2002; Wildemann, H., Dunkelmann, P., Muller, M., and Schmidt, B., A short olefin metathesis-based route to enantiomerically pure arylated dihydropyrans and a, b-unsaturated δ-valero lactones, J.Org.Chem. 68, 799-804, 2003; Murry, J.A., Synthetic methodology utilized to prepare substituted imidazole p38 MAP kinase inhibitors, Curr.Opin.Drug Discov.Devel. 6, 945-965, 2003; Reich, B.J., Justice, A.K., Beckstead, B.T., et al., Cyanide-catalyzed cyclizations via aldimine coupling, J.Org.Chem. 69, 1357-1359, 2004; Sklute, G., Oizerowich, R., Shulman, H., and Keinan, E., Antibody-catalyzed benzoin oxidation as a mechanistic probe for nucleophilic catalysis by an active site lysine, Chemistry 10, 2159-2165, 2004; Breslow, R., Determining the geometries of transition states by use of antihydrophobic additives in water, Acc.Chem.Res. 37, 471-478, 2004.
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903
ORGANIC NAME REACTIONS USEFUL IN BIOCHEMISTRY AND MOLECULAR BIOLOGY (Continued) Cannizzaro Reaction O
O
H
HO
H OH–
+
O
OH C
CH2
C
C
Cannizzaro reaction O
OH Base
CH
HO
CH2
HC
C
O
O
Glyoxal
Glycolate Internal Cannizzaro reaction H3C
+
HO
C
O
NH2
NH
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH
NH2
HO
O
C
CH
NH2
O
Lysine
Carboxymethyllysine
Base-catalyzed disproportionation of an aldehyde to yield a carboxylic acid and the corresponding alcohol; if an a-hydrogen is present, an aldol condensation is a competing reaction. See Hazlet, S.E. and Stauffer, D.A., Crossed Cannizzaro reactions, J.Org.Chem. 27, 2021-2024, 1962; Entezari, M.H. and Shameli, A.A., Phase-transfer catalysis and ultrasonic waves. I. Cannizzaro reaction, Ultrason.Sonochem. 7, 169-172, 2000; Matin, M.M., Sharma, T., Sabharwal, S.G., and Dhavale, D.D., Synthesis and evaluation of the glycosidase inhibitory activity of 5-hydroxy substituted isofagomine analogues, Org.Biomol.Chem. 3, 1702-1707, 2005; Zhang, L., Wang, S., Zhou, S., et al., Cannizzaro-type disproportionation of aromatic aldehydes to amides and alcohols by using either a stoichiometric amount or a catalytic amount of lanthanide compounds, J.Org.Chem. 71, 3149-3153, 2006. Intramolecular Cannizzaro reactions have been described (Glomb, M.A., and Monnier, V.M., Mechanism of protein modification by glyoxal and glycoaldehyde, reactive intermediates of the Maillard reaction, J.Biol.Chem. 270, 10017-10026, 1995; Russell, A.E., Miller, S.P., and Morken, J.P., Efficient Lewis acid catalyzed intramolecular Cannizzaro reaction, J.Org.Chem. 65, 8381-8383, 2000; Schramm, C. and Rinderer, B., Determination of cotton-bound glyoxal via an internal Cannizzaro reaction by means of high-performance liquid chromatography, Anal.Chem. 72, 5829-5833, 2000). Claisen Condensation CH3 C O
H3C
CH3
CH3 O
+
C O
CH2 H3C
O
CH2
Claisen condensation
9168_Book.indb 903
Base
H2C O
H3C
O
C O
CH2
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904
ORGANIC NAME REACTIONS USEFUL IN BIOCHEMISTRY AND MOLECULAR BIOLOGY (Continued) The base-catalyzed condensation of two moles of an ester to give a b-keto ester. Claisen condensations are more favorable with thioesters. This reaction is of great importance in the biosynthesis of fatty acids and polyketides (Haapalainen, A.M., Meriläinen, G., and Wierenga, R.K. The thiolase superfamily: condensing enzymes with diverse reaction specificities, Trends Biochem.Sci. 31, 64-71, 2006). For general issues see Dewar, M.J. and Dieter, K.M., Mechanism of the chain extension step in the biosynthesis of fatty acids, Biochemistry 27, 3302-3308, 1988; Clark, J.D., O’Keefe, S.J., and Knowles, J.R., Malate synthase: proof of a stepwise Claisen condensation using the double-isotope fractionation test, Biochemistry 27, 5961-5971, 1988; Nicholson, J.M., Edafiogho, I.O., Moore, J.A., et al., Cyclization reactions leading to b-hydroxyketo esters, J.Pharm.Sci. 83, 76-78, 1994; Lee, R.E., Armour, J.W., Takayama, K., et al., Mycolic acid biosynthesis: definition and targeting of the Claisen condensation step, Biochim.Biophys.Acta 1346, 275-284, 1997; Shimakata, T. and Minatogawa, Y., Essential role of trehalose in the synthesis and subsequent metabolism of corynomycolic acid is Corynebacterium matruchotil, Arch.Biochem.Biophys. 380, 331-338, 2000; Olsen, J.G., Madziola, A., von Wettstein-Knowles, P., et al., Structures of b-ketoacyl-acyl carrier protein synthase I complexed with fatty acids elucidate its catalytic machinery, Structure 9, 233-243, 2001; Klavins, M., Dipane, J., and Babre, K., Humic substances as catalysts in condensation reactions, Chemosphere 44, 737-742, 2001; Heath, R.J. and Rock, C.O., The Claisen condensation in biology, Nat.Prod.Rep. 19, 581-596, 2002; Takayama, K., Wang, C., and Besra, G.S., Pathway to synthesis and processing of mycolic acids in Mycobacterium tuberculosis, Clin.Microbiol.Rev. 18, 81-101, 2005; Ryu, Y., Kim, K.J., Roessner, C.A., and Scott, A.I., Decarboxylative Claisen condensation catalyzed by in vitro selected ribozymes, Chem.Commun. (13), 1439-1441, 2006; Kamijo, S. and Dudley, G.B., Claisen-type condensation of vinylogous acyl triflates, Org.Lett. 8, 175-177, 2006. Claisen Rearrangement R
R O
O
HO
O
O
OH
O
O
OH
OH O
CH2
OH
O
O
O OH
OH
Prephenic Acid Chorismic Acid Zhang Z. and Bruice T.C., Temperature dependence of the structure of the substrate and active site of the Thermus thermophilus chorismate mutase E-S complex, Biochemistry 45, 8562–8567, 2006 The rearrangement of an allyl vinyl ether or the nitrogen or sulfur analogue or allyl aryl ether to yield a γ, δ-unsaturated ketone or an o-allyl substituted phenol. See Hilvert, D., Carpenter, S.H., Nared, K.D., and Auditor, M.T., Catalysis of concerted reactions by antibodies: the Claisen rearrangement, Proc.Nat.Acad.Sci.USA 85, 4953-4955, 1988; Campbell, A.P., Tarasow, T.M., Massefski, W., et al., Proc.Nat.Acad.Sci.USA 90, 8663-8667, 1993; Swiss, K.A. and Firestone, R.A., Catalysis of Claisen rearrangement by low molecular weight polyethylene(1), J.Org.Chem. 64, 2158-2159, 1999; Berkowitz, D.B., Choi, S., and Maeng, J.H., Enzyme-assisted asymmetric total synthesis of (-)-podophyllotoxin and (-)-picropodophyllin, J.Org.Chem. 65, 847-860, 2000; Itami, K. and Yoshida, J., The use of hydrophilic groups in aqueous organic reactions, Chem.Rec. 2 213-224, 2002; Martin Castro, A.M., Claisen rearrangement over the past nine decades, Chem.Rev. 104, 2939-3002, 2004; Sparano, B.A., Shahi, S.P., and Koide, K., Effect of binding and conformation on fluorescence quenching in new 2’,7’-dichlorofluorescein derivatives, Org.Lett. 6, 1947-1949, 2004; Davis, C.J., Hurst, T.E., Jacob, A.M., and Moody, C.J., Microwave-mediated Claisen rearrangement followed by phenol oxidation: a simple route to naturally occurring 1,4-benzoquinones. The first synthesis of verapliquinones A and B and Panicein, A., J.Org.Chem. 70, 4414-4422, 2005; Wright, S.K., DeClue, M.S., Mandal, A., et al., Isotope effects on the enzymatic and nonenzymatic reactions of chorismate, J.Am.Chem.Soc. 127, 12957-12964, 2005; Declue, M.S., Baldridge, K.K., Kast, P., and Hilvert, D., Experimental and computational investigation of the uncatalyzed rearrangement and elimination reactions of isochorismate, J.Am.Chem.Soc. 128, 2043-2051, 2006; Zhang, X. and Bruice, T.C., Temperature dependence of the structure of the substrate and active site of the Thermus thermophilus chorismate mutase E-S complex, Biochemistry 45, 8562-8567, 2006.
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905
ORGANIC NAME REACTIONS USEFUL IN BIOCHEMISTRY AND MOLECULAR BIOLOGY (Continued) Criegee Reactions
O
R R
OH +
R’
R
O O
O Peroxyacid
R
O
R’
H
O R
R
Criegee Intermediate
O
O +
R’
OH
R
R
OH
+
R
Mostly the reaction of a peroxyacid with a tertiary alcohol to form a ketone and an alcohol. The intermediate peroxyester is an intermediate (Criegee adduct or Criegee intermediate) in the Baeyer-Villiger reaction. The Criegee intermediate is important in the ozonolysis of alkenes including fatty acids. See Leffler, J.E. and Scrivener, F.E., Jr., The decomposition of cumyl peracetate in nonpolar solvents, J.Org.Chem. 37, 1794-1796, 1978; Srisankar, E.V. and Patterson, L.K., Reactions of ozone with fatty acid monolayer: a model system for disruption of lipid molecular assemblies by ozone, Arch. Environ.Health 34, 346-349, 1979; Grammer, J.C., Loo, J.A., Edmonds, C.G., et al., Chemistry and mechanism of vanadate-promoted photooxidative cleavage of myosin, Biochemistry 35, 15582-15592, 1996; Krasutsky, P.A., Kolomitsyn, I.V., l Kiprof P., et al., Observation of a stable carbocation in a consecutive Criegee rearrangement with trifluoroperacetic acid, J.Org.Chem. 65, 3926-3992, 1996; Carlqvist, P., Eklund, P., Hult, K., and Brinck, T., Rational design of a lipase to accommodate catalysis of Baeyer-Villiger oxidation with hydrogen peroxide, J.Mol.Model. 9, 164-171, 2003; Deeth, R.J. and Bugg, T.D., A density functional investigation of the extradiol cleavage mechanism in non-heme iron catechol dioxygenease, J.Biol.Inorg.Chem. 8, 409-418, 2003; Krasutsky, P.A., Kolomitsyn, I.V., Krasutsky, S.G., and Kiprof, P., Double- and triple-consecutive O-insertion into tert-butyl and triarylmethyl structures, Org.Lett. 6, 2539-2542, 2004. Curtius Rearrangement O
O
SOCl2 R
OH
O
NaN3 Cl
R
∆ R
N3
N R
C
O
H2O H N
OH
R
CO2
NH2 R
O
The conversion of a carboxylic acid to an amine via an acid intermediate. See Inouye, K., Watanabe, K., and Shin, M., Formation and degradation of urea derivatives in the azide method of peptide synthesis. Part 1. The Curtius rearrangement and urea formation, J.Chem.Soc. (17), 1905-1911, 1977; Chorev, M., and Goodman, M., Partially modified retro-inverso peptides. Comparative Curtius rearrangements to prepare 1, 1-diaminoalkane derivatives, Int.J.Pept.Protein Res. 21, 258-268, 1983; Sasmal, S., Geyer, A., and Maier, M.E., Synthesis of cyclic peptidomimetics from aldol building blocks, J.Org.Chem. 67, 6260-6263, 2002; Kedrowski, B.L., Synthesis of orthogonally protected (R)- and (S)-2-methylcysteine via an enzymatic desymmeterization and Curtius rearrangement, J.Org.Chem. 68, 5403-5406, 2003; Englund, E.A., Gopi, H.N., and Appella, D.H., An efficient synthesis of a probe for protein function: 2,3-diaminopropionic acid with orthogonal protecting groups, Org.Lett. 6, 213-215, 2004; Spino, C., Tremblay, M.C., and Gobout, C., A stereodivergent approach to amino acids, amino alcohols, or oxazolidinones of high enantiomeric purity, Org.Lett. 6, 2801-2804, 2004; Brase, S., Gil, C., Knepper, K., and Zimmerman, V., Organic azides: an exploding diversity of a unique class of compounds, Angew.Chem.Int. Ed.Engl. 44, 5188-5240, 2005; Lebel, H. and Leogane, O., Boc-protected amines via a mild and efficient one-pot Curtius rearrangement, Org.Lett. 7, 4107-4110, 2005.
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906
ORGANIC NAME REACTIONS USEFUL IN BIOCHEMISTRY AND MOLECULAR BIOLOGY (Continued) Dakin Reaction O
R(H)
O
R(H)
OH
OH
H2O2/NaOH
or
OH
or
NH2
OH
NH2
Conversion of an aromatic ketone or aldehyde to a phenolic derivative with alkaline hydrogen peroxide. The mechanism is thought to be similar to the Baeyer-Villiger reaction possibly proceeding through a peroxyacid intermediate. The presence of an amino group or a hydroxyl group in the position para to the carbonyl function is required. See Corforth, J.W. and Elliott, D.F., Mechanism of the Dakin and West reaction, Science 112, 534-535, 1950. Dakin-West Reaction O
O H2N
CH
C
O
O
CH3
CH3
H N
OH Pyridine
H3C
CH3
O
O
C
CH
CH3
CH3
Dakin–West reaction
Conversion of amino acids to acetamidoketones via the action of acetic anhydride in base where a carboxyl group is replaced by an acyl group in a reaction proceeding through an oxazolone intermediate. This reaction has been used for the synthesis of enzyme inhibitors and receptor antagonists. See Angliker, H. Wikstrom, P., Rauber, P., et al., Synthesis and properties of peptidyl derivatives of arginylfluoromethanes, Biochem.J. 256, 481-486, 1988; Cheng, L., Goodwin, C.A., Schully, M.F., et al., Synthesis and biological activity of ketomethylene pseudopeptide analogues as thrombin inhibitors, J.Med.Chem. 35, 3364-3369, 1992; Godfrey, A.B., Brooks, D.A., Hay, L.A., et al, Application of the Dakin-West reaction for the synthesis of oxazole-containing dual PPARa/γ agonists, J.Org.Chem. 68, 2623-2632, 2003; Loksha, Y.M., el-Barbary, A.A., it-Barbary, M.A., et al., Synthesis of 2-(aminocarbonylmethylthio)-1H-imidazoles as novel Capravirine analogues, Bioorg.Med.Chem. 13, 4209-4220, 2005 Diels-Alder Condensation O HC
CH2
C
+
HC
O HC
CH2 trans-butadiene
HC
cis-butadiene diene
O
O
C
O Maleic anhydride dienophile
O
Diels–Alder condensation
A cycloaddition reaction between a conjugated diene and an alkene resulting in the formation of alkene ring; construction of a six-membered ring with multiple stereogenic centers resulting in a chiral molecule. See Wasserman, A., Diels-Alder Reactions. Organic Background and Physico-Chemical Aspects, Elsevier, Amsterdam, Netherlands, 1965; Fringuelli, F., and Taticchi, A., The Diels-Alder Reaction. Selected Practical Methods, John Wiley & Sons, Ltd., Chichester, UK, 2002; Stocking, E.M. and Williams, R.M., Chemistry and biology of biosynthetic Diels-Alder reactions, Angew.Chem.Int.Ed. 42, 3078-3115, 2003. See also Waller, R.L. and Recknagel, R.O., Determination of lipid conjugated dienes with tetracyanoethylene-14C: significance for study of the pathology of lipid peroxidation, Lipids 12, 914-921, 1977; Melucci, M., Barbarella, G., and Sotgiu, G., Solvent-free, microwave-assisted synthesis of thiophene oligomers via Suzuki coupling, J.Org.Chem. 67, 8877-8884, 2002; Breslow, R., Determining the geometries of transition states by use of antihydrophobic additives in water, Acc.Chem.Res. 37, 471-478, 2004; Conley, N.R., Hung, R.J., and Willison, C.G., A new synthetic route to authentic N-substituted aminomaleimides, J.Org.Chem. 70, 4553-4555, 2005; Boul, P.J., Reutenauer, P., and Lehn, J.M. Reversible Diels-Alder reactions for the generation of dynamic combinatorial libraries, Org.Lett. 7, 15-18, 2005. Catalytic antibodies have been used for Diels-Alder reactions (Suckling, C.J., Tedford, C.M., Proctor, G.R., et al., Catalytic antibodies: a new window on protein chemistry, Ciba Found.Symp. 159, 201-208, 1991; Meekel, A.A., Resmini, M., and Pandit, U.K., Regioselectivity and enantioselectivity in an antibody catalyzed hetero Diels-Alder reaction, Bioorg.Med.Chem. 4, 1051-1057, 1996; Romesberg, F.E., Spiller, B., Schultz, P.G., and Stevens, R.C., Immunological origins of binding and catalysis in a Diels-Alderase antibody, Science 279, 1934-1940, 1998; Romesberg, F.E. and Schultz, P.G., A mutational study of a Diels-Alderase catalytic antibody, Bioorg.Med. Chem.Lett. 9, 1741-1744, 1999; Chen, J., Deng, Q., Wang, R., et al., Shape complementarity binding-site dynamics, and transition state stabilization: a theoretical study of Diels-Alder catalysis by antibody IE9, ChemBioChem 1, 255-261, 2000; Kim, S.P., Leach, A.G., and Houk, K.N., The origins of noncovalent catalysis of intermolecular Diels-Alder reactions by cyclodextrins, self-assembling capsules, antibodies, and RNAses, J.Org.Chem. 67, 4250-4260, 2002; Cannizzaro, C.E., Ashley, J.A, Janda, K.D., and Houk, K.N., Experimental determination of the absolute enantioselectivity of an antibody-catalyzed Diels-Alder reaction and theoretical explorations of the origins of stereoselectivity, J.Am.Chem.Soc. 125, 2489-2506, 2003.
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907
ORGANIC NAME REACTIONS USEFUL IN BIOCHEMISTRY AND MOLECULAR BIOLOGY (Continued) Edman Degradation
CH3 HO
C
CH
CH3 H N
O
C
CH
CH3 H N
C
O
O
HO
C
CH
CH3 H N
O
C
CH
HO H N
S Phenylisothiocyanate CH
NH
C N
O
C S
H3O+
CH3 HO
C O
CH
C O
CH
CH3 O
CH NH
CH3 H N
N C
H3C CH3
+
NH2
CH
N NH2
C S
Phenylthiohydantoin The stepwise degradation of a peptide chain from the amino-terminal via reaction with phenylisothiocyanate. This process is used for the chemical determination of the amino acid sequence of a peptide or protein. See Edman, P., Sequence determination, Mol.Biol.Biochem.Biophys. 9, 211-255, 1970; Heinrikson, R.L., Application of automated sequence analysis to the understanding of protein structure and function, Ann.Clin.Lab.Sci. 8, 295-301, 1978; Tsugita, A., Developments in protein microsequencing, Adv.Biophys. 23, 91-113, 1987; Han, K.K. and Martinage, A., Post-translational chemical modifications of proteins – III. Current developments in analytical procedures of identification and quantitation of post-translational chemically modified amino acid(s) and its derivatives, Int.J.Biochem. 25, 957-970, 1993; Masiarz, F.R. and Malcolm, B.A., Rapid determination of endoprotease specificity using peptide mixtures and Edman degradation analysis, Methods Enzymol. 241, 302-310, 1994; Gooley, A.A., Ou, K., Russell, J., et al., A role for Edman degradation in proteome studies, Electrophoresis 18, 1068-1072, 1997; Wurzel, C., and Wittmann-Liebold, B., A wafer based micro reaction system for the Edman degradation of proteins and peptides, J.Protein Chem. 17, 561-564, 1998; Walk, T.B., Sussmuth, R., Kempter, C., et al., Identification of unusual amino acids in peptides using automated sequential Edman degradation coupled to direct detection by electrosprayionization mass spectrometry, Biopolymers 49, 329-340, 1999; Lauer-Fields, J.L., Nagase, H. and Fields, G.B., Use of Edman degradation sequence analysis and matrix-assisted laser desorption/ionization mass spectrometry in designing substrates for matrix metalloproteinases, J.Chromatog.A. 890, 117-125, 2000; Hajdu, J., Neutze, R., Sjogren, T., et al., Analyzing protein functions in four dimensions, Nat.Struct.Biol. 7, 1006-1012, 2000; Shively, J.E., The chemistry of protein sequence analysis, EXS 88, 99-117, 2000; Wang, P., Arabaci, G., and Pei, D., Rapid sequencing of library-derived peptides by partial Edman degradation and mass spectrometry, J.Comb.Chem. 3, 251-254, 2001; Brewer, M., Oost, T., Sukonpan, C., et al., Sequencing hydroxylethyleneamine-containing peptides via Edman degradation, Org.Lett. 4, 3469-3472, 2002; Sweeney, M.C. and Pei, D., An improved method for rapid sequencing of support-bound peptides by partial Edman degradation and mass spectrometry, J.Comb.Chem. 5, 218-222, 2003; Buda, F., Ensing, B., Gribnau, M.C., and Baerends, E.J., O2 evolution in the Fenton reaction, Chemistry 9, 3436-3444, 2003; Liu, Q., Berchner-Pfannschmidt, U., Moller, U., et al., A Fenton reaction at the endoplasmic reticulum is involved in the redox control of hypoxia-inducible gene expression, Proc.Nat.Acad.Sci. USA 101, 4302-4307, 2004; Maksimovic, V., Mojovic, M., Neumann, G., and Vucinic, Z., Nonenzymatic reaction of dihydroxyacetone with hydrogen peroxide enhanced via a Fenton reaction, Ann.N.Y.Acad.Sci. 1048, 461-465, 2005; Lu, C. and Koppenol, W.H., Inhibition of the Fenton reaction by nitrogen monoxide, J.Biol.Inorg.Chem. 10, 732-738, 2005; Baron, C.P., Refsgaard, H.H., Skibsted, H., and Andersen, M.L., Oxidation of bovine serum albumin initiated by the Fenton reaction—effect of EDTA, tert-butylhydroperoxide and tetrahydrofuran, Free Radic.Res. 40, 409-417, 2006; Thakkar, A., Wavreille, A.S., and Pei, D., Traceless capping agent for peptide sequencing by partial Edman degradation and mass spectrometry, Anal.Chem. 78, 5935-5939, 2006.
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908
ORGANIC NAME REACTIONS USEFUL IN BIOCHEMISTRY AND MOLECULAR BIOLOGY (Continued) Eschweiler-Clarke Reaction R
O
R
H C H
R
NH2
CH3 R
H C
N H
OH
O
Eschweiler–Clarke reaction
The reductive methylation of amines with formaldehyde in the presence of formic acid. See Lindeke, B., Anderson, B., and Jenden, D.J., Specific deuteromethylation by the Escheweiler-Clarke reaction. Synthesis of differently labelled variants of trimethylamine and their use of the preparation of labelled choline and acetylcholine, Biomed.Mass Spectrom. 3, 257-259, 1976; Boldavalli, F., Bruno, O., Mariani, E., et al., Esters of N-methyl-N-(2hydroxyethyl or 3-hydroxypropyl)-1,3,3-trimethylbicyclo[2.2.1] heptan-2-endo-amine with hypotensive activity, Farmaco 42, 175-183, 1987; Lee, S.S., Wu, W.N., Wilton, J.H., et al., Longiberine and O-methyllogiberine, dimeric protoberberine-benzyl tetrahydroisoqunioline alkaloids from Thalictrum longistrylum, J.Nat.Prod. 62, 1410-1414, 1999; Suma, R and Sai Prakash, P.K., Conversion of sertraline to N-methyl sertraline in embalming fluid: a forensic implication, J.Anal.Toxicol. 30, 395-399, 2006. The reaction can be accomplished with sodium borohydride or sodium cyanoborohydride and is related to the reductively methylation/alkylation of lysine residues in proteins (Lundblad, R.L., Chemical Reagents for the Modification of Proteins, 3rd edn., CRC Press, Boca Raton, FL, 2004). Favorskii Rearrangement O
O X
R R’’
R
–OR’’’
R’’’
R’’ O
R’
R’ O
O
OH X
NaOH
Favorskii rearrangement
The rearrangement of an a-ketone in the presence of an alkoxide to form a carboxylic ester; cyclic a-ketone undergo ring contraction. March, J. Advanced Organic Chemistry. Reactions, Mechanisms, and Structures, 3rd edn. John Wiley & Sons, New York, New York, 1985; Gardner, H.W., Simpson, T.D., and Hamberg, M., Mechanism of linoleic acid hydroperoxide reaction with alkali, Lipids 31, 1023-1028, 1996; Xiang, L., Kalaitzis, J.A., Nilsen, G. et al., Mutational analysis of the enterocin favorskii biosynthetic rearrangement, Org.Lett. 4, 957-960, 2002; Zhang, L. and Koreeda, M., Stereocontrolled synthesis of kelsoene by the homo-favorskii rearrangement, Org.Lett. 4, 3755-3788, 2002; Grainger, R.S., Owoare, R.B., Tisselli, P., and Steed, J.W., A synthetic alternative to the type-II intramolecular 4 + 3 cycloaddition, J.Org.Chem. 68, 7899-7902, 2003. Fenton Reagent/Reaction . OH + –OH
H2O2 + Fe2+
O
OH R
C
OH
. OH
O
O HO
H2 C
CH2
C OH
R
. OH
O
H C
CH2 OH
OH
OH . OH .
OH
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909
ORGANIC NAME REACTIONS USEFUL IN BIOCHEMISTRY AND MOLECULAR BIOLOGY (Continued) The reaction of ferrous ions and hydrogen peroxide to yield a hydroxyl radical. See Aust, S.D., Morehouse, L.A., and Thomas, C.E., Role of metals in oxygen radical reactions, J.Free Radic.Biol.Med. 1, 3-25, 1985; Goldstein, S., Meyerstein, D. and Czapski, G., The Fenton reagents, Free Radic.Biol.Med. 15, 435-445, 1993; Wardman, P. and Candeias, L.P., Fenton chemistry: an introduction, Radiat.Res. 145, 523-531, 1996; Held, K.D., Sylvester, F.C., Hopcia, K.L., and Biaglow, J.E., Role of Fenton chemistry in the thiol-induced toxicity and apopotosis, Radiat.Res. 145, 542-553, 1996; Merli, C., Petrucci, E., Da Pozzo, A., and Pernetti, M., Fenton-type treatment: state of the art, Ann.Chim. 93, 761-770, 2003; Groves, J.T., High-valent iron in chemical and biological oxidations, J.Inorg.Biochem. 100, 434-447, 2006. Fischer Carbene Complexes O R1Li
Cr(CO)6 Chromium carbonyl
R2X
R2
C
Cr(CO)5
R1 Fischer carbene complex
O (OC)5Me
C
R +
R’
H2C NH2
CH2
H2C
H2C
NH
CH2 (OC)5Me CH2
R’
A Fischer carbene complex consists of a transition metal with a formal carbon-metal bond containing a carbene in the singlet state; stabilization of the carbene is provided by the metal interaction. The Fischer carbene complex is electrophilic as the carbene carbon as opposed to the Schrock complex which is in the triplet state and nucleophilic at the carbene carbon. The Fischer carbene complex is high reactivity and is used in many synthetic procedures. A example is provided by the a,b-unsaturated carbenepentacarbonylchromium complex (de Meijere, A., Schirmer, H., and Duetsch, M., Fischer carbene complexes as chemical multitalents: The incredible range of products from carbenepentacarbonylmetal a,b-unsaturated complexes, Angew.Chem.Int.Ed. 39, 3964-4002, 2000). See also Salmain, M., Blais, J.C., Tran-Huy, H., et al, Reaction of hen egg white lysozyme with Fischer-type metallocarbene complexes. Chacterization of the conjugates and determination of the metal complex binding sites, Eur.J.Biochem. 268, 5479-5487, 2001; Merlic, C.A. and Doroh, B.C., Amine-catalyzed coupling of aldehydes and ketenes derived from Fischer carbene complexes: formation of beta-lactones and enol ethers, J.Org.Chem. 68, 6056-6069, 2003; Barluenga, J., Santamaria, J., and Tomas, M., Synthesis of heterocycles via group VI Fischer carbene complexes, Chem.Rev. 104, 2259-2283, 2004; Barluenga, J., Fananas-Mastral, M., and Aznar, F., A new synthesis of allyl sulfoxides via nucleophilic addition of sulfinyl carbanions to group 6 Fischer carbene complexes, Org.Lett. 7, 1235-1237, 2005; Lian, Y. and Wulff, W.D., Iron in the service of chromium: the o-benzannulation of trans,trans-dienyl Fischer carbene complexes, J.Am.Chem.Soc. 127, 17162-17163, 2005; Barluenga, J., Mendoza, A., Dieguez, A., et al., Umpolung reactivity of alkenyl Fischer carbene complexes, copper enolates, and electrophiles, Angew.Chem.Int. Ed.Engl. 45, 4848-4850, 2006; Samanta, D., Sawoo, S. and Sarkar, A., In situ generation of gold nanoparticles on a protein surface: Fischer carbene complex as reducing agent, Chem.Commun. (32), 3438-3440, 2006; Rawat, M., Prutyanov, V., and Wulff, W.D., Chromene chromium carbene complexes in the syntheses of naphthoyran and naphthopyrandione units present in photochromic materials and biologically active natural products, J.Am.Chem.Soc. 128, 11044-11053, 2006. For general information on carbenes including Fischer carbene complexes and Schrock carbene complexes, see Carbene Chemistry. From Fleeting Intermediates to Powerful Reagents, ed. G. Bertrand, Fontis Media/Marcel Dekker, New York, New York, 2002.
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ORGANIC NAME REACTIONS USEFUL IN BIOCHEMISTRY AND MOLECULAR BIOLOGY (Continued) Fischer Indole Synthesis
NH N C
CH3
Protic or Lewis acid ∆
HN
Fischer indole synthesis R NH2
R
R’
N
O
NH +
C
H2 C
C H2
NH
R’
Lewis acid or protic acid R’
R N H
The thermal conversion of arylhydrazones in the presence of a protic acid or a Lewis acid to form an indole ring. See Owellen, R.J., Fitzgerald, J.A., Fitzgerald, B.M., et al., The cyclization phase of the Fischer indole synthesis. The structure and significance of Pleininger’s intermediate, Tetrahedron Lett. 18, 1741-1746, 1967; Kim, R.M., Manna, M., Hutchins, S.M. et al., Dendrimer-supported combinatorial chemistry, Proc.Natl.Acad.Sci.USA 93, 10012-10017, 1996; Brase, S., Gil, C., and Knepper, K., The recent impact of solid-phase synthesis on medicinally relevant benzoannelated nitrogen heterocycles, Bioorg.Med.Chem. 10, 2415-2437, 2002; Rosenbaum, C., Katzka, C., Marzinzik, A., and Waldmann, H., Traceless Fischer indole synthesis on the solid phase, Chem.Commun. (15), 1822-1823, 2003; Mun, H.S., Ham, W.H., and Jeong, J.H., Synthesis of 2,3-disubstituted indole on solid phase by the Fischer indole synthesis, J.Comb.Chem. 7, 130-135, 2005; Narayana, B., Ashalatha, B.V., Vijaya Raj, K.K., et al., Synthesis of some new biologically acivie 1,3,4-oxadiazolyl nitroindole and a modified Fischer indole synthesis of ethyl nitro indole-2-carboxylates, Bioorg.Med.Chem. 13, 4638-4644, 2005; Schmidt, A.M. and Eilbracht, P., Tandem hydroformylation-hydrazone formation-Fischer indole synthesis: a novel approach to tryptamides, Org.Biomol.Chem. 3, 2333-2343, 2005; Linnepe Nee Kohling, P., Schmidt, A.M., and Eilbracht, P., 2,3-Disubstituted indoles from olefins and hydrazines via tandem hydroformylation-Fischer indole synthesis and skeletal rearrangement, Org.Biomol.Chem. 4, 302-313, 2006; Landwehr, J., George, S., Karg, E.M., et al., Design and synthesis of novel 2-amino-5-hydroxyindole derivatives that inhibit human 5-lipooxygenase, J.Med.Chem. 49, 4327-4332, 2006.
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911
ORGANIC NAME REACTIONS USEFUL IN BIOCHEMISTRY AND MOLECULAR BIOLOGY (Continued) Friedel-Crafts Reaction CH3
H2C CH3 +
AlCl3
CH2
X
Friedel-Crafts alkylation CH3 CH2
O O
+
AlCl3
CH2
X
CH3 Friedel-Crafts acylation
The alkylation of an aromatic ring by an alkyl halide (order of reactivity F>Cl>Br>I ) in the presence of a strong Lewis acid such as aluminum chloride; the acylation of an aromatic ring by an acyl halide (order of reactivity usually is I>Br>Cl>F) in the presence of a strong Lewis acid. Acids and acid anhydrides can replace the acyl halides. A related reaction is the Derzen-Nenitzescu ketone synthesis. See Olah, G.A., Friedel-Crafts Chemistry, John Wiley & Sons, New York, New York, 1973; Roberts, R.M. and Khalaf, A.A., Friedel-Krafts Alkylation Chemistry: A Century of Discovery, Marcel Dekker, New York, New York, 1989. See also Retey, J., Enzymatic catalysis by Friedel-Crafts-type reactions, Naturwissenschaften 83, 439-447, 1996; White, E.H., Darbeau, R.W., Chen, Y. et al., A new look at the Friedel-Crafts alkylation reaction(1), J.Org.Chem. 61, 7986-7987, 1996; Studer, J., Purdie, N., and Krouse, J.A., Friedel-Crafts acylation as a quality control assay for steroids, Appl.Spectrosc. 57, 791-796, 2003; Retey, J., Discovery and role of methylidene imidazolone, a highly reactive electrophilic prosthetic group, Biochim.Biophys.Acta 1647, 179-184, 2003; Bandini, M., Melloni, A., and Umani-Ronchi, A., New catalytic approaches in the stereoselective Friedel-Crafts alkylation reaction, Angew.Chem.Int.Ed.Engl. 43, 550-556, 2004; Poppe, L. and Retey, J., Friedel-Crafts-type mechanism for the enzymatic elimination of ammonia from histidine and phenylalanine, Angew.Chem.Int. Ed.Engl. 44, 3668-3688, 2005; Keni, M., and Tepe, J.J., One-pot Friedel-Crafts/Robinson-Gabriel synthesis of oxazoles using oxazolone templates, J.Org. Chem. 70, 4211-4213, 2005; Movassaghi, M. and Ondrus, A.E., Enantioselective total synthesis of tricyclic myrmicarin alkaloids, Org.Lett. 7, 44234426, 2005; Paizs, C., Katona, A., and Retey, J., The interaction of heteroaryl-acrylates and alanines with phenylalanine ammonia-lyase form parsley, Chemistry 12, 2739-2744, 2006. Cuprous ions have been observed to promote a Friedel-Crafts acylation reaction (Kozikowski, A.P. and Ames, A., Copper(I) promoted acylation reactions. A transition metal mediated version of the Friedel-Crafts reaction, J.Am.Chem.Soc. 102, 860-862, 1980) Friedländer Synthesis O C
H
Base
+
NH2
N
O
O C
O H
+
R
Base CH3
NH2
N
R
Friedlander synthesis
The base-catalyzed formation of quinoline derivatives by condensation of an o-aminobenzaldehyde with a ketone; also referred to as the Friedländer quinoline synthesis. The general utility of the reaction is somewhat limited by the availability of o-aminobenzaldehyde derivatives. See Maguire, M.P., Sheets, K.R., McVety, K., et al., A new series of PDGF receptor tyrosine kinase inhibitors: 3-substituted quinoline derivatives, J.Med.Chem. 37, 2129-2137, 1994; Lindstrom, S., Friedlander synthesis of the food carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine, Acta Chem.Scand. 49, 361-363, 1995; Gladiali, S., Chelucci, G., Mudadu, M.S., et al., Friedlander synthesis of chiral alkyl-substituted 1,10-phenanthrolines, J.Org.Chem. 66, 400-405, 2001; Patteux, C., Levacher, V., and Dupas, G., A novel traceless solid-phase Friedlander synthesis, Org.Lett. 5, 3061-3063, 2003; McNaughton, B.R. and Miller, B.L., A mild and efficient one-step synthesis of quinolines, Org.Lett. 5, 4257-4259, 2003; Yasuda, N., Hsiao, Y., Jensen, M.S., et al., An efficient synthesis of an avb3 antagonist, J.Org.Chem. 69, 1959-1966, 2004.
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912
ORGANIC NAME REACTIONS USEFUL IN BIOCHEMISTRY AND MOLECULAR BIOLOGY (Continued) Fries Rearrangement R O
O
OH
Lewis acid
O
R
hν
OH
O R
Rearrangement of a phenolic ester to yield o- and p-acylphenols. The distribution of products between the ortho and para acyl derivates depends on reaction conditions. The presence of solvent and a Lewis acid, the paraproduct is preferred; with the photolytic process or at high temperature in the absence of solvent, the ortho derivative is preferred. See Sen, A.B. and Bhattacharji, S., Fries’ rearrangement of aliphatic esters of b-naphthol, Curr.Sci. 20, 132-133, 1951; Iwasaki, S., Photochemistry of imidazolides. I. The photo-Fries-type rearrangement of N-substituted imidazoles, Helv.Chim.Acta 59, 2738-2752, 1976; Castell, J.V., Gomez, M.J., MIrabet, V., et al., Photolytic degradation of benorylate: effects of the photoproducts on cultured hepatocytes, J.Pharm.Sci. 76, 374-378,1987; Climent, M.J. and Miranda, M.A. Gas chromatographic-mass spectrometric study of photodegradation of carbamate pesticides, J.Chromatog.A. 738, 225-231, 1996; Kozhevnikova, E.F., Derouane, E.G., and Kozhevnikov, I.V., Heteropoly acid as a novel efficient catalyst for Fries rearrangement, Chem.Commun. (11), 1178-1179, 2002; Dickerson, T.J., Tremblay, M.R., Hoffman, T.Z. et al., Catalysis of the photo-Fries reaction: antibody-mediated stabilization ofhigh energy states, J.Am.Chem.Soc. 125, 15395-15401, 2003; Seijas, J.A., Vazquez-Tato, M.P., and Carballido-Reboredo, R., Solvent-free synthesis of functionalized flavones under microwave irradiation, J.Org.Chem. 70, 2855-2858, 2005;Canle Lopez, M., Fernandez, M.I., Rodriguez, S., et al., Mechanisms of direct and TiO2-photocatalyzed degradation of phenylurea herbicides, Chemphyschem 6, 2064-2074, 2005; Slana, G.B. de Azevedo, M.S., Lopes, R.S., et al., Total syntheses of oxygenated brazanquinones via regioselective homologous anionic Fries rearrangement of benzylic O-carbamates, Beilstein J.Org.Chem. 2, 1, 2006. Gabriel Synthesis
N–
R
R
K+
R
R
Cl
NH2
Gabriel synthesis The conversion of an alkyl halide to alkyl amine mediated by potassium phthalimide. The intermediate product of the reaction of the alkyl halide and phthalimide is hydrolyzed to the product amine by acid or by reflux in ethanolic hydrazine. See Mikola, H. and Hanninen, E., Introduction of aliphatic amino and hydroxy groups to keto steroids using O-substituted hydroxylamines, Bioconjugate Chem. 3, 182-186, 1992; Groutas, W.C., Chong, L.S., Venkataraman, R., et al., Mechanism-based inhibitors of serine proteinases based on the Gabriel-Colman rearrangement, Biochem.Biophys.Res. Commun. 194, 1491-1499, 1993; Konig, S., Ugi, I., and Schramm, H.J., Facile syntheses of C2-symmetrical HIV-1 protease inhibitor, Arch.Pharm. 328, 699-704, 1995; Zhang, X.X. and Lippard, S.J., Synthesis of PDK, a novel porphyrin-linked dicarboxyate ligand, J.Org.Chem. 65, 5298-5305, 2000; Scozzafava, A. Saramet, I., Banciu, M.D., and Supuran, C.T., Carbonic anhydrase activity modulators: synthesis of inhibitors and activators incorporating 2-substituted-thiazol-4-yl-methyl scaffolds, J.Enzyme Inhib. 16, 351-358, 2001; Nicolaou, K.C., Hao, J., Reddy, M.V., et al., Chemistry and biology of diazonamide A: second total synthesis and biological investigations, J.Am.Chem.Soc. 126, 12897-12906, 2004; Remond, C., Plantier-Royon, R., Aubry, N., and O’Donohue, M.J., An original chemoenzymatic rotue for the synthesis of b-D-galactofuranosides using an a-Larabinofuranosidase, Carbohydr.Res. 340, 637-644, 2005; Pulici, M., Quartieri, F., and Felder, E.R., Trifluoroacetic acid anhydride-mediated solid-phase version of the Robison-Gabriel synthesis of oxazoles, J.Comb.Chem. 7, 463-473, 2005.
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913
ORGANIC NAME REACTIONS USEFUL IN BIOCHEMISTRY AND MOLECULAR BIOLOGY (Continued) Greiss Reaction N Cl–
N+
NH2
HCl/NaNO2
NO2
NO2 Greiss reaction Greiss reaction as used for the measurement of nitrite NH2
NO2– Nitrite
H2C
+
N
NH2
N+
CH2 H3O+
HN
N-(1-naphthyl)ethylenediamine O
S
O
O
O
S
NH2
NH2
Sulfanilide
O H2N
SH
N
N
NH H2C
OH
CH2 NH2
Azo product measured at 520 nm
Diazotization of aromatic amines; used for the assay of nitrites in nitric oxide research. The assay for nitrates uses diazotization of sulfanilamide with subsequent coupling to an aromatic amine (N-1-naphthylethylenediamine) to form an chromophoric azo derivative. See Greenberg, S.S., Xie, J., Spitzer, J.J. et al., Nitro containing L-arginine analogs interfere with assays for nitrate and nitrite, Life Sci. 57, 1949-1961, 1995; Pratt, P.F., Nithipatikom, K., and Campbell, W.B., Simultaneous determination of nitrate and nitrite in biological samples by multichannel flow injection analysis, Anal.Biochem. 231, 383-386, 1995; Tang, Y., Han, C. and Wang, X., Role of nitric oxide and prostaglandins in the potentiating effects of calcitonin gene-related peptide on Lipopolysaccharide-induced interleukin-6 release from mouse peritoneal macrophages, Immunology 96, 171-175, 1999; Baines, P.B., Stanford, S., Bishop-Bailey, D., et al., Nitric oxide production in meningococcal disease is directly related to disease severity, Crit.Care. Med. 27, 1163-1165, 1999; Rabbani, G.H., Islam, S., Chowdhury, A.K., et al., Increased nitrite and nitrate concentrations in sera and urine of patients with cholera or shigellosis, Am.J.Gastroenterol. 96, 467-472, 2001; Lee, R.H., Efron, D., Tantry, U. and Barbul, A., Nitric oxide in the healing wound: a time-course study, J.Surg.Res. 101, 104-108, 2001; Stark, J.M., Khan, A.M., Chiappetta, C.L., et al, Immune and functional role of nitric oxide in a mouse model of respiratory syncytial virus infection, J.Infect.Dis. 191, 387-395, 2005; Bellows, C.F., Alder, A., Wludyka, P., and Jaffe, B.M., Modulation of macrophage nitric oxide production by prostaglandin D2, J.Surg.Res. 132, 92-97, 2006. Diazotization of aromatic amines is also used for the the modification of proteins (Lundblad, R.L., Chemical Reagents for Protein Modification, CRC Press, Boca Raton, FL, 2004; Kennedy, J.H., Kricka, L.J., and Wilding, P., Protein-protein coupling reactions and the application of protein conjugates, Clin.Chim.Acta 70, 1-31, 1976; Sinnott, M.L., Affinity labeling via deamination reactions, CRC Crit.Rev.Biochem. 12, 327-372, 1982; Blair, A.H. and Ghose, T.I., Linkage of cytotoxic agents to
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914
ORGANIC NAME REACTIONS USEFUL IN BIOCHEMISTRY AND MOLECULAR BIOLOGY (Continued) immunoglobulins, J.Immunol.Methods 59, 129-143, 1983). While alkyl azides are unstable, carbonyl azides such as diazoacetyl derivatives have been used in the modification of proteins (Lundblad, R.L. and Stein, W.H., On the reaction of diazoacetyl compounds with pepsin, J.Biol.Chem. 244, 154-160, 1969; Keilova, H. and Lapresle, C., Inhibition of cathepsin E by diazoacetyl-norleucine methyl ester, FEBS Lett. 9, 348-350, 1970; Giraldi, T. and Nisi, C., Effects of cupric ions on the antitumour activity of diazoacetyl-glycine derivatives, Chem.Biol.Interact. 11,59-61, 1975; Kaehn, K., Morr, M., and Kula, M.R., Inhibition of the acid proteinase from Neurospora crassa by diazoaetyl-DL-norleucine methyl ester, 1,2-epoxy-3-(4-nitrophenoxy) propane and pepstatin, Hoppe Seylers Z. Physiol.Chem. 360, 791-794, 1979; Ouihia, A., René, L., Guilhem, J., et al., A new diazoacylating reagent: Preparation, structure, and use of succinimidyl diazoacetate, J.Org.Chem. 58, 1641-1642, 1993. Grignard Reagent or Grignard Reaction H2 C
Br C H2
H3C
H2 C
Magnesium
Mg C H2
H3C
Dry ether
Br
Complexed with ether
O
R
H O
HO
R
H
R
R
CH2 HO
H2C
R
H2 C
CH3
C H2
H3C
R
The reaction of alkyl or aryl halides with magnesium in dry ether to yield derivatives which can be used in a variety of organic synthetic reactions. See Nagano, T. and Hayashi, T., Iron-catalyzed Grignard cross-coupling with alkyl halides possessing beta-hydrogens, Org.Lett. 6, 1297-1299, 2004; Querner, C., Reiss, P., Bleuse, J., and Pron, A., Chelating ligands for nanocrystals’ functionalization, J.Am.Chem.Soc. 126, 11574-11582, 2004; Agarwal, S. anad Knolker, H.J., A novel pyrrole synthesis, Org.Biomol.Chem. 2, 3060-3062, 2004; Hatano, M., Matsumara, T., and Ishihara, K., Highly alkylselective addition to ketones with magnesiumate complexes derived from Grignard reagents, Org.Lett. 7, 573-576, 2005; Itami, K., Higashi, S., Mineno, M., and Yoshida, J., Iron-catalyzed cross-coupling of alkenyl sulfides with Grignard reagents, Org.Lett. 7, 1219-1222, 2005; Wang, X.J., Zhang, L., Sun, X., et al., Addition of Grignard reagents to aryl chlorides: an efficient synthesis of aryl ketones, Org.Lett. 7, 5593-5595, 2005; Hoffman-Emery, F., Hilpert, H., Scalone, M., and Waldmeier, F., Efficient synthesis of novel NK1 receptor antagonists: selective 1,4-additional of Grignard reagents to 6-chloronicotinic acid derivatives, J.Org.Chem. 71, 2000-2008, 2006; Werner, T. and Barrett, A.G., Simple method for the preparation of esters from Grigard reagents and alkyl 1-imidazolecarboxylates, J.Org.Chem. 71, 4302-4304, 2006; Demel, P., Keller, M., and Breit, B., o-DPPB-directed copper mediated and –catalyzed allylic substitution with Grignard reagents, Chemistry 12, 6669-6683, 2006. Knoevenagel Reaction or Knoevenagel Condensation O
H
O +
O
OH
OH
C
O
CH2
OH Knoevenagel condensation EWG R EWG O
+
R
H2C
EWG
R EWG
R
EWG = electron-withdrawing group such as CHO, COOH, COOR, CN, NO2
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ORGANIC NAME REACTIONS USEFUL IN BIOCHEMISTRY AND MOLECULAR BIOLOGY (Continued) An amine-catalyzed reaction between active hydrogen compounds of the type Z-CH2-Z where Z can be a CHO, COOH, COOR, NO2,SOR, or related electron withdrawing groups and an aldehyde or ketone. For example, the reaction of malonic acid or malonic acid esters and an aldehyde or ketone to yield an a,b-unsaturated derivative. With malonic acid (Z is carboxyl group), decarboxylation occurs in situ. See March, J. Advanced Organic Chemistry. Reactions, Mechanisms, and Structure, 3rd edn., John Wiley & Sons, New York, New York, 1985; Klavins, M., Dipane, J., and Babre, K., Humic substances as catalysts in condensation reactions, Chemosphere 44, 737-742, 2001; Lai. S.M., Martin-Aranda, R., and Yeung, K.L., Knoevenagel condensation reaction in a membrane bioreactor, Chem.Commun. (2), 218-219, 2003; Pivonka, D.E. and Empfield, J.R., Real-time in situ Ramen analysis of microwave-assisted organic reactions, Appl.Spectrosc. 58, 41-46, 2004; Strohmeier, G.A., Haas, W., and Kappe, C.O., Synthesis of functionalized 1,3-thiazine libraries combining solid-phase synthesis and post-cleavage modification reactions, Chemistry 10, 2919-2926, 2004; Wirz, R., Ferri, D. and Baiker, A., ATR-IR spectroscopy of pendant NH2 groups on silica involved in the Knoevenagel condensation, Langmuir 22, 3698-3706, 2006. Leuckart Reaction R’
R’
Ammonium formate O
R
R
R’
H3O+ NH
R
NH2
O
H Leuckart reaction
R’
CH3 R’ +
N
O
∆
H3C R
HCOOH/H2O
O H
CH3 R
N CH3
The reductive amination of carbonyl groups by ammonium formate or amine salts of formic acid; formamides may also be used in the reaction. See Matsueda, G.R. and Stewart, J.M., A p-methylbenzhydrylamine resin for improved solid-phase synthesis of peptide amides, Peptides 2, 45-50, 1981; Agwada, V.C. and Awachie, P.I., Intermediates in the Leuckart reaction of benzophenone with formamide, Tetrahedron Lett. 23, 779-780, 1982; Loupy, A., Monteux, D., Petit, A., et al., Toward the rehabilitation of the Leuckart reductive amination reaction using microwave technology, Tetrahedron Lett. 37, 8177-8180, 1996; Adger, B.M., Dyer, U.C., Lennon, I.C., et al., A novel synthesis of tert-leucine via a Leuckart type reaction, Tetrahedron Lett. 38, 2153-2154, 1997; Lejon, T. and Helland, I., Effect of formamide in the Leuckart reaction, Acta Chem.Scand. 53, 76-78, 1999; Kitamura, M.. Lee, D., Hayashi, S., et al., Catalytic Leuckart-Wallach type reductive amination of ketones, J.Org.Chem. 67, 8685-8687, 2002; Swist, M., Wilamowski, J., and Parczewski, A., Basic and neutral route specific impurities in MDMA prepared by different synthesis methods. Comparison of impurity profiles, Forensic Sci. Int. 155, 100-111, 2005; Tournier, L. and Zard, S.Z., A practical variation on the Leuckart reaction, Tetrahedron Lett. 46, 971-973, 2005.
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916
ORGANIC NAME REACTIONS USEFUL IN BIOCHEMISTRY AND MOLECULAR BIOLOGY (Continued) Lossen Rearrangement O H N
H2 C R
O
–OH
R’
N
O
R
C
O
H2O
R’’NH2 R
R
O
O O N H
NH2
R”
C
C H2
H N
H N
H N
R
C H2
+
HO
R H2 C CH C
O
O
R’ Active Site Serine R
O O
H2 C
CH
HN
C
R
R’
O
The formation of isocyanates on heating of O-acyl derivatives of hydroxamic acids or treatment by base. The isocyanate frequently adds water in situ to form an amine one carbon shorter that the parent compound; in the presence of amines, there is the formation of ureas. Andersen, W., The synthesis of phenylcarbamoyl derivatives by Lossen rearrangement of dibenzohydroxamic acid, C.R.Trav.Lab.Carlsberg. 30, 79-103, 1956; Gallop, P.M., Seifter, S., Lukin, M., and Meilman, E. Application of the Lossen rearrangement of dintirophenylhydroxamates to analysis of of carboxyl groups in model compounds and gelatin, J.Biol.Chem. 235, 2619-2627, 1960; Hoare, D.G., Olson, A., and Koshland, D.E., Jr., The reaction of hydroxamic acids with water-soluble carbodiimides. A Lossen rearrangement, J.Am.Chem.Soc. 90, 1638-1643, 1968; Dell, D., Boreham, D.R., and Martin, B.K., Estimation of 4-butoyphenylacetohydroxamic acid utilizing the Lossen rearrangement, J.Pharm.Sci. 60, 1368-1370, 1971; Harris, R.B. and Wilson, I.B., Glutamic acid is an active site residue of angiotensin I-converting enzyme. Use of the Lossen rearrangement for identification of dicarboxylic acid residues, J.Biol. Chem. 258, 1357-1362, 1983; Libert, R., Draye, J.P., Van Hoof, F., et al., Study of reactions induced by hydroxylamine treatment of esters for organic acids and of 3-ketoacids: application to the study of urines from patients under valproate therapy, Biol.Mass.Spectrom. 20, 75-86, 1991; Neumann, U. and Gutschow, M., N-(sulfonyloxy)phthalimides and analogues are potent inactivators of serine proteases, J.Biol.Chem. 269, 21561-21567, 1994; Steinmetz, A.C., Demuth, H.U., and Ringe, D., Inactivation of subtilisin Carlsberg by N-[(t-butoxycarbonyl) alanylprolyl-phenylalanyl]-Obenzoylhydroxyl-amine: formation of a covalent enzyme-inhibitor linkage in the form of a carbamate derivative, Biochemistry 33, 10535-10544, 1994; Needs, P.W., Rigby, N.M., Ring, S.G., and MacDougall, A.J., Specific degradation of pectins via a carbodiimide-mediated Lossen rearrangement of methyl esterified galacturonic acid residues, Carbohydr.Res. 333, 47-58, 2001.
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917
ORGANIC NAME REACTIONS USEFUL IN BIOCHEMISTRY AND MOLECULAR BIOLOGY (Continued) Maillard Reaction R
R
N
HN
H
OH H
H
NH2 R
HO
CH
CHOH
CHO
HO
–H2O
H
OH
H
OH
H
HO
H
OH
H
OH
H
OH
H
OH
H
OH
H
OH
H N
H N
H N
R
R
CH
R
CH2
CH2 HO
CH2OH
CH2OH
CH2OH
OH
O
CH
HO
H
OH O HC
CH
HO
H
H
OH
H
OH
H
OH
H
OH
CH H2C
OH
CH2OH N-substituted glycosamine
CH2OH
Amadori product N-substituted 1-amino-2-deoxy-2-ketose
The reaction of amino groups with carbonyl groups resulting in the formation of complex products. This process is involved in the tanning of leather and the Browning reaction which is considered unique to the reaction of carbohydrates with proteins and is a critical aspect of food preparation. The Maillard reaction involves the nonenzymatic reaction of sugars with proteins and the formation of advanced glycation end products (AGE products). The Maillard reaction results in the formation of a number of reaction products. See Dills, W.J., Jr., Protein fructosylation: fructose and the Maillard reaction, Am.J.Clin.Nutr. 58(Suppl 5), 779S-787S, 1993; Chuyen, N.V., Maillard reaction and food processing. Application aspects, Adv.Exp.Med.Biol. 434, 213-235, 1998; van Boekel, M.A., Kinetic aspects of the Maillard reaction: a critical review, Nahrung 45, 150-159, 2001; Horvat, S. and Jakas, A., Peptide and amino acid glycation: new insights into the Maillard reaction, J.Pept.Sci. 10, 119-137, 2004; Fay, L.B. and Brevard, H., Contribution of mass spectrometry to the study of the Maillard reaction in food, Mass Spectrom.Rev. 24, 487-507, 2005; Yaylayan, V.A., Haffenden, L., Chu, F.L., and Wnorowski, A., Oxidative pyrolysis and post pyrolytic derivatization techniques for the total analysis of Maillard model systems: investigations of control parameters of Maillard reaction pathways, Ann.N.Y.Acad.Sci. 1043, 41-54, 2005; Monnier, V.M., Mustata, G.T., Biemel, K.L., et al., Cross-linked of the extracellular matrix by the Maillard reaction in aging and diabetes: an update on “a puzzle nearing resolution”, Ann.N.Y.Acad.Sci. 1043, 533-544, 2005; Matiacevich, S.B., Santagapita, P.R., and Buera, M.P., Fluorescence from the Maillard reaction and its potential applications in food science, Crit. Rev.Food Sci.Nutr. 45, 483-495, 2005; van Boekel, M.A., Formation of flavour compounds in the Maillard reaction, Biotechnol.Adv. 24, 230-233, 2006.
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918
ORGANIC NAME REACTIONS USEFUL IN BIOCHEMISTRY AND MOLECULAR BIOLOGY (Continued) Malaprade Reaction
CH2
CH2 HC
OH
HC
OH
HIO4
HC
O
HC
O
CH2
CH2
Malaprade reaction Periodic cleavage of a diol; although this term is seldom used for this extremely common reaction, it would appear to be the correct terminology. Periodic acid is used for the diol cleavage in aqueous solvent while lead tetraacetate can be used in organic solvents. The reaction also occur an amine group vicinal to a hydroxyl function. It would appear that the term Malaprade reaction has been used more in description of analytical techniques for organic diols such as gluconic acid or in the assay of periodate. See Belcher, R., Dryhurst, G., and MacDonal, A.M., Submicro-methods for analysis of organic compounds. 22. Malaprade reaction, Journal of the Chemical Society,(July) 3964, 1965; Chen, K,P., Determination of calcium gluconate by selective oxidation with periodate, J.Pharm.Sci. 73, 681-683, 1984; Verma, K.K., Gupta, D., Sanghi, S.K., and Jain, A., Spectrophotometric determination of periodate with amodiaquine dihydrochloride and its application to the indirect determination of some organic-compounds via the Malaprade reaction, Analyst 112, 1519-1522, 1987; Nevado, J.J.B. and Gonzalez, P.V., Spectrophotometric determination of periodate with salicyaldehyde guanylhydrazone – indirect determination of some organic compounds using the Malaprade reaction, Analyst 114, 243-244, 1989; Jie, N,Q., Yang, D.L., Zhang, Q.N., et al., Fluorometric determination of periodate with thiamine and its application to the determination of ethylene glycol and glycerol, Anal.Chim.Acta 359, 87-92, 1998; Guillan-Sans, R. and Guzman-Chozas, M., The thiobarbituric acid (TBA) reaction in foods, A review, Crit.Rev.Food Sci.Nutrition 38, 315-330, 1998; Pumera, M., Jelinek, I., Jindrich, J., et al., Determination of cyclodextrin content using periodate oxidation by capillary electrophoresis, J.Chromatog. A 891, 201-206, 2000; Afkhami, A. and Mosaed, F., Kinetic determination of periodate based on its reaction with ferroin and its application to the indirect determination of ethylene glycol and glycerol, Microchemical J. 68, 35-40, 2001; Afkhami, A. and Mosaed, F., Sensitive kinetic-spectrophotometric determination of trace amounts of periodate ion, J.Anal.Chem. 58, 588-593, 2003; Mihovilovic, M.D., Spina, M., Muller, B., and Stanetty, P., Synthesis of carbo- and heterocyclic aldehydes bearing an adjacent donor group – Ozonolysis versus OsO4/KIO4-oxidation, Monatshefte für Chemie 135, 899-909, 2004. Malonic Ester Synthesis CH3
CH3
H3C
H2C
Br C H2
O
O C
H2C O
O C
Base
H2C
O C
CH3 C H2
O
CH H2C
O C
CH3 C H2
O
CH3
Malonic ester synthesis
Hydrolysis
OH
O C CH2 H2C CH3
The synthesis of a variety of derivatives taking advantage of the reactivity (acidity) of the methylene carbon in malonic esters. The malonic ester synthesis is related to the acetoacetic ester synthesis and the Knoevenagel synthesis. See Mizuno, Y., Adachi, K., and Ikeda, K., Studies on condensed systems of aromatic nitrogenous series. XIII. Extension of malonic ester synthesis to the heterocyclic series, Pharm.Bull. 2, 225-234, 1954; Beres, J.A., Varner, M.G., and Bria, C., Synthesis and cyclization of dialkylmalonuric esters, J.Pharm.Sci. 69, 451-454, 1980; Kinder, D.H., Frank, S.K., and Ames, M.M. Analogues of carbamyl asparate as inhibitors of dihydroorotase: preparation of boronic acid transition-state analogues and a zinc chelator
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919
ORGANIC NAME REACTIONS USEFUL IN BIOCHEMISTRY AND MOLECULAR BIOLOGY (Continued) carbamylhomocysteine, J.Med.Chem. 33, 819-823, 1990; Groth, T. and Meldal, M., Synthesis of aldehyde building blocks protected as acid labile N-boc-N.O-acetals: toward combinatorial solid phase synthesis of novel peptide isosteres, J.Comb.Chem. 3, 34-44, 2001; Hachiya, I., Ogura, K., and Shimizu, M., Novel 2-pyridine synthesis via nucleophilic addition of malonic esters to alkynyl imines, Org.Lett. 4, 2755-2757, 2002; Strohmeier, G.A., Haas, W., and Kappe, C.O., Synthesis of functionalized 1,3-thiazine libraries combining solid-phase synthesis and post-cleavage modification methods, Chemistry 10, 2919-2926, 2004. Mannich Reaction H +
C H
HN
O
CH3
N+
CH3 Eschenmoser’s Salt
CH3 H2 C H3C
CH3 N H3C
C
CH3
H C
C
CH3
O
+ CH2
CH3
CH3 CH3
H3C O
Condensation of an amine with an carbonyl compound which can exist in an enol form, and a carbonyl compound which can not exist as an enol. The reaction frequently use formaldehyde as the carbonyl compound not existing as an enol for condensing with a secondary amine in the first phase of the reaction. See Britton, S.B., Caldwell, H.C., and Nobles, W.L., The use of 2-pipecoline in the Mannich reaction, J.Am.Pharm.Assoc.Am.Pharm.Assoc. 43, 641-643, 1954; Nobles, W.L., and Thompson, B.B., Application of the Mannich reaction to sulfones. I. Reactive methylene moiety of sulfones, J.Pharm. Sci. 54, 576-580, 1965; Thompson, B.B., The Mannich reaction. Mechanistic and technological considrations, J.Pharm.Sci. 57, 715-733, 1968; Nobles, W.L. and Potti, N.D., Studies on the mechanism of the Mannich reaction, J.Pharm.Sci. 57, 1097-1103, 1968; Delia, T.J., Scovill, J.P., Munslow, W.D., and Burckhalter, J.H., Synthesis of 5-substituted aminomethyluracils via the Mannich reaction, J.Med.Chem. 19, 344-346, 1976; List, B., Pojarliev, P., Biller, W.T., and Martin, H.J., The proline-catalyzed direct asymmetric three-component Mannich reaction: scope, optimization, and application to the highly enantioselective synthesis of 1,2-amino alcohols, J.Am.Chem.Soc. 124, 827-833, 2002; Palomo, C., Oiarbide, M., Landa, A., et al., Design and synthesis of a novel class of sugar-peptide hybrids: C-linked glyco b-amino acids through a stereoselective “acetate” Mannich reaction as the key strategic element, J.Am.Chem.Soc. 124, 8637-8643, 2002; Cordova, A., The direct catalytic asymmetric Mannich reaction, Acc.Chem.Res. 37, 102-112, 2004; Azizi, N., Torkiyan, L., and Saidi, M.R., Highly efficient one-pot three-component Mannich reaction in water catalyzed by heteropoly acids, Org.Lett. 8, 20792082, 2006; Matsuo, J., Tanaki, Y., and Ishibashi, H., Oxidative Mannich reaction of N-carbobenzoxy amines 1,3-dicarbonyl compounds, Org.Lett. 8, 4371-4374, 2006. Another important example of the Michael addition in biochemistry and molecular biology is the reaction of 4-hydroxynon-2-enal with amines and sulfydryl groups (Winter, C.K., Segall, H.J., and Haddon, W.F., Formation of cyclic adducts of deoxyguanosine with the aldehyde trans-4-hydroxy-2-hexenal and trans-4-hydroxy-2-nonenal in vitro, Cancer Res. 46, 5682-5686, 1986; Sayre, L.M., Arora, P.K., Iyer, R.S., and Salomon, R.G., Pyrrole formation from 4-hydroxyonenal and primary amines, Chem.Res.Toxicol. 6, 19-22, 1993; Hartley, D.P., Ruth, J.A., and Petersen, D.R., The hepatocellular metabolism of 4-hydroxynonenal by alcohol dehydrogenase, aldehyde dehydrogenase, and glutathione-S-transferase, Arch.Biochem. Biophys. 316, 197-205, 1995: Engle, M.R., Singh, S.P., Czernik, P.J., et al., Physiological role of mGSTA4-4, a glutathione S-transferase metabolizing 4-hydroxynonenal: generation and analysis of mGst4 null mouse, Toxicol.Appl.Pharmacol. 194, 296-308, 2004). Meerwein Reaction R’ Z R
9168_Book.indb 919
R
N2+Cl– R’
+
Z Cl
CuCl2
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920
ORGANIC NAME REACTIONS USEFUL IN BIOCHEMISTRY AND MOLECULAR BIOLOGY (Continued) The reaction of an aryl diazonium halide with an aliphatic unsaturated compound to yield an a-halo-b-phenyl alkene and alkanes. The reaction is performed in the presence of cupric ions. The presence of an electron-withdrawing group is useful in promoting the reactivity of the alkene. See Kochi, J.K., The Meerwein reaction. Catalysis by cuprous chloride, J.Am.Chem.Soc. 77, 5090, 1955; Moraes, L.A. and Eberlin, M.N., The gas-phase Meerwein reaction, Chemistry 6, 897-905, 2000; Riter, L.S., Meurer, E.C., Handberg, E.S., et al., Ion/molecule reactions peformed in a miniature cylindrical ion trap mass spectrometer, Analyst 128, 1112-1118, 2003; Meurer, E.C., Chen, H., Riter, L.S., et al., Meerwein reaction of phosphonium ions with epoxides and thioepoxides in the gas phase, J.Am.Soc.Mass Spectrom. 15, 398-405, 2004; Meurer, E.C. and Eberlin, M.N., The atmospheric pressure Meerwein reaction, J.Mass Spectrom. 41, 470-476, 2006. Michael Addition (Michael Condensation) O
O
C
C
R
C H2
R
+
O
H C
OH
C
C
H2C
O
–
CH3
C
R
CH
O
H2C
R
CH2
Michael addition/Michael condensation O CH2
H3C
CH3 O
N H3C
O
CH2 O
SH CH2 HO
C
CH
N
S
+
O
CH2
NH2
HO
C
O
CH
NH2
O Reaction of cysteine with N-ethylmaleimide as a Michael addition reaction
+ O
H O
HC
C
CH
H 2C
S
OH
H2 C
CH
CH
CH H2C
OH
C H2
C H2
H2 C C H2
CH3
CH2 CH2 CH2
H3C
HO
C
CH
NH2
O
4-HNE Formally a 1, 4 addition/conjugate addition of a resonance-stabilized carbanion (the reaction of an active methylene compound such as a malonate and an a,b-unsaturated carbonyl compound or the reaction of a nucleophile with an “activated unsaturated system; a carbanion defined as an anion with an even number of electrons). The addition of a nucleophile to a conjugated double bond. See Flavin, M. and Slaughter, C., Enzymatic elimination from a substituted four-carbon amino acid coupled to Michael addition of a b-carbon to an electrophilic double bond. Structure of the reaction product, Biochemistry 5, 1340-1350, 1966; Fitt, J.J. and Gschwend, H.W., a-Alkylation and Michael addition of amino acid—a practical method, J.Org.Chem. 42, 2639-2641, 1977; Powell, G.K., Winter, H.C., and Dekker, E.E., Michael addition of thiols with 4-methyleneglutamic acid: preparation of adducts, their properties and presence in peanuts, Biochem.Biophys.Res.Commun. 105, 1361-1367, 1982; Wang, M., Nishikawa, A. and Chung, F.L., Differential effects of thiols on DNA modifications via alkylation and Michael addition by a-acetoxy-N-nitrosopyrrolidine, Chem.Res.Toxicol. 5, 528-531, 1992; Jang, D.P., Chang, C.W., and Uang, B.J., Highly diastereoselective Michael addition of a-hydroxy acid derivatives and enantioselective synthesis of (+)-crobarbatic acid, Org.Lett. 3, 983-985, 2001;
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921
ORGANIC NAME REACTIONS USEFUL IN BIOCHEMISTRY AND MOLECULAR BIOLOGY (Continued) Naidu, B.N., Sorenson, M.E., Connolly, T.P., and Ueda, Y., Michael addition of amines and thiols to dehydroalanine amides: a remarkable rate acceleration in water, J.Org.Chem. 68, 10098-10102, 2003; Ooi, T., Doda, K., and Maruoka, K., Highly enantioselective Michael addition of silyl nitronates to a,b-unsaturated aldehydes catalyzed by designer chiral ammonium bifluorides: efficient access to optically active γ-nitro aldehydes and their enol silyl ethers, J.Am.Chem.Soc. 125, 9022-9023, 2003; Weinstein, R., Lerner, R.A., Barbas, C.F., 3rd, and Shabat, D., Antibody-catalyzed asymmetric intramolecular Michael additional of aldehydes and ketones to yield the disfavored cis-product, J.Am.Chem.Soc. 127, 13104-13105, 2005; Ding, R., Katebzadeh, K., Roman, L., et al., Expanding the scope of Lewis acid catalysis in water: remarkable ligand acceleration of aqueous ytteribium triflate catalyzed Michael addition reactions, J.Org.Chem. 71, 352-355, 2006; Pansare, S.V. and Pandya, K., Simple diamine- and triamine-protonic acid catalysts for the enantioselective Michael addition of cyclic ketones to nitroalkenes, J.Am.Chem.Soc. 128, 9624-9625, 2006; Dai, H.X., Yao, S.P., and Wang, J., Michael addition of pyrimidine with disaccharide acrylates catalyzed in organic medium with lipase M from Mucor javanicus, Biotechnol. Lett. 28, 1503-1507, 2006. One the best examples in biochemistry is the modification of cysteine residues with N-alkylmaleimide derivatives (Lundblad, R.L., Chemical Reagents for Protein Modification, 3rd edn., CRC Press, Boca Raton, FL, 2004; Heitz, J.R., Anderson, C.D., and Anderson, B.M., Inactivation of yeast alcohol dehydrogenase by N-alkylmaleimides, Arch.Biochem.Biophys. 127, 627-636, 1968; Smyth, D.B. and Tuppy, H., Acylation reactions with cyclic imides, Biochim.Biophys.Acta 168, 173-180, 1968; Lusty, C.J. and Fasold, H., Characterization of sulfhydryl groups of actin, Biochemistry 8, 2933-2939, 1969; Bowes, T.J. and Gupta, R.S., Induction of mitochondrial fusion of cysteine-alklyators ethacrynic acid and N-ethylmaleimide, J.Cell Physiol. 202, 796-804, 2005). Reformatsky Reaction
O H3C
H2 C
O C H2
C
Br + Zn
H
+
CH
OH
H2C
O
O
C Reformatsky reaction
O CH2 H3C ∆
CH HC C
O
O CH2 H3C Formation of a complex between zinc and an a-bromoester followed by condensation with an aldehyde yielding a b-hydroxyester; an a,b-unsaturated ester via dehydration following the condensation reaction. See Tanabe, K., Studies on vitamin A and its related compounds. II. Reformatsky reaction of b-cyclocitral with methyl γ-bromosenecioate, Pharm.Bull. 3, 25-31, 1955; Ross, N.A. and Bartsch, R.A., High-intensity ultrasound-promoted Reformatsky reactions, J.Org.Chem. 68, 360-366, 2003; Jung, J.C., Lee, J.H., Oh., S., Synthesis and antitumor activity of 4-hydroxycoumarin derivatives, Bioorg.Med.Chem.Lett. 14, 5527-5531,2004; Kloetzing, R.J., Thaler, T., and Knochel, P., An improved asymmetric Reformatsky reaction mediated by (-)-N,N-dimethylaminoisoborneol, Org.Lett. 8, 1125-1128, 2006; Moume, R. Laavielle, S., and Karoyan, P., Efficient synthesis of b2-amino acid by homologation of a-amino acids involving the Reformatsky reaction and Mannich-type imminium electrophile, J.Org.Chem. 71, 3332-3334, 2006.
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922
ORGANIC NAME REACTIONS USEFUL IN BIOCHEMISTRY AND MOLECULAR BIOLOGY (Continued) Rittter Reaction
R R
H+
OH
R
R
N
R +
R
C
+ R
NH
R
R
R
O
R
carbonium ion Acid-catalyzed nucleophilic addition of a nitrile to a carbenium ion generated from alcohol (usually tertiary, primary alcohols other than benzyl alcohol will not react) yielding an amide. Sanguigni, J.A. and Levine, R., Amides from nitriles and alcohols by the Ritter reaction, J.Med.Chem. 53, 573-574, 1964; Radzicka, A. and Konieczny, M., Studies on the Ritter reaction. I. Synthesis of 3-/5-bartbituryl/-1propanesulfonic acids with anti-inflammatory activity, Arch.Immunol.Ther.Exp. 30, 421-432, 1982; Van Emelen, K., De Wit, T., Hoornaert, G.J., and Compernolle, F., Diastereoselective intramolecular Ritter reaction: generation of a cis-fused hexahydro-4aH-indeno[1,2-b] pyridine ring system with 4a,9b-diangular substituents, Org. Lett. 2, 3083-3086, 2000; Concellon, J.M., Reigo, E., Suarez, J.R., et al., Synthesis of enantiopure imidazolines through a Ritter reaction of 2-(1-aminoalkyl)azirdines with nitriles, Org.Lett. 6, 4499-4501, 2004; Feske, B.D., Kaluzna, I.A., and Stewart, J.D., Enantiodivergent, biocatalytic routes to both taxol side chain antipodes, J.Org.Chem. 70, 9654-9657, 2005; Crich, D. and Patel, M., On the nitrile effect in L-rhamnopyranosylation, Carbohydr.Res. 341, 1467-1475, 2006; Fu, Q. and Li, L., Neutral loss of water from the b ions with histidine at the C-terminus and formation of the c ions involving lysine side chains, J.Mass.Spectrom. 41, 1600–1607, 2006. Schiff Base R
NH2
CH
CH2
Schiff base formation
CH2
O
CH2
R
CH2
H
CH2 NH2
CH
C
CH2
+
CH2 HO
N
CH2
O HO
Lysine O
O
C NH
C H Methylglyoxal
H3C +
N H
NH2
N
OH
R CH
Guanidine
NH
O
CH2
N
N H
NH2
CH
O
O
N
C
H3C
NH
C N
N
CH2
OH
CH2
CH
CH2
OH O N
N H
HO
CH
NH2
O
CH3
NH
C
OH N
N H O
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Organic Name Reactions Useful in Biochemistry and Molecular Biology
923
ORGANIC NAME REACTIONS USEFUL IN BIOCHEMISTRY AND MOLECULAR BIOLOGY (Continued) O R
+
H /HN3 O
OH
R
NH2
CO2
+
H+/HN3
R OH
H2O
R N
R
R
R
N
O
O R
H2O
R
C
R
N H
R
R
+ H /HN3
N
R
R
R R
R
R
R
H+/HN3
R
R
R
N
R
The formation of an unstable derivative generally between an carbonyl(usually an aldehyde) and an amino group. The Schiff base can be converted to a stable derivative by reduction with sodium borohydride or sodium cyanoborohydride; Schiff bases appear to be resistant to reduction with sulfhydrylbase reducing agents such as 2-mercaptoethanol or dithiothreitol and phosphines. Schiff bases are involved in a diverse group of biochemical events including the interaction of pyridoxal phosphate with proteins, the interaction of reducing carbohydrates with proteins in reaction leading to AGE products, and reductive alkylation of amino groups in proteins. See Feeney, R.E., Blankenhorn, G., and Dixon, H.B., Carbonyl-amine reactions in protein chemistry, Adv.Protein.Chem. 29, 135-203, 1975; Metzler, D.E. Tautomerism in pyridoxal phosphate and in enzymatic catalysis, Adv.Enzymol. Relat.Areas Mol.Biol. 50, 1-40, 1979; Puchtler, H. and Meloan, S.N., ON Schiff ’s bases and aldehyde-fuchsin: a review from H.Schiff to R.D. Lillie, Histochemistry 72, 321-332, 1981; O’Donnell, J.P., The reaction of amines with carbonyls: its significance in the nonezymatic metabolism of xenobiotics, Drug.Metab.Rev. 13, 123-159, 1982; Stadtman, E.R., Covalent modification reactions are marking steps in protein turnover, Biochemistry 29, 6232-6331, 1990; Tuma, D.J., Hoffman, T. and Sorrell, M.F., The chemistry of aldehyde-protein adducts, Alcohol Alcohol Suppl. 1, 271-276, 1991; Hargrave, P.A., Hamm, H.E., and Hofmann, K.P., Interaction of rhodopsin with the G-protein, transducin, Bioessays 15, 43-50, 1993; Chen, H. and Rhodes, J., Schiff base forming drugs: mechanisms of immune potentiation and therapeutic potential, J.Mol.Med. 74, 497-504, 1996; Yim, M.B., Yim, H.S., Lee, C., et al., Protein glycation: creation of catalytic sites for free radication generation, Ann.N.Y.Acad.Sci. 928, 48-53, 2001; Gramatikova, S., Mouratou, B., Stetefeld, J. et al., Pyridoxal-5’-phosphate-dependent catatlytic antibodies, J.Immunol.Methods 269, 99-110, 2002; Schaur, R.J., Basic aspects of the biochemical reactivity of 4-hydroxynonenal, Mol.Aspects Med. 24, 149-159, 2003; Kurtz, A.J. and Lloyd, R.S., 1, N2-deoxyguanosine adducts of acrolein, crotonaldehyde, and trans-4-hydroxynonenal cross-link to peptides via Schiff base linkage, J.Biol.Chem. 278, 5970-5975, 2003; Kandori, H., Hydration switch model for the proton transfer in the Schiff base region of bacteriorhodopsin, Biochim.Biophys.Acta 1658, 72-79, 2004; Hadjoudis, E. and Mavridis, I.M., Photochomism and thermochromism of Schiff bases in the solid state: structural aspects, Chem.Soc.Rev. 33, 579-588, 2004; Stadler, R.H., Acrylamide formation in different foods and potential strategies for reduction, Adv.Expt.Med.Biol. 561, 157-169, 2005. There is some interesting chemistry on Schiff bases in inorganic chemistry (Nakoji, M., Kanayama, T., Okino, T., and Takemoto, Y., Chiral phosphine-free Pd-mediated asymmetric allylation of prochiral enolate with a chiral phase-transfer catalyst, Org.Lett. 2, 3329-3331, 2001; Walther, D., Fugger, C. Schreer, H., et al., Reversible fixation of carbon dioxide at nickel(0) centers: a route for large organometallic rings, dimers, and tetramers, Chemistry 7, 5214-5221, 2001; Benny, P.D., Green, J.L., Engelbrecht, H.P., Reactivity and rhenium(V) oxo Schiff base complexes with phosphine ligands: rearrangement and reduction reactions, Inorg.Chem. 44, 2381-2390, 2005).
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924
ORGANIC NAME REACTIONS USEFUL IN BIOCHEMISTRY AND MOLECULAR BIOLOGY (Continued) Schmidt Reaction/Schmidt Rearrangement R2 R1
O
R1
R2
N
+ H
NH2
H O
+ N+ R3
R4
C–
OH
R1 O
R4
HN R2
N
O R3
R1
O R4
H N
N R2
R3
O
Used to describe the reaction of carboxylic acids, aldehyde and ketones(carbonyl compounds), and alcohols/alkenes with hydrazoic acid. Reaction with carboxylic acids yields amines, carbonyl compounds yield amides in a reaction involving a rearrangement, and alcohols/azides yield alkyl azides. See Rabinowitz, J.L., Chase, G.D., and Kaliner, L.F., Isotope effects of in the decarboxylation of 1-14C-dicarboxylic acids studied by means of the Schmidt reaction, Anal.Biochem. 19, 578-583, 1967; Iyengar, R., Schildknegt, K., and Aube, J., Regiocontrol in an intramolecular Schmidt reaction: total synthesis of (+)-aspidospermidine, Org.Lett. 2, 1625-1627, 2000; Sahasrabudhe, K., Gracias, V., Furness, K., et al., Asymmetric Schmidt reaction of hydroxyalkyl azides with ketones, J.Am.Chem.Soc. 125, 7914-7922, 2003; Wang, W., Mei, Y., Li, H., and Wang, J., A novel pyrrolidine imide catalyzed direct formation of a,b-unsaturated ketones from unmodified ketones and aldehydes, Org.Lett. 7, 601-604, 2005; Brase, S., Gil., C., Knepper, K., and Zimmerman, V., Organic azides: an exploding diversity of a unique class of compounds, Angew.Chem.Int.Ed.Engl. 44, 5188-5240, 2005; Lang, S. and Murphy, J.A., Azide rearrangements in electron-deficient systems, Chem.Soc.Rev. 35, 146-156, 2006; Zarghi, A., Zebardast, T., Hakimion, F., et al., Synthesis and biological evaluation of 1,3-diphenylprop-2-en-1-ones possessing a methanesulfonamido or an azido pharmacophore as cyclooxygenase-1/-2 inhibitors, Bioorg.Med.Chem. 14, 7044-7050, 2006. Ugi Condensation: A four component (aldehyde, amine, isocyanide and a carboxyl group) condensation resulting in an a-aminoacyl amide. See Liu, X.C., Clark, D.S., and Dordick, J.S., Chemoenzymatic construction of a four-component Ugi combinatorial library, Biotechnol.Bioeng. 69, 457-460, 2000; Bayer, T., Riemer, C., and Kessler, H., A new strategy for the synthesis of cyclopeptides containing diaminoglutaric acid, J.Pept.Sci. 7, 250-261, 2001; Crescenzi, V., Francescangeli, A., Renier, D., and Bellini, D., New cross-linked and sulfated derivatives of partially deacylated hyaluronan: synthesis and preliminary characterization, Biopolymers 64, 86-94, 2002; Liu, L., Ping Li, C., Cochran, S., and Ferro, V., Application of the fourcomponent Ugi condensation for the preparation of glycoconjugate libraries, Bioorg.Med.Chem.Lett. 14, 2221-2226, 2004; Bu, H., Kjoniksen, A.L., Knudsen, K.D., and Nystrom, B., Rheological and structural properties of aqueous alginate during gelation via the Ugi multicomponent condensation reaction, Biomacromolecules 5, 1470-1479, 2004; Tempest, P.A., Recent advances in heterocycle generation using the efficient Ugi multiple-component condensation reaction, Curr.Opin.Drug Discov.Devel. 8, 776-788, 2005.
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925
ORGANIC NAME REACTIONS USEFUL IN BIOCHEMISTRY AND MOLECULAR BIOLOGY (Continued) Wittig Olefination
R1
R1 +
P H
C H
R1 X
–
Base
R2
R1
R1
P+ C–
H
Wittig reaction/Wittig olefination
R1
R1
R1
R1
P R2
R2
H Ylide O R
H
R2
R Synthesis of an alkene from the reaction of an aldehyde or ketone with an ylide (internal salt) generated from a phosphophonium salt. See Jorgensen, M., Iversen, E.H., and Madsen, R., A convenient route to higher sugars by two-carbon chain elongation using Wittig/dihydroxylation reactions, J.Org. Chem. 66, 4625-4629, 2001; Magrioti, V., and Constantinou-Kokotou, V., Synthesis of (S)-a-amino oleic acid, Lipids 37, 223-228, 2002; van Staden, L.F., Gravestock, D., and Ager, D.J., New developments in the Peterson olefination reaction, Chem.Soc.Rev. 31, 195-200, 2002; Han, H., Sinha, M.K., D’Sousa, L.J., et al., Total synthesis of 34-hydroxyasimicin and its photoactive derivative for affinity labeling of the mitochondrial complex I, Chemistry 10, 2149-2158, 2004; Rhee, J.U. and Krische, M.J., Alkynes as synthetic equivalents to stabilized Wittig reagents: intra- and intermolecular carbonyl olefinations catalyzed by Ag(1), BF3, and HBF4, Org.Lett. 7, 2493-2495, 2005; Ermolenko, L. and Sasaki, N.A., Diastereoselective synthesis of all either l-hexoses from L-ascorbic acid, J.Org.Chem. 71, 693-703, 2006; Halim, R., Brimble, M.A., and Merten, J., Synthesis of the ABC tricyclic fragment of the pectenotoxins via stereocontrolled cyclization of a γ-hydroxyepoxide appended to the AB spiroacetal unit, Org.Biomol.Chem. 4, 1387-1399, 2006; Phillips, D.J., Pillinger, K.S., Li, W., et al., Desymmerization of diols by a tandem oxidation/Wittig olefination reaction, Chem.Commun. (21), 22802282, 2006; Modica, E., Compostella, F. Colombo, D., et al., Stereoselective synthesis and immunogenic activity of the C-analogue of sulfatide, Org. Lett. 8, 3255-3258, 2006.
9168_Book.indb 925
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Enzymes in Synthetic Organic Chemistrya,b The specificity of enzymes has proved useful in organic synthesis where stereochemistry is critical for success.
Aldolase/Aldol Condensation
Catalysis of Aldol Condensation
O
H
O
H
N
+
OPO3–2
HO
Dihydroxyacetone phosphate
O H3C
Rabbit Muscle Aldolase
OH
O
H
OPO3–2
N
H3C
OH
O
Austin, M.B., Izumikawa, M., Bowman, M.E., Crystal structure of a bacterial type III polyketide synthase and enzymatic control of reactive polyketide intermediates, J.Biol.Chem. 279, 45162-45174, 2004; Xiang, L., Kokaitzis, J.A, and Moore, B.S., EncM, a versatile enterocin biosynthetic enzyme involved in Favorskii oxidative rearrangement, aldol condensation, and heterocyclic-forming reactions, Proc.Nat.Acad.Sci. 101, 15609-15614, 2004; Suzuki, H., Ohnishi, Y. Fursho, Y., et al., Novel benzene ring biosynthesis from C(3) and C(4) primary metabolites by two enzymes, J.Biol.Chem. 281, 36944-36951, 2006; Zhang, W., Watanabe, K., Wang, C.C., and Tang, Y., Heterologous biosynthesis of amidated polyketides with novel cyclization regioselectivity from oxytetracycline polyketide synthase, J.Natl.Prod. 69, 1633-1636, 2006; Williams, G.J., Woodhall, T., Farnsworth, L.M., et al., Creation of a pair of stereochemically complementary biocatalysts, J.Am.Chem.Soc. 128, 16238-16247, 2006; Schetter, B. and Mahrwald, R., Model aldol methods for the total synthesis of polyketides, Angewandte Chem. Int.Ed. 45, 7506-7535, 2006; Suzuki, H., Ohnishi, Y., Furusho, Y., et al., Novel benzene ring biosynthesis from C3 and C4 primary metabolites by two enzymes, J.Biol.Chem. 281, 36944-39511, 2007. a
b
Handbook of Enzyme Biotechnology, 2nd edn., ed. A. Wiseman, Ellis Horwood, Ltd., Chichester, UK, 1985; Laskin, A.T., Enzymes and Immobilized Cells in Biotechnology, Benjamin Cummings, Menlo Park, CA, USA, 1985; Biocatalysis in Organic Media, ed. C. Laane and J. Trayser, Elsevier, Amsterdam, Nethelands, 1987; Halgaš, J. Biocatalysis in Organic Synthesis, Elsevier, Amsterdam, Netherlands, 1992; Holland, H.L., Organic Synthesis with Oxidative Enzymes, VCH Publishers, New York, NY, USA, 1992; Enzyme Catalysis in Organic Synthesis. A Comprehensive Handbook, ed. K. Drauz and H. Waldman, VCH Verlagesellschaft, Weinheim, Germany, 1995; Bioorganic Chemistry Peptides and Proteins, ed S.M. Hecht, Oxford University Press, New York, NY, USA, 1998; Faber, K., Biotransformations in Organic Chemistry, 5th edn., Springer-Verlag, Berlin, Germany, 2005 Ribozymes have been used for chiral synthesis – See Schlatterer, J.C., Stuhlman, F., and Jäschke, A., Stereoselective synthesis using immobilized Diels-Alderase ribozymes, ChemBioChem. 4, 1089-1092, 2003
927
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Handbook of Biochemistry and Molecular Biology
928
Enzymes in Synthetic Organic Chemistrya,b (Continued) Hydrolases (Esterases)
O
O
A group of enzymes which catalyze the cleavage of ester and amide bonds with addition of water. Esterases, of which lipases are a singularly important group, are important in synthetic organic chemistry. Of particular importance is the stereoselectivity of the reactionc. A racemic mixture of an ester can be resolved can be resolved into enantiomeric pairs by stereospecific hydrolysis. Butyrylcholine esterase and cocaine esterase are listed with the therapeutic enzymes. O
CH3
O
H3C
OH
H3C
HO
HO
O
O
CH3
O
O
CH3
dimethyl-beta-hydroxymethylglutarate O H2N
CH
C
OH
CH2 CH2
OH
S CH3
H2N
C
O
C
H
CH2
Acetic Anhydride
CH2 O H3C
S
O H N
CH
C
CH2 CH2
CH3
OH Stereospecific enzymatic hydrolysis
+ OH
S
O
CH3 Racemic Mixtures
H Racemization Rate increased by addition of N-acetyl aminoacid racemase
C
H N
O CH3
CH2 CH2 S CH3
c
A carbon center may be asymmetric in having four different substituents (other atoms such as sulfur can also be asymmetric centers). In the case of carbon, this can be result in a mixture of the two optical isomers resulting in a racemic mixture. Generally there is no driving force for the formation of a racemic mixture so there are equal forms of the d and l isomers. With an enzyme stereoselectivity can be achieved and the quality of an asymmetric mixture may be expressed as enantiomeric excess (enantiomeric excess = (moles of major enantiomer - moles of other enantiomer / Total moles of both enantiomers) × 100 and is usually expressed as a percentage.
9168_Book.indb 928
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Enzymes in Synthetic Organic Chemistry
929
Enzymes in Synthetic Organic Chemistrya,b (Continued) H3C
+ NH
O CH3 O
O
Cocaine
H3C
+ NH
H3C
O CH3 O
OH Methyl Ecgonine not psychoactive
O
+ NH
O OH
O
O Benzoyl ecgonine psychoactive
Venkatachalam, T.K., Samuel, P., Li, G., et al., Lipase-mediated stereoselective hydrolysis of stampidine and other phosphoroamidate derivatives of stavudine, Bioorg.Med.Chem. 12, 3371-3381, 2004; Li, Y., Aubert, S.D., Maes, E.G., and Raushel, F.M., Enzymatic resolution of chiral phosphinate esters, J.Am.Chem.Soc. 126, 8888-8889, 2004; Kim, S. and Lee, S.B., Thermostable esterase from a thermoacidophilic archaeon: purification and characterization for enzymatic resolution of a chiral compound, Biosci.Biotechnol.Biochem. 68, 2289-2298, 2004; Molinari, F., Romano, D., Gandolfi, R., et al., Newly isolated Streptomyces spp. As enantioselective biocatalysts: hydrolysis of 1,2-O-isopropylidene glycerol racemic esters, J.Appl. Microbiol. 99, 960-967, 2005; Hu, S., Martinez, C.A., Yazbeck, D.R., and Tao, J., An efficient and practical chemoenzymatic preparation of optically active secondary amines, Org.Lett. 7, 4329-4331, 2005; Nowlan, C., Li, Y., Hermann, J.C., et al., Resolution of chiral phosphate, phosphonate, and phosphinate esters by an enantioselective enzyme library, J.Am.Chem.Soc. 128, 15892-15902, 2006; Gadler, P. and Faber, K., New enzymes for biotransformations: microbial alkyl sulfatases displaying stereo- and enantioselectivity, Trends Biotechnol. 25, 83-88, 2007.
9168_Book.indb 929
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Handbook of Biochemistry and Molecular Biology
930
Enzymes in Synthetic Organic Chemistrya,b (Continued) Lipases
A group of hydrolytic enzymes that catalyze the release of fatty acids from triglycerides; more specifically the catalysis of the hydrolysis of ester bonds between alkanoic acids and glycerol. Phospholipases are a subclass which uses phospholipids as substrates. The ability to use alcohols and amines as acceptors of the fatty acid hydrolytic product permits the synthesis of chiral products. There has been recent interest in the use of lipases for the synthesis of combinatorial librariesd. R
R
H
N
H
N
O
O
O
O
+ R1OH
Lipase
H
R HN O
O
R1
O
NH2 O CH3
O +
H3C
CH3
O
ethylmethoxy acetate acetic acid, methoxy-, ethyl ester
(R,S) 1-phenylethylamine
Lipase/methyl-tert-butyl ether
O O
NH2
HN
CH3
(S)1-phenylethylamine d
CH3
CH3
(R)-phenylethylmethoxyamide
Use of lipases in the manufacture of combinatorial libraries: Liu, K.-C., Clark, D.S., and Dordick, J.S., Chemoenzymatic construction of a four-component Ugi combinatorial library, Biotechnol.Bioeng. 69, 457-460, 2000; Reetz, M.T., Lipases as practical biocatalysts, Curr.Opin.Chem.Biol. 6, 145-150, 2002; Rich, J.G., Michels, P.C., and Khmeinitsky, Y.L., Lipases as practical biocatalysts, Curr.Opin.Chem.Biol. 6, 161-167, 2002; Secundo, F., Garrea, G., De Amici, M., et al., A combinatorial biocatalysis approach to an array of cholic acid derivatives, Biotechnol.Bioeng. 81, 392-396, 2003; Kumar, R., Bruno, F., Parmar, V.S., et al., “Green”-enzymatic synthesis of pegylated phenolic macromer and polymer, Chem.Commun. (7), 862-863, 2004; Rege, K., Hu. S., Moore, J.A., Chemoenzymatic synthesis and high-throughput screening of high-affinity displacers and DNAbinding ligands, J.Am.Chem.Soc. 126, 12306-12315, 2004; Vongvilal, P., Angelin, M., Larsson, R., and Ramstrom, O., Dynamic combinatorial resolution: direct asymmetric lipase-mediated screening of a dynamic nitroaldol library, Angew.Chem.Int.Ed.Engl. 46, 948-950, 2007.
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Enzymes in Synthetic Organic Chemistry
931
Enzymes in Synthetic Organic Chemistrya,b (Continued) BnO BnO
H3C
H3C
O
O
H3C
HO
O
O Phosphate buffer, pH 7..0 10%(V/V) toluene
O
O
Lipase PS-30
BnO
BnO + isopropenyl acetate
HO OH
O
H3C heptane/tert-butylmethyl ether (10:1)
OH O
Tuomi, W.V., and Kazlauskas, R.J., Molecular basis for enantioselectivity of lipase from Pseudomonas cepacia towards primary alcohols. Modeling, kinetics, and chemical modification of tyr29 to increase or decrease enantioselectivity, J.Org.Chem. 64, 2638-2647, 1999; Ghorpade, S.R., Khani, R.K., Ioshi, R.R., et al., Desymmetrization of meso-cyclopentene-cis-1,4-diol to 4-(R)-hydroxycyclopent-2-en-1-(S)-acetate by irreversible transesterification using Chirazyme®, Tetrahedron Asymm. 10, 891-899, 1999; Chen, J.-W. and Wu, W.-T., Regeneration of immobilized Candida anartica lipase for transesterification, J.Biosci.Bioeng. 95, 466-469, 2003; Gupta, R., Gupta, N., and Rathi, P., Bacterial lipases: an overview of production, purification and biochemical properties, Appl.Microbiol.Biotechnol. 64, 763-781, 2004; Kijima, T., Sato, N., Izumi, T., Lipase-catalyzed enantioselective esterification of mono-aza-benzo-15-crown-5-ether derivatives in organic media, Biotechnol.Lett. 26, 1505-1509, 2004; Domínguez de Maria, P., Carboni-Oerlemans, C., Tuin, B., et al., Biotechnological applications of Candida antartica lipase A: state-of-the-art, J.Molec.Catal.B:Enzymatic 37, 36-46, 2005; Sharma, J., Batovska, D., Kuwamori, Y., and Asano, Y., Enzymatic chemoselective synthesis of secondary-amide surfactant from N-methylethanol amine, J.Biosci. Bioeng. 100, 662-666, 2005; Patel, R.N., Banerjee, A., Pendri, Y.R., et al., Preparation of a chiral synthon for an HBV inhibitor: enzymatic asymmetric hydrolysis of (1a,2b,3a)-2-(benzyloxymethyl)cyclopent-4-ene-1,3-diol diacetate and enzymatic asymmetric acetylation of (1a, 2b, 3a)2-(benzyloxymethyl)cyclopent-4-ene-1,3-diol, Tetrahedron Asymm. 17, 175-175, 2006; Otero, C., Lopez-Herandez, A, Garcia, H.S., et al., Continuous enzymatic transesterification of sesame oil and a fully hydrogenated fat: effects of reaction conditions on product characterization, Biotechnol.Bioeng. 94, 877-887, 2006.
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Handbook of Biochemistry and Molecular Biology
932
Enzymes in Synthetic Organic Chemistrya,b (Continued) Monooxygenase
Insertion of oxygen into organic frameworks such as the oxidation of olefins (Baeyer-Villiger Reaction); hydroxylation of alkanes and aromatics. The cytochrome P-450-dependent monooxygenase is one of the better-known examples. Monooxygenases also oxidize organic sulfur OH OH +
O
O HO
O
OH
HO
O
xylene monooxygenase
Pseudocumene
9168_Book.indb 932
3,4-dimethylbenzaldehyde
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Enzymes in Synthetic Organic Chemistry
933
Enzymes in Synthetic Organic Chemistrya,b (Continued) O
O CH3
O CH3
2-methylcyclohexanone O
O O
H3C
H3C
4-ethylcyclohexanone O CH3
CH3
O O
2-methylcyclopentadecanone
S
S
S
S O (R) 81%
S
S
S
S
O
(R) 94% O S
S
S
S
(R) 92%
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Handbook of Biochemistry and Molecular Biology
934
Enzymes in Synthetic Organic Chemistrya,b (Continued)
O S OH
O
O
O
O
S
S
OH
OH OR
(R-2) Sphingomonas sp.; 40% enantioselective
(S-2) Beauuveria bassiana; 99% enantioselective
Ogawa, J. and Shimizu, S., Microbial enzymes: new industrial applications from traditional screening methods, Trends Biotechnol. 17, 13-21, 1999; Stewart, J.D., Organic transformations catalyzed by engineered yeast cells and related systems, Curr.Opin.Biotechnol. 11, 363-368, 2000; Mihovilovic, M.D., Muller, B., and Stanetty, P., Monooxygenase-mediated Baeyer-Villiger oxidations, Eur.J.Org.Chem. (22), 3711-3730, 2002; Lee, W.H., Park, Y.C., Lee, D.H., Simultaneous biocatalyst production and Baeyer-Villiger oxidation for bioconversion of cyclohexanone by recombinant Escherichia coli expressing cyclohexanone monooxygenase, Appl.Biochem.Biotechnol. 121-124, 827-836, 2005; Han, J.H., Yoo, S.K., Seo, J.S., et al., Biomimetic alcohol oxidations by an iron (III) porphyrin complex: relevance to cytochrome P-450 catalytic oxidation and involvement of the two-state radical rebound mechanism, Dalton Trans. (2), 402-406, 2005; Kagawa, H., Tatkahashi, T., Ohta, S., and Harigaya, Y., Oxidation and rearrangements of flavanones by mammalian cytochrome P450, Xenobiotica 34, 797-810, 2004; Gillam, E.M., Exploring the potential of xenobiotic-metabolising enzymes as biocatalysts: evolving designer catalysts from polyfunctional cytochrome P450 enzymes, Clin.Exp.Pharmacol.Physiol. 32, 147-152, 2005; Bocola, M., Schultz, F., Leca, F., et al., Converting phenylacetone monooxygenase into phenylcyclohexanone monooxygenase by rational design: towards practical Baeyer-Villiger monooxygenases, Adv.Synth.Catal. 347, 979-986, 2005; Urlacher, V.B. and Eiben, S., Cytochrome P450 monooxygenases: perspectives for synthetic application, Trends Biotechnol. 24, 324-330, 2006; Iwaki, H., Wang, S. ,Grosse, S., et al., Pseudomonad cyclopentadecanone monooxygenase displaying an uncommon spectrum of Baeyer-Villiger oxidations of cyclic ketones, Appl.Environ.Microbiol. 72, 2707-2720, 2006; Mihovilovic, M.D., Enzyme mediated Baeyer-Villiger oxidations, Curr.Org.Chem. 10, 1265-1287, 2006.Application to the stereospecific oxidation of organic sulfur: Dodson, R.M., Newman, N., and Tsuchiya, H.M., Microbiological transformations. XI. The properties of optically active sulfoxides, J.Am.Chem.Soc. 27, 2707-2708, 1962; Light, D.R., Waxman, D.J., and Walsh, C., Studies on the chirality of sulfoxidation catalyzed by bacterial flavoenzyme cyclohexanone monooxygenase and hog liver flavin adenine dinucleotide containing monooxygenase, Biochemistry 21, 2490-2498, 1982; Waxman, D.J., Light, D.R., and Walsh, C., Chiral sulfoxidation catalyzed by rat live cytochrome P-450, Biochemistry 21, 2499-2507, 1982; Colonna, S., Gaggero, N., Pasta, P., and Ottolina, G., Enantioselective oxidation of sulfides to sulfoxides catalyzed by bacterial cyclohexanone monooxygenase, Chem.Commun. (20), 2303-2307, 1996; Mata, E.G., Recent advances in the synthesis of sulfoxides from sulfides, Phosphorus, Sulfur and Silicon and the Related Elements 117, 231-286, 1996; Hamman, M.A., Haehner-Daniels, B.D., Wrighton, S.A., et al., Stereoselective sulfoxidation of sulindac sulfide by flavin-containing monooxygenase-Comparison of human liver and kidney microsomes and mammalian enzymes, Biochem.Pharmacol. 60, 7-17, 2000; Reetz, M.T., Daligault, F., Brunner, B., et al., Directed evolution of cyclohexanone monooxygenases: enantioselective biocatalysts for the oxidation of prochiral thioethers, Angewandte Chem.Int. 43, 4078-4081, 2005; Legros, J., Dehli, J.R., and Bolm, C., Applications of catalytic asymmetric sulfide oxidations to the syntheses of biologically active sulfoxides, Adv.Synthes.& Catal. 19-31, 2005; Olivo, H.F., Osorio-Lozada, A., and Peeples, T.L., Microbial oxidation/amidation of benzhydrylsulfanyl acetic acid. Synthesis of (+)-modafinil, Tetrahedron Asymmetry 16, 3507-3511, 2005.
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Enzymes in Synthetic Organic Chemistry
935
Enzymes in Synthetic Organic Chemistrya,b (Continued) Dioxygenase
An enzyme activity which inserts an oxygen molecule into an organic substrate. Where hydroxyl function(s) is the terminal reaction product, the overall reaction is the sum of two separate enzymatic reactions; an oxidation followed by a reduction.
OH
OH
CH3
CH3 OH
–m-cresol
methyl hydroquinone OH
H
H
Benzene dioxygenase
(1)
OH
O2 + NADH + H+
COOH COOH Intradiol cleavage OH
O2
OH (2)
O2
OH
Extradiol cleavage COOH CHO
O O OH
OH NH N H
CH3
1H-3-hydroxy-4-oxoquinaldine
9168_Book.indb 935
O
CH3
N-acetylanthranilic acid
4/16/10 1:36 PM
Handbook of Biochemistry and Molecular Biology
936
Enzymes in Synthetic Organic Chemistrya,b (Continued) O
O HO
OH
OH
OH
OH
HO O
O cis-4,5-dihydro-4,5-dihydroxyphthalic acid
Phthalic Acid
Nakata, H., Yamuchi, T., and Fujisawa, H., Studies on the reaction intermediate of protocatechuate 3,4-dioxygenase. Formation of enzyme-product complex, Biochim.Biophys.Acta 527, 171-181, 1978; Gassner, G.T., Ludwig, M.L., Gatti, D.L., et al., Structure and mechanism of the iron-sulfur flavoprotein phthalate dioxygenase reductase, FASEB J. 9, 1411-1418, 1995; Miyauchi, K., Adachi, Y, Nagata, T., and Takagi, M., Cloning and sequencing of a novel meta-cleavage dioxygenase gene whose product is involved in degradation of g -hexachlorocyclohexane in Sphingomonas paucimobilis, J.Bacteriol. 181, 6712-6719, 1999; Calderone, V., Trabucco, M., Menin, V. et al., Cloning of human 3-hydroxyanthranilic acid dioxygenase in Escherichia coli: characterization of the purified enzyme in its in vitro inhibition by Zn2+, Biochim.Biophys.Acta 1596, 283-292, 2002; JohnsonWinters, K., Purpero, V.M., Kavana, M., et al.,(4-Hydroxyphenyl)pyruvate dioxygenase from Streptomyces avermitilis: the basis for ordered substrate addition, Biochemistry 42, 2072-2080, 2003; Frerichs-Deeken, U., Ranguelova, K., Kappl, R., et al., Dioxygenases without requirement for cofactors and their chemical model reaction: compulsory order ternary complex mechanism of 1H-3-hydroxy-4-oxyquinaldine 2,4-dioxygenase involving general base catalysis by histidine 251 and single-electron oxidation of the substrate dianion, Biochemistry 43, 14485-14499, 2004; Yin, C.X. and Finke, R.G., It is true dioxygenase or classic autoxidation catalysis? Re-investigation of a claimed dioxygenase catalyst based on a Ru(2)-incorporated, polyoxometalate precatalyst, Inorg.Chem. 44, 4175-4188, 2005; Matsumura, E., Ooi, S., Murakami, S., et al., Constitutive synthesis, purification, and characterization of catechol 1,2-dioxygenase from the aniline-assimilating bacterium Rhodococcus sp. An-22, J.Biosci.Bioeng. 98, 71-76, 2004; Lee, K., p-Hydroxylation reactions catalyzed by naphthalene dioxygenase, FEMS Microbiol.Lett. 255, 316-320, 2006; Suvorova, M.M., Solyanikova, I.P., and Gobovleva, L.A., Specificity of catechol ortho-cleavage during para-toluate degradation by Rhodococcus opacus 1cp, Biochemistry(Mosc) 71, 1316-1323, 2006. Ketone Reductases; engineered yeast cells; alcohol dehydrogenases
Enantiomeric and diastereoisomeric reductions of ketones and b-keto esters
O
R’
R’’
HO
H
R’
R’’
H
OH
R’
R’’
+
Enantioselective reactions O
O
OH
O
R’’’ R’
R’’’
O
R’
O
R’’
R’’
Diastereoselective reactions
N
H N
N
N
H N
N
Enone
O
N
N
OH (R)allylic alcohol
Stewart, J.D., Organic transformations catalyzed by engineered yeast cells and related systems, Curr.Opin.Biotechnol. 11, 363-368, 2000; Habrych, M., Rodriguez, S., and Stewart, J.D., Purification and identification of an Escherichia coli b-keto ester reductase as 2,5-diketo-d-gluconate reductase YquE, Biotechnol.Progress 18, 257-261, 2002; Katz, M., Sarvary, I., Frejd, T., et al., An improved stereoselective reduction of a bicyclic diketone by Saccharomyces cerevisiae combining process optimization and strain engineering, Appl.Microbiol.Biotechnol. 59, 641-648, 2002; Ravot, G., Wahler, D., Favre-Bulle, O., et al., High throughput discovery of alcohol dehydrogenase for industrial biocatalysis, Adv.Synthesis Catalysis 345, 691-694, 2003; Lou, W.Y., Zong, M.H., Zhang, Y.Y. and Wu, H., Efficient synthesis of optically active organosilyl alcohol via asymmetric reduction of acyl silane with immobilized yeast, Enzyme Microb.Technol. 35, 190-196, 2004; Rodrigues, J.A.R., Moran, P.J.S., Conceicao, G.J.A., and Fardelone, L.C., Recent advances in the biocatalytic asymmetric reduction of acetophenones and a,b-unsaturated carbonyl compounds, Food Technol.Biotechnol. 42, 295-303, 2004; Pollard, D.J., Telari, K., Lane, J., et al., Asymmetric reduction of a,b-unsaturated ketone to (R) allylic alcohol by Candida chilenis, Biotechnol.Bioengineer. 93, 674-686, 2006.
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Enzymes in Synthetic Organic Chemistry
937
Enzymes in Synthetic Organic Chemistrya,b (Continued) Cephalosporin Acylase
Conversion of cephalosporin C or adipyl-7-aminodesacetoxycephalosporonic acid to derivatives useful in the synthesis of semi-synthetic b-lactam antibiotics.
Sio, C.F., Otten,L.G., Cool, R.H., and Quax, W.M., Analysis of substrate specificity switch residue of cephalosporin acylase, Biochem.Biophys.Res. Commun. 312, 755-760, 2003; Sio, C.F., and Quax. W., Improved b-lactam acylases and their use as biocatalysts, Curr.Opin.Biotechnol. 15, 349-355, 2004; Sonawane, V.C., Enzymatic modifications of cephalosporins by cephalosporin acylase and other enzymes, Crit.Rev.Biotechnol.26, 95-120, 2006. Penicillin Acylase
Catalyzes the conversion of benzylpenicillin (penicillin G) to 6-aminopenicillinic acid for the production of b-lactam antibiotics. Penicillin acylase also converts cephalosporin C to 6-aminopenicillinic acid. There has been considerable work on the engineering and stabilization of the enzyme. Penicillin acylase can catalyze the reverse reaction resulting in a condensation. S
O
H3C CH3
N
N H
COOH
O Penicillin G Penicillin Acylase H3C S
O
CH3 OH
+
N
H2N
COOH
O Phenylacetic Acid
6-aminopenicillanic acid
Mahajan, P.B., Penicillin acylases. an update, Appl.Biochem.Biotechnol. 9, 538-554, 1984; Andersson, E. and Hahn-Hagerdal, B., Bioconversion in aqueous two-phase systems, Enzyme Microb.Technol. 12, 242-254, 1990; Valle, F., Balbas, P., Merino, E., and Bolivar, F., Trends Biotechnol. 16, 36-40, 1991; Zaks, A., Industrial biocatalysis, Curr.Opin.Chem.Biol. 5, 130-136, 2001; Arroyo, M. de la Mata, I., Acebal, C, and Castillon, M.P., Biotechnological application of penicillin acylases: state-of-the-art, Appl.Microbiol.Biotechnol. 60, 507-514, 2003; Albian, O., Mateo, C., Fernandez-Lorente, G., et al., Improving the industrial production of 6-APA: enzymatic hydrolysis of penicillin G in the presence of organic solvents, Biotechnol.Prog. 19, 1639-1642, 2003; Calleri, E., Temporini, C., Massolini, G., and Caccilanza, G., Penicillin G acylase-based stationary phases: analytical applications, J.Pharm.Biomed.Anal. 35, 243-258, 2004; Sio, C.F. and Quzx, W.J., Improved beta-lactam acylases and their use as industrial biocatalysts, Curr.Opin.Biotechnol. 15, 349-355, 2004; Guranda, D.T., Volovik, T.S., and Svedas, V.K., pH stability of penicillin acylase from Escherichia coli, Biochemistry 69, 1386-1390, 2004; Girelli, A.M. and Maltei, E., Application of immobilized enzyme reactor in on-line high performance liquid chromatography: a review, J.Chromatog B. Analyt.Technol.Biomed.Life Sci. 819, 3-16, 2005; Torres, R., Pessela, B., Fuentes, M., et al., Stabilization of enzymes by multipoint attachment via reversible immobilization on phenylboronic activated supports, J.Biotechnol. 120, 396-401, 2005; Nigam, V.K., Kundu, S., and Ghosh, P., Single-step conversion of cephalosporin-C to 7-aminocephalosporonic acid by free and immobilized cells of Pseudomonas diminuta, Appl.Biochem.Biotechnol. 126, 13-21, 2005; van Roon, J.L., Boom, R.M., Paasman, M.A., et al., Enzyme distribution and matrix characteristics in biocatalytic particles, J.Biotechnol. 119, 400-415, 2005; Giorando, R.C., Ribeiro, M.P., and Giordano, R.L., Kinetics of beta-lactam antibiotic synthesis by penicillin G acylase (PGA) from the view of the industrial enzyme reactor optimization, Biotechnol.Adv. 24, 27-41, 2006. Narayanan, N. Xu, Y., and Chou, G.P., High-level gene expression for recombinant penicillin acylase production using the araB promoter system in Escherichia coli, Biotechnol.Prog. 22, 1518-1523, 2006; De Leon-Rodriguez, A., Rivera-Pastrana, D., Medina-Rivero, E., et al., Production of penicillin Acylase by a recombinant Escherichia coli using cheese whey as substrate and inducer, Biomol.Eng. 23, 299-305, 2006; Wang, L., Wang, Z., Xu., J.H., et al., An eco-friendly and sustainable process for enzymatic hydrolysis of penicillin G in cloud point system, Bioprocess Biosyst.Eng. 29, 157-162, 2006; Aguilar, O., Albiter, V., Serrano-Carreon, L., and Rito-Palomares, M., Direct comparison between ion-exchange chromatography and aqueous two-phase processes for the partial purification of penicillin acylase produced by E.coli, J.Chromatog.B. Analyt.Techol. Biomed.Life Sci. 835, 77-83, 2006; Shah, S. ,Sharma, A. and Gupta, M.N., Preparation of cross-linked enzyme aggregates by using bovine serum albumin as a proteic feeder, Anal.Biochem. 351, 207-213,2006. Synthetic reaction: Nam, D.H. and Ryu, D.D., Biochemical properties of penicillin amidohydrolase from Micrococcus luteus, Appl.Environ.Microbiol. 38, 35-38, 1979; Youshko, M.I., van Langen, L.M., de Vroom, E., et al., Highly efficient synthesis of ampicillin in an “aqueous solution-precipitate” system; repetitive addition of substrates in a semicontinuous process, Biotechnol.Bioengineer. 73, 4260430, 2001; Youshko, M.I., van Langen, L.M., de Vroom, E., et al., Penicillin acylase-catalyzed ampicillin synthesis using a pH gradient: a new approach to optimization, Biotechnol.Bioeng. 78, 589-593, 2002; Goncalves, L.R., Fernandez-Lafuente, R., Guisan, J.M., et al., Inhibitory effects in the side reactions occurring during the enzymic synthesis of amoxicillin: p-hydroxyphenylglycine methyl ester and amoxicillin hydrolysis, Biotechnol. App..Biochem. 38, 77-85, 2003; Alkema, W.B., de Vries, E., Floris, R., and Janssen, D.B., Kinetics of enzyme acylation and deacylation in the penicillin acylase-catalyzed synthesis of beta-lactam antibiotics, Eur.J.Biochem. 270, 3675-3683, 2003; Alfonso, I. and Gotor, V., Biocatalytic and biomimetic aminolysis reactions: useful tools for selective transformations or polyfunctional substrates, Chem.Soc.Rev. 33, 201-209,. 2004’ Gabor, E.M. and Janssen, D.B., Increasing the synthetic performance of penicillin acylase PAS2 by structure-inspired semi-random mutagenesis, Protein Eng.Des.Sci. 17, 571579, 2004. An unusual synthetic application was removal blocking groups from synthetic insulin; Svoboda, I., Brandenburg, D., Barth, T., et al., Semisynthetic insulin analogues modified in positions B24, B25 and B29, Biol.Chem.Hoppe Seyler 375, 373-378, 1994.
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Therapeutic Enzymesa Asparaginase
Catalyzes the hydrolysis of asparagine to aspartic acid; used for the treatment of acute lymphoblastic leukemia. Normal cells can synthesize asparagine while tumor cells in acute lymphoblastic leukemia cannot synthesize asparagine
Hill, J.M., Roberts, J., Loeb, E., et al., L-Asparaginase therapy for leukemia and other malignant neoplasms. Remission in human leukemia, JAMA 202, 882-888, 1967; Broome, J.D., Studies on the mechanism of tumor inhibition by L-asparaginase. Effects of the enzyme on asparaginase levels in the blood, normal tissue, and 6C3HED lymphomas of mice: differences in asparagine formation and utilization in asparaginase-sensitive and –resistant lymphoma cells, J.Exp.Med. 127, 1055-1072, 1968; Adamson, R.H. and Fabro, S., Antitumor activity and other biologic properties of L-asparaginase (NSC-109229)-a review, Cancer Chemother.Rep. 52, 617-626, 1968; Keating, M.J., Holmes, R., Lerner, R., and Ho, D.H., L-Asparaginase and PEG asparaginase –past, present, and future, Leuk.Lymphoma 10(Suppl) 153-157, 1993; Davis, F.F., PEG-adenosine deaminase and PEG-asparaginase, Adv. Exp.Med.Biol. 519, 51-58, 2003; Pinheiro, J.P. and Boos, J., The best way to use asparaginase in childhood acute lymphatic leukemia—still to be defined?, Br.J.Haematol. 125, 119-127, 2004 Blood Coagulation Factor VIIa
Treatment of Blood Coagulation factor VIII inhibitors, potentially a general intravascular hemostatic agent
Siddiqui, M.A. and Scott, L.J., Recombinant factor VIIa (Eptacog Alfa): A review of its use in congenital or acquired haemophilia and other congenital bleeding disorders, Drugs 65, 1161-1177, 2005; Franchini, M., Zaffanello, M., and Veneri, D., Recombinant factor VIIa. An update on its clinical use, Thromb.Haemostas. 93, 1027-1035, 2005; Margaritis, P. and High, K.A., Advances in gene therapy using factor VIIa in hemophilia, Semin.Hematol. 43(Suppl 1), S101-S104, 2006; Farrugia, A., Assessing efficacy and therapeutic claims in emerging indications for recombinant Factor VIIa: regulatory perspectives, Semin.Hematol. 43(1 Suppl 1), S64-S69, 2006; Bosinski, T.J. and El Solh, A.A., Recombinant factor VIIa, its clinical properties, and the tissue factor pathway of coagulation, Mini Rev.Med.Chem. 6, 1111-1117, 2006 Butyryl cholinesterase
Detoxification of neurotoxic agents related to DFP; bioscavenger of anticholinesterase agents; Also used for treatment of cocaine overdoses
Doctor, B.P., Raveh, L., Wolfe, A.D., et al., Enzymes as pretreatment drugs for organophosphate toxicity, Neurosci.Biobehav.Rev. 15, 123-128, 1991; Broomfield, C.A., Maxwell, D.M., Solana, R.P., et al., Protection by butyrylcholinesterase against organophosphorus poisoning in nonhuman primates, J.Pharmacol.Exp.Ther. 259, 633-638, 1991; Grunwald, J., Marcus, D., Papier, Y., et al., Large-scale purification and long-term stability of human butyrylcholinesterase: a potential bioscavenger drug, J.Biochem.Biophys.Methods 34, 123-135, 1997; Lynch, T.J., Mattes, C.E., Singh, A., et al., Cocaine detoxification by human plasma butyrylcholinesterase, Toxicol.Appl.Pharmacol. 145, 363-371, 1997; Mattes, C.E., Lynch, T.J., Singh, A., et al., Therapeutic use of butyrylcholinesterase for cocaine intoxication, Toxicol.Appl. Pharmacol. 145, 372-380, 1997; Browne, S.P., Slaughter, E.A., Couch, R.A., et al., The influence of plasma butyrylcholinesterase concentration on the in vitro hydrolysis of cocaine in human plasma, Biopharm.Drug.Dispos. 19, 309-314, 1998; Yuan, H.J., Yu, W.Y., Shi, C.H., and Sun, M.J., Characteristics of recombinant human butyrylcholinesterase, Zhongguo Yao Li Xue Bao 20, 74-80, 1999; Chambers, J. and Oppenheimer, S.F., Organophosphates, serine esterase inhibition, and modeling of organophosphate toxicity, Toxicol.Sci. 77, 185-187, 2004; Guven, M., Sungur, M., Eser, B. et al., The effects of fresh frozen plasma on cholinesterase levels and outcomes in patients with organophosphate poisoning, J.Toxicol. Clin.Toxicol. 42, 617-623, 2004; Fischer, S., Arad, A., and Margalit, R., Liposome-formulated enzymes for organophosphate scavenging: butyrylcholinesterase and Demeton-S, Arch.Biochem.Biophys. 434, 108-115, 2005; Saez-Valero, J., de Gracia, J.A., and Lockridge, O. Intraperitoneal administration of 340 kDa human plasma butyrylcholinesterase increases the level of the enzyme in the cerebrospinal fluid of rats, Neurosci.Lett. 383, 93-98, 2005; Lockridge, O., Schopfer, L.M., Winger, G., and Woods, J.H., Large scale purification of butyrylcholinesterase from human plasma suitable for injection into monkeys: A potential new therapeutic for protection against cocaine and nerve agent toxicity, J.Med.Chem.Biol.Radiol.Def. 3:nihms5095, 2005; Gardiner, S.J. and Begg, E.J., Pharmacogenetics, drug-metabolizing enzymes, and clinical practice, Pharmacol.Rev. 58, 521-590, 2006; Lucic Vrdoljak, A., Calic, M., Radic, B., et al., Pretreatment with pyridinium oximes improves antidotal therapy against tabun poisoning, Toxicology 228, 41-50, 2006 Cocaine Esterase
Cocaine Detoxification
Brzezinski, M.R., Abraham, T.L., Stone, C.L., et al., Purification and characterization of a human liver cocaine carboxylesterase that catalyzes the production of benzoylecgonine and the formation of cocaethylene from alcohol and cocaine, Biochem.Pharmacol. 48, 1747-1755, 1994; Turner, J.M., Larsen, N.A., Basran, A., et al., Biochemical characterization and structural analysis of a highly proficient cocaine esterase, Biochemistry 41, 1229712307, 2002; Ascenzi, P., Clementi, E., and Polticelli, F., The Rhodococcus sp. cocaine esterase: a bacterial candidate for novel pharmacokinetic-based therapies for cocaine abuse, IUBMB Life 55, 397-402, 2003; Rogers, C.J., Mee, J.M., Kaufmann, G.F., et al., Toward cocaine esterase therapeutics, J.Am. Chem.Soc. 127, 10016-10017, 2005; Rogers, C.J., Eubanks, L.M., Dickerson, T.J., and Janda, K.D., Unexpected acetylcholinesterase activity of cocaine esterase, J.Am.Chem.Soc. 128, 15364-15365, 20006; Cooper, Z.D., Narasimhan, D., Sunahara, R.K., et al., Rapid and robust protection against cocaine-induced lethality in rates by bacterial cocaine esterase, Mol.Pharmacol. 70, 1885-1891, 2006
a
Targeting of therapeutic enzymes is a challenge and is the subject of current study. See Ribeiro, C.C. Barrias, C.C., and Barbosa, M.A., Calcium phosphate-alginate microspheres as enzyme delivery matrices, Biomaterial 25, 4363-4373, 2004; Vogler, C., Levy, B., Grubb, J.H., et al., Overcoming the blood-brain barrier with high-dose enzyme replacement therapy in murine mucopolysaccharidosis VII, Proc.Nat.Acad.Sci.USA 102, 14777-14782, 2005; Fukudo, T., Ahearn, M., Roberts, A., et al., Autophagy and mistargeting of therapeutic enzymes in skeletal muscle in Pompe disease, Mol.Ther. 14, 831-839, 2006; Lee, S., Yang, S.C., Hefferman, M.J., et al., Polyketal microparticles: a new delivery vehicle for superoxide dismutase, Bioconjug.Chem. 18, 4-7, 2007.
939
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940
Handbook of Biochemistry and Molecular Biology THERAPEUTIC ENZYMES (Continued)
DNAse
Originally used for the resolution of localized abscesses by viscosity reduction due to hydrolysis of high-molecular weight DNA arising from tissue damage. There is more recent use in the treatment of cystic fibrosis as Dornase™ alpha. A combination of streptokinase and streptodornase was developed as well and is still used as Varidaseb
Sherry, S., Johnson, A., and Tillett, W.R., The action of streptococcal desoxyribose nuclease (Streptodornase): In vitro and on purulent pleural exudations of patients, J.Clin.Invest. 29, 1094-1104, 1949; Johnson, A.J., Cytological studies in association with local injections of streptokinasestreptodornase into patients, J.Clin.Invest. 29, 1376-1386, 1950; Bryson, H.M. and Borkin, E.M., Dornase alpha. A review of its pharmacological properties and therapeutic potential in cystic fibrosis, Drugs 48, 894-906, 1994; Thomson, A.H., Human recombinant DNAse in cystic fibrosis, J.R.Soc. Med. 88 (suppl 25), 24-29, 1995; Davies, J. ,Trindale, M., Wallis, C. et al., The clinical use of rhDNAse, Pediatr.Pulmonol.Suppl. 16, 273-274, 1997; Goa, K.L. and Lamb, H., Dornase alpha. A review of pharmacoeconomic and quality-of-life aspects of its use in cystic fibrosis, Pharmacoeconomics 12, 409-422, 1997; Jones, A.F. and Wallis, C.E., Recombinant human deoxyribonuclease for cystic fibrosis, Cochrane Database Syst.Rev. (3), CD001127, 2000; Suri, R., The use of human deoxyribonuclease (rhDNAse) in the management of cystic fibrosis, BioDrugs 19, 135-144, 2005; Fayon, M., CF-emerging therapies: Modulation inflammation, Pediatr.Respir.Rev. 7 Suppl 1, S170-S174, 2006 Digestive Enzymes
Usually a crude homogenate of pancreas using to treatment pancreatic disease. Lipase is an individual enzyme which is used to treat steatorrheac. An artificial saliva is available to treat salivary gland dysfunctiond.
Gullo, L., Indication for pancreatic enzyme treatment in non-pancreatic digestive diseases, Digestion 54(suppl 2), 43-47, 1993; Kitagawa, M., Naruse, S., Ishiguro, H., and Hayakawa, T., Pharmaceutical development for treating pancreatic disease, Pancreas 16, 427-431, 1998; Nakamura, T., Takeuchi, T., and Tando, Y., Pancreatic dysfunction and treatment options, Pancreas 16, 329-336, 1998; Divisi, D., Di Tomaso, S., Salvemini, S., et al., Diet and cancer, Acta Biomed. 77, 118-123, 2006 Glucocerebrosidase (Acid β-glucosidase/ Replacement of a lysosomal enzyme deficiency which results in Gaucher’s Disease lysosomal β-glucosidase/lysosomal β-glucosidase) (Ceredase®/ Cerezyme®) Wiltink, E.H. and Hollak, C.E., Alglucerase (ceredase), Pharm.World Sci. 18, 16-19, 1996; Bijsterbosch, M.K., Donker, W., van de Bilt, H., et al., Quantitative analysis of the targeting of mannose-terminal glucocerebrosidase. Predominant uptake by liver endothelial cells, Eur.J.Biochem. 237, 344-349, 1996; Grabowski, G.A., Leslie, N., and Wenstrup, R., Enzyme therapy for Gaucher disease: the first 5 years, Blood Rev. 12,115-133, 1998; Barranger, J.A. and O’Rourke, E., Lessons learned from the development of enzyme therapy for Gaucher disease, J.Inherit.Metab.Dis. 24(suppl 2), 89-96, 2001; Charrow, J., Anderson, H.C., Kaplan, P., et al., J.Pediatr. 144, 112-120, 2004; Beutler, E., Enzyme replacement in Gaucher disease, PLoS Med. 1, e21, 2004; Connock, M., Burls, A., Frew, E., et al., The clinical effectiveness and cost-effectiveness of enzyme replacement therapy for Gaucher’s disease: a systemic review, Health Technol.Assess. 10, iii-iv, ix-134, 2006; vom Dahl, S., Poll, L., di Rocco, M., et al., Evidence-based recommendations for monitoring bone disease and the response to enzyme replacement therapy in Gaucher’s patients, Curr.Med.Res.Opin. 22, 1045-1064, 2006 Lactase
Used as an oral formulation for the treatment of lactose intolerance
Ramirez, F.C., Lee, K., and Graham, D.Y., All lactase preparations are not the same: results of a prospective, randomized placebo-controlled trial, Am.J.Gastroenterol. 89, 566-570, 1994; Gao, K.P., Mitsui, T., Fujiki, K., et al., Effect of lactase preparations in asymptomatic individuals with lactase deficiency—gastric digestion of lactose and breath hydrogen analysis, Nagoya J.Med.Sci. 65, 21-28, 2002; Erasmus, H.D., Ludwig-Auser, H.M., Paterson, P.G., et al., Enhanced weight gain in preterm infants receiving lactose-treated feeds: a randomized, double-blinded, controlled trial, J.Pediatr. 141, 532-537, 2002; Tan-Dy, C.R. and Ohlsson, A., Lactase treated feeds to promote growth and feeding tolerance in preterm infants, Cochrane Database Syst.Rev. 18, CD004591, 2005; Montalto, M., Curigliano, V., Santoro, L., et al., Management and treatment of lactose malabsorption, World J.Gastroenterol. 12, 187-191, 2006; O’Connell, S. and Walsh, G., Physicochemical characteristics of commercial lactases relevant to their application in the alleviation of lactose intolerance, Appl.Biochem.Biotechnol. 134, 179-191, 2006 b
c
d
Tillett, W.S. and Sherry, S., The effect in patients of streptococcal fibrinolysin (streptokinase) and streptococcal deoxyribonuclease on fibrinous, purulent, and sanguinous pleural exudations, J.Clin.Invest. 28, 173-190, 1949; Tillett, W.E., Sherry, S., and Read, C.T., The use of streptokinase-streptodornase in the treatment of postneumonic empyema, J.Thorac.Surg. 21, 275-297, 1951; Miller, J.M., Ginsberg, M., Lipin, R.J., and Long, P.H., Clinical experience with streptokinase and streptodornase, J.Am.Med.Assoc. 145, 620-624, 1951; Nemoto, K., Hirota, K., Ono, T., et al., Effect of varidase (streptokinase) on biofilm formed by Staphylococcus aureus, Chemotherapy 46, 111-115, 2000; Light, R.W., Nguyen, T., Mulligan, M.E., and Sasse, S.A., The in vitro efficacy of varidase versus streptokinase or urokinase for liquefying thick purulent exudative material from loculated empyema, Lung 178, 13-18, 2000; Rutter, P.M., Carpenter, B., Hill, S.S., and Locke, I.C., Varidase: the science behind the medicament, J.Wound Care 9, 223-226, 2000; Zhu, E., Hawthorne, M.L, Guo, Y., et al., Tissue plasminogen activator combined with human recombinant deoxyribonuclease is effective therapy for empyema in a rabbit model, Chest 129, 1577-1583, 2006. Greenberger, N.J., Enzymatic therapy in patients with chronic pancreatitis, Gastroenterol.Clin.North Am. 28, 687-693, 1999; Layer, P., Keller, J., and Lankisch, P.G., Pancreatic enzyme replacement therapy, Curr.Gastroenterol.Rep. 3, 101-108, 2001; DiMagno, E.P., Gastric acid suppression and treatment of severe exocrine pancreatic insufficiency, Best Pract.Res.Clin.Gastroenterol. 15, 477-486, 2001; Layer, P. and Keller, J., Lipase supplementation therapy: standards, alternatives, and perspectives, Pancreas 26, 1-7, 2003. Gaffar, A., Hunter, C.M., and Mirajkar, Y.R., Applications of polymers in dentifrices and mouthrinses, J.Clin.Dent.13, 138-148, 2002; Brennan, M.T., Shariff, G., Lockart, P.B., and Fox, F.C., Treatment of xerostomia: a systematic review of the therapeutic trials, Dent.Clin.North Am. 46, 847-856, 2002; Guggenheimer, J. and Moore, P.A., Xerostomia: etiology, recognition and treatment, J.Am.Dent.Assoc. 134, 61-69, 2003; Porter, S.R., Scully, C., and Hegarty, A.M., An update of the etiology and management of xerostomia, Oral.Surg.Oral.Med.Oral.Pathol.Radiol.Endod. 97, 28-46, 2004; Urquhart, D. and Fowler, C.E., Review of the use of polymers in saliva substitutes for symptomatic relief of xerostomia, J.Clin.Dent. 17, 29-33, 2006.
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Therapeutic Enzymes
941 THERAPEUTIC ENZYMES (Continued)
Superoxide Dismutase
Superoxide toxicity; treatment of inflammatory disorders; Also gene therapye target in amyelotropic lateral sclerosisf
Omar, B.A., Flores, S.C., and McCord, J.M., Superoxide dismutase, in Therapeutic Proteins. Pharmacokinetics and Pharmacodynamics, ed. A.H.C. King, R.A. Baughman, and J.W. Larrick, W.H. Freeman and Company, New York, New York, Chapter 14, pps. 295-315, 1993; di Napoli, M. and Papa, F., M-40403 Metaphore Pharmaceuticals, IDrugs 8, 67-76, 2005; Leite, P.F., Liberman, M., Sandoli de Brito, F., and Laurindo, F.R., Redox processes underlying the vascular repair reaction, World J.Surg. 28, 331-336, 2004; Hernanez-Savedra, D., Zhou, H., and McCord, J.M., Anti-inflammatory properties of a chimeric recombinant superoxide dismutase: SOD2/3, Biomed.Pharmacother. 59, 204-206, 2005; St. Clair, D., Zhao, Y., Chaiswing, L., and Oberly, T., Modulation of skin tumorigenesis by SOD, Biomed.Pharmacol. 59, 209-214, 2005; Emerit, J., Samuel, D., and Pavio, N., Cu-Zn superoxide dismutase as a potential antifibrotic drug for hepatitis C related fibrosis, Biomed.Pharmacother. 60, 1-4, 2006; Yasui, K. and Baba, A., Therapeutic potential of superoxide dismutase (SOD) for resolution of inflammation, Inflamm.Res. 55, 359-363, 2006 Streptokinase
Plasminogen activator derived from b-hemolytic Streptococcus (groups A,C, and G); approximately 40-50 kD; streptokinase does not have any known catalytic activity but functions by formation of a complex with plasminogen which results in plasminogen activation. Now available as a recombinant product. Streptokinase is used in combination with DNAse(streptodornase) to treat abscesses and empyemab.
de la Fuente Garcia, J. and Estrade, M.P., Experimental studies with recombinant streptokinase, in Therapeutic Proteins. Pharmacokinetics and Pharmacodynamics, ed. A.H.C. King, R.A. Baughman, and J.W. Larrick, W.H. Freeman and Company, New York, New York, Chapter 13, pps. 283-293, 1993; Konstantinides, S., Should thrombolytic therapy be used in patients with pulmonary embolism?, Am.J.Cardiovasc.Drugs. 4, 69-74, 2004; Capstick, T. and Henry, M.T., Efficacy of thrombolytic agents in the treatment of pulmonary embolism, Eur.Respir.J. 26, 664-674, 2005; Ueshima, S. and Matsuo, O., Development of new fibrinolytic agents, Curr.Pharm.Des. 12, 849-857, 2006; Caceres-Loriga, P.M., Perez-Lopez, H., Morlana-Herandez, K., Facundo-Sanchez, H., Thrombolysis as first choice therapy in prosthetic heart valve thrombosis. A study of 68 patients, J.Thromb.Thrombolysis 21, 185-190, 2006 Tissue Plasminogen Activator (tPA)
A serine protease which activate plasminogen resulting in fibrinolysis. tPA is used for the treatment of myocardial infarction and stroke. The first recombinant protein was manufactured in CHO cells; more recently an engineered form (Reteplase®) has been developed in Escherichia coli and is seeing clinical use.
Collen, D. and Lijnen, H.R., Tissue-type Plasminogen activator. Mechanisms of action and thrombolytic properties, Haemostasis 16(Suppl 3), 25-32, 1986; Anderson, J.L. Recent clinical developments in thrombolysis in acute myocardial infarction, Drugs 33(Suppl 3), 22-32, 1987; Hollander, J.J., Plasminogen activators and their potential in therapy, Crit.Rev.Biotechnol. 6, 253-271, 1987; Grossbard, E.B., Recombinant tissue plasminogen activators: a brief review, Pharm.Res. 4, 375-378, 1987; Montaner, J., Stroke biomarkers: Can they help us to guide stroke thrombolysis?, Drug News Perspect. 19, 523-532, 2006; Khaja, A.M. and Grotta, J.C., Established treatments for acute ischaemic stroke, Lancet 369, 319-330, 2007; Simpson, D., Siddiqui, M.A, Scott, L.J., and Hilleman, D.E., Spotlight on reteplase in thrombotic occlusive disorders, BioDrugs 21, 65-68, 2007 Thrombin
Therapeutic action based on the clotting of fibrinogen and aggregation of blood platelets. Thrombin is a component in fibrin sealant which is used as a tissue adhesive and is used as free-standing product as a suture support and for the treatment of vascular pseudoaneurysms.
Lundblad, R.L., Bradshaw, R.A., Gabriel, D., et al., A review of the therapeutic uses of thrombin, Thromb.Haemost. 91, 851-860, 2004; Hagberg, R.C., Safi, H.J., Sabik, J., et al., Improved intraoperative management of anastomotic bleeding during aortic reconstruction: results of a randomized controlled trial, Am.Surg. 70, 307-311, 2004; Aziz, O., Athanasiou, T., and Darzi, A., Haemostasis using a ready-to-use collagen sponge coated with activated thrombin and fibrinogen, Surg.Technol.Int. 14, 35-40, 2005; Valbonesi, M., Fibrin glues of human origin, Best Pract.Res.Clin.Haematol. 19, 191-203, 2006; Evans, L.A. and Morey, A.F., Hemostatic agents and tissue glues in urologic injuries and wound healing, Urol.Clin.North Am. 33, 1-12, 2006; Stone, P.A., AbuRhama, A.F., Flaherty, S.K., and Bates, M.C., Femoral pseudoaneurysms, Vasc.Endovascular Surg. 40, 109-117, 2006; Gabay, M., Absorbable hemostatic agents, Am.J.Health Syst.Pharm. 63, 1244-1253, 2006; Drobnic, M., Radosavljevic, D., Ravnik, D., et al., Comparison of four techniques for the fixation of a collagen scaffold in the human cadaveric knee, Osteoarthritis Cartilage 14, 337-344, 2006 Urate oxidase (Rasburicase)
Catalyzes the oxidation of urate to 5-hydroxyisourate. Used for the treatment of hyperuricemia (excess uric acid). Specifically for tumor lysis syndrome, gout.
Bessmertny, O., Robitaille, L.M., and Cairo, M.S., Rasburicase: a new approach for preventing and/or treating tumor lysis syndrome, Curr.Pharm.Des. 11, 4177-4185, 2005; Oldfield, V. and Perry, C.M., Rasburicase: a review of its use in the management of anticancer therapy-induced hyperuricaemia, Drugs 66, 529-545, 2006; Oldfield, V. and Perry, C.M., Spotlight on rasburicase in anticancer therapy-induced hyperuricemia, BioDrugs 20, 197-199, 2006; Lee, S.J. and Terkeltaub, R.A., New developments in clinically relevant mechanisms and treatment of hyperuricemia, Curr.Rheumatol.Rep. 8, 224-230, 2006; Teng, G.G., Nair, R., and Saag, K.G., Pathophysiology, clinical presentation and treatment of gout, Drugs 66, 1547-1563, 2006; Higdon, M.L. and Higdon, J.A., Treatment of oncologic emergencies, Am.Fam.Physician 74, 1873-1880, 2006; Sood, A.R., Burry, L.D., and Cheng, D.K., Clarifying the role of rasburicase in tumor lysis syndrome, Pharmacotherapy 27, 111-121, 2007 e f
In this context, gene therapy can refer to gene augmentation therapy, gene correction therapy, and RNA silencing. Xu, Z. and Xia, X.G., RNAi therapy: dominant disease gene gets silenced, Gene Ther. 12, 1159-1160, 2005; Hino, T., Yokota, T., Ito, S., et al., In vivo delivery of small interfering RNA targeting brain capillary endothelial cells, Biochem.Biophys.Res. Commun. 340, 263-267, 2006; Zemlyak, I., Nimon, V., Brooke, S., et al., Gene therapy in the nervous system with superoxide dismutase, Brain Res. 1088, 12-18, 2006; Azzouz, M., Gene therapy for ALS: progress and prospects, Biochim.Biophys.Acta 1762, 1122-1127, 2006; Xia, X., Zhou, H., Huang, T., and Zu, Z., Allele-specific RNAi selectively silences mutant SOD1 and achieves significant therapeutic benefit in vivo, Neurobiol.Dis. 23, 578586, 2006; Miller, T.M., Smith, R.A., and Cleveland , D.W., Amyelotropic lateral sclerosis and gene therapy, Nat.Clin.Pract.Neurol. 2, 462-463, 2006; Davis, A.S., Zhao, H., Sun, G.H. et al., Gene therapy using SOD1 protects striatal neurons from experimental stroke, Neurosci.Lett. 411, 32-36, 2007; Qi, X., Nauswirth, W.W., and Guy, J., Dual gene therapy with extracellular superoxide dismutase and catalase attenuates experimental optic neuritis, Mol.Vis. 13, 1-11, 2007; Epperly, M.W., Wegner, R., Kanai, A.J., Effects of MnSOD-plasmid liposome gene therapy on antioxidant levels in irradiated murine oral cavity orthotopic tumors, Radiat.Res. 167, 289-297, 2007.
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942
Handbook of Biochemistry and Molecular Biology THERAPEUTIC ENZYMES (Continued)
Urokinase
Originally isolated from urine, urokinase is now available a recombinant protein and is used for the treatment of thrombosis in myocardial infarction and stroke; tPA is more often used for stroke. Urokinase acts by converting plasminogen to plasming.
Maksimenko, A.V. and Tischenko, E.G., New thrombolytic strategy: bolus administration of tPA and urokinase-fibrinogen conjugate, J.Thromb. Thrombolysis 7, 307-312, 1999; Stepanova, V.V., and Tkachuk, V.A., Urokinase as a multidomain protein and polyfunctional cell regulation, Biochemistry 67, 109-118, 2002; Bourekas, E.C., Slivka, A.F., and Casavant, M.J., Intra-arterial thrombolysis of a distal internal carotid artery occlusion in an adolescent, Neurocrit.Care 2, 179-182, 2005; Roychoudhury, P.K., Khaparde, S.S., Mattisson, R., and Kumar, A., Synthesis, regulation and production of urokinase using mammalian cell culture: a comprehensive review, Biotechnol.Adv. 24, 514-526, 2006; Bansal, V. and Roychoudhury, P.K., Production and purification of urokinase: a comprehensive review, Protein Expr.Purif. 45, 1-14, 2006; Juttler, E., Kohrmann, M., and Schellinger, P.D., Therapy for early reperfusion after stroke, Nat.Clin.Pract.Cardiovasc.Med. 3, 656-663, 2006; Mullen, M.T., McGarvey, M.L., and Kasner, S.E., Safety and efficacy of thrombolytic therapy in postoperative cerebral infarctions, Neurol.Clin. 24, 783-793, 2006. g
Plasmin is a serine protease which digests fibrin. An acyl-plasmin was developed for therapeutic use – Smith, R.A., Dupe, R.J., English, P.D., and Green, J., Fibrinolysis with acyl-enzymes: a new approach to thrombolytic therapy, Nature 290, 505-508, 1981; Dupe, R.J., English, P.D., Smith, R.A., and Green, D.J., Acyl-enzymes as thrombolytic agents in dog models of venous thrombosis and pulmonary embolism, Thromb.Haemost. 51, 249-253, 1984; Tomiya, N., Watanabe, K., Awaya, J., et al., Modification of acylplasmin-streptokinase complex with polyethylene glycol. Reduction of sensitivity to neutralizing antibody, FEBS Lett. 193, 44-48, 1985; Kalindjian, S.B. and Smith, R.A., Reagents for reversible coupling of proteins to the active centres of trypsin-like serine proteinases, Biochem.J. 249, 409-413, 1987; Teuten, A.J., Cooper, A., Smith, R.A., and Dobson, C.M., Binding of a substrate analogue can induce co-operative structure in the plasmin-serine proteinase domain, Biochem.J. 293, 567-572, 1993; Lijnen, H.R., van Hoef, B., Smith, R.A., and Collen, D., Functional properties of p-anisolylated plasmin-staphylokinase complex, Thromb.Haemost. 70, 326-331, 1993.
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Weights of Cells and Cell Constituents
H+
PO4
50
Ca++
20 10 0
60S
K+
40 30
Ribosomes 80S
Cl–
Viruses
40S
(509 CMPDS.)
DNA
Mg++ 10 9 8 7 6 5 4 3 2 1
(67 Proteins)
Na+
No. of Proteins
No. Low M.W. Org. Compounds
60
H2O 101
102
103
104
105
106
107
Human cells Mitochondria 108
109
1010
1011
1012
1013
1014
1015
1016
Weights of Cells and Cell Constituents in Daltons
Contributed by Norman G. Anderson. This figure originally appeared in Sober, Ed., Handbook of Biochemistry and Selected Data for Molecular Biology, 2nd ed., Chemical Rubber Co., Cleveland, 1970.
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PARTICLE DIAMETER Particle Diameter Particle Diameter, Microns (µ)
0.0001
(1mµ) 0.1 0.001 0.01 2 3 456 8 2 3 456 8 2 3 456 8 10
Equivalent Sizes
10
X-Rays
Ultraviolet Fume Mist
Solid:
Gas Dispersoids
Smog
H2
Methods for Particle Size Analysis
100 2 3 456 8
105
106
2 3 456 8 4
10 Visible
Near Infrared Solar Radiation
Clay
Soil
Common Atmospheric Dispersoids
Typical Particles and Gas Dispersoids
10
2 3 456
3
10
(1cm) (1mm) 1000 10,000 2 3 456 8 2 3 456 8 2 3 107
108
Angstrom Units A
Electromagnetic Waves Technical Definitions
2
1
O2 F2
CO2 Cl2 C5H6 *Gas C4H10Molecules SO2
HCl CH4 * Molecular Dias H2O Calculated from Viscosity Data at N2 0°C CO
Microwaves (Radar, Etc.)
Far Infrared Dust
Silt
Fine Sand
Spray Coarse Sand
Clouds & Fog
Drizzle Mist Ground Fertilizer, Limestone Fly Ash Coal Dust
Gravel Rain
Rosin Smoke Oil Smokes Tobacco Smoke Metallurgical Dusts and Fumes Ammonium Cement Dust Chloride Fume Sulfuric Carbon Black Zinc Oxide Fume Colloidal Silica
Beach Sand Concentrator Mist Contact Pulverized Coal Sulfuric Mist Flotation Ores Paint Pigments Insecticide Dusts
Ground Talc Plant Spray Dried Milk Sports Alkali Fume Pollens Aitken Milled Flour Nuclei Atmospheric Dust Nebulizer Hydraulic Nozzle Drops Sea Salt Nuclei Drops Pneumatic Combustion Lung Damaging Dust Nuclei Nozzle Drops Red Blood Cell Dia (Adults): 7.5µ?? 0.3µ Viruses Bacteria Human Hair Electroformed Impingers Sieving Sieves Ultramicroscope Microscope Electron Microscope Elutriation Centrifuge Sedimentation Ultracentrifuge Turbidimetry X-Ray Diffraction Permeability Visible To Eye Adsorption Scanners Machine Tools (MIC, Calipers, Etc.) Light Scattering Nuclei Counter Electrical Conductivity
This figure originally appeared in Sober, Ed., Handbook of Biochemistry and Selected Data for Molecular Biology, 2nd ed., Chemical Rubber Co., Cleveland, 1970.
945
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Appendix A: Abbreviations and Acronyms 2D-DIGE: two-dimensional difference gel electrophoresis 2DE: two-dimensional electrophoresis A: absorbance A23187: a calcium ionophore, Calcimycin AAA: abdominal aortic aneurysm; AAA+. ATPases associated with various cellular activities: AAAA: Association Against Acronym Abuse AAG box: an upstream cis-element AAS: aminoalkylsilane; atomic absorption spectroscopy AAT: amino acid transporter; alpha-1-antitrypsin AAV: adenoassociated virus ABA: Abscisic acid, a plant hormone ABC-Transporter Proteins: ATP-Binding Cassette Transporter Proteins ABC: ATP-Binding Cassette; antigen-binding cell ABE: acetone butanol ethanol Abl: retroviral oncogene derived from Abelson murine leukemia ABRC: ABA response complex ABRE: ABA response element 7-ACA: 7-aminocephalosporanic acid ACES: 2-[(2-amino-2-oxyethyl)amino]-ethanesulfonic acid ACSF: Artificial cerebrospinal fluid ACS: active sequence collection Ach (AcCho): acetylcholine AChR (AcChoR): acetylcholine receptor ACME: arginine catabolic mobile element ACTH: adrenocorticotropin Can: acetonitrile Acrylodan: 6-acryloyl-2-(dimethylamino)-napthalene ADA: adenosine deaminase; antidrug antibody ADAM: a disintegrin and metalloproteinase ADAMTS: a subfamily of disintegrin and metalloproteinase with thrombospondin motifs. ADCC: antibody-dependent cell-mediated cytotoxicity as in NK cells attacking antibody-coated cells ADH: alcohol dehydrogenase; antidiuretic hormone ADME: adsorption, distribution, metabolism, excretion ADME-Tox: ADME-Toxicology AdoMet: S-adenosyl-l-methionine AEC: alveolar epithelial cell AFLP: amplified fragment length polymorphism AFM: atomic force microscopy AGE: advanced glycation endproducts AGO: argonaute protein family AGP: acid glycoprotein AID: activation-induced cytodine deaminase AKAP: A-kinase anchoring proteins Akt: a protein kinase Akt: a retroviral oncogene derived from AKT8 murine T cell lymphoma Alk: anaplastic lymphoma kinase; receptor member of insulin superfamily ALL: acute lymphocytic leukemia ALP: alkaline phosphatase ALS: anti-lymphocyte serum ALT: alanine aminotransferase ALV: avian leukosis virus AML: acute myeloid leukemia
AMPK: AMP-activated protein kinase AMS: accelerator mass spectrometry AMT: accurate mass tag ANDA: Abbreviated New Drug Application ANOVA: analysis of variables (factorial analysis of variables) ANS: 1-anilino-8-napthlenesulfonate; autonomic nervous system ANTH: AP180 N-terminal homology as in ANTH-domain 2-AP: 2-aminopyridine 6-APA: 6-aminopenicillanic acid APAF1: apoptotic protease activating factor 1 Apg1: a serine/threonine protein kinase required for vesicle formation which is essential for autophagy APL: acute promyelocytic leukemia ApoB: apolipoprotein B AQP: adenosine tetraphosphate ARAP3 : a dual Arf and Rho GTPase activating protein ARD: acute respiratory disease; acireductone dioxygenase; automatic relevance determination; acid rock drainage ARE: AU-rich elements ARF: ADP-ribosylation factor ARL: Arf-like ARM: arginine-rich motif ARS: automatic replicating sequence or autonomously replicating sequence ART: mono-ADP-ribosyltransferase; family of proteins, large group of A-B toxins AS: antisense ASD: alternative splicing database; http: //www.ebi.ac.uk/asd ASPP: ankyrin-repeat, SH3-domain and proline-rich-regioncontaining proteins AST: aspartate aminotransferase ATC: aspartate transcarbamylase domain ATCase: aspartate transcarbamylase ATP: adenosine-5’-triphosphate ATPg S: adenosine-5’-3-O-(thiotriphosphate) ATR-FTIR: attenuated total reflectance-Fourier transform infrared ATR-IR: attenuated total reflection infrared AVT: arginine vasotocin Axl: anexceleko; used in reference to a receptor kinase related to the Tyro 3 family BA: betaine aldehyde BAC: bacterial artificial chromosome; also blood alcohol concentration BAD: a member of the Bcl02 protein family considered to be a proapoptotic factor BADH: betaine aldehyde dehydrogenase BAEC: bovine aortic endothelial cells BAEE: Benzoyl-arginine ethyl estera BALT: bronchial associated lymph tissue BBB: blood brain barrier B-CAM: basal cell adhesion molecule BCG: bacille-Calmette-Guérin BCR: breakpoint cluster region; B-cell receptor BCR-ABL: BCR-ABL is the fused gene that results from the Philadelphia chromosome, The BCR-ABL gene produces the Bcr-Abl tyrosine kinase, Bcl-2: protein family regulating apoptosis BCIP: 5-bromo, 4-chloro, 3-indoyl phosphate 947
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BCS: biopharmaceutical classification system for describing the gastrointestinal absorption of drugs; also Budd-Chiari syndrome BDH: d-b-butyrate dehydrogenase BDNF: brain-derived growth factor BEBO: an unsymmetrical cyanine dye for binding to the minor grove of DNA; 4-[(3-methyl-6-(6-methylbenzothiazol-2-yl)-2,3,-dihydro(benzo-1,3-thiazole)2-methylidene)]-1-methyl-pyridinium iodide. BET: refers to an isotherm for adsorption phenomena in chromatography; acronym derived from Stepen Brunauer, Paul Emmet, and Edward Teller B/F: bound/free bFGF: basic fibroblast growth factor BFP: blue fluorescent protein BGE: background electrolyte Bicine: N,N-bis(2-hydroxyethyl)glycine BiFC: bimolecular fluorescence complementation BIND: biomolecular interaction network database BiP: immunoglobulin heavy chain-binding protein Bis-TRIS: 2,2-bis-(hydroxymethyl)-2,2’,2” nitriloethanol BLA: Biologic License Application BLAST: basic local alignment search tool BME: 2-mercaptoethanol; b-mercaptoethanol BMP: bone morphogenic protein BopA: a secreted protein required for biofilm formation BPTI: bovine pancreatic trypsin inhibitor BCRA-1: breast cancer 1; a tumor suppressor gene associated with breast cancer BRE-luc: a mouse embryonic stem cell line used to study bone morphogenetic protein. BRET: bioluminescence resonance energy transfer, see FRET. BrdU: bromodeoxyuridine Brig: polyoxyethylene lauryl ether BSA: bovine serum albumin bZIP: basic leucine zipper transcription factor C1INH: C1 inhibitor; inhibitor of activated complement component 1, missing in hereditary angioneurotic edema. CA125: cancer antigen 125; a glycoprotein marker used for prognosis in ovarian cancer; also referred to as MUC16 CAD: multifuntional protein and initiates and regulate de novo pyrimidine biosynthesis; caspases-activated DNAse. CAK: Cdk-activating kinase CALM: clathrin assembly lymphoid myeloid leukemia as in CALM gene CAM (CaM): calmodulin; cell adhesion molecule CAMK: Ca2+/calmodulin-dependent protein kinase CAPS: cleavable amplified polymorphic sequences; also cationic antimicrobial peptide CArG: a promoter element [CC(A/T)6G] gene for smooth muscle a-actin CASP: critical assessment of structural prediction CASPASE: cysteine-dependent asparatate-specific protease CAT: catalase; chloramphenicol acetyl transferase CATH: class, architecture, topology, homologous superfamily; hierarchical classification of protein domain structure. CATP: Chloramphenicol resistance gene, caged ATP, cation transporting p-type Cbl: a signal transducing protein downstream of a number of receptor-couple tyrosine kinases; a product of the c-cbl proto-oncogene Cbs: chromosomal breakage sequence CBz: carbobenzoxy
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948 CCC: concordance correlation coefficient CCD: charge couple device CCK: choleocystokinin CCV: clathrin-coated vesicles CD: clusters of differentiation; circular dichroism; cyclodextrin CDC: complement-dependent cytotoxicity; complement-mediated cell death CDK (cDK): cyclin-dependent kinase cDNA: complementary DNA CDR: complementary determining region CDTA: 1,2-cyclohexylenedinitriloacetic acid CE: capillary electrophoresis CEC: capillary electrochromatography CE-SDS: capillary electrophoresis in the presence of sodium dodecyl sulfate CELISA: cellular enzyme-linked immunosorbent assay; enzymelinked immunosorbent assay on live cells CERT: ceramide transport protein CEPH: Centre d’Etude du Polymorphisme Humain CEX: cation exchange CFA: complete Freund’s adjuvant CFP: Cyan Fluorescent Protein CFTR: cystic fibrosis transmembrane conductance region Cfuc: colony forming unit CGE: capillary gel electrophoresis CGH: comparative genome hybridization CGN: cis-Golgi network CH: calponin homology CHAPS: 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid CHCA: a-cyano-4-hydroxycinnamic acid CHEF: chelation-enhanced fluorescence CHES: 2-(N-cyclohexylamino)ethanesulfonic acid ChiP: chromatin immunoprecipitation CHO: chinese hamster overy; carbohydrate CID: collision-induced dissociation; collision-induced dimerization CIDEP: chemically induced dynamic electron polarization CIDNP: chemically induced dynamic nuclear polarization CIEEL: chemically initiated electron exchange luminescence CLIP: class-II-associated invariant chain (Ii) peptide CLT: clotvinazole [1-(a2-chlorotrityl)imidazole] CLUSTALW: A general purpose program for structural alignment of proteins and nucleic acids http: //www.ebi. ac.uk/clustalw/ cM: centimorgan CM: carboxymethyl CaMK: calmodulin kinase, isoforms I, II, III CMCA : competitive metal capture analysis CML: chronic myelogenous leukemia CML: carboxymethyl lysine CAN: bacterial cell wall collagen-binding protein Cn: calcineurin CNC: Cap’n’Collar family of basic leucine zipper proteins CNE: conserved non-coding elements dCNE: duplicated CNE COACH: comparison of alignments by constructing hidden Markow models CoA: coenzyme A COFFEE: consistency based objective function for alignment evaluation COFRADIC: combined fractional diagonal chromatography COG: conserved oligomeric Golgi; cluster of orthologous groups COPD: chronic obstructive pulmonary disease
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COX: cytochrome C oxidase Cp: ceruloplasmin CPA: carboxypeptidase A CPB: carboxypeptidase B CPD: cyclobutane pyrimidine dimer CPDK: calcium-dependent protein kinase CpG: cytosine-phosphate-guanine CpG-C: cytosine-phosophate-guanine class C Cdpk4: a Ca2- protein kinase CPP: cell penetrating peptide; combinatorial protein pattern CPSase: carbamoyl-phosphate synthetase CPY: carboxypeptidase Y CRAC: calcium-release activated calcium (channels) CRE: cyclicAMP response element Cre1: cytokine response 1; a membrane kinase CREA: creatinine CREB: cAMP-response element binding protein CRM: certified reference material CRP: C-reactive protein; also cAMP receptor protein CRY: chaperone CS: chondroitin sulfate CSF: colony stimulating factor CSP: cold-shock protein CSR: cluster-situated regulator; class-switch recombination CSSL: chromosome segment substitution lines Ct: chloroplast CT: charge transfer CTB: cholera toxin B subunit CTD: C-terminal domain CTL: cytotoxic T lymphocytes CTLA: cytotoxic T lymphocyte-associated antigen CTLL: cytotoxic T-cell lines CTPSase: CTP synthetase CtrA: a master regulator of cell cycle progression CV: coefficient of variation Cvt: cytosome to vacuole targeting CW: continuous wave(non-pulsed source of electromagnetic radiation) CYP: cytochrome P450 enzyme Cst3: cystatin 3 CZE: capillary zone electrophoresis D: diffusion Dax: axial dispersion coefficient DAB(p-dab): p-dimethyl amino azo benzene dABs: domain antibodies DABSYL: N,N-dimethylaminoazobenzene-4’-sulfonyl-usually as the chloride, DABSYL chloride DAD: diaphanous-autoregulatory domain DAF: decay accelerating factor DAG: diacyl glycerol DALI: distance matrix alignment; http: //www2.ebi.ac.uk/dali/ DANSYL: 5-dimethylaminonapthalene-1-sulfonyl; usually as the chloride (DANSYL chloride) DAP: DNAX-activation protein; also diaminopimelic acid; DAP12: DNAX activating protein of 12kDa mass DAS: distributed annotated system; downstream activation site DBMB: Dulbecco’s modified Eagles Medium DBD-PyNCS: 4-(3-isocyanatopyrrolidin-1-yl)-7-(N,N-dimethylaminosulfonyl)-2-benzoxadiazole DBTC: “Stains All”; 4,5,4’,5’-dibenzo-3,3’-diethyl-9-methylthiacarbocyanine bromide DC: dendritic cell DCC: dicyclohexylcarbodiimide
9168_Book.indb 949
949 DCCD: N,N’-dicyclohexylcarbodimide DDBJ: DNA Databank of Japan; http: //www.ddbj.nig.ac.jp DDRs: discoidin domain receptors (DDR1, DDR2) DDR1: discoidin domain receptor1, CAK, CD167a, PTK3, Mck10 DDR2: discoidin domain receptor2, NTRK3, TKT, Tyro10 DEAE: diethylaminoethyl DEG: differentially expressed gene(s) DEX: dendritic-cell-derived exosomes DFF: DNA fragmentation factor DFP: diisopropylfluorophosphate; diisopropylphosphorofluoridate DHFR: dihydrofolate reductase DHO: dihydroorotase domain DHOase: dihydroorotase DHPLC(dHPLC): denaturing HPLC DHS: DNase I hypersensitivity site DIP: database of interacting proteins - http: //dip.doe-mbi.ucla. edu; also dictionary of interfaces in proteins – http: // drug-redesign.de/superposition.html Dipso: 3-[N,N.-bis(2-hydroxyethyl)amino]-2-hydroxypropanesulfonic acid DLS: dynamic light scattering DM: an accessory protein located in the lysosome associated with MHC-class-II antigen presentation. It is located in the endosomal/lysosomal system of APC DMBA: 7,12-dimethylbenzo[a]anthracene DMD: Duchenne muscular dystrophy; also Doctor of Dental Medicine DMF: dimethylformamide; decayed, missing, filled(denistry) DMS: dimethyl sulfate DMSO: dimethyl sulfoxide DMT1: divalent metal transporter 1 ssDNA: single-stranded DNA DNAa: a bacterial replication initiation factor DNAX: DNAase III, tau and gamma subunits dNPT: deoxynucleoside triphosphate DO: an accessory protein located in the lysosome associated with MHC-class-II antigen presentation. DO has an accessory role to DM DOTA: tetraazacyclodedecanetetraacetic acid DPE: downstream promoter element DPI: dual polarization interferometry DPM: disintegrations per minute DPN: diphosphopyridine dinucleotide (currently NAD); DPPC: dipalmitoylphosphatidylcholine DPPE: 1,2-dipalmitoyl-sn-glycerol-3-phosphoethanolamine DPTA: diethylenetriaminepentaacetic acid dsDNA: double-stranded DNA dsRNA: double-stranded RNA dsRBD: double-stranded RNA binding domain DRE: dehydration response element; dioxin response element DRT: dimensionless retention time (a value for chromatography) DSC: differential scanning calorimetry DSP: downstream processing DTAF: dichlorotriazinyl aminofluorescein DTE: dithioerythritol DTNB: 5,5’-dithio-bis(2-nitrobenzoic acid) Ellman’s Reagent DTT: dithiothreitol DUP: a duplicated yeast gene family DVDF: polyvinyl difluoride E1: ubiquitin-activating enzyme E2: ubiquitin carrier protein E3: ubiquitin-protein isopeptide ligase
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E-64: trans-epoxysuccinyl-l-leucylamino-(4-guanidino)-butane, proteolytic enzyme inhibitor EAA: excitatory amino acid EBA: expanded bead adsorption EBV: Epstein-Barr virus EDC(EADC): 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide; N-ethyl-N’-(3-dimethyl-aminopropyl) carbodiimide ECF: extracytoplasmic factor; extracellular fluid ECM: extracellular matrix EDC: 1-ethyl-(3-dimethylaminopropyl)-carbodiimide EDI: electrodeionization EDTA: ethylenediaminetetraacetic acid, Versene, (ethylenedinitrilo) tetraacetic acid; EEO: electroendoosmosis EEOF: electroendoosmotic flow EF: electrofiltration EGF: epidermal growth factor EGFR: epidermal growth factor receptor; Erbb-1; HER1 EGTA: ethyleneglycol-bis(b-aminoethylether)-N,N,N’,N’-tetraacetic acid; eIF: eukaryotic initiation factor EK: electrokinetic EKLF: erythroid Krüppel-like factor ELISA: enzyme-linked immunosorbant assay EMBL: European Molecular Biology Laboratory EMCV: encephalomyocarditis virus EMF: electromotive force EMMA: enhanced mismatch mutation analysis EMSA: electrophoretic mobility shift assay ENaC: epithelial Na channel EndoG: endonuclease G ENTH: epsin N-terminal homology as ENTH-domain ENU: N-ethyl-N-nitrosourea EO: ethylene oxide EOF: electroosmotic flow Eph: a family of receptor tyrosine kinases; function as receptors/ ligands for ephrins EPL: expressed protein ligation Epps: 4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid EPR: electron paramagnetic resonance ER: endoplasmic reticulum ERAD: endoplasmic reticulum-associated protein degradation ErbB2: epidermal Growth Factor Receptor, HER2 ErbB3: epidermal Growth Factor Receptor, HER3 ErbB4: epidermal Growth Factor Receptor, HER4 ERK: Extracellular-regulated kinase Erk ½: P 42/44 extracellular signal-regulated kinase Ero1p: a thiol oxidase which generates disulfide bonds inside in the endoplasmic reticulum ERSE: endoplasmic reticulum(ER) stress response element ES: embryonic stem as in embryonic stem cell ESI: electrospray ionization ESR: electron spin resonance; also erthyrocyte sedimentation rate ESS: exonic splicing silencer EST: expressed sequence tag ETAAS: electrothermal atomic absorption 5,6-ETE: 5,6-epoxyeicosatrienoic acid ETS: family of transcription factors EUROFAN: European functional analysis network - http: // mips.gsf.de/proj/eurofan/; also European Programme for the Study and Prevention of Violence in Sport Exo1: exonuclease 1
9168_Book.indb 950
950 EXP1: expansion gene FAAH: fatty acid amide hydrolase Fab: antigen binding fragment from immunoglobulin FAB: fast atom bombardment FAB-MS/MS: fast atom bombardment-mass spectrometry/ mass spectrometry FACE: fluorophore-assisted carbohydrate electrophoresis FACS: fluorescence-activated cell sorting FADD: Fas association death domain FAD: flavin adeninine dinucleotide FAK: focal adhesion kinase FBS: fetal bovine serum Fc: Fc region of an immunoglobulin representing the C-terminal region FcgR: cell surface receptor for the Fc domain of IgG FDA: fluorescein diacetate FDC: follicular dendritic cells FCCP: carbonyl cyanide p-trifluoromethoxyphenyl-hydrazine FEAU: 2’-fluoro-2’-deoxy-b-D-arabinofuranosyl-5-ethyluracil FERM: as in FERM-domain (four-point-one; ezrin, radixin, moesin). FFAT: two phenylalanyl residues in an acidic tract FIAU: 2’-fluoro-2’-deoxy-b-D-arabinofuranosyl-5-iodouracil FecA: ferric citrate transporter FEN: flap endonuclease Fes: retroviral oncogene derived from ST and GA feline sarcoma FFPE: formalin-fixed, paraffin-embedded FGF: fibroblast growth factor FGFR: fibroblast growth factor receptor Fgr: retroviral oncogene derived from GR feline sarcoma FIGE: field-inversion gel electrophoresis FITC: fluoroscein isothiocyanate FTIR: Fourier-transformed infrared reflection FTIR-ATR: Fourier-transformed infrared reflection-attenuated total reflection FLAG™: an epitope “tag” which can be used as a fusion partner for recombinant protein expression and purification. FlhB: a component of the flagellum-specific export apparatus in bacteria. FLIP: fluorescence loss in photobleaching FLK-1: vascular endothelial growth factor receptor (VEGFR) FLT-1: vascular endothelial growth factor receptor (VEGFR) fMLP(FMLP): N-formyl methionine leucine phenylalanine fMOC: 9-Fluorenzylmethyloxycarbonyl Fms: retroviral oncogene derived from SM feline sarcoma Fok1: a type IIS restriction endonuclease derived from Flavobacterium okeanokoites Fos: retroviral oncogene derived from FBJ murine osteosarcoma FOX: forkhead box FpA: fibrinopeptide A FPC: fingerprinted contigs Fps: retroviral oncogene from Fujiami avian sarcoma FRAP: fluorescence recovery after photobleaching FRET: fluorescence resonance energy transfer; Förster resonance energy transfer FT: Fourier Transform FSSP: fold classification based on structure alignment of proteins; http: //www2.ebi.ac.uk/dali/fssp/fssp.html FU: aluorescence unit 5-Fu: 5-fluorouracil Fur: ferric uptake receptor Fur: gene for Fur
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FYVE: a zinc-binding motif; acronym derived from four proteins containing this domain G: guanine Ga: heterotrimeric G protein, a-subunit Gb: heterotrimeric G protein, b-subunit Gg: heterotrimeric G protein, g-subunit G-6-PD: glucose-6-phosphate dehydrogenase GABA: gamma (g)-aminobutyric acid GAG: glycosaminoglycan GALT: gut-associated lymphoid tissues GalNac: N-acetylgalactosamine GAPDH: glyceraldehayde 3-phosphate dehydrogenase GAPS: GTPase activating proteins GAS6: a protein, member of the vitamin K-dependent protein family GASP: Genome Annotation Assessment Project; http: //www. fruitfly.org/GASP1/; also growth advantage in stationary phase GBD: GTPase binding domain GC: gas chromatography; granular compartment GC-MS: gas chromatography-mass spectroscopy GC-MSD: gas chromatography-mass selective detector GcrA: A master regulator of cell cycle progression G-CSF: granulocyte colony stimulating factor GCP: Good Clinical Practice GDH: glutamate Dehydrogenase GDNF: glial-derived neurotrophic factor GdnHCl: guanidine hydrochloride GEFs: guanine nucleotide exchange factors GF-AAS: graphite furnace atomic absorption spectroscopy GFP: green fluorescent protein GGDEF: a protein family GGT: gamma-glutamyl transferase GGTC: German Gene Trap Consortium; a reference library of gene trap sequence tags (GTST) http: //www. genetrap.de/ GHG: greenhouse gas GI: gastrointestinal; genomic islands cGK: cyclic GMP (cGMP)-dependent protein kinase GlcNac: N-acetylglucosamine GLD: gelsolin-like domain GLP: Good Laboratory Practice(s) GlpD: glyceraldehyde-3-phosphate dehydrogenase GLUT: a protein family involves in the transport of hexoses into mammalian tissues Glut4: facilitative glucose transporter which is insulin-sensitive Glut5: a fructose transporter, catalyzes the uptake of fructose GM: genetically modified GMP-PDE(cyclic GMP-PDE): cyclic GMP-phosphodiesterase GM-CSF: granulocyte-macrophage colony stimulating factor cGMP: current Good Manufacturing Practice GNSO: 5-nitrosoglutathione GPC: gel permeation chromatography GPCR: G-protein coupled receptor GPI: glycosyl phosphatidylinositol GRIP: a Golgi-targeting protein domain GRP: glucose-regulated protein Grp78: a glucose regulated protein; identical with BiP GSH: glutathione GST: glutathione-S- transferase; gene trap sequencing tag GTF: general Transcription factor GTST(GST): gene trap sequence tags GUS: beta-glucuronidase
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951 GXP(s): A generic acronymy for good ………. practices including but not limited to good clinical practice, good laboratory practice, good manufacturing processes. HA: hemaglutin-A; hyaluronic acid; hydroxyapaptite, Ca10(PO4)6(OH)2 HABA: [2-(4’-hydroxyazobenzene)] benzoic acid; HAS: human serum albumin; hyaluron synthase HAT: histone acetyltransferase; hypoxanthine, aminopterin and thymidine HBSS: Hanks Balanced Salt Solution H/D: hydrogen/deuterium exchange HAD: heteroduplex analysis HDAC: histone deacetylase HDL: high-density lipoprotein HDLA: human leukocyte differentiation antigen HD-ZIP: homeodomain-leucine zipper proteins HEPT: height equivalent to plate number HERV: human endogenous retrovirus 20-HETE: 20-hydroxyeicosatetranenoic acid HETP: plate height (chromatography); HexNac: N-acetylhexosamine HGP: human genome project HH: hereditary hemochromatosis HHM: hidden Markov models His-Tag(His6; H6): histidine tag – a hexahistidine sequence HLA: human leukocyte associated antigen HLA-DM: enzyme responsible for loading peptides onto MHC class II molecules HLA-DO: protein factor which modulates the action of HLA-DM HMGR: 3-hydroxy-3-methylglutamyl-coenzyme A reductase HMP: herbal medicinal product(s) HMT: histone hnRNA: heterologous nuclear RNA HOG: high-osmolarity glycerol HOPE: Hepes-glutaminic acid-buffer mediated organic solvent protein effect HOX(HOX, hox): describing a family of transcription factors HPAED-PAD: high performance anion-exchange chromatography-pulsed amperometric detection 5-HPETE: 5-hydroperoxyeicosatetranenoic acid HPRD: human protein reference database HPRT: hypoxanthine phosphoribosyl transferase HRP: horse radish peroxidase HS: heparan sulfate HSB: homologous synteny blocks HSC: hematopoietic stem cell HSCQ: heteronuclear single quantum correlation HSE: heat shock element Hsp: heat-shock protein Hsp70: heat shock protein 70 5-HT: 5-hydroxytryptamine HTF: HpaII tiny fragments; distinct fragments from the HpaII digestion of DNA; HpaII is a restriction endonuclease HTH: helix-turn-helix HTS: high-throughput screening htSNP: haplotype single nucleotide polymorphism HUGO: human genome organization HUVEC: human umbilical vein endothelial cells IAA: iodoacetic acid IAEDANS: N-iodoacetyl-N’-(5-sulfo-1-napthyl) ethylenediamine IBD: identical-by-descent; also inflammatory bowel disease IC: ion chromatography
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ICAM: intercellular adhesion molecule ICAT: isotope-coded affinity tag ICH: intracerebral hemorrhage; a gene related to Ice involved in programmed cell death; historically, international chick unit; International Conference for Harmonisation ICPMS: inductively coupled plasma mass spectrometry ID: internal diameter IDA: interaction defective allele IDMS: isotope dilution mass spectrometry IEC: ion-exchange chromatography IEF: isoelectric focusing IES: internal eliminated sequences IFE: immunofixation electrophoresis IFN: interferon Ig: immunoglobulin IGF: insulin-like growth factor IGFR: insulin-like growth factor receptor Ihh: indian hedgehog IkB: NF-kB inhibitor IkK: IkB kinase IL: Interleukin iLAP: integrated lysis and purification ILGF: insulin-like growth factor ILGFR: insulin-like growth factor receptor ILK: integrin-linked kinase IMAC: immobilized metal-affinity chromatography IMINO: Na+ -dependent alanine-insensitive proline uptake system (SLC6A20) IMP: integrin-mobilferrin pathway membrane protein system involves in the transport of ferric iron; also inosine-5’- monophosphate IPG: immobilized pH gradient IPTH: isopropylthio-b-d-galactopyranoside IP3: inositol 1,4,5-triphosphate IPTG: Isopropylthio-b-d-galactosidase IR: inverted repeat; insulin receptor IRES: internal ribosome entry site IRS: insulin receptor substrate ISE: ion-specific electrode ISO: International Standards Organization ISS: immunostimulatory sequence; also intronic splicing silencer ISS-ODN: immunostimulatory sequence-oligodeoxynucleotide ISSR: inter-simple sequence repeats IT: isotocin ITAF: IRES trans-acting factor ITAM: immunoreceptor tyrosine-based activation motif ITC: isothermal titration calorimetry iTRAQ: isobaric tags for relative and absolute quantitation of proteins in proteomic research JAK: Janus Kinase JNK: c-Jun N-terminal kinase KARAP: killer cell activating receptor-associated protein Kb, kb : Kilobase KDR: kinase insert domain-containing receptor; KDR is the human homolog of the mouse FLK-1 receptor. The KDR and FLK-1 receptors are also known as VEGFR2. See VEGFR Kit: mast/stem cell growth factor receptor, CD 117 Kit: retroviral oncogene derived from HZ4 feline sarcoma KLF5: Kruppel-like factor 5, a transcription factor LAK: lymphokine-activated killer cells LATE-PCR: linear-after-the-expotential-PCR LB: Luria-Bertani
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952 Lck: member of the Src family of protein kinases LC50: median lethan concentration in air LC-MS: liquid chromatography-mass spectrometry LCR: low-copy repeat; locus control region; low complexity region LCST: lower critical solution temperature LD: as in LD motif, a leucine/aspartic acid-rich protein-binding domain; also used to refers to peptidases without sterospecificity; also longin domain; linkage disequilibrium; lactate dehydrogenase LD50 : median lethal dose LDL: low-density lipoprotein LECE: ligand exchange capillary electrophoresis LED: light emitting diode Lek: lymphocyte-specific protein tyrosine kinase LFA: lymphocyte function-associated antigen LGIC: ligand-gated ion channel LH: luteinizing hormone LIF: laser-induced fluorescence LIM : a domain involved in protein-protein interaction, originally described in transcription factors LIN1, ISL1, and MED3. LINE: long interspersed nuclear element LLE: liqud-liquid extraction LLOD: lower limit of detection LLOQ: lower limit of quantification Lnr: initiator element lnRNP: large nuclear ribonucleoprotein LOD: limit of detection; log10 of odds LOLA: list of lists - annotated LOQ: limit of quantitation LP: lysophospholipid LPA: lysophosphatidic acid LPH: lipotropic hormone LPS: lipopolysaccharide LTB4: leukotriene B4 LTH: luteotropic hormone Ltk: leukocyte tyrosine kinase LRP: low-density lipoprotein receptor-related protein LSPR: localized surface plasmon resonance LTR: long terminal repeat LUCA: last universal cellular ancestor M13: a bacteriophage used in phage display MФ: macrophage Mab, MABq: monoclonal antibody MAC: membrane attack complex MAD: multiwavelength anomalous diffraction Maf: retroviral oncogene derived from AS42 avian sarcoma MAGE: microarray and gene expression MALDI-TOF: matrix-assisted laser desorption ionization time of flight MAP: mitogen-activated protein; usually referring to a protein kinase such MAP-kinase MAPK: MAP-kinase MAPKK: MAP-kinase kinase MAPKKK: MAP-kinase kinase kinase MAR: matrix attachment region Mb, mb: megabase (10 6) MB: molecular beacon MBL: mannose-binding lectin; maltose-binding protein MBP: myelin basic protein MCA: 4-methylcoumaryl-7-acetyl MCAT: mass coded abundance tag
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MCD: magnetic circular dichroism MCM: mini-chromosome maintenance MCS: multiple cloning site M-CSF: M-colony stimulating factor (Macrophage-colony stimulating factor) MDA: malondialdehyde MDMA: 3,4-methylenedioxymethamphetamine MDCK: Madin-Darby canine kidney MEF: mouse embryonic fibroblasts MEF-2: myocyte enhancer factor 2 MEGA-8: octanoyl-N-methylglucamide MEGA-10: decanoyl-N-methylglucamide MEK: mitogen-activated protein kinase/extracellular signalregulated kinase kinase; also methylethyl ketone MELC: microemulsion liquid chromatography MELK: multi-epitope-ligand-kartographie MEM: minimal essential medium Mer: a receptor protein kinase; also Mertk, Mer tyrosine kinase MES: 2-(N-morpholinoethanesulfonic acid) Met: receptor for hepatocyte growth factor MFB: membrane fusion protein MGO: methylglyoxal MGUS: monoclonal gammopathy of undetermined significance MHC: major histocompatibility complex MIAME: minimum information about a microarray experiment Mil: retroviral oncogene derived from Mill Hill-2 chicken carcinoma MIP: molecularly imprinted polymer; macrophage inflammatory protein; methylation induced premeiotically MIPS: Munich Information Center for Protein Sequences MIS: Mullerian Inhibiting Substance MLCK: myosin light chain kinase MLCP: myosin light chain phosphatase MMP: matrix metalloproteinase MMR: mismatch repair MMTV: mouse mammary tumor virus MOPS: 3-(N-morpholino)propanesulfonic acid; 4-morpholinopropanesulfonic acid MOPSo: 3-(N-morpholino)-2-hydroxypropanesulfonic acid Mos: retroviral oncogene derived from Moloney murine sarcoma MPD: 2-methyl-2,4-pentanediol MPSS: massively parallel signature sequencing MR: magnetic resonance MRI: magnetic resonance imaging mRNA: messenger RNA MRP: migratory inhibitory factor-related protein MRTF: myocardin-related transcription factor MS: mass spectrometry, also mechanosensitive (receptors), multiple sclerosis MS/MS: mass spectrometry/mass spectrometry MS3: tandem mass spectrometry/mass spectrometry/mass spectrometry MSP: macrophage stimulating protein Mt: mitochondrial mt-DNA: mitochondrial DNA MTBE: methyl-t-butyl ether MTOC: microtubule organizing center mTOR: a eukaryotic regulatory of cell growth and proliferation. See TOR MTSP: membrane type serine proteases MTT: methylthiazoletetrazolium MTX: methotrexate
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953 MU: Miller Units Mu: Mutator MUSK: muscle skeletal receptor tyrosine kinase MuDPiT: multidimensional protein identification technology MuLV: Muloney leukemia virus MWCO: molecular weight cut-off My: million years Myb : retroviral oncogene derived from avian myeloblastosis Myc: retroviral oncogene derived from MC29 avian myelocytomatosis MYPT: myosin phosphatase-targeting Mys: myristoylation site NAA: neutron activation analysis Nabs: neutralizing antibodies nAChR (nAcChoR): nicotinic acetylcholine receptor NAD: nicotinamide adenine dinucleotide (DPN) NADP: nicotinamide adeninine dinucleotide phosphate (TPN) NAO: non-animal origin NAT: nucleic acid amplification testing; nucleic acid testing. Nbs2: Ellman’s Reagent; 5,5’-dithiobis(2-nitrobenzene acid) NBD: nucleotide-binding domain NBD-PyNCS: 4-(3-isothiocyanato pyrrolidin-1-yl)-7-nitro-2,1,3benzoxadiazole NBS: N-bromosuccinimide NBT: nitroblue tetrazolium NCBI: National Center for Biotechnology Information NCED: 9-cis-epoxycarotenoid dioxygenase NDA: New Drug Application NDB: nucleic acid databank NDMA: N-methyl-d-aspartate NDSB: 3-(1-pyridinio)-1-propanesulfonate (non-detergent sulfobetaine) NEM: N-ethylmaleimide NEO: Neopterin NEP: nucleous-encoded polymerase (RNA polymerase) NeuAc: N-acetylneuraminic acid NeuGc: N-glycolylneuraminic acid NF: National Formulary NFAT: nuclear factor of activated T cells, a transcription factor NF-kB: nuclear factor kappa B, a nuclear transcription factor NGF: nerve growth factor NGFR: nerve growth factor receptor NHS: N-hydroxysuccinimide NK: natural killer (as in cytotoxic T cell) NKCF: natural killer cytotoxic factor NKF: N-formylkynurenine Ni-NTA: Ni2+ -nitriloacetate NIR: near infrared NIRF: near infrared fluorescence NIST: National Institute of Standards and Technology NMDA: N-methyl-d-aspartate NMM: nicotinamide mononucleotide NMR: nuclear magnetic resonance NO: nitric oxide NOE: nuclear Overhauser effect NOESY: nuclear Overhauser effect spectroscopy NOHA: Nw-hydroxy-l-arginine NORs: specific chromosomal sites of nuclear reformulation NOS: nitric oxide synthetase iNOS: inducible oxide synthetase NPC: nuclear pore complex pNPP: p-Nitrophenyl phosphate
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NSAID: non-steroid anti-inflammatory drug(s) NSF: N-ethylmaleimide sensitive factor; National Science Foundation; N-ethylmaleimide-sensitive fusion Nt,nt: nucleotide NTA: nitriloacetic acid NTPDases: nucleoside triphosphate diphosphohydrolases; also known as apyrases, E-ATPases NuSAP: nucleolar spindle-associated protein OCED: Organization for Economic Cooperation and Development ODMR: optically detected magnetic resonance ODN: oligodeoxynucleotide OFAGE: orthogonal-field-alternation gel electrophoresis OHQ: 8-hydroxyquinoline OMG: Object Management Group OMIM: online Mendelian Inheritance in Man (database) OMIM220100 located at http: //www.ncbi.nlm.nih.gov OMP: outer membrane protein; A protein family associated with membranes OMT: outer membrane transport OPG: Osteoprotegerin ORC: origin recognition complex ORD: optical rotatory dispersion ORF: open reading frame ORFan: orphan open-reading frame ORFeome: the protein-coding ORFs of an organism uORF: upstream open reading frame OSBP: oxysterol-binding proteins OVA: Ovalbumin OXPHOS: oxidative phosphorylation OYE: Old Yellow Enzyme p53: A nuclear phosphoprotein which functions as a tumor suppressor. PA: peptide amphiphile PAC: P1-derived artificial chromosome PACAP: pituitary adenyl cyclase-activating polypeptide PAD: peptidylarginine deiminase; protein argininine deiminase (EC 3.5.5.15) PADGEM: platelet activator-dependent granule external membrane protein; GMP-140 PAGE: polyacrylamide gel electrophoresis PAH: polycyclic aromatic hydrocarbon PAK: P21-activated kinase PAO: A redundant gene family (seripaoparin) PAR: protease-activated receptor PAS: preautophagosomal structure PAT1: H+ -coupled amino acid transporter (slc36a1) PAZ: a protein interaction domain; PIWI-argonaute-zwille PBS: phosphate-buffered saline PBST: phosphate-buffered saline with Tween-20 PBP: periplasmic binding protein PC: polycystin; phosphatidyl choline PCAF: p300/CBP-associated factor, a histone acetyltransferase PCNA: proliferating Cell Nuclear Antigen; processing factor PDB: Protein Data Bank PDE: phosphodiesterase PDGF: platelet-derived growth factor PDGFR: platelet-derived growth factor receptor pDNA : plasmid DNA PDI: protein disulfide isomerase PDMA: polydimethylacrylamide PDMS: polydimethylsiloxane PE: phycoerythrin; polyethylene PEC: photoelectrochemistry
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954 PECAM-1: platelet/endothelial cell adhesion molecule-1 PEI: polyethyleneimine PEND protein: a DNA-binding protein in the inner envelope membrane of the developing chloroplast PEP: phosphoenol pyruvate PEPCK-C: phosphoenolpyruvate carboxykinase, cytosolic form PERK: double-stranded RNA-activated protein kinase-like ER kinase PES: photoelectron spectroscopy PET: positron emission tomography PEP: plastid-encoded polymerase (RNA polymerase) Pfam: a protein family database; protein families database of alignments PFGE: pulsed-field gel electrophoresis; PFK: phosphofructokinase PFU: plaque forming unit PG: phosphatidyl glycerol; prostaglandin, 3-PGA: 3-phospho-d-glycerate PGO: phenylglyoxal PGP-Me: archaetidylglycerol methyl phosphate PGT box: an upstream cis-element PGx(PGX): pharmacogenetics (PGx) is the use of genetic information to guide drug choice; prostaglandins(PGX) include thromboxanes and prostacyclins. PH: pleckstrin homology PHD: plant homeodomain pHB(p-HB): 4-hydoxybenzoic acid (p-hydroxybenzoate) PI: propidium iodide PIC: pre-initiation complex – complex of GTFs PINCH: PINCH-protein; particularly interesting cis-his-rich protein PIP3: phosphatidylinositol-3,4,5-triphosphate PIPn: polyinositol polyphosphate PIPnS: polyinositol polyphosphates Pipes: 1,4-piperzainediethanesulfonic acid PIRLb: paired immunoglobulin-like type 2 receptor b PKA: protein kinase A; cAMP-dependent kinase; pKa, acid dissociation constant PKC: protein kinase C Pkl: paxillin kinase linker PLL: poly-l-Lysine PLP: pyridoxal-5-phosphate PMA: phenyl mercuric acetate; phorbol-12-myristate-13 acetate; PMCA: plasma membrane Ca2+ as PMCA-ATPase, a PMCA pump PMSF: phenylmethylsulfonyl fluoride PNA: peptide nucleic acid; p-nitroanilide PNGase: endoglycosidase PNP: p-nitrophenol (4-nitrophenol) POD: peroxidase POET: pooled ORF expression technology POINT: prediction of interactome database Pol II: RNA polymerase II POTRA: polypeptide translocation associated PP: polypropylene PPAR: peroxisome proliferator activated receptor PPase: phosphoprotein phosphatase PQL: protein quantity loci PS: position shift polymorphism PSG: pregnancy-specific glycoprotein(s) PS-1: presenilin-1 PSI: photosystem I
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PSI-BLAST: position specific interative BLAST; position-shife iterated BLAST (software program) PSII: photosystem II PTB: polypyrimidine-tract-binding protein; a repressive regulator of protein splicing; also pulmonary tuberculosis PTD: protein transduction domain PTEN: phosphatase and tensin homolog deleted on chromosome 10 PTFE: polytetrafluoroethylene PTH: phenylthiohydantoin PTGS: post-transcriptional gene silencing PTK: protein-tyrosine kinase PTPase: protein tyrosine phosphatase PVA: polyvinyl alcohol PVDF: polyvinylidine difluoride QA: quality assurance QC: quality control QSAR: Quantitive Strucure-Activity Relationship(s) Q-TOF: quadrupole time-of-flight QTL: quantitative trait loci R f: retardation factor RA: rheumatoid Arthritis RAB-GAP: Rab-GTPase-activing protein RACE: rapid amplification of cDNA ends Raf : retroviral oncogene derived from 3611 murine sarcoma RAGE: receptors for Advanced Glycation Endproducts; receptors for AGE; recombinase-activated gene expression RAMP: receptor activity modified protein RANK: receptor activator of NF-kB RANK-L: receptor activator of NF-kB ligard Rap: a family of GTPase-coupled signal transduction factors which are part of the RAS superfamily Rap1: a small GTPase involved in integrin activation and cell adhesion RAPD : randomly amplified polymorphic DNA RARE: RecA-assisted restriction endonuclease RAS: GTP-binding signal transducers H-ras: retroviral oncogene derived from Harvey murine sarcoma K-ras: retroviral oncogene derived from Kirsten murine sarcoma RC: recombinant cogenic RCA: rolling circle amplification RCCX: RP-C4-CYP21-TNX module RCFP: reef coral fluorescent protein RCR: rolling circle replication RDP: receptor component protein rDNA: ribosomal DNA REA: restriction enzyme analysis Rel: avian reticuloendotheliosis REMI: restriction enzyme-mediated integration RET: receptor for the GDNF family RF: a transcription factor, RFX family Rfactor: final crystallographic residual RFID: radio frequency identification device RFLP: restriction fragment length polymorphism RGD: a signature peptide sequence-arginine-glycine-aspartic acid found in protein which bind integrins RGS: regulator of G-protein signaling RHD: Rel homology domain Rheb: Ras homologue enriched in brain RhoA: Ras homologous; signaling pathway RI: random integration RIP: repeat-induced point mutation RIS: radioimmunoscintigraphy
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955 RISC: RNA-induced silencing complex RIT: radioimmunotherapy RM: reference material RNAi: RNA interference dsRNA: double-stranded RNA hpRNAi: hairpin RNA interference ncRNA: non-coding RNA rRNA: ribosomal RNA shRNA: small hairpin RNA siRNA: small interfering RNA snRNA: small nuclear RNA snoRNA: small nuceolar RNA stRNA: small temporal RNA RNAse/RNAase: ribonuclease RNAse III: a family of ribonucleases (RNAses) RNC: ribosome-nascent chain complex snRNP: small nuclear ribonucleoprotein particle RNS: reactive nitrogen species RO: reverse osmosis ROCK (ROK): Rho kinase ROESY: rotating frame Overhauser effect spectroscopy Ron: receptor for macrophage stimulating protein ROS: reactive oxygen species Ros: retroviral oncogenes derived from UR2 avian sarcoma RP: reverse-phase; also a nuclear serine/threonine protein kinase RP-CEC: reverse-phase capillary electrochromatography RP-HPLC: reverse-phase high performance liquid chromatography RPA: replication protein A RPEL: a protein motif involved in the cytoskeleton RPC: reverse-phase chromatography RPMC: reverse phase microcapillary liquid chromatography RPMI 1640: growth media for eukaryotic cells RPTP: receptor protein-tyrosine kinase RRM: RNA-recognition motif RRS: Ras recruitment system; resonance Raleigh scattering R,S: designating optical activity of chiral compounds where R is rectus (right) and S is sinester (left) RSD: root square deviation RT: reverse transcriptase; also room temperature RTD: residence time distribution RTK: receptor tyrosine kinase RT-PCR: reverse transcriptase-polymerase chain reaction RTX: repeat in toxins; pore-forming toxin of E.Coli type (RTX toxin); also rituximab, resiniteratoxin, renal transplantation Rub1: a ubiquitin-like protein, Nedd8 S1P: sphingosine-1-phosphate S100 : S100 protein family SA: salicylic acid SAGE: serial analysis of gene expression SALIP: saposin-like proteins SAM: self-assembling monolayers SAMK: a plant MAP kinase SAMPL: selective amplification of microsatellite polymorphic loci Sap: Saposin SAP: sphingolipid activator protein; also serum amyloid P, shrimp alkaline phosphatase SAR: scaffold associated region; structure-activity relationship SATP: heterobifunctional crosslinker; N-succinimidyl-Sacetylthiopropionate SAXS: small angle x-ray scattering
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scFv: single chain Fv fragment of an antibody SCID: severe combined immunodeficiency SCOP: structural classification of proteins; http://scop.mrc-lmb. cam.ac.uk/scop SCOPE: structure-based combinatorial protein engineering SDS: sodium dodecyl sulfate SEC: secondary emission chamber for pulse radiolysis; size exclusion chromatography Sec-: secretory – usually related to protein translocation SELDI: surface-enhanced laser desorption/ionization SELEX: systematic evolution of ligands by expotential enrichment SERCA: sarco/endoplasmic reticulum Ca2+ as in SERCAATPase, a calcium pump SFC: supercritical fluid SHAP: serum-derived hyaluron-associated protein SH2: Src homology domain 2 SH3: Src homology domain 3 Shh: sonic hedgehog shRNA: small hairpin RNA SHO: yeast osmosensor SILAC: stable-isotope labeling with amino acids in cell culture SIMK: a plant MAP kinase SINE: short interspersed nuclear element SINS: sequenced insertion sites SIPK: salicylic-acid induced protein kinase Sis: retroviral oncogene derived from simian sarcoma SISDC: Sequence-independent site-directed chimeragenesis Ski: retroviral oncogene derived from avian SK77 Skp: a chaperone protein SLAC: serial Lectin Affinity Chromatography SLE: systemic lupus erythematoses SLN1: yeast osmosensor S/MAR: scaffold and matrix attachment region SMC: smooth muscle cell SNAREs: soluble N-ethylmaleimide-sensitive fusion (NSF; N-ethylmaleimide-sensitive factor) protein attachment protein receptors; can be either R-SNAREs or Q-SNARES depending on sequence homologies SNM: SNARE motif SNP: Single nucleotide polymorphism snRNA: small nuclear RNA snoRNA: small nucleolar RNA snRNP: small nuclear ribonucleoprotein particle SOC: soil organic carbon; store-operated channel SOCS: suppressors of cytokine signalling SOD: superoxide dismutase SOD1s: CuZn-SOD enzyme (Intracellular) SOP: standard operating procedure SOS: response of a cell to DNA damage; salt overly sensitive(usually plants); son of Sevenless (signaling cascade protein) SPA: scintillation proximity assay SPC: statistical process control SPECT: sporozite mineneme protein essential for transversal; also single-photon emission computed tomography SPIN: surface properties of protein-protein interfaces (database) SPR: surface plasmon resonance SQL: structured query language SR: as in the SR protein family (serine- and arginine-rich proteins); also sarcoplasmic reticulum; also scavenger receptor SRCD: synchrotron radiation circular dichroism SRP: signal recognition particle SRF: serum response factor, a ubiquitous transcription factor
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956 SRPK: SR protein kinase SRS: sequence retrieval system; SOS recruitment system SRWC: short rotation woody crop SSC: saline sodium citrate ssDNA: single-stranded DNA SSLP: simple sequence length polymorphism SSR: simple sequence repeats STAT: signal transducers and activators of transcription STC: sequence-tagged connector STM: sequence-tagged mutagenesis STORM: systematic tailored ORF-data retrieval and management STR: short tandem repeats STREX: stress-axix related exon stRNA: small temporal RNA SUMO: small ubiquitin-like (UBL) modifier; small ubiquitinrelated modifier; sentrin SurA: a chaperone protein SV40: simian virus 40 SVS: seminal vesicle secretion Sw,20: sedimentation coefficient corrected to water at 20°C SWI/SNF: Switch/sucrose-non fermenting TAC: transcription-competent artificial chromosome TACE: tumor necrosis factor –a-converting enzyme; also transcatheter arterial chemoembolization TAFs: TBP-associated factors TAFE: transversely alternating-field electrophoresis TAG: triacyl glycerol TAME: tosyl-arginine methyl ester TAP: tandem affinity purification; also transporter associated with antigen processing TAR: transformation-associated recombination; trans-activation response region TAT: trans-activator of transcription TATA: as in the TATA box which is a TATA-rich region located upstream from the initiation RNA-synthesis initiation site in eukaryotes and within the promoter region for the gene in question. Analogous to the Pribnow box in prokaryotes. TBA-Cl: tetrabutylammonium chloride TBP: TATA-binding protein; telomere-binding protein TCA: trichloroacetic acid; tricarboxylic acid TCR: T-cell receptor TE: therapeutic equivalence; transposable elements TEA: triethylamine TEAA: triethylammonium acetate TEF: toxic equivalency factor TEM: transmission electron microscopy TEMED (TMPD): N,N,N’,N’-tetramethylethylenediamine TEV: tobacco etch protease TF: tissue factor; transcription factor TFA: trifluoroacetic acid TFIIIA: transcription factor IIIA TGN: trans-Golgi network TGS: transcriptional gene silencing TH: thyroid hormone THF: tetrahydrofuran TIGR: The Institute for Genomic Research TIM: translocase of inner mitochondrial membrane TIP: tonoplast intrinsic protein(s) TIR: toll/IL-1 receptor TI-VAMP: tetanus neurotoxin-insensitive VAMP TLCK: tosyl-lysyl chloromethyl ketone
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TLR: toll-like receptor Tm: tubular membrane TM: transmembrane TMAO: trimethylamine oxide TMD: trans-membrane domain TMS: trimethylsilyl; thimersol TMV: tobacco mosaic virus TNA: treose nucleic acid TNB: 5-thio-2-nitrobenzoate TNBS: trinitrobenzenesulfonic acid TnC: troponin C TNF: tumor necrosis factor Tnl: troponin l TnT: troponin T TNF-a (TNFa): tumor necrosis factor-a TNR: transferrin receptor TNX: tenascin-X TPD: temperature programmed desorption TOC: total organic carbon TOCSY: total correlated spectroscopy TOF: time-of-flight TOP: 5’ tandem oligopyrimidine (terminal oligopyrimidine) tract TOPRIN: Topoisomerase and Primase in reference to a domain TOR: target of rapamycin; mTOR, mammalian target of rapamycin; dTOR, Drosophila target of rapamycin TOX: toxicology TPCK: tosylphenylalanylchloromethyl ketone TPEN: N’,N’-tetrakis-(2-pyridyl-methyl)ethylenediamine TPN: triphosphopyridine dinucleotide (now NADP) TRADD: a scaffold protein TRAP: tagging and recovery of associated proteins as in RNATRAP; also thrombin receptor activation peptide TRE: thyroid hormone response elements TRH: thyrotropin-releasing hormone TRI: as in TRI reagents such as TRIZOL™ reagents used for RNA purification from cells and tissues Tricine: N-(2-hydroxy-1,1-bis(hydroxymethyl)ethyl) glycine TRIF: TIR domain-containing adaptor-inducing interferon-b Tris: tris-(hydroxymethyl)aminomethyl methane; 2-amino-2hydroxymethyl-1,3-propanediol bis-Tris: 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl) propane-1,3-diol Trk: neurotrophic tyrosine kinase receptor TRL: time-resolved luminescence TRP: transient receptor potential as in TRP-protein TRs: thyroid receptors TSP: thrombospondin; traveling salesman problem TTSP: transmembrane type serine proteases TUSC: Trait Utility System for Corn Tween: polyoxyethylsorbitan monolaurate TX: thromboxane, also treatment TyroBP: tyro protein tyrosine kinase binding protein, DNAXactivation protein 12, DAP12, KARAP UAS: upstream activation site UBL: ubiquitin-like modifiers UCDS: universal conditions direct sequencing UDP: ubiquitin-domain proteins; uridine diphosphate
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957 UDP-GlcNAc: uidine-5’-diphospho-N-acetylglucosamine UNG: uracil DNA glycosylase UPA: universal protein array; urokinse-like plasminogen activator UPR: unfolded protein response URL: uniform resource locator URS: upstream repression site USP: United States Pharmacopeia USPS: ubiquitin-based split protein sensor UTR: untranslated region VAMP: vesicle-associated membrane protein VAP: VAMP-associated protein VDAC: voltage-dependent anion-selective channel VCAM: vascular cellular adhesion molecule VDJ: variable diversity joining; regions of DNA joined in recombination during lymphocyte development; see VDJ recombination. VDR: vitamin D receptor VEGF: vascular endothelial growth factor VEGFR: vascular endothelial growth factor receptor VGH: non-acronymial use; a neuronal peptide V H: variable heavy chain domain VICKZ: a family of RNA-binding proteins recognizing specific cis-acting elements VIGS: virus-induced gene silencing VIP: vasoactive intestinal peptide VLP: virus-like particle VLDL: very low density lipoprotein VNC (VNBC): viable, but not-cultivatable (bacteria) VNTR: variable number tandem repeat; variable number of tandem repeats VOC: volatile organic carbon VPAC: VIP PACAP receptors VSG: variable surface glycoproteins VSP: vesicular sorting pathway Vsp10: a type I transmembrane receptor responsible for delivery of protein to lysozyme/vacuole vsp10: gene for Vsp10 WGA: whole-genome amplification WT, Wt: wild type XBP: x-box binding protein XO: xanthine oxidase Y2H: yeast two-hybrid YAC: yeast artificial chromosome YCp: yeast centromere plasmid YEp: yeast episomal plasmid YFP: yellow fluorescent protein Z: benzyloxycarbonyl ZDF: Zucker diabetic factor Zif: zinc finger domain peptides(i.e. Zif-1, Zif-3) ZIP: leucine zipper b-ZIP: basic leucine zipper ZZ Domain: a tandem repeat dimer of the immunoglobulinbinding protein A from Staphylococcus aureus a
All compounds are the l isomer unless otherwise indicated
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Appendix b: Glossary of Terms Useful in Biochemistry Abbreviated New Drug Application (ANDA) This document contains data that, when submitted to FDA’s Center for Drug Evaluation and Research (CDER), Office of Generic Drugs, provide for the review and ultimate approval of a generic drug product. This document does not contain preclinical or clinical data but must demonstrate that the drug is question is bioequivalent to the currently licensed drug which is also referred to as the innovator drug. See http://www.fda.gov/cder/ drugsatfda/glossary.htm
ABC transporter The ATP-binding cassette transporter family consists of a large number of membrane proteins involved in the transport of variety of substances including ions, steroids, metabolites and drugs across extracellular and intracellular membranes. A defect in an ABC transporters is important in cystic fibrosis. See Schwiebert, E.M., ABC transporter-facilitated ATP conductive transport, Am.J.Physiol. 276, C1-C8, 1999; Dean, M., Rzhetsky, A., and Allikmets, R., The human ATP-binding cassette (ABC) transporter superfamily, Genome Res. 11, 1156-1166, 2001; Dean, M., Hamon, Y., and Chimini, G., The human ATP-binding cassette (ABC) transporter superfamily, J.Lipid Res. 42, 1007-1017, 2001; Georujon, C., Orelle, C., Steinfels, E. et al., A common mechanism for ATP hydrolysis in ABC transporter and helicase superfamilies, Trends Biochem.Sci. 26, 539-544, 2001; Schmitt, L., The first view of an ABC transporter: the X-ray crystal structure of MsbA from E.coli, Chembiochem 3, 161-165, 2002; Holland, I.B., Schmitt, L., and Young, J., Type 1 protein secretion in bacteria, the ABC-transporter dependent pathway, Mol.Membr.Biol. 22, 29-39, 2005; Blemans-Oldehinkel, E., Doeven, M.K., and Poolman, B., ABC transporter architechture and regulatory roles of accessory domains, FEBS Lett. 580, 1023-1035, 2006; Frelet, A. and Klein, M., Insight in eukaryotic ABC transporter function by mutation analysis, FEBS Lett. 580, 1064-1084, 2006; Crouzet, J., Trombik, T., Fraysse, A.S., and Boutry, M., Organization and function of the plant pleiotropic drug resistance ABC transporter family, FEBS Lett. 580, 1123-1130, 2006.
Ablation A multifunctional word derived from the latin ablatus (to carry away). In medicine, refers to the surgical removal of tissue or the elimination of cells by irradiation or immunological approaches. The surgery approach is used extensively in cardiology (Gillinov, A.M. and Wolf, R.K., Surgical ablation of atrial fibrillation, Prog.Cardiovasc.Dis. 48, 169-177, 2005) while irradiation or immunological approaches are used in oncology (Appelbaum, F.R., Badger, C.C., Bernstein, I.D., et al., Is there a better way to delivery total body irradiation, Bone Marrow Transplantation 10, Suppl. 1., 77-81, 1992; van Bekkum, D.W., Immune ablation and stem-cell therapy in autoimmune disease. Experimental basis for autologous stem-cell transplantation, Arthritis Res. 2, 281-284, 2000). It also refers to the reduction of particles into smaller sizes during erosion by other particles or the surrounding fluid (see Lindner, H., Koch, J., Niema, K., Production of ultrafine particles by nanosecond laser sampling
using orthogonal prepulse laser breakdown, Anal.Chem. 77, 7528-7533, 2005). It also has a definition in aerospace technology for the dissipation of heat generated by atmospheric friction upon reentry of a space vehicle.
Abscisic Acid A plant hormone. See Leung, J. and Giraudet, J., Abscisic acid signal transduction, Annu.Rev.Plant Physiol.Plant Mol.Biol. 25, 199-221, 1998; Finkelstein, R.R., Gampala, S.S. and Rock, C.D, Abscisic acid signaling in sees and seedlings, Plant Cell 14 Suppl., S15-S45, 2002.
Absorption Generally refers to the ability of a material to absorb another substance (hydration) or energy (the ability of a substance to absorb) light. See adsorption.
Absolute Oils See essential oils
Abzymes See catalytic antibodies
Accuracy The difference between the measured value for an analyte and the true value. Absolute error is the difference between the measured value and the true value while the relative error is that fraction that the absolute error is of the measured amount and is usually expressed as a percentage or at ppt/ppm. See Handbook of Analytical Chemistry, ed. L. Meites, McGraw-Hill, New York, New York, 1963; Analytical Chemistry Handbook, J.A. Dean, McGrawHill, New York, New York, 1995; Dean’s Analytical Chemistry Handbook, McGraw-Hill, New York, New York, 2005.
Active Ingredient Any component of a final drug product that provides pharmacological activity or other direct effect in the diagnosis, cure, mitigation, treatment, or prevention of disease or to affect the structure on any function of the body of man or animals. Sometimes referred to as the active pharmaceutical ingredient (API). See http://www.fda.gov/cber; http://www.ich.org – See Q7, Good Manufacturing Guide for Active Pharmaceutical Ingredients.
Activity-based Proteomics Identification of proteins in the proteome by the use of reagents which measure biological activity. Frequently the activity is measured by the incorporation of a “tag” into the active site of the enzyme. The earliest probes were derivatives of alkylflurophosphonates which were well-understood inhibitors of 959
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Accurate Mass Tag (AMT) serine proteases. The technical approach is related to enzyme histochemistry/histocytochemistry. Most often used for enzymes where functional families of proteins can be identified. See See Liu, Y., Patricelli, M.P., and Cravatt, B.F., Activitybased protein profiling: the serine hydrolases, Proc.Nat.Acad. Sci.USA 96, 14694-14699, 1999; Adam, G.C., Sorensen, E.J., and Carvatt, B.F., Chemical strategies for functional proteomics, Mol.Cell.Proteomics 1, 781-790, 2002; Speers, A.E. and Cravatt, B.F., Chemical strategies for activity-based proteomics, Chembiochem. 5, 41-47, 2004; Kumar, S., Zhou, B., Liang, F., Activity-based probes for protein tyrosine phosphatases, Proc. Natl.Acad.Sci.USA 101, 7943-7948, 2004; Berger, A.B., Vitorino, P.M., and Bogyo, M., Activity-based protein profiling: applications to biomarker discovery, in vivo imaging and drug discovery, Am.J.Pharmacogenomics 4, 371-381, 2004; Willams, S.J., Hekmat, O., and Withers, S.G., Synthesis and testing of mechanism-based protein-profiling probes for retaining endo-glycosidases, Chembiochem 7, 116-124, 2006; Sieber, S.A. and Cravatt, B.F., Analytical platforms for activity-based protein profiling – exploiting the versatility of chemistry for functional proteomics, Chem.Commun. (22), 2311-2318, 2006; Schmidinger, H., Hermetter, A., and Birner-Gruenberger, R., Activity-based proteomics: enzymatic activity profiling in complex proteomes, Amino Acids 30, 333-350, 2006.
Accurate Mass Tag (AMT) A peptide of sufficiently distinctive and accurate mass and elution time from liquid chromatography which can be used a single identifier of a protein. See Conrads, T.P., Anderson, G.A., Veenstra, T.D. et al., Anal.Chem. 72, 3349-3354, 2000; Smith, R.D., Anderson, G.A., Lipton, M.S., et al., An accurate mass tag strategy for quantitative and high-throughput proteome measurements, Proteomics 2, 513-523, 2002; Strittmatter, E.F., Ferguson, P.L., Tang, K., and Smith, R.D., Proteome analyses using accurate mass and elution time peptide tags with capillary LC time-of-flight mass spectrometry, J.Am.Soc.Mass Spectrom. 14, 980-991, 2003; Shen, Y., Tolic, N., Masselon, C. et al., Nanoscale proteomics, Anal.Bioanal.Chem. 378, 1037-1045, 2004; Zimmer, J.S., Monroe, M.E., Qian, W.J., and Smith, R.D., Advances in proteomics data analysis and display using an accurate mass and time tag approach, Mass Spectrom.Rev. 25, 450-482, 2006.
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concentration increases (or decreases) by 25% or more during certain inflammatory disorders. Acute phase proteins include C-reactive protein, fibrinogen, and a-1-acid glycoprotein. Acute phase proteins are part of the acute phase response. Some acute phase proteins have been used for diagnostic of specific disorders such as C-reactive protein and cardiovascular disease. See Bowman, B.H., Hepatic Plasma Proteins: Mechanisms of Function and Regulation, Academic Press, San Diego, CA, USA, 1993; Mackiewicz, A. and Kushner, I., Acute Phase Proteins: Molecular Biology, Biochemistry, and Clinical Applications, CRC Press, Boca Raton, FL, 1993; Kerr, M.A. and Thorpe, R., Immunochemistry Labfax, Bios Scientific Publishers, Oxford, UK, 1994; Sutton, H.E., The haptoglobins, Prog.Med.Genet. 7, 163-216, 1970; Gordon, A.H., Acute-phase proteins in wound healing, Ciba Found.Symp. 9, 73-90, 1972; Black, S., Kushner, I., and Samols, D., C-reactive protein, J.Biol.Chem. 279, 4848748490, 2004; Du Clos, T.W. and Mold, C., C-reactive protein: an activator of innate immunity and a modulator of adaptive immunity, Immunol.Res. 30, 261-277, 2004; Garlanda, C., Bottazzi, B., Bastone, A., and Mantovani, A., Pentraxins at the crossroads between innate immunity, inflammation, matrix deposition, and female fertility, Annu.Rev.Immunol. 23, 337-366, 2005; Ceron, J.J., Eckersall, P.D., and Martynez-Subiela, S., Acute phase proteins in dogs and cats: current knowledge and future perspectives, Vet.Clin.Pathol. 34, 85-99, 2005; Sargent, P.J., Farnaud, S., and Evans, R.W., Structure/function overview of proteins involved in iron storage and transport, Curr.Med.Chem. 12, 2683-2693, 2005; Bottazzi, B., Garlanda, C., Salvatori, G., et al., Pentraxins as a key component on innate immunity, Curr.Opin. Immunol. 18, 10-15, 2006; Vidt, D.G., Inflammation in renal disease, Am.J.Cardiol. 97, 20A-27A, 2006; Armstrong, E.J., Morrow, D.A., and Sabatine, M.S., Inflammatory biomarkers in acute coronary syndromes: part II: acute-phase reactants and biomarkers of endothelial cell activation, Circulation 113, e152-e155, 2006. See also heat shock proteins.
ADAMTS
A collection of active protein sequences or protein fragments or subsequences, collected in the form of function-oriented databases, http://bioinformatica.isa.cnr.it/ACS/ AIRS – Autoimmune Related Sequences BAC – Bioactive Peptides CHAMSE – Chameleon Sequences; sequences which can adopt both an alpha helix and beta sheet conformation DORRS – Database of RGD Related Sequences DVP – Delivery Vector Peptides SSP – Structure Solved Peptides TRANSIT – Transglutamination Sites
A disintegrin and metalloproteinase with thrombospondin motifs. A family of multidomain metalloproteinases with a variety of biological activities. ADMETS are part of the reprolysin family. ADAMTS13 which is involved in the processing of von Willebrand Factor is the best known member of this family. See Hooper, N.M., Families of zinc metalloproteases, FEBS Lett. 354, 1-6, 1994; Hurskainen, T.L., Hirohata, S., Seldin, M.F., and Apte, S.S., ADAM-TS5, ADAM-TS6, and ADAM-TS7, novel members of a new family of zinc metalloproteases. General features and genomic distribution of the ADAM-TS family, J.Biol.Chem. 274, 2555-2563, 1999; Sandy, J.D. and Verscharen, C., Analysis of aggrecan in human knee cartilage and synovial fluid indicates that aggrecanase (ADAMTS) activity is responsible fo the catabolic turnover and loss of aggrecan whereas other protease activity is required for C-terminal processing in vivo., Biochem.J. 358, 615-626, 2001; Fox, J.W. and Serrano S.M., Structural considerations of the snake venom metalloproteinases, key members of the M12 reprolysin family of metalloproteinases, Toxicon 45, 969-985, 2005.
Acute Phase Proteins
Adjuvant
Proteins which are either de novo or markedly elevated after challenge by infectious disease, inflammation or other challenge to homeostasis. Another definition is any protein whose blood
A substance which increases an immune response. Frequently a component of the excipients in the formulation of vaccine. See Brown, L.E. and Jackson, D.C., Lipid-based self-adjuvanting
Active Sequence Collection (ACS)
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Adrenomedullin vaccines, Curr.Drug Deliv. 2, 283-393, 2005; Gluck, R., Burri, K.G., and Metcalfe, I., Anjuvant and antigen delivery properties of virosomes, Curr.Drug Deliv. 2, 395-400, 2005; Topics in Vaccine Adjuvant Research, ed. D.R. Spriggs and W.C. Koff, CRC Press, Boca Raton, FL, 1991; Vaccine Design: The Subunit and Adjuvant Approach, ed. M.F. Powell, Plenum Press, New York, NY, 1995; Therapeutic Proteins: Methods and Protocols, ed. M.C. Smales and D.C. James, Humana Press, Totowa, NJ, 2005; Immunopotentiation in Modern Vaccines, ed. V.E.J.C. Schijns and D.T. O’Hagan, Elsevier, Amsterdam, Netherlands, 2006.
Adrenomedullin Adrenomedullin is a peptide originally isolated from a phenochromocytoma (Kitamura, K., Kangawa, K., Kawamoto, M., et al., Adenomedullin: a novel hypotensive peptide isolated from human phenochromocytoma, Biochem.Biophys.Res.Commun. 192, 553560, 1993). Adrenomedullin elevated intracellular cAMP in platelets and caused hypotension. Since its discovery, adrenomedullin has been found in a variety of cells and tissues (Hinson, J.P., Kapas, S., and Smith, D.M., Adrenomedullin, a multifunctional regulatory peptide, Endocrine Rev. 21, 138-167, 2000). Adrenomedullin has been suggested to have a variety of physiological activities. See Poyner, D., Pharmacology of receptors for calcitonin generelated peptide and amylin, Trends Pharmacol.Sci. 16, 424-428, 1995; Muff, R., Born, W., and Fischer, J.A., Calcitonin, calcitonin gene-related peptide, adrenomedullin and amylin: homologous peptides, separate receptors and overlapping biological actions, Eur.J.Endocrinol. 133, 17-20, 1995; Richards, A.M., Nichools, M.G., Lewis, L., and Lainchbury, J.G., Adrenomedullin, Clin. Sci. 91, 3-16, 1996; Massart, P.E., Hodeige, D., and Donckier, J., Adrenomedullin: view on a novel vasodialatory peptide with naturetic properties, Acta Cardiol. 51, 259-269, 1996; Hay, D.L. and Smith, D.M., Adrenomedullin receptors: molecular identity and function, Peptides 22, 1753-1763, 2001; Julian, M., Cacho, M., Garcia, M.A., et al., Adrenomedullin: a new target for the design of small molecule modulators with promising pharmacological activities, Eur.J.Med.Chem. 40, 737-750, 2005; Shimosawa, T. and Fujita, T., Adrenomedullin and its related peptides, Endocr.J. 52, 1-10, 2005; Zudaire, E., Portal-Núñez. S., and Cuttitta, F., The central role of adrenomedullin in host defense, J.Leuk.Biol. 80, 237244, 2006. Hamid, S.A. and Baxter, G.F., A critical cytoprotective role of endogenous adrenomedullin in acute myocardial infarction, J.Mol.Cell Cardiol. 41, 360-363, 2006.
Adsorption The transfer of a substance from one medium to another such as the adsorption of a substance from a fluid onto a surface. The adsorbent is the substrate onto which material is adsorbed. The adsorbate is the material adsorbed onto a matrix.
Advanced Glycation Endproducts (AGE) A heterogeneous group of products resulting from a series of chemical reactions starting with the formation of adducts between reducing sugars and protein nucleophiles such as nitrogen bases. Reaction with nucleic acid is also possible but has not been extensively described. The reactions involved are complex involving the Amadori reaction and the Maillard reaction/Some products include triosidines, Nε-carboxymethyl-lysine, and pentosidine-adducts. These products can undergo further reactions to form cross-linked products; advanced glycation
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endproducts are involved in the generation of reactive oxygen species (ROS). See Deyl, Z. and Mikšík, I., Post-translational non-enzymatic modification of proteins. I. Chromatography of marker adducts with special emphasis to glycation reactions, J.Chromatog. 699, 287-309, 1997; Bonnefont-Rousselot, D. D., Glucose and reactive oxygen species, Curr.Opin.Clin.Nutr. 5, 561-568, 2002; Tessier, F.J., Monnier, V.M., Sayre, L.M., and Kornfield, J.A., Triosidines: novel Maillard reaction products and cross-links from the reaction of triose sugars with lysine and arginine residues, Biochem.J., 369, 705-710, 2003: Thornally, P.J., Battah, S., Ahmed, N., Karachalias, N., Agalou, S., Babaei-Jadidi, R. and Dawnay, A., Quantiative screening of advanced glycation endproducts in cellular and extracellular proteins by tandem mass spectrometry, Biochem. J. 375, 581-592, 2003; Ahmed, N., Advanced glycation endproducts—role in pathology of diabetic complications, Diabetes Res. Clin.Pract. 67, 3-21, 2005.
Aeration The dispersion and/or dissolution of a gas into a liquid; generally refers to the process of dispersing air or an oxygen-gas mixture into a liquid such as culture media (Wang, D.I. and Humphrey, A.E., Developments in agitation and aeration of fermentation systems, Prog.Ind.Microbiol. 8, 1-34, 1968; Papoutsakis, E.T., Media additives for protecting freely suspended animal cells against agitation and aeration damage, Trends Biotechnol. 9, 316324, 1991; Barberel, S.I. and Walker, J.R., The effect of aeration upon the secondary metabolism of microorganisms, Biotechnol. Genet.Eng.Rev. 17, 281-323, 2000). Also refers to the process of air dispersion in the pulmonary system which can include both the inspiratory process and the exchange between the pulmonary system and the vascular bed; most frequently the later (Newman, B. and Oh, K.S., Abnormal pulmonary aeration in infants and children, Radiol.Clin.North Am. 26, 323-339, 1988; Kothari, N.A. and Kramer, S.S., Bronchial diseases and lung aeration in children, J.Thorac.Imaging 16, 207-223, 2001).
Aerosol A colloid-like dispersion of a liquid or solid material into a gas. There is considerable interest in the use of aerosols as a drug delivery vehicle. See Aerosol Science, ed. C.N. Davies, Academic Press, London, UK, 1996; Sanders, P.A., Aerosol Science, Van Nostrand Reinhold, New York, NY, 1970; Sanders, P.A., Handbook of Aerosol Techology, Van Nostrand Reinhold, New York, NY, 1979; Adjei, A.L. and Gupta, P.K., Inhalation Delivery of Therapeutic Peptides and Proteins, Marcel Dekker, New York, NY, 1997; Macalady, D.L., Perspectives in Environmental Chemistry, Oxford University Press, New York, NY, 1998; Hinds, W.C., Aerosol Technology: Properties, Behavior, and Measurement of Airborne Particles, Wiley,. New York, NY, 1999; Roche, N. and Huchon, G.J., Rationale for the choice of an aerosol delivery system, J.Aerosol.Med. 13, 393-404, 2000; Gautam, A., Waldrep, J.C., and Densmore, C.L., Aerosol gene therapy, Mol. Biotechnol. 23, 51-60, 2003; Densmore, C.L., The re-emergence of aerosol gene delivery: a viable approach to lung cancer therapy, Curr.Cancer Drug Targets 3, 275-286, 2003. See also colloid.
Affibody A phage-selected protein developed using a scaffold domain from Protein A. Such a protein can be selected for specific binding characteristics. See Ronnmark, J., Hansson, M., Nguyen, T.,
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Affinity Proteomics Uhlen, M., Robert, A., Stahl, S., and Nygren, P.A., Construction and characterization of affibody-Fc chimeras produced in Escherichia coli, J.Immunol.Meth. 261, 199-211, 2002; Eklund, M., Axelsson, L., Uhlen, M., and Nygren, P.A., Anti-idiotypic protein domains selected from protein A-based affibody libraries, Proteins 48, 454-462, 2002; Renberg, B., Shiroyama, I., Engfeldt, T., Nygren, P.A., and Karlström, A.E., Affibody protein capture microarrays: synthesis and evaluation of random and directed immobilization of affibody molecules, Anal.Biochem. 341, 334-343, 2005; Orlova, A., Nilsson, F.Y., Wikman, M., et al., Comparative in vivo evaluation of technetium and iodine labels on a n anti-HER2 affibody for single-photon imaging of HER2 expression in tumors, J.Nucl.Med. 47, 512-519, 2006; Wahlberg, E. and Hard, T., Conformational stabilization of an engineered binding protein, J.Am.Chem.Soc. 128, 7651-7660, 2006; Lendel, C., Dogan, J., and Hard, T., Structural basis of molecular recognition in an affibody: affibody complex, J.Mol.Biol., 359, 1293– 1304, 2006.
Affinity Proteomics The use of affinity reagents for the study of the proteome. The concept of the design and use of affinity labels for the study of proteins is well understood (see Plapp, B.V. and Chen, W.S., Affinity labeling with omega-bromoacetamido fatty acids and analogs, Methods Enzymol. 72, 587-591, 1981; Plapp, B.V., Application of affinity labeling for studying structure and function of enzymes, Methods Enzymol. 87, 469-499, 1982; Fan, F. and Plapp,B.V., Probing the affinity and specificity of yeast alcohol dehydrogenase I for coenzymes, Arch.Biochem.Biophys. 367, 240-249, 1999). For application of affinity technology to proteomics, see Larsson, T., Bergstrom, J., Nilsson, C. and Karlsson, K.A., Use of an affinity proteomics approach for the identification of low-abundant bacterial adhesins as applied on the Lewis(b)-binding adhesin of Helicobacter pylori, FEBS Lett 469, 155-158, 2000; Agaton, C., Falk, R., Hoiden Guthenberg, I., Gostring, L., Uhlen, M., and Hober, S., Selective enrichment of monospecific polyclonal antibodies for antibody-based proteomics efforts, J.Chromatog. A, 1043, 33-40, 2004; Stults, J.T. and Arnott, D., Proteomics, Methods Enzymol. 402, 245-289, 2005; Monti, M., Orru, S., Pagnozzi, D., and Pucci, P., Interaction proteomics, Biosci.Rep. 25, 45-56, 2005; Schou, C. and Heegaard, N.H., Recent applications of affinity interactions in capillary electrophoresis, Electrophoresis 27, 44-59, 2006; Niwayama, S., Proteomics in medicinal chemistry, Mini Rev.Med.Chem. 6, 241246, 2006; Capillary Electrophoresis of Proteins and Peptides, ed. M.A.Strege and Lagu, A.L., Humana Press, Totowa, NJ, 2004; Chemical Genomics: Reviews and Protocols, ed. E.D. Zanders, Humana Press, Totowa, NJ, 2005; New and Emerging Proteomics Techniques, Humana Press, Totowa, NJ, 2006. See also activitybased proteomics
Agar/Agarose Agar is a heterogeneous natural product derived from algae/ seaweed. It is used a “thickening” agent in cooking as a gelatinlike material. Agar is also used as matrix for growing microorganisms. See Johnstone, K.I., Micromanipulation of Bacteria: The Cultivation of Single Bacteria and Their Spores by the Agar Gel Dissection Techniques, Churchill-Livingston, Edinburgh, UK, 1973; Watanabe, T., Pictorial Atlas of Soil and Seed Funfi: Morphologies of Cultured Fungi and Key to Species, CRC Press, Boca Raton, FL, 1973; Wilkinson, M.H.F., Digital Image Analysis
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of Microbes: Imaging, Morphometry, Fluorometry, and Motility Techniques and Applications, Wiley, Chichester, UK, 1988; Turner, H.A., Theory of assays performed by diffusion in agar gel. I: General considerations, J.New Drugs 41, 221-226, 1963; Rees, D.A., Structure, conformation, and mechanism in the formation of polysaccharide gels and networks, Adv.Carbohydr. Chem.biochem. 24, 267-332, 1969; Metcalf., D., Clinical applications of the agar culture technique for haematopoietic cells, Rev.Eur.Etud.Clin.Biol. 16, 855-859, 1971; Holt, H.M., GahrnHansen, B., and Bruun, B., Shewanella algae and Shewanella putrefaciens: clinical and microbiological characteristics, Clin. Microbiol.Infect. 11, 347-352, 2005; Discher, D.E., Janmey, P. and Wang, Y.L., Tissue cells feel and respond to the stiffness of their substrate, Science 310, 1139-1143, 2005. Agar is composed of two primary components, agarose which is a gelling component and agaropectin which is a sulfated, non-gelling component. Agarose is used as a matrix for the separation of large molecules such as DNA. See Electrophoresis of Large DNA Molecules: Theory and Applications, ed. Lai, E.H.C. and Birren,B.W., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1990; Birren, B.W. and Lai, E.H.C., Pulsed Field Gel Electrophoresis: A Practical Guide, Academic Press, San Diego, CA, 1993; Bickerstaff, G.F., Immobilization of Enzymes and Cells, Humana Press, Totowa, NJ, 1997; Electrophoresis in Practice: A Guide to Methods and Applications of DNA and Protein Separations, Wiley-VCH, Weinheim, Germany, 2001.
Aggregation The process of forming a ordered or disordered group of particles, molecules, bubbles, drops, or other physical components which bind together in an undefined fashion; a common physical analogy is concrete or brick. Aggregation is used to measure macromolecular interactions and the interactions of cells such as platelets and frequently involves nephelometry. Agglutination is a term used to described the aggregation or clumping of blood cells or bacteria caused by antibody or other biological or chemical factors. Aggregation of proteins is thought to involved in the pathogenesis of diseases such as Parkinson’s disease and Alzheimer’s disease; these diseases are thought to be conformation diseases of proteins resulting in disorder structure and aggregation. Aggregation of blood platelets is an initial step in the hemostatic response. See Born, G.V., Inhibition of thrombogenesis by inhibition of platelet aggregation, Thromb.Diath. Haemorrh.Suppl. 21, 159-166, 1966; Zucker, M.B., ADP- and collagen-induced platelet aggregation in vivo and in vitro, Thromb.Diath.Haemorrh.Suppl 26, 175-184, 1967; Luscher, E.F., Pfueller, S.L., and Massini, P., Platelet aggregation by large molecules, Ser.Haematol. 6, 382-391, 1973; Harris, R.H. and Mitchell, R., The role of polymers in microbial aggregation, Ann.Rev.Microbiol. 27, 27-50, 1973; Harrington, R.A., Kleimna, N.S., Granger, C.B., et al., Relation between inhibition of platelet aggregation and clinical outcomes, Am.Heart J. 136(4 Pt 2 Su), S43-S50, 1998; Holyaerts, M.F., Oury, C., Toth-Zamboki, E., and Vermylen, J., ADP receptors in platelet activation and aggregation, Platelets 11, 307-309, 2000; Kopito, R.R., Aggresomes, inclusion bodies and protein aggregation, Trends Cell Biol. 10, 524-530, 2000; Savage, B., Cattaneo, M., and Ruggeri, Z.M., Mechanisms of platelet aggregation, Curr.Opin.Hematol. 8, 270-276, 2001; Valente, J.J., Payne, R.W., Manning, M.C., et al., Colloidal behavior of proteins: effects of the second virial coefficient on solubility, crystallization and aggregation of proteins in aqueous solution, Curr.Pharm.Biotechnol. 6, 427-436, 2005;
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Agonist Schwarzinger, S., Horn, A.H., Ziegler, J., Sticht, H., Rare large scale subdomain motions in prion protein can initiate aggregation, J.Biomol.Struct.Dyn. 23, 581-590, 2006; Elllis, R.J. and Minton, A.P., Protein aggregation in crowded environments, Bio. Chem. 387, 485-497, 2006; Estada, L.D. and Soto, C., Inhibition of protein misfolding and aggregation by small rationally-designed peptides, Curr.Pharm.Des. 12, 2557-2567, 2006.
Agonist Generally a compound or substance which binds to a receptor site which could be on a cell membrane or a protein and elicits a positive physiological response. See Gowing, L., Ali, R., and White, J., Opioid antagonists with minimal sedation for opioid withdrawal, Cochrane Database Syst.Rev. (2), CD002021, 2002; Bernardo, A. and Minghetti, L., PPAR-gamma agonists as regulators of microglial activation and brain inflammation, Curr.Pharm.Des. 12, 93-109, 2006; Bonuccelli, U. and Pavese, N., Dopamine agonists in the treatment of Parkinson’s disease, Expert Rev. Neurother. 6, 81-89, 2006; Thobois, S., Proposed dose equivalence for rapid switch between dopamine receptor agonists in Parkinson’s diseases: a review of the literature, Clin. Ther. 28, 1-12, 2006; Schwartz, T.W. And Holst, B., Ago-allosteric modulation and other types of allostery in dimeric 7TM receptors, J.Recept.Signal Transduct.Res. 26, 107-128, 2006.
Algorithm The underlying iterative method or mathematic theory for any particular computer programming technique; a precisely described routine process that can be applied and systematically followed through to a conclusion; a step-by-step procedure for solving a problem or accomplishing some end. There is a variety of algorithms ranging from defining clinical treatment protocols to aligning and predicting sequences of biopolymers. See Rose, G.D. and Seltzer, J.P., A new algorithm for finding the peptide chain turns in a globular protein, J.Mol. Biol. 113, 153-164, 1977; Gotoh, O., An improved algorithm for matching biological sequences, J.Mol.Biol. 162, 705-708, 1982; Dandekar, T. and Argos, P., Folding the main chain of small proteins with the genetic algorithm, J.Mol.Biol. 236, 844-861, 1994; Rarey, M., Kramer, B., Langauer, T., and Klebe, G., A fast, flexible docking method using an incremental construction algorithm, J.Mol.Biol. 261, 470-489, 1996; Jones, G., Willett, P., Glen, R.C. et al., Development and validation of a genetic algorithm for flexible docking, J.Mol.Biol. 267, 427-448, 1997; Samudrala, R. and Moult, J., A graph-theoretic algorithm for comparative modeling of protein structure, J.Mol.Biol. 279, 287-302, 1998; Chacon, P. Diaz, J.F., Moran, F., and Andreu, J.M., Reconstruction of protein form with X-ray solution scattering and a genetic algorithm, J.Mol.Biol. 299, 1289-1302, 2000; Mathews, D.H. and Turner, D.H., Dyalign: an algorithm for finding the secondary structure common to two RNA sequences, J.Mol.Biol. 317, 191-203, 2002; Herrmann, T., Guntert, P., and Wuthrich, K., Protein NMR structure determination with automated NOE assignment using the new software CANDID and the torsion angle dynamics algorithm DYANA, J.Mol.Biol. 319, 209-227, 2002; Andronescu, M., Fejes, A.P., Hutter, F., et al., A new algorithm for RNA secondary structure design, J.Mol.Biol. 336, 607-624, 2004; Fang, Q. and Shortle, D., Protein refolding in silico with atom-based statistical potentials and conformational search using a simple genetic algorithm, J.Mol.Biol. 359, 1456-1467, 2006.
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Albumin
963
Albumin A protein, most notably derived from plasma or serum and secondarily from egg (ovalbumin). It is the most abundant protein in blood/plasma constituting approximately half of the total plasma protein. It functions in establishing plasma colloid strength which preserves the fluid balance between the intravascular and extravascular space (Starling, E.H., On the absorption of fluids from the connective tissue spaces, J.Physiol. 19, 312326, 1896). Albumin, particularly bovine serum albumin (BSA), is used as a model protein and as a standard for the measurement of protein concentration. See Foster, J.F., Plasma albumin, in The Plasma Proteins, Vol. 1 ed. F.W. Putnam, Academic Press, New York, NY, Chapter 6, pp. 179-239, 1960; Tanford, C., Protein Denaturation, Adv.Protein Chem. 23, 121-282, 1968; Peters, T., Jr., Serum albumin, Adv.Clin.Chem. 13, 37-111, 1970; Gillette, J.R., Overview of drug-protein binding, Ann.N.Y.Acad.Sci. 226, 6-17, 1973; Peters, T., All about Albumin: Biochemistry, Genetics, and Medical Applications, Academic Press, San Diego, CA, 1996; Vo-Dinh, T., Protein nanotechnology: the new frontier in biosciences, Methods Mol.Biol. 300, 1-13, 2005; Quinlan, G.J., Martin, G.S., and Evans, T.W., Albumin: biochemical properties and therapeutic potential, Hepatology 41, 1211-1219, 2005; Rasnik, I., McKenney, S.A., and Ha, T., Surfaces and orientation: much to FRET about? Acc.Chem.Res. 38, 542-548, 2005; Therapeutic Proteins: Methods and Protocols, ed. C.M. Smales and D.C., James, Humana Press, Totowa, NJ, 2005; Yamakura, F. and Ikeda, K., Modification of tryptophan and tryptophan residues in proteins by reactive nitrogen species, Nitric Oxide 14, 152-161, 2006; Chuang, V.T. and Otagiri, M., Stereoselective binding of human serum albumin, Chirality 18, 159-166, 2006; Ascenzi, P., Bocedi, A., Notari, S., et al., Allosteric modulation of drug binding to human serum albumin, Mini Rev.Med.Chem. 6, 483-489, 2006. Albumin was the first protein biopharmaceutical (Newhauser, L.R. and Loznen, E.L., Studies on human albumin in military medicine: the standard Army-Navy package of serum albumin (concentrated), U.S.Navy Med.Bull. 40, 796-799, 1942; Heyl, J.T., Gibson, J.G., 2nd, and Janeway, C.W., Studies on the plasma proteins. V. The effect of concentrated solutions of human and bovine serum albumin in man, J.Clin.Invest. 22, 763-773, 1943) and is used for a variety of clinical indications (Albumin and the Systemic Circulation, ed. Blauhut, B. and Lundsgaard-Hansen, P., Karger, Berlin, 1986), including use in extracorporeal circulation as “bridge-to-transplant (Sen, S. and Williams, R., New liver support devices in acute liver failure: a critical evaluation, Semin.Liver Dis. 23, 283-294, 2003; Tan, H.K., Molecular absorbent recirculating system (MARS), Ann.Acad.Med.Singapore 33, 329-335, 2004; George, J., Artificial liver support systems, J.Assoc.Physicians India 52, 719-722, 2004; Barshes, N.R., Gay, A.N., Williams, B., et al., Support for the acutely failing liver: a comprehensive review of historic and contemporary strategies, J.Am.Coll.Surg. 201, 458-476, 2005. Albumin is also noted for its ability to interact with various dyes and the binding of bromcresol green is an example of a clinical assay method for albumin (Rodkey, F.L., Direct spectrophotometric determination of albumin in human serum, Clin.Chem. 11, 478-487, 1965; Hill, P.G., The measurement of albumin in serum and plasma, Ann.Clin.Biochem. 22, 565-578, 1985; Doumas, B.T. and Peter, T., Jr., Serum and urine albumin: a progress report on their measurement and clinical significance, Clin.Chim.Acta 258, 3-20, 1997; Duly, E.B., Grimason, S., Grimaon, P. et al., Measurement of serum albumin by capillary zone electrophoresis, bromocresol green, bromocresol purple, and immunoassay methods,
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Alloantibody J.Clin.Pathol. 56, 780-781, 2003). Albumin is a general designation to describe a fraction of simple proteins which are soluble in water and dilute salt solutions as opposed to the globulin fraction which is insoluble in water but soluble in dilute salt solutions. This is a old classification has many exceptions(Taylor, J.F., The isolation of proteins, in The Proteins. Chemistry, Biological Activity and Methods, Volume I. Pt.A, ed. H.Neurath and K.Bailey, Chapter 1, pp. 1-85, 1953; Albumins also migrate faster than globulins on electrophoresis which resulted in the development of the classification of plasma proteins as albumins and globulins (Cooper, G.R., Electrophoretic and ultracentrifugal analysis of normal human serum, in The Plasma Proteins, ed. F.W. Putman, Academic Press, New York, NY, 1960, Chapter 3, pp. 51-103, 1960.
Alloantibody Also an isoantibody. An antibody directed against a cell or tissue from an individual of the same species. Transplantation antibodies, transfusion antibodies and antibodies against blood coagulation factors such as factor VIII inhibitors are examples of alloantibodies. See Glotz, D., Antoine, C., and Duboust, A., Antidonor antibodies and transplantation: how to deal with them before and after transplantation, Transplantation 79 (Suppl 3), S30-S32, 2005; Colvin, R.B. and Smith, R.N., Antibody-mediated organ-allograft rejection, Nat.Rev.Immunol. 5, 807-817, 2005; Moll, S. and Pascual, M., Humoral rejection of organ allografts, Am.J.Transplant. 5, 2611-2618, 2005; Waanders, M.M., Roelen, D.L, Brand, A., and Class, F.H., The putative mechanism for the immunomodulating effect of HLA-DR shared allogeneic blood transfusion on the alloimmune response, Transfus.Med.Rev. 19, 281-287, 2005.
Alloantigen An antigen present in some, but not all members of a species or strain. The histocompatitibility locus antigens (HLA) is an example. See Schiffman, G. and Marcus, D.M., Chemistry of the ABH blood group substances, Prog.Hematol. 27, 97-116, 1964; Race, R.R., Contributions of blood groups to human genetics, Proc.R.Soc.Lond.B.Biol.Sci. 163, 151-168, 1965; Dausset, J., Leucocye and tissue groups, Vox Sang. 11, 263-275, 1966; Amos, B., Immunologic factors in organ transplantation, Am.J.Med. 55, 767-775, 1968; March, D.M., The ABO and Lewis bloodgroup system. Immunochemistry, genetics and relation to human disease, N.Engl.J.Med. 280, 994-1006, 1969; Bach, F.H., Histocompatibility in man – genetic and practical considerations, Prog.Med.Genet. 6, 201-240, 1969; Drozina, G., Kohoutek, J., Janrane-Ferrat, N., and Peterlin, B.M., Expression of MHC II genes, Curr.Top.Microbiol.Immunol. 290, 147-170, 2005; Serrano, N.C., Millan, P., and Paez, M.C., Non-HLA associations with autoimmune diseases, Autoimmun.Rev. 5, 209-214, 2006; Koehn, B., Gangappa, S., Miller, J.D., Ahmed, R., and Larsen, C.P., Patients, pathogens, and protective immunity: the relevance of virus-induced alloreactivity in transplantation, J.Immunol. 176, 2691-2696, 2006. Turesson, C. and Matteson, E.L., Genetics of rheumatoid arthritis, Mayo Clin.Proc. 81, 94-101, 2006.
Allosteric Originally a term which described the interaction of small molecules with an enzyme at a site physically distant from the
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active site where such interaction influenced enzyme activity. These small molecules were generally related to the substrate or product of the enzyme action. More recently, it has been used to describe the modulation of enzyme activity by the binding of a large or small molecule to a site distant from the active site. See Changeux, J.-P. and Kvamme, E., Regulation of Enyzme Activity and Allosteric Interactions, Academic Press, New York, 1968; Kurganov, B.I., Allosteric Enzymes: Kinetic Behavior, J.Wiley, Chichester, UK, 1982; Perutz, M.F., Mechanisms of Cooperativity and Allosteric Regulation in Proteins, Cambridge University Press, Cambridge, UK, 1990; Segal, L.A., Biological Kinetics, Cambridge University Press, Cambridge, UK, 1991; Changeux, J.P., Allosteric interactions interpreted in terms of quaternary structure, Brookhaven Symp.Biol. 17, 232-249, 1964; Monod, J., From enzymatic adaptation to allosteric transitions, Science 154, 475-483, 1966; Stadtman, E.R., Allosteric regulation of enzyme activity, Adv.Enzymol.Relat.Areas Mol.Biol. 28, 41-154, 1966; Frieden, C., Protein-protein interaction and enzymatic activity, Annu.Rev.Biochem. 40, 653-696, 1971; Matthews, B.W. and Bernhard, S.A., Structure and symmetry of oligomeric enzymes, Annu.Rev.Biophys.Bioeng. 2, 257-317, 1973; Hammes, G.G. and Wu, C.W., Kinetics of allosteric enzymes, Annu.Rev.Biophys. Bioeng. 3, 1-33, 1974; Ostermeier, M., Engineering allosteric protein switches by domain insertion, Protein Eng.Des.Sel. 18, 359-264, 2005; Horovitz, A. and Willison, K.R., Allosteric regulation of chaperonins, Curr.Opin.Struct.Biol. 15, 646-651, 2005; Ascenzi, P., Bocedi, A., Notari, S., et al., Allosteric modulation of drug binding to human serum albumin, Mini Rev.Med.Chem. 6, 483-489, 2006.
Alternative Splicing Alternative splicing is a process by which biological diversity can be increased without change in DNA content. Alternative splicing is a mechanism by a single pre-mRNA is processed is different ways (different splicing sites) to yield a diverse group of messenger RNA molecules. See Choi, E., Kuehl, M. and Wall, R., RNA splicing generates a variant light chain from an aberrantly rearranged kapp gene, Nature 286, 776-779, 1980; Mariamn, E.C., van Beek-Reinders, R.J., and van Venrooij, W.J., J.Mol. Biol. 163, 239-256, 1983; Lerivray, R., Mereau, A., and Osborne, H.B., Our favorite alternative splice site, Biol.Cell. 98, 317-321, 2006; Florea, L., Bioinformatics of alternative splicing and its regulation, Brief Bioinform. 7, 55-69, 2006; Xing, Y. and Lee, C., Alternative splicing and RNA selection pressure – evolutionary consequences for eukaryotic genomes, Nat.Rev.Genet. 7, 499-509, 2006. Alternative trans-splicing has also been demonstrated. See Maniatis, T. and Tasic, B., Alternative pre-mRNA splicing and proteome expansion in metazoans, Nature 418, 236-243, 2002; Garcia-Blanco, M.A., Messenger RNA reprogramming by spliceosome-mediated RNA trans-splicing, J.Clin.Invest. 112, 474-480, 2003; Kornblihtt, A.R., de la Mata, M., Fededa, J.P., et al., Multiple links between transcription and splicing, RNA 10, 1489-1498, 2004; Horiuchi, T. and Aigaki, T., Alternative transsplicing: a novel mode of pre-mRNA processing, Biol.Chem. 98, 135-140, 2006. The production of variants of fibronectin (is one of the better known examples of alternative splicing. See Schwarzbauer, J.E., Paul, J.T., and Hynes, R.O., On the origin of species of fibronectin, Proc.Natl.Acad.USA 82, 1424-1428, 1985; ffrench-Constant, C., Alternative splicing of fibronectin—many different proteins but few different functions.
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Aminophospholipids
Aminophospholipids Amino-containing phospholipids such as phosphatidyl ethanolamine and phosphatidyl serine. Phosphatidyl serine is involved in specific membrane functions and change in membrane distribution producing asymmetry are considered important for function. These are enzymes described as flippases, floppases, transporters, scramblaese, and aminophospholipid translocase which are responsible for this asymmetry which results in aminophospholipids on the cytoplasmic side of the membrane and cholines and sphingolipids on the outer surface. See Devaux, P.F., Protein involvement in transmembrane lipid asymmetry, Annu.Rev.Biophys.Biomol.Struct. 21, 417-439, 1992; Schelgel, R.A., Callahan, M.K., and Williamson, Ann.N.Y.Acad.Sci. 926, 271-225, 2000; Daleke, D.L. and Lyles, J.V., Identification and purification of aminophospholipid flippases, Biochem.Biophys. Acta. 1486, 108-127, 200; Balasubramanian, K., and Schroit, A.J., Aminophospholipid asymmetry: a matter of life and death, Annu.Rev.Physiol. 65, 701-734, 2003; Daleke, D.L.. Regulation of transbilayer plasma membrane phospholipid asymmetry, J.Lipid.Res. 44, 233-242, 2003.
Ambisense A genome or genome segment which contains regions that are positive-sense for some genes and negative-sense(antisense) for other genes as in an ambisense RNA as viral ssRNA genome or genome segment. See Bishop, D.H., Ambisense RNA viruses: positive and negative polarities combined in RNA virus genomes, Microbiol.Sci. 3, 183-187, 1986; Ngugen, M. and Naenni, A.L., Expression strategies of ambisense viruses, Virus Res. 93, 141150, 2003; van Knippenberg, I., Goldbach, R., and Kormelink, R., Tomato spotted wilt virus S-segment mRNAs have overlapping 3’-ends containing a predicted stem-loop structure and conserved sequence motif, Virus Res. 110, 125-131, 2005; Barr, J.N., Rodgers, J.W., and Wertz, G.W., The Bunyamwera virus mRNA transcription signal resides within both the 3’ and the 5’ terminal regions and allows ambisense transcription from a model RNA segment, J.Virol. 79, 12602-12607, 2005.
Amorphous Powder A solid form of a material which does not have a definite form such as a crystal structure. Differing from a crystal form, an amorphous form is thermodynamically unstable and does not have a defined melting point. The physical characteristics of an amorphous powder make it the desired physical state for drug after lyophilization. See Jennings, T.A., Lyophilization Introduction and Basic Principles, Interpharm Press, Denver, Colorado, 1999; Izutsu, K., Yoshioka, S., and Kojima, S., Increased stabilizing effects of amphiphilic excipients on freezedrying of lactate dehydrogenase (LDH) by dispersion into sugar matrices, Pharm.Res. 12, 838-843, 1995; Royall, P.G., Huang, C.Y., Tang, S.W., et al., The development of DMA for the detection of amorphous content in pharmaceutical powdered material, Int.J.Pharm. 301, 181-191, 2005; Stevenson, C.L., Bennett, D.B., and Lechuga-Ballesteros, D., Pharmaceutical liquid crystals: the relevance of partially ordered systems, J.Pharm.Sci. 94, 1861-1880, 2005; Skakle, J., Applications of X-ray power diffraction in materials chemistry, Chem.Rec. 5, 252-262, 2005; Farber, L., Tardos, G.I., and Michaels, J.N., Micro-mechanical properties of drying material bridges of pharmaceutical excipients,
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Amphipathic (amphiphilic)
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Int.J.Pharm. 306, 41-55, 2005; Jovanovic, N., Bouchard, A., Hofland, G.W., et al., Distinct effects of sucrose and trehalose on protein stability during supercritical flud drying and freezedrying, Eur.J.Pharm.Sci. 27, 336-345, 2006; Reverchon, E. and Atanacci, A., Cyclodextrins micrometric powders obtained by supercritical fluid processing, Biotechnol.Bioeng., 94, 753– 761, 2006; Jorgensen, A.C., Miroshnyk, I., Karjalainen, M., et al., Multivariate data analysis as a fast tool in evaluation of solid state phenomena, J.Pharm.Sci. 95, 906-916, 2006; Shah, S., Sharma, A., and Gupta, M.N., Preparation of cross-linked enzyme aggregates by using bovine serum albumin as a proteic feeder, Anal.Biochem. 351, 207-213, 2006.
Amplicon (Usually) the DNA product of a PCR reaction, usually an amplified segment of a gene or DNA. An RNA amplicon would be an RNA sequence can be obtained by transcription mediated amplification. See Bustin, S.A., Benes, V., Nolan, T., and Pfaffi, M.W., Quantitative real-time RT-PCR – a perspective, J.Mol. Endocrinol. 34, 597-601, 2005; Sarrazin, C., Highly sensitive hepatitis C virus RNA detection methods: molecular backgrounds and clinical significance, J.Clin.Virol. 25, S23-S29, 2002). This also refers to herpesvirus vectors for gene therapy (Oehmig, A., Fraefel, C., and Breakfield, X.O., Update on herpesvirus amplicon vectors, Molecular Therapy 10, 630-643, 2004).
Amphipathic (amphiphilic) A compound which has both hydrophilic (lyophilic) and hydrophobic (lyophobic) properties. This property is important for the interaction of protein with lipids and for the properties of cell-penetrating peptides. Detergents are amphipathic molecules. See Fasman, G.D., Prediction of Protein Structures and the Principles of Protein Conformation, Plenum Press, New York, NY, 1989; Epand, R.M., The Amphipathic Helix, CRC Press, Boca Raton, FL, 1993; Langel, U., Cell-Penetrating Peptides: Processes and Applications, CRC Press, Boca Raton, FL, 2002; Peptide-Lipid Interactions, ed. S.A. Simon and T.J. McIntosh, Academic Press, San Diego, CA, 2002; Scow, R.O., BlanchetteMackie, E.J., and Smith, L.C., Transport of lipid across capillary endothelium, Fed.Proc. 39, 2610-2617, 1980; Corr, P.B., Gross, R.W., and Sobel, B.E., Amphipathic metabolites and membrane dysfunction in ischemic myocardium, Circ.Res. 55, 135-154, 1984; Anantharamaiah, G.M., Brouillette, C.G., Engler, J.A., et al., Role of amphipathic helixes in HDL structure/function, Adv.Exp.Med.Biol. 285, 131-140, 1991; Segrest, J.P., Garber, D.W., Brouillette, C.G., et al., The amphipathic alpha helix: a multifunctional structural motif in plasma apolipoproteins, Adv.Protein Chem. 45, 303-369, 1994; Lester, J.B. and Scott, J.D., Anchoring and scaffold proteins for kinases and phosphatases, Recent Prog.Horm.Res. 52, 409-429, 1997; Lesieur, C., VecseySemjen, B., Abrami, L. et al., Membrane insertion: the strategies of toxins, Mol.Membr.Biol. 14, 45-64, 1997; Johnson, J.E. and Cornell, R.B., Amphitropic proteins: regulation by reversible membrane interactions, Mol.Membr.Biol. 16, 217-235, 1999; Tossi, A., Sandri, L., and Giangaspero, A., Amphipathic, alphahelical antimicrobial peptides, Biopolymers 55, 4-30, 2000; Garavito, R.M. and Ferguson-Miller, S., Detergents as tools in membrane biochemistry, J.Biol.Chem. 276, 32403-32406, 2001; El-Andaloussi, S., Holm. T., and Langel, U., Cell-penetrating
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Ampholyte peptides: mechanisms and applications, Curr.Pharm.Des. 11, 3597-3611, 2005; Deshayes, S., Morris, M.C., Divita, G., and Heitz, F., Interactions of primary amphipathic cell penetrating peptides with model membranes: consequences on the mechanism of intracellular delivery of therapeutics, Curr.Pharm.Des. 11, 3629-3638, 2005.
Ampholyte An amphoteric electrolyte. In proteomics, this term is used to describe small multi-charged organic buffer used to establish pH gradients in isoelectric focusing. See Righetti, P.G., Isoelectric focusing as the crow flies, J.Biochem.Biophys.Methods 16, 99-108, 1988; Patton, W.F., Pluskal, M.G., Skea, W.M., et al., Development of a dedicated two-dimensional gel electrophoresis system that provides optimal pattern reproducibility and polypeptide resolution, Biotechniques 8, 518-527, 1990; Hanash, S.M., Strahler, J.R., Neel, J.V., et al., Highly resolving two-dimensional gels for protein sequencing, Proc.Natl.Acad. Sci.USA 88, 5709-5713, 1991; Cade-Treyer, D., Cade, A.,Darjo, A., and Jouvion-Moreno, M., Isoelectric focusing and titration curves in biomedicine and in agrofood industries: a multimedia teaching program, Electrophoresis 17, 479-482, 1996; Stoyanov, A.V. and Pawliszyn, J., Buffer composition changes in background electrolyte during electrophoretic run in capillary zone electrophoresis, Analyst 129, 979-982, 2004; Gorg, A. Weiss, W. and Dunn, M.J., Current two-dimensional technology for proteomics, Proteomics 4, 3665-3685, 2004; Kim, S.H., Miyatake, H., Ueno, T., et al., Development of a novel ampholyte buffer for isoelectric focusing: electric charge-separation of protein samples for X-ray crystallography using free-flow isoelectric focusing, Acta Crystallogr.D Biol.Crystallogr. 61, 799-802, 2005; Righetti, P.G., The Alpher, Bethe, Gamow of isoelectric focusing, the alpha-Centaury of electrokinetic methods, Electrophoresis 27, 923-938, 2006.
Amphoteric Referring to a molecule such as protein, peptide, amino acid capable of having a positive charge, negative charge, or zero net charge. When at a zero net charge, it is also referred to as a zwitterion. See Haynes, D., The action of salts and non-electrolytcs upon buffer solutions and amphoteric electrolytes anad the relation of these effects to the permeability of the cell, Biochem.J. 15, 440-461, 1921; Akabori, S., Tani, H., and Noguchi, J., A synthetic amphoteric polypeptide, Nature 167, 1591-160, 1951; Coway-Jacobs, A. and Lewin, L.M., Isoelectric focusing in acrylamide gels: use of amphoteric dyes as internal markers for determination of isoelectric points, Anal.Biochem. 43, 294-400, 1971; Chiari, M. Pagani, L. and Righetti, P.G., Phsico-chemical properties of amphoteric, isoelectric, macroreticulate buffers, J. Biochem.Biophys.Methods 23, 115-130, 1991; Blanco, S., Clifton, M.J., Joly, J.L., and Peltre, G., Protein separation by electrophoresis in a nonsieving amphoteric medium, Electrophoresis 17, 1126-1133, 1996; Tulp, A., Verwoerd, D., and Hart, A.A., Density gradient isoelectric focusing of proteins in artificial pH gradients made up of binary mixtures of amphoteric buffers, Electrophoresis 18, 767-773, 1997; Akahoshi, A., Sato, K., Nawa, Y. et al., Novel approach for large-scale, biocompatible, and lowcost fractionation of peptides in proteolytic digest of food protein based on the amphoteric nature of peptides, J.Agric.Food Chem. 48, 1955-1959, 2000; Matsumoto, H., Koyama, Y., and Tanioka, A., Interaction of proteins with weak amphoteric charged
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membrane surfaces: effect of pH, J.Colloid Interface Sci. 264, 82-88, 2003; Fortis, F., Girot, P., Brieau, O., et al., Amphoteric, buffering chromatographic beads for proteome prefractionation. I: theoretical model, Proteomics 5, 620-628, 2005; Kitano, H., Takaha, K., and Gemmei-Ide, M., Raman spectroscopic study of the structure of water in aqueous solutions of amphoteric polymers, Phys.Chem.Chem.Phys. 8, 1178-1185, 2006.
Amyloid A wax-like translucent insoluble material consisting largely of protein and may or may not contain carbohydrate and is associated with tissue degeneration. Amyloid peptides/proteins are thought to be associated with Alzheimer’s disease. Glenner, G.G., The pathogenetic and therapeutic implications of the discovery of the immunoglobulin origin of amyloid fibrils, Hum. Pathol. 3, 157-162, 1972; Franklin, E.C. and Zucker-Franklin, D., Current concepts of amyloid, Adv.Immunol. 15, 249304, 1972; Glenner, G.G. and Terry, W.D., Characterization of amyloid, Annu.Rev.Med. 25, 131-135, 1974; Glenner, G.G. and Page, D.L., Amyloid, amyloidosis, and amyloidogenesis, Int.Rev.Exp.Pathol. 15, 1-92, 1976; Gorevic, P.D., Cleveland, A.B., and Franklin, E.C., The biologic significance of amyloid, Ann.N.Y.Acad.Sci. 389, 380-394, 1982; Reinhard, C., Herbert, S.S., and De Strooper, B., The amyloid-beta precursor protein: integrating structure with biological function, EMBO J. 24, 3996-4006, 2005; Meersman, F. and Dobson, C.M., Probing the pressure-temperature stability of amyloid fibrils provides new insights into their molecular properties, Biochem. Biophys.Acta 1764, 452-460, 2006; Tycko, R., Solid-state NMR as a probe of amyloid structure, Protein Pept.Lett. 13, 229-234, 2006; Torrent, J., Balny, C. and Lange, R., High pressure modulates amyloid formation, Protein Pept.Lett. 13, 271-277, 2006; Gorbenko, G.P. and Kinnuen, P.K., The role of lipid-protein interactions in amyloid-type protein fibril formation, Chem. Phys.Lipids 141, 72-82, 2006; Catalano, S.M., Dodson, E.C., Henze, D.A., et al., The role of amyloid- beta derived diffusible ligands (ADDLs) in Alzheimer’s disease, Curr.Top.Med.Chem. 6, 597-608, 2006.
Anaphylatoxin(s) Fragment(s) of complement proteins released during complement activation. See Corbeil, L.B., Role of the complement system in immunity and immunopathology, Vet.Clin.North Am.8, 585-611, 1978; Hugli, T.E. and Muller-Eberhard, H.J., Anaphylatoxins: C3a and C5a, Adv.Immunol. 26, 1-53, 1978; Hugli, T.E., The structural basis for anaphylatoxin and chemotactic functions of C3a, C4a, and C5a, Crit.Rev.Immunol. 1, 321-366, 1981; Hawlisch, H., Wills-Karp, M, Karp, C.L, and Kohl, J., The anaphylatoxins bridge innate and adaptive immune responses in allergic asthma, Mol.Immunol. 41, 123-131, 2004; Ali, H. and Panettieri, R.A., Jr., Anaphylatoxin C3a receptors in asthma, Respir.Res. 6, 19, 2005; Sunyer, J.O., Boshra, H., and Li, J., Evolution of anaphylatoxins, their diversity and novel roles in innate immunity: insights from the study of fish complement, Vet.Immunol.Immunopathol. 108, 77-89, 2005; Schmidt, R.E. and Gessner, J.E., Fc receptors and their interactions with complement in autoimmunity, Immunol. Lett. 100, 56-67, 2005; Chaplin, H., Jr., Review: the burgeoning history of the complement system 1888-2005, Immunohematol. 21, 85-93, 2005; Lambrecht, B.N., An unexpected role for the analphylatoxin C5a receptor in allergic sensitization, J.Clin. Invest. 116, 626-632, 2006.
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Anergy
Anergy Lack of an immune response to an allergen (antigen); can refer to an individual cell such as a B-cell or a T-cell, tissue, or intact organism; however, it is used most frequently with respect to B-cells or T-cells and immunological tolerance. See. Kantor, F.S., Infection, anergy, and cell-mediated immunity, N.Engl.J.Med. 292, 629-634, 1975; Bullock, W.E., Anergy and infection, Adv. Intern.Med. 21, 149-173, 1976; Dwyer, J.M., Anergy. The mysterious loss of immunological energy, Prog.Allergy 35, 15-92, 1984; Brennan, P.J., Saouaf, S.J., Greene, M.I., and Shen. Y., Anergy and suppression as coexistent mechanisms for the maintenance of peripheral T cell tolerance, Immunol.Res. 27, 295-302, 2003; Macian, F., Im, S.H., Garcia-Cozar, F.J., and Rao, A., T-cell anergy, Curr.Opin.Immunol. 16, 209-216, 2004; Mueller, D.L., E3 ubiquitin ligases as T cell anergy factors, Nat.Immunol. 5, 883-890, 2004; Faria, A.M. and Weiner, H.L., Immunol.Rev. 206, 232-259, 2005; Akdis, M., Blaser, K., and Akdis, C.A., T regulatory cells in allergy, Chem.Immunol.Allergy 91, 159-173, 2006; Ferry, H.. Leung, J.C., Lewis , G. et al., B-cell tolerance, Transplantation 81, 308-315, 2006.
Angiopoietin A protein family that binds to endothelial cells; specific for Tie2 receptor kinase. See Plank. M.J., Sleeman, B.D., and Jones, P.F., The role of the angiopoietins in tumor angiogenesis, Growth Factors 22, 1-11, 2004; Oike, Y., Yasunaga, K., and Suda, T., Angiopoietin-related/angiopoietin-like proteins regulate angiogenesis Int.J.Hematol. 80, 21-28, 2004; Giuliani, N., Colla, S., Morandi, F., and Rizzoli, V., Angiopoietin-1 and myelomainduced angiognesis, Leuk.Lymphoma 46, 29033, 2005; Dhanabal, M., Jeffers, M., LaRochelle, W.J., and Lichenstein, R.S., Angioarrestin: a unique angiopoietin-related protein iwht anti-angiogenic properties, Biochem.Biophys.Res.Commu. 333, 308-315, 2005; Endothelial/pericyte interactions, Circ.Res. 97, 512-523, 2005.
Anisotropy A difference in a physical property such as melting point when measured in different principle directions; antonym, isotropy. Anisotropy is also defined as the property of being anisotropic as in the case of light transmission where different values are obtained when along axes in different directions Time-resolved fluorescence anisotropy decay measures the time dependence of the depolarization of light emitted from a fluorophore experiencing angular motions. In botany, anisotropy is defined as assuming different positions in response to the action of external stimuli. See Kinosita, K., Jr., Kawato, S., and Ikegami, A., Dynamic structure of biological and model membranes: analysis by optical anisotropy decay measurement, Adv.Biophys. 17, 147203, 1984; Kinosita, K., Jr. and Ikegami, A., Dynamic structure of membranes and subcellular components revealed by optical anisotropy decay methods, Subcell.Biochem. 13, 55-88, 1988; Bucci, E. and Steiner, R.F., Anisotropy decay of fluorescence as an experimental approach to proteins, Biophys.Chem. 30, 199224, 1988; Matko, J., Jenei, A., Matyus, L. Ameloot, M., and Damjanovich, S., Mapping of cell surface protein-patterns by combined fluorescence anisotropy and energy transfer measurements, J.Photochem.Photobiol.B 19, 69-73, 1993; Rachofsky, E.L. and Laws, W.R., Kinetic methods and data analysis methods for fluorescence anisotropy decay, Methods Enzymol. 321, 216-238,
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2000; Santos, N.C., Prieto, M., and Castanho, M.A., Quantifying molecular partition into model systems of biomembranes: an emphasis on optical spectroscopic methods, Biochim.Biophys. Acta 1612, 123-135, 2003; Vrielink, A. and Sampson, N., Subangstrom resolution x-ray structures: is seeing believing?, Curr. Opin.Struct.Biol. 13, 709-715, 2003; Wang, J., Cao, Z., Jiang, Y., et al., Molecular signaling aptamers for real-time fluorescence analysis of proteins, IUBMB Life 57, 123-128, 2005; Dmitrienko, V.E., Ishida, K., Kirfel, A., and Ovchinnikova, E.N., Polarization anisotropy of X-ray atomic factors and ‘forbidden’ resonant reflections, Acta Crystallogr.A 61, 481-493, 2005; Baskin, T.I., Anisotropic expansion of the plant cell wall, Annu.Rev.Cell Dev. Biol. 21, 203-222, 2005; Heilker, R., Zemanova, L., Valler, M.J., and Nienhaus, G.U., Confocal fluorescence microscopy for highthroughput screening of G-protein coupled receptors, Curr.Med. Chem. 12, 2551-2559, 2005; Guthrie, J.W., Hamula, C.L., Zhang, H., and Le, X.C., Assays for cytokines using aptamers, Methods 38, 324-330, 2006.
Ankyrin-repeat domains/proteins A domain or motif, named after ankydrin, a cytoskeletal protein, is found in a large number of proteins. This domain which was first described in a yeast cell cycle regulator (Swi6/cdc 10) and Drosphilia (notch protein) consists of approximately 30 amino acids and is involved in protein-protein interactions. See Liou, H.C. and Baltimore, D., Regulation of the NF-kappa b/rel transcription factor and I kappa B inhibitor system, Curr.Opin.Cell Biol. 5, 477-487, 1993; Dedhar, S. and Hannigan, G.E., Integrin cytoplasmic interactions and bidirectional transmembrane signalling, Curr.Opin.Cell Biol. 8, 657-669, 1996; Sedgwick, S.G. and Smerdon, S.J., The ankyrin repeat: a diversity of interactions on a common structural framework, Trends Biochem.Sci. 24, 311-316, 1999: Yoganathan, T.N., Costello, P., Chen, X., et al., Integrin-linked kinase (ILK): a “hot” therapeutic target, Biochem. Pharmacol. 60, 1115-1119, 2000; Hryniewicz-Jankowska, A., Czogalla, A., Bok, E., and Sikorsk, A.F., Ankyrins, multifunctional proteins involved in many cellular pathways, Folia Histochem.Cytobiol. 40, 239-249, 2002; Lubman, O.Y., Korolev, S.V., and Kopan, R., Anchoring notch genetics and biochemistry; structural analysis of the ankyrin domain sheds light on existing data, Mol.Cell. 13, 619-626, 2004; Mosavi, L.K., Cammett, T.J., Desosiers, D.C., and Peng, Z.Y., The ankyrin repeat as molecular architecture for protein recognition, Protein Sci. 13, 1435-1448, 2004; Tanke, H.J., Dirks, R.W., and Raap, T., FISH and immunocytochemistry: toward visualizing single target molecules in living cells, Curr.Opin.Biotechnol. 16, 49-54, 2005; Trigiante, G. and Lu, X., ASPPs and cancer, Nat.Rev.Cancer 6, 217-226, 2006; Legate, K.R., Montañez, E., Kudlacek, O. and Fässler, R., ILK, PINCH and parvin: the tIPP of integrin signalling, Nat.Rev.Mol. Cell Biol. 7, 20-31, 2006.
Annotation Information added to a subject after the initial overall definition. Most frequently used in molecular biology for the addition of information regarding function to the initial description of a gene/ gene sequence in a genome. See Brent, M.R., Genome annotation past, present, and future: how to define an ORF at each locus, Genome Res. 15, 177-1786, 2005; Boutros, P.C. and Okey, A.B., Unsupervised pattern recognition: an introduction to the whys and wherefores of clustering microarray data, Brief Bioinform. 6, 331-343, 2005; Boeckman, B., Blatter, M.C., Famiglietti, L., et al.,
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Anoikis Protein variety and functional diversity: Swiss-Prot annotation in its biological context, C.R.Biol. 328, 882-899, 2005; Koonin, E.V., Orthologs, paralogs, and evolutionary genomics, Annu.Rev. Genet. 39, 309-338, 2005; Cahan, P., Ahmed, A.M., Burke, H., et al., List of list-annotated (LOLA): a database for annotation and comparison of published microarray gene lists, Gene 360, 78-82, 2005; Dong, Q., Kroiss, L., Oakley, F.D., Wang, B.B., and Brendel, V., Comparative EST analyses in plant systems, Methods Enzymol. 395, 400-418, 2005; Crockett, D.K., Seiler, C.E., 3rd, Elenitoba-Johnson, K.S., and Kim, M.S., J.Biomed.Tech. 16, 341346, 2005; Hermida, L., Schaad, O., Demougin, P., Descombes, P., and Primig, M., MIMAS: an innovative tool for network-based high density oligonucleotide microarray data management and annotation, BMC Bioinformatics 7, 190, 2006; Huang, D., Wei, P., and Pan, W., Combining gene annotation and gene expression data in model-based clustering weighted method, OMICS 10, 28, 2006; Snyder, K.A., Feldman, H.J., Dumontier, M., Salama, J.J., and Hogue, C.W., Domain-based small molecule binding site annotation, BMC Bioinformatics 7, 152, 2006.
Anoikis Apoptosis following loss of attachment to a matrix or specific anchorage site. See Grossman, J., Molecular mechanisms of “detachment-induced apoptosis—Anoikis.”, Apoptosis 7, 247260, 2002; Zvibel, I., Smets, F., and Soriano, H., Anoikis: roadblock to cell transplantation? Cell Transplant. 11, 621-630, 2002; Valentijn, A.J., Zouq, N., and Gilmore, A.P., Anoikis, Biochem. Soc.Trans. 32, 421-425, 2004; Zhan, M., Zhao, H., and Han, Z.C., Histol.Histopathol. 19, 973-983, 2004; Reddig, P.J. and Juliano, R.L., Clinging to life: cell to matrix adhesion and cell survival, Cancer Metastasis Rev. 24, 425-439, 2005; Rennebeck, G., Martelli, M., and Kyprianou, N., Anoikis and survival connections in the tumor microenvironment: is there a role in prostate cancer metastasis? Cancer Res. 65, 11230-11235, 2005.
ANTH-Domain A protein domain similar to the ENTH-domain and contained in proteins involved in endocytotic processes. See Stahelin, R.V., Long, F., Petter, B.J., et al., Contrasting membrane interaction mechanisms of AP180 N-terminal homology (ANTH) and epsin N-terminal homology (ENTH) domains, J.Biol.Chem. 278, 28993-28999, 2003; Sun, Y., Kaksonen, M., Madden, D.T., et al., Interaction of Slap2p’s ANTH domain with PtdIns(4,5)P2 is important for actin-dependent endocytotic internalization, Mol. Biol.Cell 16, 717-730, 2005; Yao, P.J., Bushlin, I., and Petralia, R.S., Partially overlapping distribution of epsin1 and HIP1 at the synapse: analysis by immunoelectron microscopy, J.Comp. Neurol. 494, 368-379, 2006.
Antibody A protein synthesized and secreted by a plasma cell. A plasma cell or antibody-secreting cell is derived from an undifferentiated B-cell. Antibodies are designated as the humoral immune response as opposed to the cellular immune response. Antibodies are usually synthesized and secreted in response to a foreign protein or bacteria. Natural antibody preparation are polyclonal in that such preparations are derived from a population of plasma cells. A monoclonal antibody is derived from a signal plasma cell clone. Antibodies can be formed against self; such antibodies are referred to as autoantibodies. Disease resulting from the
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Antibody-dependent cellular cytotoxicity (Adcc) formation of antibodies are called autoimmune diseases and can result from disorders of the humoral immune system or the cellular immune system Antibodies are classified as IgG, IgM, IgE, IgA, and IgD. There are unusual naturally-occuring antibodies such as camelid antibodies and artificial derivatives such as Fab fragments and scFv fragments.
Antibody Valency Antibody valency refers to the number of antigen binding sites there are on a single antibody molecule. An IgG molecule which consists of two heavy chain and two light chains (a dimer of heterodimers) has two antibody binding sites and hence is bivalent have a valency of two. IgM which is a pentamer of IgG has a valency of 10. An scFv fragment is monovalent. An antibody with increased valence is considered to have greater avidity. See Marrack, J.R., Hoch, H., and Johns, R.G., The valency of antibodies, Biochem.J. 48, xxi-xxii, 1951; Sela, M., Antibodies: shapes, homogeneity, and valency, FEBS Lett. 1, 83-85, 1968; van Regenmortel, M.H., Which value of antigenic valency should be used in antibody avidity calculations with multivalent antigens, Mol.Immunol. 25, 565-567, 1988; Gerdes, M., Meusel, M., and Spener, F., Influence of antibody valency in a displacement immunoassay for the quantitation of 2,4-dichlorophenoxyacetic acid, J.Immunol.Methods 223, 217-226, 1999; Hudson, P.J. and Kortt, A.A., High avidity scFv multimers: diabodies and triabodies, J.Immunol.Methods 231, 177-189, 1999; Hard, S.A. and Dimmock, N.J., Valency of antibody binding to virions and its determination by surface plasmon resonance, Rev.Med.Virol. 14, 123-135, 2004; Scallon, B., Cai, A., Radewonuk, J., and Naso, M., Addition of an extra immunoglobulin domain to two antirodent TNF monoclonal antibodies substantially increased their potency, Mol.Immunol. 41, 73-80, 2004; Adams, G.P., Tai, M.S., McCartney, J.E. et al., Avidity-mediated enhancement of in vivo tumor targeting by single-chain Fv dimers, Clin.Cancer Res. 12, 1599-1605, 2006.
Antibody-dependent cellular cytotoxicity (Adcc) The process of cell death. This is usually the process by which an organism destroys bacterial and viral pathogens but also is the mechanism by which tumor cells are lysed secondary to treatment with antibodies. The process involves the recognition of epitopes by the Fab region of the IgG on the target cell surface resulting in the binding of the antibody. The Fc domain is then recognized by a phagocytic cell such as a natural killer (NK) cell. The Fc region is critical for this process. See Dallegri, F. and Ottonello, D., Neutrophil—mediated cytotoxicity against tumor cells: state of the art, Arch.Immunol.Ther.Exp. 40, 39-42, 1992; Muller-Eberhard, H.J., The molecular basis of target cell killing by human lymphocytes and of killer cell self-protection, Immunol.Rev. 103, 87-98, 1981; Morretta, L., Moretta, A., Canonica, G.W., et al., Receptors for immunoglobulins on resting and activated human T cells, Immunol.Rev. 56, 141-162, 1981; Santonine, A., Herberman, R.B., and Holden, H.T., Correlation between natural and antibody-dependent cell-mediated cytotoxicity against tumor targets in the mouse. II. Characterization of the effector cells, J.Natl.Cancer Inst. 63, 995-1003, 1979; Sissons, J.G. and Oldstone, M.B., Antibody-mediated destruction of virus-infected cells, Adv.Immunol. 29, 209-260, 2000; Perussia, B. and Loza, M.J., Assays for antibody-directed cellmediated cytotoxicity (ADCC) and reverse ADCC (redirected
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Antibody-Proteomics/Antibody Based Proteomics cytotoxicity) in human natural killer cells, Methods Mol.Biol. 121, 179-192, 2000; Villamor, N., Montserrat, E., and Colomer, D., Mechanism of action and resistance to monoclonal antibody therapy, Semin.Oncol. 30, 424-433, 2003; Casadevall, A and Pirofski, L.A., Antibody-mediated regulation of cellular immunity and the inflammatory response, Trends Immunol. 24, 474-478, 2003; Mellstedt, H., Monoclonal antibodies in human cancer, Drugs Today 39 Supp C, 1-16, 2003; Gelderman, K.A., Tomlinson, S., Ross, G.D., and Gorter, A., Complement function in mAb-mediated cancer immunotherapy, Trends Immunol. 25, 158-164, 2004; Schmidt, R.E. and Gessner, J.E., Fc receptors and their interaction with complement in autoimmunity, Immunol.Lett. 100, 56-67, 2005; Iannello, A. and Ahmad, A. Role of antibody-dependent cell-mediated cytotoxicity in the efficacy of therapeutic anti-cancer monoclonal antibodies, Cancer Metastasis Rev. 24, 487-499, 2005.
Antibody-Proteomics/Antibody Based Proteomics The systematic generation and use of antibodies for the analysis of the proteome. An example would be the use of an antibodybased protein microarray. See Agaton, C., Falk, R., Holden Guthenberg, I. et al., Selective enrichment of monospecific polyclonal antibodies for antibody-based proteomics efforts, J.Chromatog.A 1043, 33-40, 2004; Nielsen, A.B. and Geierstanger, B.H., Multiplexed sandwich assays in a microwave format. J.Immunol.Methods 290, 107-120, 2004; Uhlen, M. and Ponten, F., Antibody-based proteomics for human tissue profiling, Mol.Cell.Proteom. 4, 384-393, 2005; Stenvall, M., Steen, J., Uhlen, M., et al., High-throughput solubility assay for purified recombinant protein immunogens, Biochim.Biophys.Acta. 1752, 6-10, 2005; Uhlen, M., Bjorling, E., Agaton, C. et al., A human protein atlas for normal and cancer tissues based on antibody proteomics, Mol.Cell.Proteomics 4, 1920-1932, 2005. See also immunoproteomics.
Antigen A material which can be of diverse substance and origin such as protein or microorganism which elicits an immune response. An immune response can be the formation of an antibody directed against the antigen (humoral response; B-cell response) as well as a cellular response (T cell response). Antigens can be separated into immunogens (complete antigens) which can elicit an immune response and haptens or incomplete immunogens which do not by themselves elicit and immune response but can reaction with antibodies. Haptens require combination with a larger molecule such as protein to elicit antibody formation. See Nossal, G.J.V., Antigens, Lymphoid Cells, and the Immune Response, Academic Press, New York, USA, 1971; Langone, J.J., Antibodies, Antigens, and Molecular Mimicry, Academic Press, San Diego, USA, 1989; Fundamental Immunology, ed. W.Paul, Raven Press, New York, 1993; Cruse, J.M., Lewis, R.E., and Wang, H., Immunology Guidebook, Elsevier, Amsterdam, Netherlands, 2004.
Antigenic Determinant An antigenic determinant is also an epitope; this is the region of an antigen which binds to the reactive site of an antibody which is referred to as a paratope. The antigenic determinant which elicits the antibody response. There are linear or continuous
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determinants which would be a continuous amino acid sequence in a protein antigen and conformation or discontinuous determinants where, for example, with a protein, the epitope is formed by protein folding. A linear determinant is recognized by T-cells as well as B-cell and antibodies while a discontinuous determinant is recognized only by B-cells and antibodies. See Eisen, H.N., The immune response to a simple antigenic determinant, Harvey Lect. 60, 1-34, 1966; Kabat, E.A., The nature of an antigenic determinant, J.Immunol. 97, 1-11, 1966; Franks, D., Antigens as markers on cultured mammalian cells, Biol.Rev.Camb.Philos. Soc. 43, 17-50, 1968; Stevanovic, S., Antigen processing is predictable: from genes to T cell epitopes, Transpl.Immunol. 14, 171-174, 2005; McRobert, E.A., Tikoo, A., Gallicchio, M.A., Cooper, M.E., and Bach, L.A., Localization of the ezrin binding epitope for glycated proteins, Ann.N.Y.Acad.Sci. 1043, 617-624, 2005; Lovtich, S.B., and Unanue,E.R., Conformational isomers of a peptide-class II major histocompatibility complex, Immunol. Rev. 207, 293-313, 2005; Phillips, W.J., Smith, D.J., Bona, C.A., Bot, A., and Zaghouani, H., Recombinant immunoglobulinbased epitope delivery: a novel class of autoimmune regulators, Int.Rev.Immunol. 24, 501-517, 2005; De Groot, A.S., Knopp, P.M., and Martin, W., De-immunization of therapeutic proteins by T-cell epitope modification, Dev.Biol.(Basel) 122, 171-194, 2005; Burlet-Schlitz, O., Claverol, S., Gairin, J.E., and Monsarrat, B., The use of mass spectrometry to identify antigens from proteosome processing, Methods Enzymol. 405, 264-300, 2005.
Anti-idiotypic Usually in reference to antibodies whose specificity is directed against the idiotypic region of an antibody; most frequently with naturally occurring antibodies. Since receptors and antibodies share common binding characteristics, this term is sometimes used to described antibodies directed against receptors. See Couraud, P.O. and Strosberg, A.D., Anti-idiotypic antibodies against hormone and neurotransmitter receptors, Biochem.Soc. Trans. 19, 147-151, 1991; Erlanger, B.F., Antibodies to receptors by an auto-anti-idiotypic strategy, Biochem.Soc.Trans. 19, 138143, 1991; Greally, J.M. Physiology of anti-idiotypic interactions: from clonal to paratopic selection, Clin.Immunol.Immunopathol. 60, 1-12, 1991; Friboulet, A., Izadyar, L., Avalle, B., Abzyme generation using an anti-idiotypic antibody as the “internal image” of an enzyme active site, Appl.Biochem.Biotechnol. 47, 229-237, 1994; Hebert, J. and Boutin, Y., Anti-idiotypic antibodies in the treatment of allergies, Adv.Exp.Med.Biol. 409, 431-437, 1996.
Antisense Generally refers a nucleotide sequence that is complementary to a sequence of messenger RNA which is the product of the non-coding sequence of DNA. It also refers to the peptide products from the antisense sequence referred to as antisense peptides. Antisense peptides have been investigated for biological activity. siRNA are based on the processing of antisense RNA. See Korneev, S. and O’Shea, M., Natural antisense RNAs in the nervous system, Rev.Neurosci. 16, 213-222, 2005. See also MicroRNA, siRNA, antisense peptides. aptamers
Antisense Peptides The products from the translation of antisense RNA. Some antisense peptides have been demonstrated to show affinity properties that appear to be unique to that sequence and not seen in
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scrambled sequences. See Schwabe, C., New thoughts on the evolution of hormone-receptor systems, Comp.Biochem.Physiol.A 97, 101-106, 1990; Chaiken, I., Interactions and uses of antisense peptides in affinity technology, J.Chromatog. 597, 29-36, 1992; Labrou, N. and Clonis, Y.D., The affinity technology in downstream processing, J.Biotechnol. 36, 95-119, 1994; Root-Bernstein, R.S. and Holsworth, D.D., Antisense peptides: critical minireview, J.Theoret.Biol. 190, 107-1199, 1998; Siemion, I.Z., Cebrat, M., and Kluczyk, A., The problem of amino acid complementarity and antisense peptides, Curr.Protein Pept.Sci. 5, 507-527, 2004.
Apical In reference to a differentiated cell, that portion or apex of the cell that is pointed toward the lumen as for example in endothelial cells. The membrane protein distribution is frequently different between the apical domain and the basolateral domain. See basolateral. See Alfalah, M., Wetzel, G., Fischer, I. et al., A novel type of detergentresistant may contribute to an early protein sorting event in epithelial cells, J.Biol.Chem. 280, 42636-42643, 2005; Kellett, G.L., and Brot-Laroche, E., Apical GLUT2: a major pathway of intestinal sugar absorption, Diabetes 54, 3056-3062, 2005; Ito, K., Suszuki, H., Horie, T., and Sugiyama, Y., Apical/basolater surface expression of drug transporters and its role in vectorial drug transport, Pharm.Res. 22, 1559-1577, 2005; Anderson, J.M., Van Itallie, C.M., and Fanning, A.S., Setting up a selective barrier at the apical junction complex, Curr.Opin.Cell Biol. 16, 140-145, 2004.
Apoptosis Programmed cell death; an organized process by which cell undergo degradation and elimination. See Tomei, L.D. and Cope, F.O., Apoptosis: The Molecular Basis of Cell Death, Cold Spring Harbor Laboratory Press, Plainview, NY, 1991; Studzinski, G.P., Cell Growth and Apoptosis: A Practial Approach, IRL Press ata Oxford University Press, Oxford, UK, 1995; Christopher, G.D., Apoptosis and the Immune Response, Wiley-Liss, New York, NY,1995; Kumar. S., Apoptosis: Mechanisms and Role in Disease, Springer, Berlin, 1998; Lockshin, R.A. and Zakeri, Z., When Cells Die: A Comprehensive Evaluation of Apoptosis and Programmed Cell Death, Wiley-Liss, New York, NY, 1998; Jacobson, M.D., and McCarthy, N.J., Apoptosis, Oxford University Press, Oxford, UK, 2002; Apoptosis Techniques and Protocols, 2nd edn., ed. A.C. LeBlanc, Humana Press, Totowa, NJ, 2002; Cell Proliferation and Apoptosis, ed. D. Hughes and Hm Mehmet, Bios, Oxford, UK, 2003; Potten, C.S. and Wilson, J.W., Apoptosis: The Life and Death of Cells, Cambridge Universtiy Press, Cambridge, UK, 2004.
Apoptosome A multiprotein complex which contains capases-9 and is thought to represent a holoenzyme involved in apoptosis. See Tsujimoto, Y., Role of Bcl-2 family proteins in apoptosis: apoptosomes or mitochondria, Genes Cells, 3, 697-707,1998; Adrain, C. and Martin, S.J., The mitochondrial apoptosome: a killer unleashed by the cytochrome seas, Trends Biochem.Sci. 26, 390-397, 2001; Salvesen, G.S. and Renatus, M., Apoptosome: the seven-spoked death machine, Dev.Cell. 2, 256-257, 2002; Cain, K., Bratton, S.B., and Cohen, G.M., The Apaf-1 apoptosome: a large caspase-activating complex, Biochemie 84, 203-214, 2002; Shi, Y., Apoptosome: the cellular engine for the activation of caspase-9, Structure 10, 285-288, 2002; Reed, J.C., Apoptosis-based therapies, Nat. Rev.Drug Disc. 1, 111-121, 2002; Adams, J.M., and Cory, S.,
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Apoptosomes: engines for caspase activation, Curr.Opin.Cell Biol. 14, 715-720, 2002; Hajra, K.M. and Liu, J.R., Apoptosome dysfunction in human cancer, Apoptosis 9, 691-704, 2004.
Aprotinin A small protein (single chain protein, MW 6.5 kDa; 58 amino acids) also known as basic pancreatic trypsin inhibitor (BPTI) or the Kunitz pancreatic trypsin inhibitor. It is best known as an inhibitor of tryptic-like serine proteases such as plasma kallikrein and plasmin. Aprotinin is also used as model for protein folding. See Kellermeyer, R.W. and Graham, J.C., Jr., Kinins-possible physiologic and pathologic roles in man, N.Eng.J.Med. 279, 754-759, 1968; Schachter, M., Kallikreins (kininogenases) – a group of serine proteases with bioregulatory actions, Pharmacol.Rev. 31, 1-17, 1979; Creighton, T.E., Experimental studies of protein folding and unfolding, Prog.Biophys.Mol.Biol. 33, 231-297, 1978; Fritz, H. and Wunderer, G., Biochemistry and applications of Aprotinin, the kallikrein inhibitor from bovine organs, Arzneimittelforschung 33, 479-494, 1983; Sharpe, S., De Meester, I., Hendriks, D. et al., Proteases and their inhibitors: today and tomorrows, Biochimie 73, 121-126, 1991; Creighton, T.E., Protein folding pathways determined using disulphide bonds, Bioessays 14, 195-199, 1992; Day, R. and Daggett, V., All-atom simulations of protein folding and unfolding, Adv.Protein Chem. 66, 373-403, 2003.
Aptamer Aptamers are relatively short oligonucleotides (generally 100 bp or less) which have the property of acting as relatively specific ligands to a broad range of targets. Aptamers are generally selected by combinatorial chemistry techniques. See Ellington, A.D. and Szostak, J.W., In vitro selection of RNA molecules that bind specific ligands, Nature 346, 818-822, 1990; Burke, J.M., and Berzal-Herranz, A., In vitro selection and evolution of RNA: applications for catalytic RNA, molecular recognition, and drug discovery, FASEB J. 7, 106-112, 1993; Stull, R.A. and Szoka, F.C., Jr., Antigene, ribozyme and aptamer nucleic acid drugs: progress and prospects, Pharm.Res. 12, 465-483, 1995; Uphoff, K.W., Bell, S.D., and Ellington, A.D., In vitro slection of aptamers: the dearth of pure reason, Curr.Opin.Struct.Biol. 6, 281-288, 1996; Collett, J.R., Cho, E.J. and Ellington, A.D., Production and processing aptamers microarrays, Methods 37, 4-15, 2005; Nutiu, R. and Li, Y., Aptamers with fluorescence-signaling properties, Methods 37, 16-25, 2005; Proske, D.,Blank, M., Buhmann, R., and Resch, A., Aptamers—basic research, drug development, and clinical applications, Appl.Microbiol.Biotechnol. 69, 367-374, 2005; Pestourie, C., Tavitian, B., and Duconge, F., Aptamers against extracellular targets for in vivo applications, Biochimie 87, 921-930, 2005. It is noted that the term intramer is used to describe intracellular aptamers (see Famulok, M., Blind, M., and Mayer, G., Intramers as promising new tools in functional proteomics, Chem.Biol. 8, 931-939, 2001; Famulok, M. and Mayer, G., Intramers and aptamers: applications in protein-function analyses and potential for drug screening, ChemBioChem 6, 19-26, 2005).
Aquaporin Water-specific membrane pores facilities osmosis. See van Lieburg, A.F., Knoers, N.V., and Deen, P.M., Discovery of aquaporins: a breakthrough in research on renal water transport, Ped. Nephrol. 9, 228-234, 1995; Sabolic, I. and Brown, D., Water transport in renal tubules is mediated by aquaporins, Clin.Investig.
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72, 689-700, 1994; King, L.S. and Agre, P. Pathophysiology of the aquaporin water channels, Annu.Rev.Physiol. 58, 619-648, 1996; Chaumont, F., Moshelion, M., and Daniels, M.J., Regulation of plant aquaporin activity, Biol.Cell. 97, 749-764, 2005; Castle, N.A., Aquaporins as targets for drug discovery, Drug.Discov.Today 10, 485-493, 2005.
Arabidopsis thaliana A small plant in the mustard family that is the model for studies of the plant genome. Meinke, D.W., Cheng, D.M., Dean, C., Rounsley, S.D., and Koorneeft, M., Arabidopis thaliana: A model plant for genome analysis, Science 282, 662-682, 1998; Methods in Plant Molecular Biology and Biotechnology, ed. B.R.Glick and J.E.Thompson, CRC Press, Boca Raton, FL, 1993; Aribidopsis, ed. E.R. Meyerowitz and C.R. Somerville, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1994; Aribidopsis, ed. M. Anderson and J.A. Roberts, Sheffield Academic Press, Sheffield, UK, 1998; Aribidopsis Protocols, ed. J. Salinas and J.J. Sánchez-Serrano, Humana Press, Totowa, NJ, 2006.
Arf Family GTPases The ADP-ribosylation family of GTPases. The ADP-ribosylation factor small GTPases have a role in the regulation of vesicular function via the recruitment of coat proteins and regulation of phospholipid metabolism. See Goud, B., Small GTP-binding proteins as compartmental markers, Semin.Cell Biol. 3, 301-307, 1992; Kjeldgaard, M., Nyborg, J., and Clark, B.F., The GTP binding motif: variations on a theme, FASEB J. 10, 1347-1368, 1996; Donaldson, J.G. and Jackson, C.L., Regulators and effectors of the ARF GTPases, Curr.Opin.Cell Biol. 12, 475-482, 2000; Takai, Y., Sasaki, T., and Matozaki, T., Small GTP-binding proteins, Physiol.Rev. 81, 153-208, 2001; ARF Family GTPases, ed. R.A., Kahn, Kluwer Academic Publisher, Dordrecht, Netherlands, 2003; Munro, S., The Arf-like GTPase Arl1 qnd its role in membrane traffic, Biochem.Soc.Trans. 33, 601-605, 2005; Kahn, R.A., Cherfils, J., Elias, M., et al., Nomenclature for the human Arf family of GTP-binding proteins: ARF, ARL, and SAR proteins, J.Cell. Biol. 172, 645-650, 2006; Nie, Z. and Randazzo, P.A., Arf GAPs and membrane traffic, J.Cell Sci. 119, 1203-1211, 2006; D’SouzaSchorey, C. and Chavrier, P., ARF proteins: roles of membrane traffic and beyond, Nat.Rev.Mol.Cell Biol. 7, 347-358, 2006. Also includes ARL and SAR proteins.
Arrhenius Energy of Activation An operationally defined quantity that relates rate constants to temperature by the following equation: k = Ae-Ea/R where k is a rate constant; A is a constant, R is the gas constant, T the absolute temperature. A plot of ln k vs 1/T (Arrhenius plot) yields the Arrhenius energy of activation. See Van Tol, A., On the occurrence of a temperature coefficient (Q10) of 18 and a discontinuous Arrhenius plot for homogeneous rabbit muscle fructosediphosphatase, Biochem.Biophys.Res.Communn. 62, 750-756, 1975; Ceuterick, F., Peeters, J., Heremans, K., et al., Involvement of lipids in the break of the Arrhenius plot of Azobacter nitrogenase, Arch.Int.Physiol. Biochim. 84, 587-588, 1976; Ceuterick, F., Peeters, J., Heremans, K., et al., Effect of high pressure, detergents and Phospholipase on the break in the Arrhenius plot of Azobacter nitrogenase, Eur.J.Biochem. 87, 401-407, 1978; Stanley, K.K. and Luzio, J.P., The Arrhenius plot behaviour of rat liver 5’-nucleotidase in different lipid environments, Biochim.Biophys.Acta 514, 198-205, 1978; De
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Smedt, H., Borghgraef, R., Ceuterick, F., and Heremans, K., The role of lipid-protein interactions in the occurrence of a non-linear Arrhenius plot for (sodium-potassium)-activated ATPase, Arch. Int.Physiol.Biochim. 87, 169-170, 1979; Biosca, J.A., Travers, F., and Barman, T.E., A jump in an Arrhenius plot can be the consequence of a phase transition. The binding of ATP to myosin subfragment 1, FEBS Lett. 153, 217-220, 1983; Haeffner, E.W. and Friedel, R., Induction of an endothermic transition in the Arrhenius plot of fatty acid uptake by lipid-depleted ascites tumor cells, Biochim. Biophys.Acta 1005, 27-33, 1989; Muench, J.L., Kruuv, J., and Lepock, J.R., A two-step reversible-irrreversible model can account for a negative activation energy in an Arrhenius plot, Cryobiology 33, 253-259, 1996; Rudzinski, W., Borowieki, T.., Panczyk, T., and Dominko, A., On the applicability of Arrhenius plot methods to determine surface energetic heterogeneity of adsorbents and catalysts surfaces from experimental TPD spectra, Adv.Colloid Interface Sci. 84, 1-26, 2000.
Atomic Force Microscopy A high resolution form of microscopy which involves a probe or tip moving over a surface (alternatively the sample can move with a static tip; the detection method is the same) and as the probe changes position in response to sample topography, the movement is tracked deflection of a laser beam which is recorded by a detector (Gadegaard, N., Atomic force microscopy in biology: technology and techniques, Biotechnic and Histochem. 81, 87-97, 2006). See Hansma, P.K., Elings, V.B., Marti, O., and Bracker, C.E., Scanning tunneling microscopy: application to biology and technology, Science 242, 209-216, 1988; Yang, J., Tamm, L.K., Somlyo, A.P., and Shao, Z., Promises and problems of biological atomic force microscopy, J.Microscop. 171, 183-198, 1993; Hansma, H.G. and Hoh, J.H., Biomolecular imaging with the atomic force microscope, Annu.Rev.Biophys. Biomol. Struct 23, 115-139, 1994; Tendler, S.J., Davies, M.C., and Roberts, C.J., Molecules under the microscope, J.Pharm.Pharmacol. 48, 2-8, 1996; Lekka, M., Lekki, J., Shoulyarenko, A.P., et al., Scanning force microscopy of biological samples, Pol.J.Pathol. 47, 51-55, 1996; Ivanov, Y.D., Govorum, V.M., Bykov, V.A., and Archakov, A.I., Nanotechnologies in proteomics, Proteomics 6, 1399-1414, 2006; Connell, S.D. and Smith, D.A., The atomic force microscope as a tool for studying phase separation in lipid membranes, Mol.Membr.Biol. 23, 17-28, 2006; Bai, L., Santangelo, T.J., and Wang, M.D., Single-molecule analysis of RNA polymerase transcription, Annu.Rev.Biophys.Biomol. Struct. 35, 343-360, 2006; Guzman, C., Jeney, S., Kreplak, L., et al., Exploring the mechanical properties of single vimentin intermediate filaments by atomic force microscopy, J.Mol.Biol. 360, 623630, 2006; De Jong, K.L., Incledon, B., Yip, C.M., and Defelippis, M.R., Amyloid fibrils of glucagon characterized by high-resolution atomic force microscopy, Biophys.J. 91, 1905-1914, 2006; Xu, H., Zhao, X., Grant, C. et al., Orientation of a monoclonal antibody adsorbed at the solid/solution interface: a combined study using atomic force microscopy and neutron reflectivity, Langmuir 22, 6313-6320, 2006.
Atomic Radius A measurement of an atom which is not considered precise; generally half the distance between adjacent atoms of the same type in a crystal or molecule. It may be further described as a covalent radius, an ionic radius or metallic radius. The inability of cysteine to effectively substitute for serine in serine proteases is due, in part, to the increased atomic radius of sulfur
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compared to oxygen. See Alterman, M.A., Chaurasia, C.S., Lu, P., and Hanzlik, R.P., Heteroatom substitution shifts regioselectivity of lauric acid metabolism from omega-hydroxylation to (omega-1)-oxidation, Biochem.Biophys.Res.Commun. 214, 10891094, 1995; Zhang, R., Villeret, V., Lipscomb, W.N., and Fromm, H.J., Kinetics and mechanisms of activation and inhibition of porcine liver fructose-1,6-bisphosphatase by monovalent cations, Biochemistry 35, 3038-3043, 1996; Wachter, R.M. and Brachaud, B.P., Thiols as mechanistic probes for catalysis by the free radical enzyme galactose oxidase, Biochemistry 35, 14425-14435, 1996; Wagner, M.A., Trickey, P., Chen, Z.W., et al., Monomeric sarcosine oxidase: 1. Flavin reactivity and active site binding determinants, Biochemistry 39, 8813-8824, 2000; Lack, J.G., Chaudhuri, S.K., Kelly, S.D. et al., Immobilization of radionucleotides and heavy metals through anaerobic bio-oxidation of Fe(II), Appl. Environ.Microbiol. 68, 2704-2710, 2002; Hamm, M.L., Rajguru, S., Downs, A.M., and Cholera, R., Base pair stability of 8-chloroand 8-iodo-2’-deoxyguanosine opposite 2’-deoxycytidine: implications regarding the bioactivity of 8-oxo-2’-deoxyguanosine, J.Am.Chem.Soc. 127, 12220-12221, 2005.
Autoantigen A component of self which is able to elicit an immune response, an autoimmune reaction; frequently with pathological complications such as the destruction of pancreatic beta cells (Islets of Langerhans) resulting in Type 1 diabetes. See Sigurdsson, E. and Baekkeskov, S., The 64-kDa beta cell membrane autoantigen and other target molecules of humoral autoimmunity in insulin-dependent diabetes mellitus, Curr.Top.Microbiol.Immunol. 164, 143-168, 1990; Werdelin, O., Autoantigen processing and the mechanisms of tolerance to self, Immunol.Ser. 52, 1-9, 1990; Manfredi, A.A., Protti, M.P., Bellone, M., et al., Molecular anatomy of an autoantigen: T and B epitopeson the nicotinic acetylcholine receptor in myasthenia gravis, J.Lab.Clin.Med. 120, 13-21, 1992; Sedgwick, J.D., Immune surveillance and autoantigen recognition in the central nervous system, Aust.N.E.J.Med. 25, 784-792, 1995; Utz, P.J., Gensler, T.J., and Anderson, P., Death, autoantigen modifications, and tolerance, Arthritis Res. 2, 101-114, 2000; Narendran, P., Mannering, S.I., and Harrison, L.C., Proinsulin-a pathogenic autoantigen in type 1 diabetes, Autoimmun.Rev. 2, 204-210, 2003; Gentile, F., Conte, M., and Formisano, S., Thyroglobulin as an autoantigen: what can we learn about immunopathogenicity from the correlation of antigenic properties with protein structure?. Immunology 112, 13-25, 2004; Pendergraft, W.F., 3rd, Pressler, E.M., Jennette, J.C., et al., Autoantigen complementarity: a new theory implicating complementary proteins as initiators of autoimmune disease, J.Mol. Med. 83, 12-25, 2005; Wu, C.T., Gershwin, M.E., and Davis, P.A., What makes an autoantigen an autoantigen? Ann.N.Y.Acad.Sci. 1050, 134-1045, 2005; Wong, F.S., Insulin-a primary autoantigen in type 1 diabetes?, Trends Mol.Med. 11, 445-448, 2005; Jasinski, J.M. and Eisenbarth, G.S., Insulin as a primary autoantigen for type 1A diabetes, Clin.Dev.Immuol. 12, 181-186, 2005.
Autocoid A internal, physiologically secretion of uncertain or unknown classification. Adenosine is one of the better examples as, apart from its role as purine base in RNA and DNA, it has diverse physiologic functions. See Yan. L., Burbiel, J.C., Maass, A., and Muller, C.E., Adenosine receptor agonists: from basic medicinal chemical to clinical development, Expert Opin.Emerg.Drugs 8, 537-576,
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2003; Tan, D.X., Manchester, L.C., Hadeland, R., Lopez-Burillo, S., Mayo, J.C., Sainz, R.M., and Reiter, R.J., Melatonin: a hormone, a tissue factor, an autocoid, a paracoid, and an antioxidant vitamin, J.Pineal Res. 34, 75-78, 2003; Boyan, B.D., Schwartz, Z., and Swain, L.D., Cell maturation-specific autocrine/paracrine regulation of matrix vesicles, Bone Miner. 17, 263-268, 1992; Polosa, R., Holgate, S.T. and Church, M,K., Adenosine as a pro-inflammatory mediator in asthma, Pulm.Pharmacol. 2, 21-26, 1989.
Autocrine Usually in reference to a hormone or other biological effector such as a peptide growth factor or cytokine which has an effect on the cell or tissue responsible for the synthesis of the given compound. Differentiated from endocrine or paracrine phenomena. See Sporn, M.B. and Roberts, A.B., Autocrine, paracrine, and endocrine mechanisms of growth control, Cancer Surv. 4, 627-632, 1985; Heldin, C.H. and Westermark, B., PDGF-like growth factors in autocrine stimulation of growth, J.Cell Physiol.(Suppl 5), 31-34, 1987; Ortenzi, C., Miceli, C. Bradshaw, R.A., and Luporini, P., Identification and initial characterization of an autocrine pheromone receptor in the protozoan cilitate Euplotes raikovi, J.Cell.Biol. 111, 607-614, 1990; Vallesi, A., Giuli, G., Bradshaw, R.A., and Luporini, P., Autocrine mitogenic activity of pheromones produced by the protozoan ciliate Euplotes raikovi, Nature 376, 522-524, 1995; Bischof, P., Meissner, A., and Campana, A., Paracrine and autocrine regulators of trophoblast invasion—a review, Placenta 21(Suppl. A), S55-S60, 2000; Bilezikjian, L.M., Blount, A.L., Leal, A.M., et al., Autocrine/paracrine regulation of pituitary function of activin, inhibin and folllistatin, Mol. Cell.Endocrinol. 225, 29-36, 2004; Singh, A.B. and Harris, R.C., Autocrine, paracrine and juxtacrine signaling by EGFR ligands, Cell Signal. 17, 1183-1193, 2005; Ventura, C. and Branzi, A., Autocrine and intracrine signaling for cardiogenesis in embryonic stem cells: a clue for the development of novel differentiating agents, Handb.Exp.Pharmacol. (174), 123-146, 2006.
Autophagy A pathway for the physiological degradation of cellular macromolecules and subcellular structures mediated by intracellular organelles such as lysosomes. It can be considered to be a process by which there is a membrane reorganization to separate or sequester a portion of the cytoplasm or cytoplasmic contents for subsequent delivery to an intracellular organelle such as a lysosome for degradation. This pathway of “self-destruction” is separate from proteosome-mediated degradation of macromolecules internalized from outside the cell. See Kroemer, G. and Jaattela, M., Lysosomes and autophagy in cell death control, Nat.Rev.Cancer 5, 886-897, 2005; Deretic, V., Autophagy in innage and adaptive immunity, Trends Immunol. 26, 523-528, 2005; Baehrecke, E.H., Autophagy: dual roles in life and death? Nat.Rev.Mol.Cell Biol. 6, 505-510, 2005; Wang, C-W., and Klianksy, D.J., The molecular mechanism of autophagy, Molec. Med. 9, 65-76, 2003. Klinosky, D.J., Autophagy, Curr.Biol. 15, R282-F283, 2005;
Autophosphorylation A process by which a substrate protein, usually a receptor, catalyzes self-phosphorylation usually at a tyrosine residue. The mechanism can be either intramolecular (cis) or intermolecular (trans) although at least one system has been described with both
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B-Lymphocytes Basolateral 973 cis and trans processes. See Cobb, M.H., Sang, B.-C., Gonzalez, R., Goldsmith, E., and Ellis, L., Autophosphorylation activates the soluble cytoplasmic domain of the insulin receptor in an intermolecular reaction, J.Biol.Chem. 264, 18701-18706, 1989; Frattali, A.L., Treadway, J.L., and Pessin, J.E., Transmembrane signaling by the human insulin receptor kinase. Relationship between intramolecular b subunit trans- and cis- autophosphorylation and substrate kinase activation, J.Biol.Chem. 267, 19521-19528, 1992; Rim, J., Faurobert, E., Hurley, J.B., and Oprian, D.D., In vitro assay for trans-phosphorylation of rhodopsin by rhodopsin kinase, Biochemistry 36, 7064-7070, 1997; Cann, A.D., Bishop, S.M., Ablooglu, A.J., and Kobanski, R.A., Partial activation of the insulin receptor kinase domain by juxtamembrane autophosphorylation, Biochemistry 37, 11289-11300, 1998; Iwasaki, Y., Nishiyama, H., Suzuki, K., and Koizumi, S., Sequential cis/trans autophosphorylation in TrkB tyrosine kinase, Biochemistry 36, 2694-2700, 1997; Cohen, P., The regulation of protein function by multisite phosphorylation – a 25 year update, Trends in Biochem.Sci. 25, 596-601, 2000; Wick, M.J., Ramos, F.J., Chen, H., Quon, M.J., Dong, L.Q., and Liu, F., Mouse 3-phosphoinositide-dependent protein kinase-1 undergoes dimerization and trans-phosphorylation in the activation loop, J.Biol.Chem. 278, 42913-42919, 2003; Wu, S. and Kaufman, R.J., trans-Autophosphorylation by the isolated kinase domain is not sufficient for dimerization or activation of the dsRNA-activated protein kinase PKR, Biochemistry 43, 11027-11034, 2004.
B-Lymphocytes Also called B-cells, named derived from original studies involving the cells from the bursa of chickens. B-cells are best known for the production of antibodies but recent studies are showing increased complexity of function. See T and B Lymphocytes: Origins, Properties and Roles in Immune Responses, ed. M.F. Greaves, J.J.T. Owen, and M.C. Raff, Excerpta Medica, New York, NY, 1973; B and T Cells in Immune Recognition, ed. Loor, F. and Roelants, G.E. Wiley, New York, NY, 1977; Cells of Immunoglobulin Synthesis, ed. B. Pernis and H.J. Vogel, Academic Press, New York, NY, 1979; T and B Lymphocytes: Recognition and Function, ed. F.H. Bach, Academic Press, New York, NY, 1979; Cambier, J.C., B-Lymphocyte Differentiation, CRC Press, Boca Raton, FL, 1986; B Lymphocytes and Autoimmunity, ed. N. Chiorazzi, New York Academy of Sciences, New York, NY, 1997; Cruse, J.M. and Lewis, R.E., Atlas of Immunology, CRC Press, Boca Raton, FL, 1999; Fundamental Immunology, ed. W.E. Paul, Lippincott, Williams, and Wilkins, Philadelphia, PA, 2003.
Backflushing or Back Flushing A method for cleaning filters involving reverse flow through the membrane; is occasionally used for cleaning large-scale chromatographic columns. See Tamai, G., Yoshida, H., and Imai, H., High-performance liquid chromatographic drug analysis by direct injection of whole blood samples. III. Determination of hydrophobic drugs adsorbed on blood cell membranes, J.Chromatog. 423, 163-168, 1987; Kim, B.S. and Chang, H.N., Effects of periodic backflushing on ultrafiltration performance, Bioseparation 2, 23-29, 1991; Dai, X.P., Luo, R.G. and Sirkar, K.K., Pressure and flux profiles in bead-filled ultrafiltration/microfiltration hollow fiber membrane modules, Biotechnol.Prog. 16, 1044-1054, 2000; Seghatchian, J. and Krailadsiri, P., Validation of different enrichment strategies for analysis of leucocyte subpopulations: development and application of a new approach,
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based on leucofiltration, Transfus.Apher.Sci. 26, 61-72, 2002; Kang, I.J., Yoon, S.H. and Lee, C.H., Comparison of the filtration characteristics or organic and inorganic membranes in a membrane-coupled anaerobic bioreactor, Water Res. 36, 1803-1813, 2002; Nemade, P.R. and Davis, R.H., Secondary membranes for flux optimization in membrane filtration of biologic suspensions, Appl.Biochem.Biotechnol. 113-116, 417-432, 2004.
Bacterial Artificial Chromosome A bacterial artificial chromosome (BAC) is a DNA construct based on a fertility plasma and is used for transforming and cloing in bacterial. It has an average insert size of 150 kbp with a range of approximately 100 kbp to 300 kbp. Bacterial artificial chromosomes are frequently used to sequence genomes where the PCR reaction is used to prepare a region of genomic DNA and then sequenced; in other words, a BAC is a vehicle based on the bacteria Escherichia coli that is used to copy, or clone, fragments of DNA that are 150,000 to 180,000 base pairs (bp) long. These DNA fragments are used as starting material for DNA.sequencing. See Schalkwyk, L.C., Francis, F., and Lehrach, H., Techniques in mammalian genome mapping, Curr.Opin.Biotechnol. 6, 37-43, 1995; Zhang, M.B. and Wing, R.A., Physical mapping of the rice genome with BACs, Plant Mol.Biol. 35, 115-127, 1997; Zhu, J., Use of PCR in library screening. An overview, Methods Mol.Biol. 192, 353-358, 2002; Ball, K.D. and Trevors, J.T., Bacterial genomics: the use of DNA microarrays and bacterial artificial chromosomes, J.Microbiol.Methods 49, 275-284, 2002; Miyake, T. and Amemiya, C.T., BAC libraries and comparative genomics of aquatic chordate species, Comp.Biochem.Physiol.C Toxicol.Pharmacol. 138, 233244, 2004; Ylatra, B., van den Ijssel, P., Carvalho, B., Brakenhoff, R.H., and Maijer, G.A., BAC to the future! or oligonucleotides: a perspective for micro array comparative genomic hybridization (array CGH), Nucleic Acids Res. 34, 445-450, 2006.
Balanced Translocation A chromosomal relocation which does not involved the net gain or loss of DNA; also referred to as reciprocal translocation. See Fraccaro, M., Chromosome abnormalities and gamete production in man, Differentiation 23(Suppl), S40-S43, 1983; Davis, J.R., Rogers, B.B., Hagaman, R.M., Thies, C.A., and Veomett, I.C., Balanced reciprocal translocations: risk factors for aneuploid segregant viability, Clin.Genet. 27, 1-19, 1985; Greaves, M.F., Biological models for leukaemia and lymphoma, IARC Sci. Publ. 157, 351-372, 2004; Benet, J., Oliver-Bonet, M., Cifuentes, P., Templado, C. and Navarro, J., Segregation of chromosomes in sperm of reciprocal translocation carriers: a review, Cytogenet. Genome Res. 111, 281-290, 2005. Aplan, P.D., Causes of oncogenic chromosomal translocation, Trends Genet. 22, 46-55, 2006.
Basolateral Literally located on the bottom opposite from the apical end of a differentiated cell. See apical. See Terada, T. and Inui, K., Peptide transporters: structure, function, regulation and application for drug delivery, Curr.Drug.Metab. 5, 85-94, 2004; Brone, B. and Eggermont, J., PDZ proteins retain and regulate membrane transporters in polarized epithelial cell membranes, Am.J.Physiol.Cell Physiol. 288, C20-C29, 2005; RodriquezBoulan, E and Musch, A., Protein sorting in the Golgi complex: shifting paradigms, Biochim.Biophys.Acta 1744, 455-464, 2005; Vinciguerra, M., Mordasini, D., Vandewalle, A., and Feraille, E.,
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Bathochromic Shift Bioinformatics 974 Hormonal and nonhormonal mechanisms of regulation of the NA,K-pump in collecting duct priniciple cells, Semin.Nephrol. 25, 312-321, 2005.
Bathochromic Shift A shift in the absorption/emission of light to a longer wavelength ( l >l o); a “red” shift. See Waleh, A. and Ingraham, L.L., A molecular orbital study of the protein-controlled bathochromic shift in a model of rhodopsin, Arch.Biochem.Biophys. 156, 261-266, 1973; Heathcote, P., Vermeglio, A., and Clayton, R.K., The carotenoid band shift in reaction centers from the Rhodopseudomonas sphaeroides, Biochim.Biophys.Acta 461, 358-364, 1977; Kliger, D.S., Milder, S.J., and Dratz, E.A., Solvent effects on the spectra of retinal Schiff bases—I. models for the bathochromic shift of the chromophore spectrum in visual pigments, Photochem. Photobiol. 25, 277-286, 1977; Cannella, C., Berni, R., Rosato, N., and Finazzi-Agro, A., Active site modifications quench intrinsic fluorescence of rhodanese by different mechanisms, Biochemistry 25, 7319-7323, 1986; Hermel, H., Holtje, H.D., Bergemann, S., et al, Band-shifting through polypeptide beta-sheet structures in the cyanine UV-Vis spectrum, Biochim.Biophys.Acta 1252, 79-86, 1995; Zagalsky, P.F., b-Crustacyanin, the blue-purple carotenoprotein of lobster carapace: consideration of the bathochromic shift of the protein-bound astaxanthin, Acta Chrystallogr. D Biol.Chrystallogr. 59, 1529-1531, 2003.
Betaine Glycine betaine, (carboxymethyl)trimethylamonnium inner salt, Cystadane®. Derived from choline; serves as methyl donor in the synthesis of methionine from homocysteine. Also functions as an osmoprotectant and this function is similar to trehalose in plants. See Chambers, S.T., Betaines: their significance for bacteria and the renal tract, Clin.Sci. 88, 25-27, 1995; Nuccio, M.L., Rhodes, D., McNeil, S.D., and Hanson, A.D., Metabolic engineering of plants for osmotic stress resistance, Curr.Opin.Plant Biol. 2, 129134, 1999; Zou, C.G. and Banerjee, R., Homocysteine and redox signaling, Antioxid.Redox.Signal. 7, 547-559, 2005; Craig, S.A., Betaine in human nutrition, Am.J.Clin.Nutr. 80, 539-549, 2004; Fowler, B., Homocysteine: overview of biochemistry, molecular biology, and role in disease processes, Semin.Vasc.Med. 5, 77-86, 2005; Ueland, P.M., Holm, P.I. and Hustad, S., Betaine: a key modulator of one-carbon metabolism and homocysteine status, Clin. Chem.Lab.Med. 43, 1069-1075 2005.
2001; Lynch, J. and Desplan, C., Evolution of development: beyond bicoid, Curr.Biol. 12, R557-R559, 2003.
BIND Biomolecular Interaction Data Base which is designed to store full descriptions of interactions, molecular complexes, and metabolic pathways. See Bader, G.D., Donaldson, I., Wolting, C., Ouellette, B.F., Pawson, T., and Hogue, C.W., BIND—the biomolecular interaction network database, Nucleic Acids Res. 29, 242-245, 2001; Alfarano, C. Andrade, C.E., Anthony, K. et al., The biomolecular interaction network database and related tools 2005 update, Nucleic Acids Res. 33, D418-D424, 2005; Shah, S.P., Huang, Y., Xu, T., Yuen, M.M. Ling, J., and Ouellette, B.F., Atlas – a data warehouse for integrative bioinformatics, BMC Bioinformatics 6, 34, 2005; Aytuna, A.S., Gursoy, A., Keskin, O., Prediction of protein-protein interactions by combining structure and sequence conservation in protein interfaces, Bioinformatics 21,2850-2855, 2005; Gilbert, D., Biomolecular interaction network database, Brief Bioinform. 6, 194-198, 2005.
Bioassay Generally used to describe an assay for a drug/biologic after administration to subject. As such, a bioassay usually involves the sampling of a biological fluid such as blood. The term bioassay is also used to describe an assay which uses a biological substrate such as a cell or organism. The term bioassay does not define a technology. See Yamamoto, S. Urano, K., and Nomura, T., Validation of transgenic mice harboring the human prototype c-Ha-ras gene as a bioassay model for rapid carcinogenicity testing, Toxicol.Lett. 28, 102-103, 1998; Colburn, W.A. and Lee, J.W., Biomarkers, validation and pharmacokinetic-pharmacodynamic modelling, Clin.Pharmacokinet. 42, 997-1022, 2003; Tuomela, M., Stanescu, I., and Krohn, K., Validation overview of bio-analytical methods, Gene Ther. 22 (Suppl 1), S131-138, 2005; Indelicato, S.R., Bradshaw, S.L., Chapman, J.W. and Weiner, S.H., Evaluation of standard and state of the art analytical technology-bioassays, Dev.Biol.(Basal) 122, 102-114, 2005.
Bioequivalence
One scFv fragment coupled to the C-terminus of the CH1 domain of a Fab fragment. See Schoonjans, R., Willems, A., Schoonooghe, S., et al., Fab chains as an efficient heterodimerization scaffold for the production of recombinant bispecific and trispecific antibody derivatives, J.Immunol. 165, 7050-7057, 2000. See also tribody, diabody.
Similarity of biological properties; used in the characterization of pharmaceuticals to demonstrate therapeutic equivalence. See Levy, R.A., Therapeutic inequivalence of pharmaceutical alternatives, Am.Pharm. NS23, 28-39, 1985; Durrleman, S. and Simon, R., Planning and monitoring of equivalence studies, Biometrics 46, 329-336, 1990; Schellekens, H., Bioequivalance and the immunogenicity of biopharmaceuticals, Nat.Rev.Drug.Disc. 1, 457-462, 2002; Lennernas, H. and Abrahamsson, B., The use of biopharmaceutical classification of drugs in drug discovery and development: current status and future extension, J.Pharm.Pharmacol. 57, 273-285, 2005; Bolton, S., Bioequivalence studies for levothyroxine, AAPS J. 7, E47-E53, 2005.
Bicoid Protein
Bioinformatics
A transcription-factor protein produced in Drosophila. See background to bicoid, Cell 54, 1-2, 1988; Stephenson, E.C. and Pokrywka, N.J., Localization of bicoid message during Drosophila oogenesis, Curr.Top.Dev.Biol. 26, 23-34, 1992; Johnstone, O. and Lasko, P., Translational regulation and RNA localization in Drosophila oocytes and embryos, Annu.Rev.Genet. 35, 365-406,
The use of information technology to analyze data obtained from proteomic analysis. An example is the use of data bases such as SWISSPROT to identify proteins from sequence information determined by the mass spectrometric analysis of peptides. See Baxevanis, A.D. and Ouellette, B.F.F., Bioinformatics: A Practical Guide to the Analysis of Genes and Proteins, Wiley, Hoboken, NJ.,
Bibody
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Biologicals Bone 975 Morphogenetic Protein(s) (BMP) USA, 2005; Buehler, L.K. and Rashidi, H.H., Bioinformaics basics: applications in biological sciences and medicine, Taylor & Francis, Boca Raton, Florida, USA, 2005; Evans, W.J., Statistical methods in bioinformatics: an introduction, Springer, New York, New York, USA, 2005; Wang, J.T.L., Data mining in bioinformatics, Springer, London, United Kingdom, 2005; Lesk, A.M., Introduction to bioinformatics, Oxford, New York, New York, 2005; Englbrecvht, C.C. and Facius, A., Bioinformatics challenges in proteomics, Comb.Chem.High Throughput Screen. 8, 705-715, 2005; Chandonia, J.M. and Brenner, S.E., The impact of structural genomics: expectations and outcomes, Science 311, 347-351, 2006; Chalkley, R.J., Hansen, K.C., and Baldwin, M.A., Bioinformatic methods to exploit mass spectrometric data for proteomic applications, Methods Enzymol. 402, 289-312, 2005; Allison, D.B, Cui, X., Page, G.P., and Sabripour, M., Microarray data analysis: from disarray to consolidation and consensus, Nat.Rev.Genet. 7, 55-65, 2006; Brent, M.R., Genome annotation past, present, and future: how to define an ORF at each locus, Genome Res. 15, 1777-1786, 2005.
Biologicals A biological product is any virus, serum, toxin, antitoxin, blood, blood component or derivative, allergenic product, or analogous product applicable to the prevention, treatment, or cure of diseases or injury. Biologic produts are a subset of “drug products” distinguished by their manufacturing processes (biological process vs. a chemical process). In general, the term “drugs” includes biological products. Within the United States, the regulation of biological is the purview of the FDA Center for Biologicals Evaluation and Research (CBER) and drugs within the FDA Center for Drug Evaluation and Research (CDER). There has been a recent shift of some drug products which were traditionally in CBER such as monoclonal antibodies and peptide growth factors to CDER. Vincent-Gattis, M., Webb, C., and Foote, M., Clinical research strategies in biotechnology, Biotechnol.Annu.Rev. 5, 229267, 2000; Steinberg, F.M. and Raso, J., Biotech pharmaceuticals and biotherapy: an overview, J.Pharm.Pharm.Sci. 1, 48-59, 1998; Stein, K.E. and Webber, K.O., The regulation of biologic products derived from bioengineered plants, Curr.Opin.Biotechnol. 12, 308-311, 2001; Morrow, K.S. and Slater, J.E., Regulatory aspects of allergen vaccines in the US, Clin.Rev.Allergy Immunol. 21, 141152, 2001; Hudson, P.J. and Souriau, C., Recombinant antibodies for cancer diagnosis and therapy, Expert Opin. Biol.Ther. 1, 845-855, 2001; Monahan, T.R., Vaccine industry perspective of current issues of good manufacturing practices regarding product inspections and stability testing, Clin.Infect.Dis. 33(Suppl.4), S356-S361, 2001; Hsueh, E.C. and Morton, D.L., Angiten-based immunotherapy of melanoma: Canvaxin therapeutic polyvalent cancer vaccine, Semin.Cancer Biol. 13, 401-407, 2003; Miller, D.L. and Ross, J.J., Vaccine INDs: review of clinical holds, Vaccine 23, 1099-1101, 2005; Sobell, J.M., Overview of biologic agents in medicine and dermatology, Semin.Cutan.Med.Surg. 23, 2-9, 2005; Morenweiser, R., Downstream processing of viral vectors and vaccines, Gene Ther. 12 (Suppl 1), S103-S110, 2005.
Biomarker A change in response to an underlying pathology; current examples of molecular changes include C-reactive protein, fibrin D-dimer, and troponin; the term biomarker is also used to include higher level responses such as behavior changes or anatomic changes. See Tronick, E.Z., The neonatal behavioral assessment
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scale as a biomarker of the effects of environmental agents on the newborn, Environ.Health Perspect. 74, 185-189, 1987; Salvaggio, J.E., Use and misuse of biomarker tests in “environmental conditions”, J.Allergy Clin.Immunol. 94, 380-384, 1994; Den Besten, P.K., Dental fluorosis: its use as a biomarker, Adv.Dent.Res. 8, 105-110, 1994; Lohmander, L.S. and Eyre, D.R., From biomarker to surrogate outcome to osteoarthritis-what are the challenges?, J.Rheumatol. 32, 1142-1143, 2005; Vineis, P. and HusgafvelPursiainen, K., Air pollution and cancer: biomarker studies in human populations, Carcinogenesis 26, 1846-1855, 2005; Seligson, D.B., The tissue micro-array as a translational research tool for biomarker profiling and validation, Biomarkers 10(Suppl 1), S77S82, 2005; Danna, E.A. and Nolan, G.P., Transcending the biomarker minset: deciphering disease mechanisms at the single cell level, Curr.Opin.Chem.Biol. 10, 20-27, 2006; Felker, G.M., Cuculich, P.S., and Gheorghiade, M., The Valsalva maneuver: a bedside “biomarker” test for heart failure, Am.J.Med. 119, 117122, 2006; Allam, A. and Kabelitz, D., TCR trans-rearrangments: biological significance in antigen recognition vs the role as lymphoma biomarker, J.Immunol. 176, 5707-5712, 2006.
Biopharmaceutical classification system The biopharmaceutical classification system (BCS) provides a classification of gastrointestinal absorption. See Amidon, G., Lennernas, H., Shah, V.P., and Crison, J.A., A theoretical basis for a biopharmaceutic drug classification: the correlation of in vitro drug product dissolution and in vivo bioavailability, Pharm.Res. 12, 413-420, 1995; Wilding, I.R., Evolution of the biopharmaceutics classification system (BCS) to oral modified release (MR) formulations: what do we need to consider? Eur.J.Pharm.Sci. 8, 157-159, 1999; Dressman, J.B. and Reppas, C., In vitro-in vivo correlations for lipophilic, poorly water-soluble drugs, Eur.J.Pharm. Sci. 11(Suppl 2), S73-S80, 2000; Taub, M.E., Kristensen, L. and Frokjaer, S., Optimized conditions for MDCK permeability and turbidimetric solubility studies using compounds representative of BCS class I-IV, Eur.J.Pharmaceut.Sci. 15, 311-340, 2002; Huebert, N.D., Dasgupta, M., and Chen, Y.M., Using in vitro human tissues-to predict pharmacokinetic properties, Curr.Opin. Drug.Disc.Dev. 7, 69-74, 2004; Lennernas, H. and Abrahamsson, B., The use of biopharmaceutic classification of drugs in drug discovery and development: current status and future extension, J. Pharm.Pharmcol. 57, 273-285, 2005.
Bone Morphogenetic Protein(s) (BMP) A group of peptide/proteins which are multifunctional growth factors that are members of the TGFb superfamily. There are multiple forms of bone morphogenetic proteins which all function as differentiation factors for the maturation of mesenchymal cells into chondrocytes and osteoblasts. See Hauschka, P.V., Chen, T.L., and Mavrakos, A.E., Polypeptide growth factors in bone matrix, Ciba Found.Symp. 136, 207-225, 1988; Wozney, J.M., Bone morphogenetic proteins, Prog.Growth Factor Res. 1, 267-280, 1989; Rosen, V. and Thies, R.S., The BMP proteins in bone formation and repair, Trends Genet. 8, 97-102, 1992; Wang, E.A., Bone morphogenetic proteins (BMPs): therapeutic potential in healing bony defects, Trends Biotechnol. 11, 379-383, 1993; Kirker-Head, C.A., Recombinant bone morphogenetic proteins: novel substances for enhancing bone healing, Vet.Surg. 24, 408-419, 1995; Ramoshibi, L.N., Matsaba, J., Teare, L., et al., Tissue engineering: TGF-b superfamily members and delivery systems in bone regeneration, Expert Rev.Mol.Med. 2002, 1-11, 2002; Monteiro, R.M.,
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de Sousa Lopez, S.M., Korchynskyi, O., et al., Spatio-temporal activation of Smad1 and Smad5 in vivo: monitoring transcriptional activity of Smad proteins, J.Cell Sci. 117, 4653-4663, 2004; Canalis, E., Deregowski, V., Pereira, R.C., and Gazzerro, E., Signals that determine the fate of osteoblastic cells, J.Endocrinol.Invest. 28(Suppl 8), 3-7, 2005; Franceschi, R.T., Biological approaches to bone regeneration by gene therapy, J.Dent.Res. 84, 1093-1103, 2005; Ripamonti, U., Teare, J., and Petit, J.C., Pleiotropism of bone morphogenetic proteins: from bone induction to cementogenesis and periodontal ligament regeneration, J.Int.Acad.Periodontol. 8, 23-32, 2006; Logeart-Avramoglou, D., Bourguignon, M., Oudina, K., Ten Dijke, P., and Petite, H., An assay for the determination of biologically active bone morphogenetic proteins using cells transfected with an inhibitor of differentiation promoter-luciferase construct, Anal.Biochem. 349, 78-86, 2006.
Bottom-up Proteomics Identification of unknown proteins by analysis of peptides obtained from unknown proteins by enzymatic (usually trypsin) hydrolysis. , See Brock, A., Horn, D.M., Peters, E.C., et al., An automated matrix-assisted laser desorption/ionization quadrupole Fourier transform ion cyclotron resonance mass spcctrometer for “bottom-up” proteomics, Anal.Chem. 75, 3419-3428, 2003; Wennder, B.R. and Lynn, B.C., Factors that affect ion trap data-dependent MS/MS in proteomics, J.Am.Soc.Mass Spectrom. 15, 150-157, 2004; Amoutzias, G.D., Robertson, D.L., Oliver, S.G., and Bornberg-Bauer, E., Convergent evolution of gene networks by single-gene duplications in higher eukaryotes, EMBO Rep. 5, 274-279, 2004; Ren, D., Julka, S., Inerowicz, H.D., and Regnier, F.E., Enrichment of cysteine-containing peptides from tryptic digests using a quaternary amine tag, Anal.Chem. 76, 4522-4530, 2004; Listgarten, J. and Emili, A., Statistical and computational methods for comparative proteomic profiling using liquid chromatography-tandem mass spectrometry, Mol.Cell.Proteomics 4, 419-434, 2005; Slysz, G.W. and Schriemer, D.C., Blending protein separation and peptide analysis through real-time proteolytic digestion, Anal.Chem. 77, 1572-1579, 2005; Zhong, H., Marcus, S.L., and Li, L., Microwave-assisted acid hydrolysis of proteins combined with liquid chromatography MALDI MS/MS for protein identification, J.Am.Soc.Mass Spectrom. 16, 471-481, 2005; Putz, S., Reinders, J., Reinders, Y., and Sickmann, A., Mass spectrometry-based peptide quantification: applications and limitations, Expert Rev. Proteomics 2, 381-392, 2005; Riter, L.S., Gooding, K.M., Hodge, B.D., and Julian, R.K., Jr., Comparison of the Paul ion trap to the linear ion trap for use in global proteomics, Proteomics 6, 1735-1740, 2006.
Brand Name Drugs A brand name drug is a drug marketed under a proprietary, trademarked-protected name.
BRET Bioluminesence Resonance Energy Transfer. Similar to FRET in BRET is technique which can be used to measure physical interactions between molecules. Intrinsic bioluminescence is used in this procedure such as different fluorescent protein (e.g. green fluorescent protein and blue fluorescent protein). See De, A. and Gambhir, S.S., Noninvasive imaging of protein-protein interactions from live cells and living subjects using bioluminescence resonance energy transfer, FASEB J. 19, 2017-2019, 2005.
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Cadherins
Brownian Movement The random movement of small particles in a suspension where the force of collision between particles is not lost but retained in part by the particle. The practical effect is to set the lower limit of particle size for settling from a suspension. Brownian movements is usually restricted to particles 1 µm in diameter and is not observed with particles of 5 µm.
Bulk Solution Bulk solution which would be defined as any macroscropic volume of a substance. In the case of an electrolyte. a bulk solution is charge neutral; intracellular and extracellular solutions possess a neutral charge even the presence of a membrane potential. The term bulk solution is also used to describe the difference between water structure in the hydration layer immediately around a macromolecule such as protein and the bulk solvent space. See Nakasako, M., Large-scale networks of hydration water molecules around proteins investigated by cryogenic X-ray crystallography, Cell.Mol.Biol. 47, 767-790, 2001; Lever, M., Blunt, J.W., and Maclagan, R.G., Some ways of looking at compensatory kosmotropes and different water environment, Comp.Biochem.Physiol. A Mol. Integr.Physiol. 130, 471-486, 2001; Halle, B., Protein hydration dynamics in solution: a critical survey, Philos.Trans.R.Soc. Biol.Sci. 359, 1207-1223, 2004; Levicky, R. and Horgan, A., Physicochemical perspectives on DNA microarray and biosensor technologies, Trends Biotechnol. 23, 143-149, 2005.
CAD A multifunctional protein that initiates and regulates de novo pyrimidine biosynthesis. See Carrey, E.A., Phosphorylation, allosteric effectors and inter-domain contacts in CAD: their role in regulation of early steps of pyrimidine biosynthesis, Biochem. Soc.Trans. 21, 191-195, 1993; Davidson, J.N., Chen, K.C., Jamison, R.S., Musmanno, L.A., and Kern, C.B., The evolutionary history of the first three steps in pyrimidine biosynthesis, Bioessays 15, 157-164, 1993; Evans, D.R. and Guy, H.I., Mammalian pyrimidine biosynthesis: fresh insights into an ancient pathway, J.Biol.Chem. 279, 33035-33038, 2005.
Cadherins A group of cell adhesion proteins which enable cells to interact with other cells and extracellular matrix components. See Obrink, B., Epithelial cell adhesion molecules, Exp.Cell Res. 163, 1-21, 1986; Takeichi, M., Cadherins: a molecular family important in selective cell-cell adhesion, Annu.Rev.Biochem. 59, 237252, 1990; Geiger, B. and Ayalon, O., Cadherins, Annu.Rev.Cell Biol. 8, 307-332, 1992; Tanoue, T. and Takeichi, M., New insights into fat cadherins, J. Cell Sci. 118, 2347-2353, 2005; Lecuit, T., Cell adhesion: sorting out cell mixing with echinoid? Curr.Biol. 15, R505-R507, 2005; Gumbiner, B.M., Regulation of cadherin-mediated adhesion in morphogenesis, Nat.Rev.Mol.Cell Biol. 6, 622634, 2005; Bamji, S.X., Cadherins: actin with the cytoskeleton to form synapses, Neuron 47, 175-178, 2005; Junghans, D., Hass, I.G., and Kemler, R., Mammalian cadherins and protocadherins: about cell death, synapses and processing, Curr.Opin.Cell Biol. 17, 446452, 2005; Cavallaro, U., Liebner, S., and Dejana, E., Endothelial cadherins and tumor angiogenesis, Exp.Cell Res. 312, 659-667, 2006; Redies, C., Vanhalst, K., and Roy, F., delta-Protocadherins: unique structures and functions, Cell Mol.Life Sci. 62, 2840-2852,
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2005; Collona, M., Cytolytic responses: cadherins put out the fire, J.Exp.Med. 203, 289-295, 2006; Chan, A.O., E-cadherin in gastric cancer, World J. Gastroenterol. 12, 199-203, 2006.
Caenorhabditis elegans A free-living roundworm which has been used extensively for genomic studies. It is notable for the discovery of RNA silencing/RNA interference (Fire, A., Xu, S., Montgomery, M.K., et al., Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans, Nature 391, 806-811, 1998). For general aspects of Caenorhabditis elegans see Zuckerman, B.M. and Merton, B., Nematodes as Biological Models, Academic Press, New York, 1980; C.elegans see Emmons, S.W., Mechanisms of C.elegans development, Cell 51, 881-883, 1987; Blumenthal, T. and Thomas, J., Cis and trans splicing in C.elegans, Trends Genet. 4, 305-308, 1988; Wood, W.B., The Nematode Caenorhabditis elegans, Cold Spring Harbor Labortory Press, Cold Spring Harbor, NY, 1988; Greenwald, I., Cell-cell interactions that specify certain cell fates in C.elegans development, Trends Genet. 5, 237241, 1989; Coulson, A., Kozono, Y., Lutterbach, B., et al., YACs and the C.elegans genome, Bioessays 13, 413-417, 1991; Plasterk, R.H., Reverse genetics of Caenorhabditis elegans, Bioessays 14, 629-633, 1992; Burglin, T.R. and Ruvkun, G., The Caenorhabditis elegans homeobox gene cluster, Curr.Opin.Genet.Dev. 3, 615620, 1993; Selfors, L.M. and Stern, M.J., MAP kinase function in C.elegans, Bioessays 16, 301-304, 1994; Stern, M.J. and DeVore, D.L., Extending and connecting signaling pathways in C.elegans, Dev.Biol. 166, 443-459, 1994; Kayne, P.S. and Sternberg, P.W., Ras pathways in Caehorhabditis elegans, Curr.Opin.Genet.Dev. 5, 38-43, 1995; Hope, I.A., C. elegans; A Practical Approach, Oxford University Press, Oxford, UK, 1999; Brown, A., In the Beginning was the Worm: Finding the Secrets of Life in a Tiny Hermaphrodite, Columbia University Press, NY, 2003; Filipowicz, W., RNAi: the nuts and bolts of the RISC machine, Cell 122, 17-20, 2005; Grishok, A., RNAi mechanisms in Caenorhabditis elegans, FEBS Lett. 579, 5932-5939, 2005. Hobert, O. and Loria, P., Uses of GFP in Caenorhabditis elegans, Methods Biochem.Anal. 47, 203-226, 2006; Hillier, L.W., Coulson, A., Murray, J.I., et al., Genomics in C.elegans; so many genes, such a little worm, Genome Res. 15, 1651-1660, 2005; http://www.nematodes.org/Caenorhabditis; http://www.wormbook.org; http://elegans.swmed.edu.
Calcineurin A protein phosphatase which is involved in the activation of IL-2 transcription; IL-2 stimulated the T-Cell response. Calcineurin is inhibited by immuosuppressive drugs such as cyclosporine and FK506(tacrolimus). See Pallen, C.J. and Wang, J.H., A multifunctional calmodulin-stimulated phosphatase, Arch. Biochem.Biophys. 237, 281-291, 1985; Klee, C.B., Draetta, G.F., and Hubbard, M.J., Calcineurin, Adv.Enzymol.Relat.Areas Mol. Biol. 61, 149-200, 1988; Siekierka, J.J. and Sigal, N.H., FK-506 and cyclosporine A: immunosuppressive mechanism of action and beyond, Curr.Opin.Immunol. 4, 484-552, 1992;Groenendyk, J., Lynch, J., and Michalak, M., Calreticulin, Ca 2+, and calcineurin – signaling from the endoplasmic reticulum, Mol.Cells 30, 383389, 2004; Michel, R.N., Dunn, S.E. and Chin, E.R., Calcineurin and skeletal muscle growth, Proc.Nutr.Soc. 63, 341-349, 2004; Im, S.H. and Rao, A., Activation and deactivation of gene expression by Ca2+/calcineurin-NFAT-mediated signaling, Mol.Cells 16, 1-9, 2004; Bandyopadhyay, J., Lee, J. and Bandopadhyay, A., Regulation of calcineurin, a calcium/calmodulin-dependent
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Calnexin
protein phosphatase in C.elegans, Mol.Cells 18, 10-16, 2004; Taylor, A.L., Watson, C.J., and Bradley, J.A., Immunosuppresive agents in solid organ transplantation: mechanisms of action and therapeutic efficacy, Crit.Rev.Oncol.Hematol. 56, 23-46, 2005; Crespo-Leiro, M.G., Calcineurin inhibitors in heart transplantation, Transplant.Proc.37, 4018-4020, 2005.
Calcium Transients Physiology phenomena resulting from changes in calcium concentration across membranes such as the diversity in Ca++ -stimulated transcriptional phenomona. See Spitzer, N.C., Lautermilch, N.J., Smith, R.D., and Gomez, T.M., Coding of neuronal differentiation by calcium transients, Bioessays 22, 811-817, 2000; Afroze, T. and Husain, M., Cell cycle dependent regulation of intracellular calcium concentration in vascular smooth muscle cells: a potential target for drug therapy, Curr.Drug Targets Cardiovasc.Haematol. Disord. 1, 23-40, 2001; Thorner, M.O., Holl, R.W., and Leong, D.A., The somatotrope: an endocrine cell with functional calcium transients, J.Exp.Biol. 139, 169-179, 1988; Morgan, K.G., Bradley, A., and DeFeo, T.T., Calcium transients in smooth muscle, Ann.N.Y.Acad.Sci. 522, 328-337, 1988; Fumagalli, G., Zacchetti, D., Lorenzon, P., and Grohovaz, F., Fluorimetric approaches to the study of calcium transients in living cells, Cytotechnology (Suppl 5) 1, 99-102, 1991; Komura, H., and Kumada, T., Ca 2+ transients control CNS neuronal migration, Cell Calcium 37, 387393, 2005; Fossier, P., Tauc, L. and Baux, G., Calcium transients and neurotransmitter release at an identified synapse, Trends Neurosci. 22, 161-166, 1999.
CALM-domain/CALM protein Clathrin assembly lymphoid myeloid-domain, related to ANTHdomain proteins and involved in endocytosis, formation of clathrin-coated pits; binds to lipids. See Kim, J.A., Kim, S.R., Jung, Y.K., et al., Properties of GST-CALM expressed in E.coli. Exp. Mol.Med. 32, 93-99, 2000; Kusner, L. and Carlin, C., Potential role for a novel AP180-related protein during endocytosis in MDCK cells, Am.J.Physiol.Cell Physiol. 285, C995-C1008, 2003; Archangelo, L.F>. Glasner, J., Krause, A., and Bohlander, S.K., The novel CALM interactor CATS influences the subcellular localization of the leukemogenic fusion protein CALM/AF10, Oncogene, 25, 4099–4109, 2006.
Calnexin A lectin protein associated with the endoplasmic reticulum which functions as a chaperone. See Cresswell, P., Androlewicz, M.J. and Ortmann, B., Assembly and transport of class I MHC-peptide complexes, Ciba Found.Symp. 187, 150-162, 1994; Bergeron, J.J., Brenner, M.B., Thomas, D.Y., and Williams, D.B., Calnexin: a membrane-bound chaperone or the endoplasmic reticulum, Trends Biochem.Sci. 19, 124-128, 1995;, Parham, P., Functions for MHC class I carbohydrates inside and outside the cell, Trends Biochem.Sci. 21, 472-433, 1996; Trombetta, E.S. and Helenius, A., Lectins as chaperones in glycoprotein folding, Curr.Opin.Struct. Biol. 8, 587-592, 1998; Huari, H.. Appenzeller, C., Kuhn, F., and Nufer, O., Lectins and traffic in the secretory pathway, FEBS Lett. 476, 32-37, 2000; Ellgaard, L. and Frickel, E.M., Calnexin, calreticulin, and ERp57: teammates in glycoprotein folding, Cell. Biochem.Biophys. 39, 223-247, 2003; Spiro, R.G., Role of N-linked polymannose oligosaccharides in targeting glycoproteins for endoplasmic reticulum-associated degradation, Cell.Mol.Life
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Calponin
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Sci. 61, 1025-1041, 2004; Bedard, K., Szabo, E., Michalak, M., and Opas, M., Cellular functions of endoplasmic reticulum chaperones calreticulin, calnexin, and ERp57, Int.Rev.Cytol. 245, 91-121, 2005; Ito, Y., Hagihara, S., Matsuo, I., and Totani, K., Structural approaches to the study of oligosaccharides in glycoprotein quality control, Curr.Opin.Struct.Biol.15, 481-489, 2005.
Calponin A family of actin-binding proteins which exist in various isoforms. As with other protein isoforms or isoenzymes, the expression of the isoforms is tissue-specific. The interaction of calponin with actin inhibits the actomyosin Mg-ATPase activity. See Winder, S and Walsh, M., Inhibition of the actinomyosin MgATPase by chicken gizzard calponin, Prog.Clin.Biol.Res. 327, 141-148, 1990; Winder, S.J., Sutherland, C. and Walsh, M.P., Biochemical and functional characterization of smooth muscle calponin, Adv.Exp.Med.Biol. 304, 37-51, 1991; Winder, S.J. and Walsh, M.P., Calponin: thin filament-linked regulation of smooth muscle contraction, Cell Signal. 5, 677-686, 1993; el-Mezgueldi, M., Calponin, Int.J.Biochem.Cell Biol. 28, 1185-1189, 1996; Szymanski, P.T., Calponin (CaP) as a latch-bridge protein—a new concept in regulation of contractility in smooth muscle, J.Muscle Res.Cell Motil. 25, 7-19, 2004; Lehman, W. Craig, R., Kendrick-Jones, J., and Sutherland-Smith, A.J., An open or closed case for the conformation of calponin homology domains on F-actin?, J.Muscle Res. Cell Motil. 25, 351358, 2004; Ferjani, I., Fattoum, A., Maciver, S.K. et al., A direct interaction with calponin inhibits the actin-nucleating activity of gelsolin, Biochem.J. 396, 461-468, 2006.
Calreticulin A 50-60 kDa protein found in the endoplasmic reticulum. Calreticulin binds calcium ions tightly and it thought to play a role in calcium homeostasis. Calreticulin also functions as chaperone. See Koch, G.L. and Smith, M.J., The analysis of glycoproteins in cells and tissues by two-dimensional polyacrylamide gel electrophoresis, Electrophoresis 11, 213-219, 1990; Krause, K.H., Ca(2 +)-storage organelles, FEBS Lett. 285, 225-229, 1991; Herbert, D.N., Simons, J.F., Peterson, J.R., and Helenius, A., Calnexin, calreticulin, and Bip/Kar2p in protein folding, Cold Spring Harbor Symp.Quant.Biol. 60, 405-415, 1995; Groenendyk, J., Lynch, J. and Michalak, M., Calreticulin, Ca 2+, and calcineurin – signaling from the endoplasmic reticulum, Mol.Cells 17, 383389, 2004; Michalak, M., Guo, L., Robertson, M., Lozak, M., and Opas, M., Calreticulin in the heart, Mol.Cell.Biochem. 263, 137142, 2004; Gelebart, P., Opas, M., and Michalak, M., Calreticulin, a Ca2+ -binding chaperone of the endoplasmic reticulum,, Int.J.Biochem.Cell Biol. 37, 260-266, 2005; Bedard, K., Szabo, E., Michalak, M., and Opas, M., Cellular functions of endoplasmic reticulum chaperones calreticulin, calnexin, and ERp57, Int. Rev.Cytol. 245, 91-121, 2005; Ito, Y,, Hagihara, S., Matsuo, I., and Totani, K., Structural approaches to the study of oligosaccharides in glycoprotein quality control, Curr.Opin.Struct.Biol. 15, 481-489, 2005; Garbi, N., Tanaka, S., van den Broek, M. et al., Accessory molecules in the assembly of major histocompatibility complex class I/peptide complexes: how essential are they for CD(+) T-cell immune responses? Immunol.Rev. 207, 77-88, 2005; Cribb, A.E., Peyrou, M., Muruganandan, S., and Schneider, L., The endoplasmic reticulum in xenobiotic toxicity, Drug.Metab. Rev. 37, 405-442, 2005; Hansson, M., Olsson, I., and Nauseef, W.M., Biosynthesis, processing and sorting of human myeloperoxidase, Archs.Biochem.Biophys. 445, 214-224, 2006.
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Cathepsins
Camelid Antibodies Unique antibodies from members of the Camelidae family. The antibody structure consists of a heavy chain consisting of a variable region VHH but no light chain. The structure also misses the the first constant domain (CH1) but retains the other constants regions C-terminal from the hinge region. Thus, while a classical IgG is a dimer of heterodimers, the camelid antibody described herein is a homodimer. See Ghahroudi, M.A., Desmyter, A., Wyns, L., Hamers, R., and Muyldermans, S., Selection and identification of single domain antibody fragments from camel heavy-chain antibodies, FEBS Lett. 414, 521-526, 1997; Nguyen, V.K., Desmyter, A., and Muylderman, S., Functional heavy-chain antibodies in Camelidae, Adv.Immunol. 79, 261-296, 2001; Muyldermans, S., Single domain camel antibodies: current status, J.Biotechnol. 74, 277-302, 2001; Nguyen, V.K., Su., C., Muyldermans, S., and van der Loo, W., Heavy-chain antibodies in Camelidae, a case of evolutionary innovation, Immunogenetics 54, 39-47, 2002; Conrath, K.E., Wernery, U., Mulydermans, S., and Nguyen, V.K., Emergence and evolution of functional heavy-chain antibodies in Camelidae, Dev.Comp.Immunol. 27, 87-103, 2003; Rahbarisadeh, F., Rasaee, M.J., Forouzandeh, M., et al., The production and characterization of novel heavy-chain antibodies against the tandem repeat region of MUC1 mucin, Immunol.Invest. 34, 431-452, 2005.
Cap Structure at the 5’-end of eukaryotic RNA, introduced after transcription by linking the terminal phosphate of 5’-GTP to the terminal base of the mRNA. The guanine base can be nucleated. 7MeG5’-ppp5Np See Banerjee, A.K., 5’-terminal cap structure in eukaryotic messenger ribonucleic acids, Microbiol.Rev. 44, 175-205, 1980; Miura, K., The cap structure in eukaryotic RNA as a mark of a strand carrying protein information, Adv.Biophys. 14, 205-238, 1981; Lewin, B., Genes IV, Oxford University Press, Oxford, United Kingdom, 1990; Cougot, N., van Dijk, E., Babajko, S., and Seraphin, B., ‘Cap-tabolism’, Trends Biochem.Sci. 29, 436-444, 2004; Gu, M. and Lima, C.D., Processing the message: structural insights into capping and decapping mRNA, Curr.Opin.Struct. Biol. 15, 99-106, 2005; Bentley, D.L., Rules of engagement: co-transcriptional recruitment of pre-mRNA processing factors, Curr.Opin.Cell Biol. 17, 251-256, 2005; Liu, H. and Kiledjian, M., Decapping the message: a beginning or an end, Biochem.Soc.Trans. 34, 35-38, 2006; Simon, E., Camier, S., and Seraphin, B., Trends Biochem.Sci. 31, 241-243, 2006. CAP is also acronym for catabolic activator protein.
Catabolic activator protein A transcription-regulating protein which binds to DNA in the promoter loop. See Benoff, B., Yang, H., Lawson, C.L., et al., Structural basis of transcription activation: the CAP-alpha CTD-DNA complex, Science 297, 1562-1566, 2002; Balaeff, A., Mahadevan, L., and Schulten, K., Structural basis for cooperative DNA binding by CAP and lac repressor, Structure 12, 123-132, 2004; Akaboshi, E., Dynamic profiles of DNA: analysis of CAPand LexA protein-binding regions with endonucleases, DNA Cell Biol. 24, 161-172, 2005.
Cathepsins A family of intracellular thiol proteases involved in lysosomal digestion of proteins. See Janoff, A., Mediators of tissue damage in human polymorphonuclear neutrophils, Ser.Haematol.
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CASP
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3, 96-130, 1970; Harris, E.D., Jr. and Krane, S.M., Collagenases, N.Engl.J.Med. 291, 605-609, 1974; Larzarus, G.S., Hatcher, V.B., and Levine, N., Lysosomes and the skin, J.Invest.Dermatol. 65, 259-271, 1975; Ballard, F.J., Intracellular protein degradation, Essays Biochem. 13, 1-37, 1977; Barrett, A.J., Cathepsin D: the lysosomal aspartic proteinase, Ciba Found.Symp. (75), 37-50, 1979; Barrettt, A.J. and Kischeke, H., Cathepsin B, Cathepsin H, and Cathepsin L, Methods Enzymol. 80, 535-561, 1981; Groutas, W.C., Inhibitors of leukocyte elastase and leukocyte cathepsin G. Agents for the treatment of emphysema and related ailments, Med.Res.Rev. 7, 227-241, 1987; Stoka, V., Turk, B., and Turk, V., Lysosomal cathepsin proteases: structural features and their role in apoptosis, IUBMB Life 57, 347-353, 2005; Roberts, R., Lysosomal cysteine proteases: structure, function and inhibition of cathepsins, Drug News Perspect. 18, 605-614, 2005; Chwieralski, C.E., Welte, T., and Buhling, F., Cathepsin-regulated apoptosis, Apoptosis 11, 143-149, 2006. There is particular interest in the role of cathepsins in antigen processing (Honey, K. and Rudensky, A.Y., Lysosomal cysteine proteases regulate antigen presentation, Nat.Rev.Immunol. 3, 472-482, 2003; Bryant, P. and Ploegh, H., Class II MHC peptide loading by the professionals, Curr.Opin.Immunol. 16, 96-102, 2004; Liu, W. and Spero, D.M., Cysteine protease cathepsin S as a key step in antigen presentation, Drug.News Perspect. 17, 357-363, 2004; Hsing, L.C. and Rudensky, A.Y., The lysosomal cysteine proteases in MHC Class II antigen presentation, Immunol.Rev. 207, 229-241, 2005.
CASP Critical Assessment of Structure Prediction describes a process for the evaluation of protein model building. See Moult, J. Predicting protein three-dimensional structure, Curr.Opin. Biotechnol. 10, 583-588, 1999; Moult, J., Fidelis, K., Rost, B., Hubbard, T., and Tramontano, A., Critical assessment of methods of protein structure prediction (CASP) – round 6, Proteins 61 Suppl. 7, 3-7, 2005; Giorgetti, A., Raimondo, D., Miele, A.E., and Tramontano, A., Evaluting the usefulness of protein structure models for molecular replacement, Bioinformatics 21(Suppl. 2), ii72-ii76, 2005; Espejo, F. and Patarroyo, M.E., Determining the 3D structure of human ASC2 protein involved in apoptosis and inflammation, Biochem.Biophys.Res.Commun. 340, 860-864, 2006; Moult, J., Rigorous performance evaluation in protein structure modeling and implications for computational biology, Philos.Trans.R.Soc.Lond. B Biol.Sci. 361, 453-458, 2006.
Caspases A family of intracellular cysteine proteases that are involved in the process of apoptosis (programmed cell death). Caspases are synthesized as precursor or zymogen forms which required activation prior to function. One such activation process involves granzymes. Caspases also function in other intracellular processes. See Jacobson. M.D. and Evan, G.I., Apoptosis. Breaking the ice, Curr.Biol. 4, 337-340, 1994; Patel, T., Gores, G.BJ., and Kaufmann, S.H., The role of proteases during apoptosis, FASEB J. 10, 587-597, 1996; Alnemri, E.S., Mammalian cell death proteases: a family of highly conserved aspartate specific cysteine proteases, J.Cell Biochem. 64, 33-42, 1997; Zhivotovsky, B., Caspases: the enzymes of death, Essays Biochem. 39, 25-40, 2003; Twomey, C. and McCarthy, J.V., Pathways of apoptosis and importance in development, J.Cell Mol.Med. 9, 345-359, 2005; Ashton-Rickardt, P.G., The granule pathway of programmed cell death, Crit.Rev. Immunol. 25, 161-182, 2005; Yan, N. and Shi, Y., Mechanisms of
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apoptosis through structural biology, Annu.Rev.Cell Dev.Biol. 21, 35-56, 2005; Harwood, S.M., Yaqoob, M.M., and Allen, D.A., Caspase and calpain function in cell death: bridging the gap between apoptosis and necrosis, Ann.Clin.Biochem. 42, 415-431, 2005; Ho, P.K. and Hawkins, C.J., Mammalian initiator apoptotic caspases, FEBS J. 272, 5436-5453, 2005; Fardeel, B. and Orrenius, S., Apoptosis: a basic biological phenomenon with wide-ranging implications in human disease, J.Intern.Med. 258, 479-517, 2005; Cathelin, S., Rébe. C., Haddaoui, L., et al., Identification of proteins cleaved downstream of caspases activation in monocytes undergoing macrophage differentiation, J.Biol.Chem. 281, 1777917788, 2006.
Catalomics The study of the enzymes in a proteome; the study of catalysis in a proteome. See Hu, Y., Uttamchandani, M., and Yao, S.Q., Microarray: a versatile platform for high-throughput functional proteomics, Comb.Chem.High Throughput Screen. 9, 201-212, 2006.
Catalytic Antibodies Antibodies which demonstrate catalytic activity. The early development of these antibodies was based on the use of haptens which mirrored transition state intermediates for enzyme-catalyzed reactions. Catalytic antibodies can be referred to as abzymes. See Kraut, J., How do enzymes work? Science 242, 533-540, 1988; Lerner, R.A. and Tramontano, A., Catalytic antibodies, Sci.Am. 258, 65-70, 1988; Green, B.S., Catalytic antibodies and biomimetics, Curr.Opin.Biotechnol. 2, 395-400, 1991; Jacobs, J.W., New perspectives on catalytic antibodies, Biotechnology 9, 258-262, 1991; Blackburn, G.M., Kingsbury, G., Jayaweera, S., and Burton, D.R., Expanded transition state analogues, Ciba Found.Symp. 159, 211-222, 1991; O’Kennedy, R. and Roben, P., Antibody engineering: an overview, Essays Biochem. 26, 59-75, 1991; Stewart, J.D., Krebs, J.F., Siuzdak, G., et al., Dissection of an antibody-catalyzed reaction, Proc.Nat.Acad.Sci.USA 91, 7404-7409, 1994; Posner, B., Smiley, J., Lee, I., and Benkovic, S., Catalytic antibodies: perusing combinatorial libraries, Trends Biochem.Sci. 19, 145-150, 1994; Kikuchi, K. and Hilvert, D., Antibody catalysis via strategic use of hepatenic charge, Acta Chem.Scand. 50, 333-336, 1996; Wentworth, P., Jr., and Janda, K.D., Catalytic antibodies: structure and function, Cell. Biochem.Biophys. 35, 63-87, 2001; Ostler, E.L., Resmini, M., Brocklehurst, K., and Gallacher, G., Polyclonal catalytic antibodies, J.Immunol.Methods 269, 111-124, 2002; Hanson, C.V., Nishiyama, Y., and Paul, S., Catalytic antibodies and their applications, Curr.Opin.Biotechnol. 16, 631-666, 2005. There has also been considerable interest in catalytic antibodies in pathological processes and as potential therapeutic agents. See LacroixDemazes, S., Kazatchkine, M.D. and Kaveri, S.V., Catalytic antibodies to factor VIII in haemophilia A., Blood Coag.Fibrinol. 14(Suppl 1), S31-S34, 2003; Poloukhina, D.I., Kanyshkova, T.G., Doronin, B.M., et al., Hydrolysis of myelin basic protein by polyclonal catalytic IgGs from the sera of patients with multiple sclerosis, J.Cell.Mol.Med. 8, 359-368, 2004; Paul, S., Nishiyama, Y.,. Planque, S., et al., Antibodies as defensive enzymes, Springer Semin.Immunopathol. 26, 485-503, 2005; Ponomarenko, N.A., Vorobiev, I.I., Alexandrova, E.S., et al., Induction of a proteintargeted catalytic response in autoimmune prone mice: antibodymediated cleavage of HIV-1 glycoprotein GP120, Biochemistry 45, 324-330, 2006; Lacroix-Desazes, S., Wootla, B., Delignat, S., et al., Pathophysiology of catalytic antibodies, Immunol.Lett. 103, 3-7, 2006.
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CATH
980
CATH
Cell Culture
Enzyme-linked immunoassay on live cells. See Geraghyty, R.J., Jogger, C.P., and Spear, P.B, Cellular expression of alphaherpesvirus gD interferes with entry of homologous and heterologous alphaviruses by blocking access to a shared gD receptor, Virology 68, 147-156, 2000; Lee, R.B., Hassone, D.C., Cottle, D.L., and Picket, C., Interactions of Campylobacter jejuni cytolethal distending toxin subunits Cdta and Cdtc with HeLa cells, Infect. Immun. 71, 4883-4890, 2003.
chemical fixed with glutaraldehyde or similar reagents, or pelleted onto the surface. Hoffman, T. and Herberman, R.B., Enzymelinked immunosorbent assay for screening monoclonal antibody production: use of intact cells as antigen, J.Immunol.Methods 39, 309-316, 1980; Krakauer, H., Hartman, R.J., and Johnson, A.H., Monoclonal antibodies specific for human polymorphic cell surface antigens. I. Evaluation of methodology. Report of a workshop, Human Immunol. 4, 167-181, 1982; Bishara, A., Brautbar, C., Marbach, A., Bonvida, B., and Nelken, D., Enzyme-linked immunosorbent assay for HLA determination on fresh and dried lymphocytes, J.Immunol.Methods 62, 265-271, 1983; Sharon, R., Duke-Cohan, J.S., and Galili, U., Determination of ABO blood group zygosity by an antiglobulin resetting technique and cellbased enzyme immunoassay, Vox Sang. 50, 245-249, 1986; Zhao, Q., LU, H., Schols, D., de Clercq, E., and Jiang, S., Development of a cell-based enzyme-linked immunosorbent assay for highthroughput screening of HIV type enzyme inhibitors targeting the coreceptor CXCR4, Aids Res.Human Retrovirus 19, 947-955, 2003; Yang, X.Y., Chen, E., Jiang, H. et al., Development of a quantitative cell-based ELISA, for a humanized anti-IL-2/IL-15 receptor beta antibody (HuMikbeta(1)), and correlation with functional activity using an antigen-transferred murine cell line. In some cases, a cell homogenate could be used as the sample. See Franciotta, D., Martino, G., Brambilla, E., et al., TE671 cell-based ELISA for anti-acetylcholine receptor antibody determination in myasthenia gravis, Clin.Chem. 45, 400-405, 1999. The cell-based ELISA is distinct from the ELISPOT assay where there a capture antibody on the membrane (Arvilommi, H., Elispot for detecting antibody-secreting cells in response to infections and vaccination, APMIS 104, 401-410, 1996).
Cell-based assays
Cell Culture
This is a broad classification for assays where cells are used as the substrate or indictor for the action of a drug. Examples include platelet aggregation, cell-based ELISA(see below), gene expression assays, receptor-ligand interactions, etc. See Nuttall, M.E., Drug discovery and target validation, Cells Tissues Organs 169, 265-271, 2001; Bhadriraju, K. and Chen, C.S., Engineering cellular microenvironments to improve cell-based drug testing, Drug Discov. Today 7, 612-620, 2002; Indelicato, S.R., Bradshaw, S.L., Chapman, J.W., and Weiner, S.H., Evaluation of standard and state of the art analytical technology-bioassays, Dev.Biol. 122, 103-114, 2005; Stacey, G.N., Standardisation of cell lines, Dev.Biol. 111, 259-272, 2002; Qureshi, S.A., Sanders, P., Zeh, K., et al., A onearm homologous recombination approach for developing nuclear receptor assays in somatic cells, Assay Drug Dev.Technol. 1, 767776, 2003; Wei, X., Swanson, S.J., and Gupta, S., Development and validation of a cell-based bioassay for th detection of neutralizing antibodies against recombinant human erythropoietin in clinical studies, J.Immunol.Methods 293, 115-126, 2004; Pietrak, B.L., Crouthamel, M.C., Tugusheva, K., et al., Biochemical and cell-based assays for characterization of BACE-1 inhibitors, Anal. Biochem. 342, 144-151, 2005; Chen. T,, Hansen, G., Beske, O., et al., Analysis of cellular events using Cell Card System in cellbased high-content multiplexed assays, Expert Rev. Mol.Diagn. 5, 817-829, 2005.
The maintenance of dispersed animal or plant cells in a specialized media (cell culture media). In biotechnology manufacturing, cell culture is used for the production of protein biopharmaceuticals using cells such as Chinese hamster ovary (CHO) cells or baby hamster kidney (BHK) cells. The use of the term cell culture differentiates such a process from fermentation. See Mantell, S.H. and Smith, H., Plant Biotechnology, Cambridge University Press, Cambridge, UK, 1983; Applications of Plant Cell and Tissue Culture, Chichester, UK, 1988; Freshney, R.I., Animal Cell Culture: A Practical Approach, IRL Press at Oxford University Press, Oxford, UK, 1992; Morgan, S.J. and Darling, D.C., Animal Cell Culture, Bios/Biochemical Society, London, UK, 1993; Davis, J.M., Basic Cell Culture: A Practical Approach, IRL Press at Oxford University Press, Oxford, UK, 1994; Dodds, J.H. and Roberts, L.W., Experiments in Plant Tissue Culture, Cambridge University Press, Cambridge, UK, 1995; Spier, R., Encyclopedia of Cell Technology, Wiley-Interscience, New York, NY, 2000; Hesse, F. and Wagner, R., Development and improvements in the manufacture of human therapeutics wit mammalian cell culture, Trends Biotechnol. 18, 173-180, 2000; James, E. and Lee, J.M., The production of foreign proteins from genetically modified plant cells, Adv.Biochem.Eng.Biotechnol. 72, 127-156, 2001; Kaeffer, B., Mammalian intestinal epithelial cells in primary culture: a mini-review, In Vitro Cell Dev.Biol.-Animal 38, 128-134, 2002; Ikonomou, L., Schneider, Y.J., and Agathos, S.N., Insect cell culture for industrial production of recombinant proteins, Appl.Microbiol.Biotechnol. 62, 1-20, 2003; Kallos, M.S., Sen, A., and Behie, L.A., Large-scale expansion of mammalian neural stem cells: a review, Med.Biol.Eng.Comput. 41, 271-282, 2003; Schiff, L.J., Review: production, characterization, and testing of
A classification process for protein domain structures based on class(C), architecture(A), topology(T), and homology superfamily (H). See Orengo, C.A., Michie, A.D., Jones, S., Jones, D.T., Swindells, M.B., and Thornton, J.M., CATH –a hierarchic classification of protein domain structures, Structure 5, 1093-1108, 1997; Bray, J.E., Todd, A.E., Pearl, F.M., Thornton, J.M., and Orengo, C.A., The CATH dictionary of homologous superfamilies (DHS): a consensus approach for identifying distant structural homologues, Protein Eng. 13, 153-165, 2000; Ranea, J.A., Buchan, D.W., Thornton, J.M., and Orengo, C.A., Evolution of protein superfamilies and bacterial genome size, J.Mol.Biol. 336, 871-887, 2004; Velazquez-Muriel, J.A., Sorzano, C.O., Scheres, S.H., and Carazo, J.M., SPI-EM: towards a tool for predicting CATH superfamilies in 3D-EM maps, J.Mol.Biol. 345, 759-771, 2005. Sillitoe, I., Dibley, M., Bray, J., Addou, S., and Orengo, C., Assessing strategies for improved superfamily recognition, Protein Sci. 14, 1800-1810, 2005.
CELISA
Cell-based ELISA Cell-based ELISA are indirect or direct ELISA systems which use intact cells as the antigen sample. Cells may be dried onto the microplate surface or a microplate surface treated with polylysine,
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Cell Penetrating Peptide
981
banked mammalian cell substrates used to produce biological products, In Vitro Cell Dev.Biol.-Animal 41, 65-70, 2005; Evan, M.S., Sandusky, C.B. and Barnard, N.D., Serum-free hybridoma culture: ethical, scientific and safety considerations, Trends Biotechnol. 24, 105-108, 2006.
Cell Penetrating Peptide Cell-penetrating peptides are relatively small peptides, usually less than 30 amino acids in length, which have the ability to pass through or translocate the cellular membrane in via a mechanisms which appears to be both receptor-independent as well as distinct from an endocytotic process. Such peptides have been demonstrated to “transport” diverse cargo and are being evaluated for drug delivery. See Lundberg, P. and Langel, U., A brief introduction to cell-penetrating peptides; Temsamani, J. and Vidal, P., The use of cell-penetrating peptides for drug delivery, Drug Discovery Today 9, 1012-1019, 2004; Gupta, B., Levchenko, T.S., and Torchilin, V.P., Intracellular delivery of large molecules and small particles by cell-penetrating proteins and peptides, Adv.Drug.Deliv.Rev. 57, 637-651, 2005; Deshayes, S., Morris, M.C., Divta, G., and Heitz, F., Cell-penetrating peptides: tools for intracellular delivery of therapeutics, Cell.Mol.Life.Sci. 62, 1839-1849, 2005. See also amphipathic.
CentiRay A measure of the frequency of chromosome breakage between DNA markers in radiation-reduced somatic cell hybrids (radiation hybrids). One centiRay is equivalent to a 1% probability that a chromosome break (CentiRay distances are generally proportional to physical distance and are measured in centimorgans. See Hukriede, N.A., Joly, L., Tsang, M., et al., Radiation hybrid mapping of the zebrafish genome, Proc.Nat.Acad.Sci.USA 96, 97459750, 1999; Hamasima, N. Suzuki, H., Mikawa, A. Morozumi, T., Plastow, G., and Mitsuhashi, T., Construction of a new porcine whole-genome framework map using a radiation hybrid panel, Anim.Genet. 34, 216-220, 2003; Voigt, C., Moller, S., Ibrahim, S.M., and Serrano-Fernandez, P., Non-linear conversion between genetic and physical chromosomal distances, Bioinformatics 20, 1966-1977, 2004.
Centimorgan
Chemical Biology
1996; Mezei, M., Chameleon sequences in the PDB, Protein.Eng. 11, 411-414, 1998; Tidow, H., et al., The solution structure of a chimeric LEKTI domain reveals a chameleon sequence, Biochemistry 43, 11238-11247, 2004.
Chaotropic Describing a reagent which disrupts the structure of water and macromolecules such as proteins. Chaotropic is sometimes confined to uncharged molecules such as urea or thiourea but is usually extended to include reagents such as guanidine hydrochloride and sodium thiocyanate. See Dandliker, W.B., Alonso, R., de Saussure, V.A., Kierszenbaum, F., et al., The effect of chaotropic ions on the dissociation of antigen-antibody complexes, Biochemistry 6, 1460-1467, 1967; Hanstein, W.G., Davis, K.A., and Hatefi, Y., Water structure and the chaotropic properties of haloacetates, Arch.Biochem.Biophys. 147, 534-544, 1971; Sawyer, W.H. and Puckridge, J., The dissociation of proteins by chaotropic salts, J.Biol.Chem. 248, 8429-8433, 1973; Hatefi, Y. and Hanstein, W.G., Destablization of membranes with chaotropic ions, Methods Enzymol. 31, 770-790, 1974; McLaughlin, S., Bruder, A., Chen, S., and Moser, C., Chaotropic anions and the surface potential of bilayer membranes, Biochim.Biophys. Acta 394, 304-313, 1975; Stein, M., Lazaro, J.J., and Wolsiuk, R.A., Concerted action of cosolvents, chaotropic anions and thioredoxin on chloroplast fructose-1,6-bisphosphatase. Reactivity to iodoacetate, Eur,J.Biochem. 185, 425-431, 1989; Lever, M., Blunt, J.W., and MacLagan, R.G., Some ways of looking at compensatory kosmotropes and different water environments, Comp. Biochem.Physiol.A Integr.Physiol. 130, 471-486, 2001; Pilorz, K. and Choma, I., Isocratic reversed-phase high-performance liquid chromatographic separation of tetracyclines and flumequine controlled by a chaotropic effect, J.Chromatog.A. 1031, 303-306, 2004; Moelbert, S., Normand, B., and De Los Rios, P., Kosmotropes and chaotropes: modelling preferential exclusion, binding and aggregate stability, Biophys.Chem. 112, 45-57, 2004; Salvi, G., De Los Rios, P., and Vendruscolo, M., Effective interactions between chaotropic agents and proteins, Proteins 61, 492499, 2005; LoBrutto, R. and Kazakevich, Y.V., Chaotropic effects in RP-HPLC, Adv.Chromatog. 44, 291-315, 2006.
Chaperone
A measure of genetic distance that tells how far apart physically two genes are based on the frequency of recombination or crossover between the two gene loci. A frequency of 1% recombination in meiosis is one centimorgan and equals about 1 million base pairs. See Southern, E.M., Prospects for a complete molecular map of the human genome, Philos.Trans.R.Soc.Lond. B Biol.Sci. 319, 299-307, 1988; White, R., Lalauel, J.M., Leppert, M., Lathrop, M., Nakamura, Y., and O’Connell, P., Linkage maps of human chromosomes, Genome 31, 1066-1072, 1989; Smith, L.H., Jr., Overview of hemochromatosis, West.J.Med. 153, 296-308, 1990; Crabbe, J.C., Alcohol and genetics: new models, Am.J.Med.Genet. 114, 969-974, 2002.
An intracellular factor, most frequently a protein, which guides the intracellular folding/assembly of another protein. Examples include heat shock proteins, chaperoinins, See Gregerson, N.. Bolund, L., and Bross, P., Protein misfolding, aggregation, and degradation in disease, Mol.Biotechnol. 31, 141-150, 2005; Anken, E., Braakman, I. and Craig, E., Versatility of the endoplasmic reticulum protein folding factory, Crit.Rev.Biochem.Mol.Biol. 40, 191-288, 2005; Macario, A.J., and Conway de Marcario, E., Sick chaperones, cellular stress, and disease, New Eng.J.Med. 353, 1489-1501, 2005; Weibezahn, J., Schlieker, C., Tessarz, P., Mogk, A., and Bukau, B., Novel insights into the mechanism of chaperone-assisted protein disaggregation, Biol.Chem. 386, 739-744, 2006.
Chameleon Sequences
Chemical Biology
Identical sequences in a protein which can adopt either an alpha helical conformation or a beta sheet conformation: see Minor, D.L., Jr. and Kim, P.S., Context-dependent secondary structure formation of a designed peptide sequence, Nature 380, 730-734,
The application of chemical techniques to problems in biology – the emphasis is directed toward study of the interaction of small molecules with proteins and other macromolecules. See Li, C.H., Current concepts on the chemical biology of pituitary hormones,
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Chemical Proteomics
982
Perspect.Biol.Med. 11, 498-521, 1968; Malmstrom, B.G. and Leckner, J., The chemical biology of copper, Curr.Opin.Chem. Biol. 2, 286-292, 1998; Bertini, I. and Luchinat, C., New applications of paramagnetic NMR in chemical biology, Curr.Opin. Chem.Biol. 3, 145-151, 1999; Volkert, M., Wagner, M., Peters, C. and Waldmann, H., The chemical biology of Ras lipidation, Biol.Chem. 382, 1133-1145, 2001; Hahn, M.E. and Muir, T.W., Manipulating proteins with chemistry: a cross-section of chemical biology, Trends Biochem.Sci. 30, 26-34, 2005; Cambell-Valois, F.X., and Michnick, S., Chemical biology on PINs and NeeDLes, Curr.Opin.Chem.Biol. 9, 31-37, 2005; Doudna, J.A., Chemical biology at the crossroads of molecular structure and mechanism, Nat.Chem.Biol. 1, 300-303, 2005.
Chemical Proteomics Use of chemical modification to identify enzymes in the proteome and to identify signalling pathways. See Jeffery, D.A. and Bogyo, M., Chemical proteomics and its application to drug discovery, Curr.Opin.Biotechnol. 14, 87-95, 2003; Daub, H., Godl, K., Brehmer, D., et al., Evaluation of kinase inhibitor selectivity by chemical proteomics, Assay Drug Dev.Technol. 2, 215-224, 2004; Piggott, A.M. and Karuso, P., Quality, not quantity: the role of natural products and chemical proteomics in modern drug discovery, Comb.Chem.High Throughput Screen. 7, 607-630, 2004; Beillard, E and Witte, O.N., Unraveling kinase signaling pathways with chemical genetic and chemical proteomic approaches, Cell Cycle 4, 434-437, 2005; Sem, D.S., Chemical proteomics from a nuclear magnetic resonance spectroscopy perspective, Expert Rev. Proteomics 1, 165-178, 2004; Daub, H., Characterization of kinase-selective inhibitors by chemical proteomics, Biochim. Biophys.Acta 1754, 183-190, 2005; Verdoes, M., Berkers, C.R., Florea, B.I., et al., Chemical proteomics profiling of proteosome activity, Methods Mol.Biol. 328, 51-69, 2006.
Chemokines A large family of cytokines having a wide variety of biological actions but are generally associated with inducing mobilization and activation of immune cells; a contraction of chemotactic cytokines. See Horuk, R., Chemokine Receptors, Academic Press, San Diego, CA, USA, 1997; Vaddi, K., Keller, M., and Newton, R.C., The Chemokine Factbook, Academic Press, San Diego, CA, USA, 1997; Hebert, C., Chemokines in Disease: Biology and Clinical Research, Humana Press, Totowa, NJ, USA, 1999; Proudfoot, A.E.I. and Well, T.N.C., Chemokine Protocols, Humana Press, Totowa, NJ, USA, 2000; Schwiebert, L.M., Chemokines, Chemokine Receptors, and Disease, Elsevier, Amsterdam, NL, 2005; Atkins, P.C. and Wasserman, S.I., Chemotactic mediators, Clin.Rev.Allergy 1, 385395, 1983; Hayashi, H., Honda, M., Shimokawa, Y., and Hirashima, M., Chemotactic factors associated with leukocyte emigration in immune tissue injury: their separation, characterization, and functional specificity, Int.Rev.Cytol. 89, 179-250, 1984; Bignold, L.P., Measurement of chemotaxis of polymorphonuclear leukocytes in vitro. The problems of the control of gradients of chemotactic factors, of the control of the cells and of the separation of chemotaxis from chemokinesis, J.Immunol.Methods 108, 1-18, 1988; Schwarz, M.K., and Wells, T.N.C., New therapeutics that modulate chemokine networks, Nat.Rev.Drug.Disc. 1, 342-358, 2002; White, F.A., Bhangoo, S.K., and Miller, R.J., Chemokines: integrators or pain and inflammation, Nat.Rev.Drug Discov. 4, 834-844, 2005; Steinke, J.W. and Borish, L., Cytokines and chemokines, J.Allergy Clin.Immunol. 117 (Suppl 2), S441-S445, 2006;
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Chromatin Remodeling
Charo, I.F., and Ranosohoff, R.M., The many roles of chemokines and chemokine receptors in inflammation, N.Engl.J.Med. 354, 610-621, 2006; Laudanna, C. and Alon, R., Right on the spot. Chemokine triggering of integrin-mediated arrest of rolling leukocytes, Thromb.Haemostas. 95, 5-11, 2006.
Chemoproteomics The use of small molecules as affinity materials for the discovery of specific binding proteins in the proteome; the application of chemogenomics for proteomic research. See Beroza, P., Villar, H.O., Wick, M.M., and Martin, G.R., Chemoproteomics as a basis for post-genomic drug discovery, Drug Discov.Today 7, 807-814, 2002; Gagna, C.E., Winokur, D., Lambert, W.C., Cell biology, chemogenomics and chemoproteomics, Cell Biol.Int. 28, 755-764, 2004; Shin, D., Heo, Y.S., Lee, K.J., et al, Structural chemoproteomics and drug discovery, Biopolymers 80, 258-263, 2005; Hall, S.E., Chemoproteomics-driven drug discovery: addressing high attrition rates, Drug Discov.Today 11, 495-502, 2006.
Chondrocyte A cartilage cell. See von der Mark, K. and Conrad, G., Cartilage cell differentiation: review, Clin.Orthop.Relat.Res. (139), 195-205, 1979; Serni, U. and Mannoni, A., Chondrocyte physiopathology and drug efficacy, Drug Exp.Clin.Res. 17, 75-79, 1991; Urban, J.P., The chondrocytes: a cell under pressure, Br.J.Rheumatol. 33, 901908, 1994; Yates, K.E., Shortkroff, S., and Reish, R.G., Wnt influence on chondrocyte differentiation and cartilage function, DNA Cell Biol. 24, 446-457, 2005; Wendt, D., Jakob, M., and Martin, I., Bioreactor-based engineering of osteochondral grafts: from model systems to tissue manufacturing, J.Biosci.Bioeng. 100, 489-494, 2005; Goldring, M.B., Tsduchmochi, K., and Ijiri, K., The control of chondrogenesis, J. Cell Biochem. 97, 33-44, 2006; Ruano-Ravina, A. and Diaz, M.J., Autologous chondrocytes implantation: a systematic review, Osteoarthritis Cartilage 14, 47-51, 2006; Toh, W.S., Yang, Z., Heng, B.C., and Cao, T., New perspectives in chondrogenic differentiation of stem cells for cartilage repair, ScientificWorldJournal 6, 361-364, 2006.
Chromatin Chromatin consists of a repeating fundamental nucleoprotein complex, the nucleosome; DNA wrapped around histones where the histones mediate the folding of DNA into chromatin. see Wolfe, A., Chromatin. Structure and Function, 3rd Ed., Academic Press, San Diego, CA, 1998; Woodcock, C.L., Chromatin architecture, Curr.Opin.Struct.Biol. 16, 213-220, 2006; Aligianni, S. and Varga-Weisz, P., Chromatin-remodelling factors and the maintenance of transcriptional states through DNA replication, Biochem.Soc.Symp. (73), 97-108, 2006; de la Serna, I.L., Ohkawa, Y., and Imbalzano, A.N., Chromatin remodelling in mammalian differentiation: lessons from ATP-dependent remodellers, Nat. Rev.Genet. 7, 461-473, 2006; Mersfelder, E.L., and Parthun, M.R., The tail beyond the tail: histone core domain modifications and the regulation of chromatin structure, Nucleic Acids Res. 34, 2653-2662, 2006.
Chromatin Remodeling The dynamic structural change in chromatin by nucleosome sliding or post-translational modifications (acetylation, methylation) of the histones. See Becker, P.B., The chromatin accessibility
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complex: chromatin dynamics through nucleosome sliding, Cold Spring Harb.Symp.Quant.Biol. 69, 281-287, 2004; Henikoff, S. and Ahmed, K., Assembly of variant histones into chromatin, Annu.Rev.Cell Dev.Biol. 21, 133-153, 2005; Dhananjayan, S.C., Ismail, A., and Nawaz, Z., Ubiquitin and control of transcription, Essays Biochem. 41, 69-80, 2005; Lucchesi, J.C., Kelly, W.G., and Panning, B., Chromatin remodeling in dosage compensation, Annu.Rev.Genet. 39, 615-651, 2005; Saha, A., Wittmeyer, J.. and Cairns, B.R., Chromatin remodelling: the industrial revolution of DNA around histones, Nat.Rev.Mol.Cell Biol. 7, 437-447, 2006.
Chromatography The physical separation of two or more components of a solution mixture based on the distribution of said individual components between a stationary phase and a mobile phase. Chromatography can occur within a enclosed column or tube (column chromatography, gas chromatography being a variant of column chromatography with a gaseous mobile phase) or a planar surface as in paper chromatography or thin-layer chromatography. A chromatogram is (usually) a graphical representation of specific solute concentration at a given moment either in time or elution volume. In the case of planar chromatography, the term chromatography can refer to the actual paper or layer on which separation has occurred. The stationary phase may be a solid, gel, or liquid adsorbed onto a solid matrix. The mobile phase may be liquid or gaseous in nature. See Ettre, L.S., Nomenclature for chromatography, Pure Appl.Chem. 65, 819-872, 1993; Lederer, E. and Lederer, M., Chromatography: A Review of Principles and Applications, Elsevier, Amsterdam, 1957; Bobbit, J.M., ThinLayer Chromatography, Reinhold, New York, NY, USA, 1963; Zweig, G. and Sherma, J., CRC Handbook of Chromatography, CRC Press, Cleveland, OH, USA, 1972; Snyder, L.R., Kirkland, J.J., and Glajch, J.L., Practical HPLC- Method Development, 2nd edn., John Wiley & Sons, Inc., New York, NY, USA, 1997; Miller, J.M., Chromatography: Concepts and Contrasts, Wiley, New York, NY, USA, 2005; Wall, P.E., Thin-Layer Chromatography: A Modern Practical Approach, Royal Society of Chemistry, Cambridge, UK, 2005; Cazes, J., Encyclopedia of Chromatography, Taylor & Francis, Boca Raton, FL, USA, 2005; Perssen, P., Gustavsson, P.-E., Zacchi, G., and Nilsson, B., Aspects of estimating parameter dependencies in a detailed chromatography model based on frontal experiments, Process Biochem. 41, 1812-1821, 2006; Alpert, A.J., Chromatography of difficult and water-soluble proteins with organic solvents, Adv.Chromatog. 44, 317-329, 2006; Lundanes, E. and Greibrokk, T., Temperature effects in liquid chromatography, Adv.Chromatog. 44, 45-77, 2006.
Circadian Used to describe an approximate 24 hour period; a phenomena has demonstrates a circadian variation if it occurs with a certain frequency within an approximate 24 hour period. See Mills, J.N., Human circadian rhythms, Physiol.Rev. 46, 128-171, 1966; Brady, J., How are insect circadian rhythms controlled?, Nature 223, 781-784, 1969; Menaker, M., Takahashi, J.S., and Eskin, A., The physiology of circadian pacemakers, Annu.Rev.Physiol. 40, 501-526, 1978; Soriano, V., The circadian rhythm embraces the variability that occurs within 24 hours, Int.J.Neurol. 15, 7-16, 1981; Gardner, M.J., Hubbard, K.E., Hatta, C.T., et al., How plants tell the time, Biochem.J. 397, 15-24, 2006; McClung, C.R., Plant circadian rhythms, Plant Cell 18, 792-803, 2006; Brunner, M. and Schafmeier, T., Transcriptional and post-transcriptional
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regulation of the circadian clock of cyanobacteria and Neurospora, Genes Dev. 20, 1061-1074, 2006; Hardin, P.E. and Yu, W., Circadian transcription: passing the HAT to CLOCK, Cell 125, 424-426, 2006; Lewy, A.J., Emens, J., Jackman, A., and Yuhas, K., Circadian uses of melatonin in humans, Chronobiol. Int. 23, 403-412, 2006; Rosato, E., Tauber, E., and Kyriacou, C.P., Molecular genetics of the fruit-fly circadian clock, Eur.J.Hum. Genet. 14, 729-738, 2006.
Cis-element; cis-locus; cis-factors A region or regions on a DNA molecule which affects activity of DNA sequences on its own DNA molecule; an intramolecular effect; usually but not always does not code for the expression of protein; A cis-element or regulatory region can be complex and may contain several regulatory sequences. See Gluzman, Y., Eukaryotic Transcription: The Role of Cis- and Trans-Acting Elements in Initiation, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, USA, 1985; Hames, B.D. and Higgins, S.J., Gene Transcription: A Practical Approach, IRL Press at Oxford, Oxford, UK, 1993; Tanaka, N. and Taniguchi, T., Cytokine gene regulation: regulatory cis-elements and DNA binding factors involved in the interferon system, Adv.Immunol. 52, 263-281, 1992; Galson, D.L., Blanchard, K.L., Fandrey, J., Goldberg, M.A., and Bunn, H.F., Cis elements that regulate the erythropoietin gene, Ann.N.Y.Acad.Sci. 718, 21-30, 1994; Hapgood, J.P., Riedemann, J., and Scherer, S.D., Regulation of gene expression by GC-rich DNA cis-elements, Cell Biol. Int.25, 71-31, 2001; Tumpel, S., Maconochie, M., Wiedmann, L.M., and Krumlauf, R., Conservation and diversity in the cis-regulatory networks that integrate information controlling expression of Hoxa2 in hindbrain and cranial neural crest cells in vertebrates, Dev.Biol. 246, 45-56, 2002; Moolla, N., Kew, M., and Arbuthnot, P., Regulatory elements of hepatitis B virus transcription, J.Viral Hepat. 9, 323-331, 2002; Manna, P.R., Wang, X.J., and Stocco, D.M., Involvement of multiple transcription factors in the regulation of steroidogenic acute regulatory protein gene expression, Steroids 68, 1125-1134, 2003; Gambari, R., New trends in the development of transcription factor decoy (TFD) pharmacotherapy, Curr.Drug Targets 5,419-430, 2004; McBride, D.J. and Kleinjan, D.A.. Rounding up active cis-elements in the triple C corral: combining conservation, cleavage and conformation capture for the analysis of regulatory gene domains, Brief Funct. Genomic Proteomic 3, 267-279, 2004.
Circular Dichroism The differential absorption of plane polarized light passing through a solution and is expression as molar ellipticity [θ]m. See Greenfield, N.J., Analysis of circular dichroism data. Meth. Enzymol, 383, 282-317, 2004; Bayer, T.M., Booth, L.N., Knudsen, S.M., and Ellington, A.D., Arginine-rich motifs present multiple interfaces for specific binding by RNA, RNA 11, 1848-1857, 2005; Miles, A.J., and Wallace, B.A., Synchrotron radiation circular dichroism spectroscopy of proteins and applications in structural and functional genomics, Chem.Soc.Rev. 35, 39-51, 2006; Paramonov, S.E., Jun, H.W., and Hartgerink, J.D., Modulation of peptide-amphiphile nanofibers via phospholipid inclusions, Biomacromolecules 7, 24-26, 2006; Harrington, A., Darboe, N., Kenjale, R., et al., Characterization of the interaction of single tryptophan containing mutants of IpaC from Shingella flexneri with phospholipid membranes, Biochemistry 45, 626-636, 2006.
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Claisen Condensation
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Coefficient of Linear Thermal Expansion (Clte)
Claisen Condensation
Clone
Base-catalyzed reaction of an ester with an a-carbon hydrogen with another ester (same or different) to yield a b-keto ester. A model for thiolase reactions. See Claisen, L and Lowman, O., Berichte 20, 651, 1887; Clark, J.D., O’Keefe, S.J., and Knowles, J.R., Malate synthase: proof of a stepwise Claisen condensation using the double-isotope fractionation test, Biochemistry 27, 5961-5971, 1988; Modia, Y. and Wierenga, R.K., A biosynthetic thiolase in complex with a reaction intermediate: the crystal structure provides new insight into the catalytic mechanism, Structure 7, 1279-1290, 1999; Watanabe, A. and Ebizuka, Y., Unprecedented mechanism of chain length determination in fungal aromatic polyketide synthases, Chem.Biol. 11, 1101-1106, 2004; Veyron-Churlet, R., Bigot, S., Guerrini, O. et al., The biosynthesis of mycolic acids in Mycobaceterium tuberculosis relies on multiple specialized elongation complexes interconnected by specific protein-protein interactions, J.Mol.Biol. 353, 847-858, 2005; von Wettstein-Knowles, P., Olsen, J.G., McGuire, K.A, and Henriksen, A., Fatty acid synthesis. Role of active site histidines and lysine in Cys-His-His-type beta-ketoacyl-acyl carrier protein synthases, FEBS J. 273, 695-710, 2006; Ryu, Y., Kim, K.J., Rosennser, C.A., and Scott, A., Decarboxylative Claisen condensation catalyzed by in vitro selected ribozymes, Chem.Commun. 7, 1439-1441, 2006.
A cell or organism descended from and genetically identical to a single common ancestor. Clone is also used to refer to a DNA sequence encoding a product or an entire gene sequence from an organism which is replicated by genetic engineering. Such material can be transferred to another organism for the expression of such cDNA or gene. See Cunningham, A.J., Antibody formation studied at the single-cell level, Prog.Allergy 17, 5-50, 1973; Hamer, D.H. and Thomas, C.A., Jr., Molecular cloning, Adv. Pathobiol. (6), 306-319, 1977; von Boehmer, H., Haas, W., Pohlit, H., Hengartner, H., and Nabholz, M., T cell clones: their use for the study of specificity, induction, and effector-function of T cells, Springer Semin.Immunopathol. 3, 23-37, 1980; Fung, J.J., Gleason, K., Ward. R., and Kohler, H., Maturation of B-cell clones, Prog. Clin.Biol.Res. 42, 203-214, 1980; Veitia, R.A., Stochasticity or the fatal ‘imperfection’ of cloning, J.Biosci. 30, 21-30, 2005; Kettman, J.R., from clones of cells to cloned genes and their proteinpaedia, Scand.J.Immunol. 62, Suppl 1, 119-122, 2005; Vats, A., Bielby, R.C., Tolley, N.S., Nerem, R., and Polak, J.M., Stem cells, Lancet 366, 592-602, 2005; Wells, D.N., Animal cloning: problems and prospects, Rev.Sci.Tech. 24, 251-264, 2005; Diep, B.A., Gill, S.R., Chang, R.F., et al., Complete genome sequence of USA300, an epidemic clone of community-acquired methicillin-resistant Staphylococcus aureus, Lancet 367, 731-739, 2006.
Class Switch Recombination A process by which one constant region gene segment is switched with another gene segment during B-cell development when immunoglobulin production changes from IgM to IgA, IgE, or IgG. See Davis, M.M., Kim, S.K., and Hood, L.E., DNA sequences mediating class switching in alpha-immunoglobulin, Science 209, 1360-1365, 1980; Geha, R.S., Jabara, H.H., and Brodeur, S.R., The regulation of immunoglobulin E class-switch recombination, Nat.Rev.Immunol. 3, 721-732, 2003; Yu, K. and Lieber, M.R., Nucleic acid structures and enzymes in the immunoglobulin class switch recombination mechanism, DNA Repair 2, 1163-1174, 2003; Diamant, E. and Melamed, D., Class switch recombination in B lymphopoiesis: a potential pathway for B cell autoimmunity, Autoimmun.Rev. 3, 464-469, 2004; Min, I.M. and Selsing, E., Antibody class switch recombination: roles for switch sequence and mismatch repair proteins, Adv.Immunol. 87, 297-328, 2005.
Classical Proteomics Proteomic analysis based on the direct analysis of the expressed proteome such an extract obtained from lysis of a cell; also referred to as forward proteomics as compared to reverse proteomics. More generally, classically proteomics is taken to mean protein separation followed by characterization. See Klade, C.S., Proteomics approaches toward antigen discovery and vaccine development, Curr.Opin.Mol.Ther. 4, 216-223, 2002; Vondriska, T.M. and Ping, P., Functional proteomics to study protection of the ischaemic myocardium, Expert Opin.Ther.Targets 6, 563570, 2002; Thiede, B. and Rudel, T., Proteome analysis of apoptotic cells, Mass Spectrom.Rev. 23, 333-349, 2004; Gottlieb, D.M., Schultz, J., Bruun, S.W., et al., Multivariate approaches in plant science, Phytochemistry 65, 1531-1548, 2004.
Clinomics Application of oncogenomics to cancer care. See Workman, P. and Clarke, P.A., Innovative cancer drug targets: genomics, transcriptomics, and clinomics, Expert Opin.Pharmacother. 2, 911915, 2001.
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Clonal Selection Literally, the selection of a clone. Most often used to describe the process by which a B-cell is challenged by a specific antigen to produce a committed plasma cell or the differentiation of T-cells. More generally, the selection a stem cell to become committed to a specific antigen. See Williamson, A.R., The biological origin of antibody diversity, Annu.Rev.Biochem. 45, 467-500, 1976; D’Eustachio, P., Rutishauser, U.S., and Edelman, G.M., Clonal selection and the ontogeny of the immune response, Int.Rev.Cytol. Suppl. (5), 1-60, 1977; Coutinho, A., Beyond clonal selection and network, Immunol.Rev. 110, 63-87, 1989; Cohen, I.R., Antigenic mimicry, clonal selection and autoimmunity, J.Autoimmun. 16, 337-340, 2001; Defrance, T., Casamayor-Palleja, M., and Krammer, P.H., The life and death of a B cell, Adv.Cancer Res. 86, 195-225, 2002; van Boehmer, H., Aifantis, I., Gounari, F., et al., Thymic selection revisited: how essential is it?, Immunol. Rev. 191, 62-78, 2003, McHeyzer-Williams, L.J. and McHeyzerWilliams, M.G., Antigen-specific memory B cell development, Annu.Rev.Immunol. 23, 487-513, 2005; Bock, K.W. and Kohle, C., Ah receptor- and TCDD-mediated liver tumor promotion: clonal selection and expansion of cells evading growth arrest and apoptosis, Biochem.Pharmacol. 69, 1403-1408, 2005; Burnet, F.M., The Clonal Selection Theory of Acquired Immunity, Vanderbilt University Press, Nashville, TN, USA, 1959; Mazumdar, P.M.H., Immunology 1930-1980: Essays on the History of Immunology, Wall & Thompson, Toronto, Canada, 1989; Podolsky, S.H. and Tauber, A.I., The Generation of Diversity: Clonal Selection Theory and ther Rise of Molecular Immunology, Harvard University Press, Cambridge, MA, USA, 1997.
Coefficient of Linear Thermal Expansion (Clte) Ration of the change in length per degree C to length at O°C. The coefficient of linear thermal expansion (CTLE) is used to described the changes in the structure of proteins and other
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polymers as a function of temperature; the CTLE has also been used to describe thermal changes in micelles. See Frauenfelder, H., Hartmann, H., Karplus, M., et al., Thermal expansion of a protein, Biochemistry 26, 254-261, 1987; Schulenberg, P.J., Rohr, M., Gartner, W., and Braslavsky, S.E., Photoinduced volume changes associated with the early transformations of bacteriorhodopsin: a laser-induced optoacoustic spectroscopy study, Biophys.J. 66, 838-843, 1994; Marsh, D., Intrinsic curvature in normal and inverted lipid structures and I membranes, Biophys.J. 70, 22482255, 1996; Daniels, B.V., Schoenborn, B.P., and Korszun, Z.R., A low-resolution low-temperature neutron diffraction study of myoglobin, Acta Crystallogr.D.Biol. Crystallogr. 53, 544-550, 1997; Cordier, F. and Grzesiek, S., Temperature-dependence of protein hydrogen bond properties as studied by high-resolution NMR, J.Mol.Biol. 317, 739-752, 2002; Pereira, F.R., Machado, J.C., and Foster, F.S., Ultrasound characterization of coronary artery wall in vitro using temperature-dependent wave speed, IEEE Trans Ultrason. Ferroelectr.Freq.Control 50, 1474-1485, 2003; Bhardwaj, R., Mohanty, A.K., Drzal, L.T., et al., Renewable resource-based composities from recycled cellulose fiber and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) bioplastic, Biomacromolecules 7, 2044-2051, 2006.
Cold-chain product A product or reagent which must be kept cold during transit and storage; most often between 4° and 8°C. See Elliott, M.A. and Halbert, G.W., Maintaining the cold chain shipping envioronment for phase I clinical trial distribution, Int.J.Pharm. 299, 49-54, 2005; Streatfield, S.J., Mucosal immunization using recombinant plant-based oral vaccines, Methods 38, 150-157, 2005.
Cold Shock Protein A group of proteins which are synthesized by plant cells, prokaryotic and eukaryotic cells in response to cold stress. It has been suggested that cold shock proteins (CSPs) function as “chaperones” for mRNA. Graumann, P.L. and Marshiel, M.A., A superfamily of proteins that contain the cold-shock domain, Trends.Biochem.Sci. 23, 286-290, 1998; Phadtare, S., Alsina, J., and Inouye, M., Cold-shock response and cold-shock proteins, Curr.Opin.Microbiol. 2, 175-180, 1999; Sommerville, J., Activities of cold-shock domain proteins in translational control, Bioessays 21, 319-325, 1999; Graumann, P.L. and Marahiel, M.A., Cold shock response in Bacillus subtilis, J.Mol.Microbiol. Biotechnol. 1, 203-209, 1999; Loa, D.A. and Murata, N., Responses to cold shock in cyanobacteria, J.Mol.Microbiol. Biotechnol. 1, 221-230, 1999; Ermolenko, D.N. and Makhatadze, G.I., Bacterial coldshock proteins, Cell.Mol.Life.Sci. 59, 1902-1913, 2002; Alfageeh, M.B., Marchant, R.J., Carden, M.J., and Smales, C.M., The cold-shock response in cultured mammalian cells: harnessing the response for the improvement of recombinant protein production, Biotechnol.Bioeng. 93, 829-835, 2006; Al-Fageeh, M.B. and Smales, C.M., Control and regulation of the cellular response to cold shock: the responses in yeast and mammalian systems, Biochem.J. 397, 247-259, 2006; Fraser, K.r., Tuite, N.L., Bhagwat, A., and O’byrne, C.P., Global effects of homocysteine on transcription in Escherichia coli; induction of the gene for the major cold-shock protein, CspA, Microbiology 152, 221-2231, 2006; Magg, C., Kubelka, J., Holtermann, G., et al., Specificity of the initial collapsein the folding of the cold shock protein, J.Mol. Biol. 360, 1067-1080, 2006; Sauvageot, N., Beaufils, S., Maze, A., Cloning and characterization of a gene encoding a cold-shock
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Complement
protein in Lactobacillus casei, FEMS Microbiol.Lett. 254, 55-62, 2006; Narberhaus, F., Waldminghous, T., and Chowdhury, S., RNA thermometers, FEMS Microbiol.Lett. 30, 3-16, 2006.
Colloid The term colloid refers to a particle with dimensions between 1 nm and 1 μm although it is not necessary for all three dimensions to be in this size range. For example, a thin fiber might only have two dimensions in this size range. A colloidal dispersion is a system where colloid particles are dispersed in a continuous phase of a different composition such as a suspension (particles in a liquid), a emulsion (colloids of one liquid are suspended in another liquid where the two liquids are immiscible such as oil and water), a foam (gas dispersed in a liquid or gel), or an aerosol (a colloid in a gas such as air; a fog is a liquid colloid dispersed in a gas). See Tolson, N.D., Boothroyd, B., and Hopkins, C.R., Cell surface labelling with gold colloid particulates: the use of aviden and staphylococcal protein A-coated gold in conjunction with biotin and fc-bearing ligands, J.Microsc. 123, 215-226, 1981; Rowe, A.J., Probing hydration and the stability of protein solution—a colloid science approach, Biophys.Chem. 93, 93-101, 2001; Bolhuis, P.G., Meijer, E.J., and Louis, A.A., Colloid-polymer mixtures in the protein limit, Phys.Rev.Lett. 90:068304, 2003; Zhang, Z and van Duijneveldt, J.S., Experimental phase diagram of a model colloidpolymer mixture in the protein limit, Langmuir 22, 63-66, 2006; Xu, L.C. and Logan, B.E., Adhesion forces between functionalized latex microspheres and protein-coated surfaces evaluated using colloid probe atomic force microscopy, Colloids Surf. B. Biointerfaces 48, 84-94, 2006.
Colloid Osmotic Strength/Colloid Osmotic Pressure Combination Electrode An ion-selective electrode and an external reference electrode combined into a single functional unit. A separate reference electrode is not required.
Combination Product A regulatory term used to describe a final drug product composed of, for example, two separate drugs, a drug and a biologic or a drug and a device. See Leyden, J.J., Hickman, J.G., Jarratt, M.T., et al., The efficacy and safety of a combination benzoyl peroxide/ clindemycin topical gel compared with benzoyl peroxide alone and a benzoyl peroxide/erythromycin combination product, J.Cutan.Med.Surg. 5, 37-42, 2001; Bays, H.E., Extended-release niacin/lovastatin: the first combination product for dyslipidemia, Expert Rev.Cardiovasc.Ther. 2, 485-501, 2004; anon, Definition of the primary mode of action of a combination product. Final rule, Fed.Regist. 70, 49848-49862, 2005.
Complement A combination or system of plasma/serum proteins which interact to form a membrane attack complex which results in the lysis of bacterial pathogens and other cell targets such as tumor cells. There are three pathways of complement activation; the classical pathway, the alternative pathway, and the MBLectin (mannosebinding lectin; a plasma protein) pathway. The classical pathway is activated by an antigen-antibody complex (free antibody does not activate complement) via the Fc domain of the antibody; there are other mechanisms for classical pathway activation
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which make minor contributions. The alternative pathway is activated by direct recognition of foreign materials in an antibodyindependent manner and is driven by the autocatalytic action of C3b. The alternative pathway is thought the oldest of the three pathways is phyllogenetic development. The MBlectin pathway is initiated by the interaction of the MBlectin with a bacterial cell surface polysaccharide. The activation of complement component C3 is common to all three pathways. It is noted that there similarities to the blood coagulation cascade. See Prodinger, W.M., Würznen, R., Erdei, A., and Dierich, M.P., Complement, in Fundamental Immunology, ed. W.E.Paul, Lippincott-Raven, Philadelphia, USA, Chapter 29, pps. 967-995, 1999; Activators and Inhibitors of Complement, ed. R.B. Sim, Kluwer Academic, Dordrecht, Netherlands, 1993; Complement in Health and Disease, 2nd edn., ed. K Whaley, M. Loos, and J. Weiler, Kluwer Academic, Dordrecht, Netherlands, 1993; The Complement System, 2nd edn., ed., K. Rother, G.O. Till, and G.M Hansch, Springer, Berlin, 1998; The Human Complement System in Health and Disease, ed. J.E. Volanakis and MM. Frank, Marcel Dekker, New York, 1998; Therapeutic Interventions in the Complement System, ed. J.D. Lambis and K.M., Holer, Humana, Totowa, New Jersey, 2000; Szebeni, J., The Complement System Novel Roles in Health and Disease, Kluwer Academic, Boston, 2004.
Complement Fixation Complement fixation refers to the binding of the first component of the complement pathway, C1, to an IgG- or IgM-antigen complex. The antigen is usually a cell surface protein. Free antibody does not fix complement. Productive binding of the antigenantibody complex (binding involves the Fc portion of the antibody and a minimum of two Fc domains is required; thus two intact antibody molecules) results in complement activation. An antibody that activates complements is describes as having fixed complement. Complement fixation has formed the basis for many serological tests but most have been replaced by ELISA assays for the diagnosis of infectious disease. See Juji, T., Saji, H., Sataki, M., and Tukinaga, K., Typing for human platelet alloantigens, Rev.Immunogenet. 1, 239-254, 1999; Pappagianus, D., Serological studies in coccidiomycosis, Semin.Respir.Infect. 16, 242-250, 2001; Nielsen, K., Diagnosis of brucellosis by serology, Vet.Microbiol. 90, 447-459, 20002; Al-Dahouk, S., Tomaso, H., Nackler, E. et al., Laboratory-based diagnosis of brucellosis - review of the literature. Part I. Techniques for direct detection and identification of Brucella sp., Clin.Labl. 49, 387-404, 2003; Taggart, E.W., Hill, H.R., Martins, T.B., and Litwin, C.M., Comparison of complement fixation with two enzyme-linked immunosorbent assays for the detection of antibodies to respiratory viral antigens, Amer.J.Clin.Path. 125, 460-466, 2006. Complement fixation is usually measured by the lysis of sensitized cells (e.g hemolysis of sensitized sheep red blood cells; CH50 assay), See Morgen, P.B., Complement, in Immunochemistry, ed. C.J. van Oss and M.C.H., van Regenmortel, Marcel Dekker, New York, 1994, Chapter 34, pp. 903-923, 1994. The concept of complement fixation is still discussed with respected to in vivo antigen-antibody reactions such as those seen with transplantation antigens and alloantibodies. See Feucht, H.E., Felber, E., Gokel, M.J., et al., Vascular deposition of complement-split products in kidney allografts with cellmediated rejection, Clin.Exp.Immunol. 86, 464-470, 1991; Feucht, H.E., Complement C4d in graft capillaries—the missing link in the recognition of humoral alloreactivity, Am.J.Transplant. 3, 646-652, 2003; Colvin, R.B. and Smith, R.N., Antibody-mediated organ-allograft rejection, Nat.Rev.Immunol. 5, 807-817, 2005;
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Conjugate Vaccine
Rickert, R.C., Regulation of B lymphocyte activation by complement C3 and the B cell coreceptor complex, Curr.Opin.Immunol. 17, 237-243, 2005.
Confocal Microscopy A fluorescent microscopy technique which uses a highly focused beam of light with suppression of fluorescence above and below the point of optimum focus. An image is obtained by oving the excitation beam and measurement aperature over the sample with point-by-point measurement. See Cherry, R.J., New Techniques of Optical Microscropy and Microspectroscopy, CRC Press, Boca Raton, FL, 1991; Stelzer, E.H., Wacker, I., and De Mey, J.R., Confocal fluorescence microscopy in modern cell biology, Sermin.Cell Biol. 2, 145-152, 1991; Stevens, J.K. and Mills, L.R., Three-Dimensional Confocal Microscopy: Volume Investigation of Biological Specimens, Academic Press, San Diego, CA, 1994; Smith, R.F., Microscopy and Photomicrography: A Working Manual, CRC Press, Boca Raton, FL, 1994; Pawley, J.B., Handbook of Biological Confocal Microscropy, Plenum Press, New York, NY, 1995; Fay, F.S., Optical methods in cell physiology, in Handbook of Physiology, Section 14, Cell Physiology, ed. J.F. Hoffman and J.D. Jamieson, Oxford University Press (for the American Physiological Society), New York, NY, 1997; Paddock, S.W., Confocal Microscopy Methods and Protocols, Humana Press, Totowa, NJ, 1999; Brelje, T.C. Wessendorf, M.W., and Sorenson, R.L., Multicolor laser scanning confocal immunofluorescence microscopy: practical application and limitations, Methods Cell Biol. 70, 165-244, 2002; Bacia, K. and Schwille, P., A dynamic view of cellular processes by in vivo fluorescence auto- and cross-correlation spectroscopy, Methods 29, 74-85, 2003; Miyashita, T., Confocal microscopy for intracellular co-localization of proteins, Methods Mol.Biol. 261, 399-410, 2004; Heilker, R., Zemanova, L., Valler, M.J., and Nienhaus, G.U., Confocal fluorescence microscopy for high-throughput screening of G-protein coupled receptors, Curr.Med.Chem. 12, 2551-2559, 2005; Becker, B.E. and Gard, D.L., Visualization of the cytoskeleton in Xenopus oocytes and eggs by confocal immunofluorescence microscopy, Methods Mol.Biol. 322, 69-86, 2006.
Conjugate Vaccine Coupling of a weak immunogen such as a polysaccharide to a protein to improve/enhance immunogenicity. See Cryz, S.J., Jr., Furer, E., Sadoff, J.C., et al., Use of Pseudomonas aeruginosa toxin A in the construction of conjugate vaccines and immunotoxins, Rev.Infect.Dis. 9(Suppl. 5), S644-S649, 1987; Garner, C.V. and Pier, G.B. Immunologic considerations for the development of conjugate vaccines, Contrib.Microbiol.Immunol. 10, 11-17, 1989; Dintzis, R.Z., Rational design of conjugate vaccines, Pediatr.Res. 32, 376-385, 1992; Ellis, R.W. and Douglas, R.G., Jr., New vaccine technologies, JAMA 272, 929-931, 1994; Lindberg, A.A. and Pillai, S., Recent trends in the developments of bacterial vaccines, Dev.Biol.Stand. 87, 59-71, 1996; Zimmer, S.M. and Stephens, D.S., Meningococcal conjugate vaccines, Expert Opin. Pharmacother. 5, 855-863, 2004; Finn, A., Bacterial polysaccharide-protein conjugate vaccines, Br.Med.Bull. 70, 1-14, 2004; Shape, M.D. and Pollard, A.J., Meningococcal polysaccharideprotein conjugate vaccines, Lancet Infect.Dis. 5, 21-30, 2005; Finn, A. and Heath, P., Conjugate vaccines, Arch.Dis.Child. 90, 667-669, 2005; Jones, C., NMR assays for carbohydrate-based vaccines, J.Pharm.Biomed.Anal. 38, 840-850, 2005; Whitney, C.G, Impact of conjugate pneumococcal vaccines, Pediatr.Infect. Dis. 24, 729-730, 2005; Lee, C.J., Lee, L.H., and Gu, X.X., Mucosal
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Connexins
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immunity induced by pneumococcal glycoconjugate, Crit.Rev. Microbiol. 31, 137-144, 2005.
Connexins A protein subunit of connexon which form gap junctions critical for intercellular communication. Mutations in the connexins are responsible for a diversity of diseases, including deafness, skin disorders and idiopathic atrial fibrillation. Connexins have been designated by their molecular mass while another system separate connexins on the basis of sequence homology. See Beyer, E.C., Paul, D.L., and Goodenough, D.A., Connexin family of gap junction proteins, J.Membr.Biol. 116, 187-194, 1990; Revel, J.P., Nicholson, B.J., and Yancey, S.B., Chemistry of gap junctions, Annu.Rev.Physiol. 47, 263279, 1985; Revel, J.P., Yancey, S.B., Nicholson, B., and Hoh, J., Sequence diversity of gap junction proteins, Ciba Found.Symp. 125, 108-127, 1987; Gollob, M.H., Cardiac connexins as candidate genes for idiopathic atrial fibrillation, Curr.Opin.Cardiol. 21, 155-158, 2006; Stains, J.P. and Civitelli, R., Gap junctions in skeletal development and function, Biochim.Biophys.Acta 1719, 69-81, 2005; Anand, R.J., and Hackam, D.J., The role of gap junctions in health and disease, Crit.Care Med. 33 (Suppl 12), S535-S535, 2005; Michon, L., Nlend Nlend, R., Bavamian, S., et al., Involvement of gap junctional communication in secretion, Biochim.Biophys.Acta 1719, 82-101, 2005; Vinken, M., Vanhaecke, T., Papeleu, P., et al., Connexins and their channels in cell growth and cell death, Cell Signal. 18, 592-600, 2006; Petit, C., From deafness genes to hearing mechanisms: harmony and counterpoint, Trends Mol.Med. 12, 57-64, 2006; Evans, W.H., De Vuyst, E., and Leybaert, L., The gap junction cellular internet: connexin hemichannels enter the signalling limelight, Biochem.J. 397, 1-14, 2006.
Contig The term contig was originally defined as a set of overlapping DNA sequences and has been expanded to include a set of overlapping DNA clones. Specifically, it refers to a set of gel bands which can be related to each other by overlaps sequences – see http:// staden.sourceforge.net/contig.html See Staden, R., A new computer method for the storage any manipulation of DNA gel reading data, Nucleic Acids Res. 8, 3673-3694, 1980; Presting, G.G., Budiman, M.A., Wood, T., et al., A framework for sequencing the rice genome, Novartis Found.Symp. 236, 13-24, 2001; Dodgson, J.B., Chicken genome sequence: a centennial gift to poultry genetics, Cytogenet.Genome Res. 102, 291-296, 2003; Schalkwyk, L.C., Francis, F., and Lehrach, H., Techniques in mammalian genome mapping, Curr.Opin.Biotechnol. 6, 37-43, 1995; Carrano, A.V., de Jong, P.J., Branscomb, E., et al., Constructing chromosome- and region-specific cosmid maps fo the human genome, Genome 31, 1059-1065, 1989.
Contour Length End-to-end length of a stretched DNA molecule (see Wellauer, P., Weber, R., and Wyler, T., Electron microscopic study of the influence of the preparative conditions on contour length and structure of mitrochondrial DNA of mouse liver, J.Ultrastruct.Res. 42, 377393, 1973; Geller, K. and Reinert, K.E., Evidence for an increase of DNA contour length at low ionic strength, Nucleic Acids Res. 8, 2807-2822, 1980; Motejlek, K., Schindler, D., Assum, G., and Krone, W.. Increased amount and contour length distribution of
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Cosolvent
small polydisperse circular DNA (spcDNA) in Fanconi anemia, Mutat.Res. 293, 205-214, 1993; Gast, F.U. and Sanger, H.L., Gel dependence of electrophoretic mobilites of double-stranded and viroid RNA and estimation of the contour length of a viroid by gel electrophoresis, Electrophoresis 15, 1493-1498, 1994; SanchezSevilla, A., Thimonier, J., Marilley, M., et al., Accuracy of AFM measurements of the contour length of DNA fragments adsorbed on mica in air and in aqueous buffer, Ultramicroscopy 92, 151158, 2002) although the term has been used to describe very long proteins such as titan (Helmes, M., Trombitas, K., Centner, T., et al., Mechanically driven contour-length adjustment in rat cardiac titin’s unique N2B sequence: titin is an adjustable spring, Circ. Res. 84, 1339-1352, 1999).
Core Promoter A region immediately (+/–30 bp) around the transcription start site that contain consensus sequence elements (TATA boxes, lnr, DPEs); in vitro, the core promoter is the minimal required sequence that is recognized by general transcription factors that activate correct transcription by RNA polymerase II. See Gill, G., Transcriptional initiation, Curr.Biol. 4, 374-376, 1994; Gill, G., Regulation of the initiation of eukaryotic transcription, Essays Biochem. 37, 33-43,2001; Butler, J.E. and Kadonaga, J.T., The RNA polymerase II core promoter: a key component in the regulation of gene expression, Genes Dev. 16, 2583-2592, 2002; Kadnoaga, J.T., The DPE, a core promoter element for transcription by RNA polymerase II, Exp.Mol.Med. 34, 259-264, 2002; Smale, C.T. and Kadonaga, J.T., The RNA polymerase II core promoter, Annu.Rev. Biochem. 72, 449–479, 2003; Lewis, B.A. and Reinberg, D., The mediator coactivator complex: functional and physical roles in transcriptional regulation, J.Cell Sci. 116, 3667-3675, 2003; Mulle, F. and Tora, L., The multicolored world of promoter recognition complexes, EMBO J. 23, 2-8, 2004; Chen, K., Organization of MAO A and MAO B promoters and regulation of gene expression, Neurotoxicity 25, 31-36, 2004; Hasselbach, L., Haase, S., Fischer, D., Kolberg, H.C., and Sturzbecher, H.W., Characterization of the promoter region of the human DNA-repair gene Rad51, Eur.J.Gynecol. Oncol. 26, 589-598, 2005.
Cosolvent A miscible solvent added a primary solvent to enhance salvation or stability of a specific solute. Such solvents have been used extensively in studies on enzymes where cosolvents were required to dissolve the substrate. Cosolvents are also used in the formulation of pharmaceuticals and in liquid chromatography. See Tan, K.H. and Lovrien, R., Enzymology in aqueousorganic cosovlent binary mixtures, J.Biol.Chem. 247, 3278-3285, 1972; Richardson, N.E. and Meaekin, B.J., The influence of cosolvents and substrate substituents on the sorption of benzoic acid derivatives by polyamides, J.Pharm.Pharmcol. 27, 145-151, 1975; Pescheck, P.S. and Lovrien, R.E., Cosolvent control of substrate inhibition I cosolvent stimulation of beta-glucuronidase activity, Biochem.Biophys.Res.Commun. 79, 417-421, 1977; Bulone, D., Cupane, A. and Cordone, L, Conformational and functional properties of hemoglobin in water-organic cosolvent mixtures: effect of ethylene glycol and glycerol on oxygen affinity, Biopolymers 22, 119-123, 1983; Rubino, J.T. and Berryhill, W.S., Effects of solvent polarity on the acid dissociation constants of benzoic acids, J.Pharm.Sci. 75, 182-186, 1986; Buck, M., Trifluoroethanol and colleagues: cosolvents come of age. Recent studies with peptides and proteins, Q.Rev.Biophys. 31, 297-355,
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Coupled Enzyme Systems
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1998; Jouyban-Gharamaleki, A., Valaee, L., Barzegar-Jalali, M., et al., Comparison of various cosolvency models for calculating solute solubility in water-cosolvent mixtures, Int.J.Pharm. 177, 93-101, 1999; Lee, J.C., Biopharmaceutical formulation, Curr.Opin.Biotechnol. 11, 81-84, 2000; Moelbert, S., Normand, B, and de los Rios, P., Kosmotropes and chaotropes: modeling preferential exclusion, binding and aggregate stability, Biophys. Chem. 112, 45-57, 2004; Scharnagl, C., Reif, M., and Friedrich, J., Stability of proteins: temperature, pressure and the role of solvent, Biochim.Biophys.Acta. 1749, 17-213, 2005.
Coupled Enzyme Systems Most metabolic systems are composed of enzymes in a pathway where there is the sequential transformation of a substrate into a product through a series of separate enzyme-catalyzed reactions. One of the more simple coupled systems is the detoxification of ethyl alcohol (Plapp, B.V., Rate-limiting steps in ethanol metabolism and approaches to changing these rates biochemically, Adv.Expt.Biol.Med. 56, 77-109, 1975) or more complex (Brooks, S.P.J., Enzymes in the cell. What’s really going on? in Function and Metabolism, ed. K.B. Storey, WileyLiss, Hoboken, NJ, USA, Chapter 3, pp. 55-86, 2004). Coupled enzyme systems are also used extensively in clinical chemistry where they are also referred to an indicator enzyme systems (Russell, C.D. and Cotlove, E., Serum glutamic-oxaloacetic transaminase: evaluation of a coupled-reaction enzyme assay by means of kinetic theory, Clin.Chem. 17, 1114-1122, 1971; Bais, R. and Pateghini, M., Principles of clinical enzymology, in Tietz Textbook of Clinical Chemistry and Molecular Diagnostics, ed. C.A. Burtis, E.R. Ashwood, and D.E. Bruns, Elsevier/Saunders, St. Louis, MO, USA, Chapter 9, pp. 191-218, 2006). The assay for creatine kinase is a coupled enzyme system as are some of the assays for glucose oxidase. An enzyme assay system is coupled to an immunological reaction in many solid-phase immunoassays such as ELISA assays (Kircks, LJ., Selected strategies for improving sensitivity and reliability of immunoassays, Clin.Chem. 40, 347-357, 1994). See Wimmer, M.C., Artiss, J.D., and Zak, B., Peroxidase-coupled method for kinetic colorimetry of total creatine kinase activity in serum, Clin.Chem. 31, 1616-1620, 1965; Shin, T., Murao, S., and Matsumura, E., A chromogenic oxidative coupling reaction of laccase: applications for laccase and angiotensin I converting enzyme assay, Anal.Biochem. 166, 380-388, 1987.
Creatine A nitrogenous compound which is synthesized from arginine, glycine and S-adenosylmethionine (Van Pilsum J.F., Stephens, G.C., and Taylor, D., Distribution of creatine, guanidinoacetate and the enzymes for their biosynthesis in the animal kingdom, Biochem.J. 126, 325-345, 1972; Walker, J.B. and Hannan, J.K., Creatine biosynthesis during embryonic development. False feedback suppression of liver amidinotransferase by N-acetimdoylsarcosine and 1-carboxymethy-2-iminoimdazolidine (Cyclocreatine), Biochemistry 15, 2519-2522, 1976; Walker, J.B., Creatine: biosynthesis, regulation, and function, The Enzymes 50 (ed. A.Meister, Academic Press, New York, NY), 177-242, 1979; Wyss, M. and Wallimann, T., Creatine metabolism and the consequences of creatine depletion in muscle, Mol.Cell.Biochem. 133-134, 51-66, 1994; Wu, G. and Morris, S.M., Jr., Arginine metabolism: nitric oxide and beyond, Biochem.J. 336, 1-17, 1998; Brosnan, M.E. and Brosnan, J.T., Renal arginine metabolism, J.Nutr. 134(Suppl
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Creatinine
10), 2791S-2795S, 1994; Morris, S.M., Jr., Enzymes of arginine metabolism, J.Nutr.(Suppl. 10), 2743S-2747S, 1994). Creatine is used as a biomarker for erthyrocytes (Beyer, C. and Alting, I.H., Enzymatic measurement of creatine in erythrocytes, Clin. Chem. 42, 313-318, 1996; Jiao, Y., Okumiya, T., Saibara, T., et al., An enzymatic assay for erythrocyte creatine as an index of the erythrocyte life time, Clin.Biochem. 31, 59-65, 1998; Takemoto, Y., Okumiya, T., Tsuchida, K., et al., Erythrocyte creatine as an index of the erythrocyte life span and erythropoiesis, Nephron 86, 513-514, 2000; Okumiya, T., Ishikawa-Nishi, M., Doi, T., et al., Evaluation of intravascular hemolysis with erythrocyte creatine in patients with cardiac valve prostheses, Chest 125, 21152120, 2004). There is increased use of creatine as a nutritional supplement (Korzun, W.J., Oral creatine supplements lower plasma homocysteine concentrations in humans, Clin.Lab.Sci. 17, 102-106, 2004; Pearlman, J.P. and Fielding, R.A., Creatine monohydrate as a therapeutic aid in muscular dysthrophy, Nutr. Rev. 64, 80-88, 2006; Hespel, P., Maughan, R.J., and Greenhaff, P.L., Dietary supplements for football, J.Sports Sci. 24, 749-761, 2006; Shao, A. and Hathcock, J.N., Risk assessment for creatine monohydrate, Regul.Toxicol. Pharmacol., 45, 242–251, 2006.
Creatine Kinase Adenosine triphosphate: creatine N-phosphotransferase (EC 2.7.3.2) also creatine phosphokinase. Creatine kinase is found in muscle and is responsible for the formation of creatine phosphate from creatine and adenosine triphosphate; creatine phosphate is a higher energy source for muscle contraction. Creatine kinase is elevated in all forms of muscular dystrophy. Creatine kinase is dimer and is present as isozymes (CK-1, BB; CK-2, MB; CK-3, MM) and Ck-mt(mitochondrial). Creatine kinase is also used for measure cardiac muscle damage in myocardial infarction. See Bais, R. and Edwards, J.B., Creatine kinase, CRC Crit. Rev.Clin. Lab.Sci. 16, 291-355, 1982; McLeish, M.J. and Kenyon, G.L., Relating structure to mechanism in creatine kinase, Crit. Rev.Biochem.Mol.Biol. 40, 1-20, 2005.
Creatinine A catabolic product of creatine which should be in blood as a constant quantity. An increase in urinary creatinine is associated with a loss of kidney function. See Hodgkinison, A. and Edwards, N.A., Laboratory determinations of renal function, Biochem.Clin. 2, 77-86, 1963; Blainey, J.D., The renal excretion of higher molecular weight substances, Curr.Probl.Clin.Biochem. 2, 85-100, 1968; Cook, J.G., Factors influencing the assay of creatinine, Ann.Clin. Biochem. 12, 219-232, 1975; Greenberg, N., Smith, T.A., and VanBrunt, N., Interference in the Vitros CREA method when measuring urine creatinine on samples acidified with acetic acid, Clin.Chem. 50, 1273-1275, 2004. Price, C.P., Newall, R.G., and Boyd, J.C., Prediction of significant proteinuria: a systematic review, Clin.Chem. 51, 1577-1586, 2005; Verhoeven, N.M., Salmons, G.S., and Jakobs, C., Laboratory diagnosis of defects of creatine biosynthesis and transport, Clin.Chim.Acta 361, 1-9, 2005; Wishart, D.S., Metabolomics: the principles and potential applications to transplantation, Am.J.Transplant. 5, 2814-2820, 2005; Seron, D., Fulladosa, X., and Moreso, F., Risk factors associated with the deterioration of renal function after kidney transplantation, Kidney Int.Suppl. 99, S113-S117, 2005; Schrier, R.W., Role of diminished renal function in cardiovascular mortality: marker or pathogenic factor? J.Am. Coll.Cardiol. 47, 1-8, 2006.
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Critical Pressure
989
Critical Pressure The minimum pressure required to condense gas to liquid at the critical temperature.
Critical Temperature The critical point (end of a vapor pressure curve in a phase diagram); above this temperature, a gas cannot be liquefied.
Crowding A term used to described general effect of polymers including proteins and polysacchardies on the solution properties of proteins. See Zimmerman, S.B., Macromolecular crowding effects on macromolecular interactions: some implications for genome structure and function, Biochim.Biophys.Acta 1216, 175-185, 1993; Minton, A.P., Molecular crowding: analysis of effects of high concentrations of inert cosolutes on biochemical equilibria and rates in terms of volume exclusion, Methods Enzymol. 295, 127-149, 1998; Johansson, H.O., Brooks, D.E., and Haynes, C.A., Macromolecular crowding and its consequences, Int.Rev.Cytol. 192, 155-170, 2000; Ellis, R.J., Macromolecular crowding: obvious but underappreciated, Trends Biochem.Sci. 26, 597-604, 2001; Bernardo, P., Garcia de la Torre, J., and Pons, M., Macromolecular crowding in biological systems: hydrodynamic and NMR methods, J.Mol.Recognit. 17, 397-407, 2004; Martin, J., Chaperon function – effects of crowding and confinement, J.Mol.Recognit. 17, 465-472, 2004; Minton, A.P., Influence of macromolecular crowding upon the stability and state of association of proteins: predictions and observations, J.Pharm.Sci. 94, 1668-1675, 2005; del Alamo, M., Rivas, G., and Mateu, M.G., Effect of macromolecular crowding agents on human immunodeficiency virus type 1 capsid protein assembly in vitro, J.Virol. 79, 14271-14281, 2005; Despa, F., Orgill, D.P., and Lee, R.C., Molecular crowding effects on protein stability, Ann.N.Y.Acad.Sci. 1066, 54-66, 2006; Szymanski, J., Patkowski, A., Gapinski, J. et al., Movement of proteins in an environment crowded by surfactant micelles: anomalous versus normal diffusion, J.Phys.Chem.B.Condens.Matter Mater.Surf.Interfaces Biophys. 110, 7367-7373, 2006; Derham, B.K. and Harding, J.J., The effect of the presence of globular proteins and elongated polymers on enzyme activity, Biochim. Biophys.Acta 1764, 1000-1006, 2006; Grailhe, R., Merola, F., Ridard, J. et al., Monitoring protein interactions in the living cell through the fluorescence decays of the cyan fluorescent protein, Chemphyschem. 7, 1442-1454, 2006. McPhie, P., Ni, Y.S., and Minton, A.P., Macromolecular crowding stabilizes the molten globule form of apomyoglobin with respect to both cold and heat unfolding, J.Mol.Biol. 361, 7-10, 2006.
Crown Gall Disease/Crown Gall Tumors Crown Gall is caused by a bacteria (Agrobacterium tumefaciens). These galls begin with tumor-like cell growth at or just below the soil’s surface, near the base of the plant and commonly on bud unions. Galls usually begin as green, pliable tissue; then develop into dark, crusty growths. Crown Gall Disease has been used to study transformation with relevance to tumor formation. See Knoft, U.C., Crown-gall and Agrobacterium tumefaciens: survey of a plant-cell-transformation system of interest to medicine and agriculture, Subcell.Biochem. 6, 143-173, 1978; Zhu, J., Oger, P.M., Schrammeijer, B. et al., The bases of crown gall tumorigenesis, J.Bacteriol. 182, 3885-3895, 2000; Escobar, M.A., and
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Cyanine Dyes (CyDyes)
Dadekar, A.M., Agrobacterium tumefaciens as an agent of disease, Trends Plant.Sci. 8, 380-386, 2003; Brencic, A. and Winans, S.C., Detection of and response to signals involved in host-microbe interactions by plant-associated bacteria. Micobiol.Mol.Biol.Rev. 69, 155-194, 2005;
Cryosection A tissue section cut from a frozen specimen; in this situation, ice i)s the supporting matrix. See Yamada, E. and Watanabe, H., High voltage electron microscopy of critical-point dried cryosection, J.Electron Microsc. 26(Suppl), 339-342, 1977; Maddox, P.H., Tay, S.K., and Jenkins, D., A new fixed cryosection technique for the simultaneous immunocytochemical demonstration of T6 and S100 antigens, Histochem.J. 19, 35-38, 1987; Sod, E.W., Crooker, A.R. and Morrison, G.H., Biological cryosection preparation and practical ion yield evaluation for ion microscopic analysis, J.Microsc. 160, 55-65, 1990; Lewis Carl, S.A., Gillete-Ferguson, I., and Ferguson, D.G., An indirect immunofluorescence procedure for staining the same cryosection with two mouse monoclonal primary antibodies, J.Histochem.Cytochem. 41, 1273-1278, 1993; Jensen, H.L. and Norrild, B., Easy and reliable double-immunogold labelling of herpes simplex virus type-1 infected cells using primary antibodies and studied by cryosection electron microscopy, Histochem.J 31, 525-533, 1999; Gou, D. and Catchpoole, D.R., Isolation of intact RNA following cryosection of archived frozen tissue, Biotechniques 34, 48-50, 2003; Rieppo, J., Hyttinen, M.M. Jurvelin, J.S., and Helminen, H.J., Reference sample method reduces the error caused by variable cryosection thickness in Fourier transform infrared imaging, Appl.Spectrosc. 58, 137-140, 2004; Takizawa, T. and Robinson, J.M., Thin is better! Ultrathin cryosection immunocytochemistry, J.Nippon Med.Sch. 71, 306307, 2004.
Cyanine Dyes (CyDyes) A family of fluorescent polymethine dyes contain containing a -CH= group linking two nitrogen-containing heterocyclic rings; developed as sensitizer for photographic emulsions. Used in biochemistry and molecular biology on nucleic acid probes for DNA microarrays and for labeling proteins for electrophoretic analysis. See Ernst, L.A., Gupta, R.K., Mujumdar, R.B., and Waggoner, A.S., Cyanine dye labeling reagents for sulfydryl groups, Cytometry 10, 3-10, 1989; Mujumdar, P.S., Ernst, L.A., Mujumdar, S.R., and Waggoner, A.S., Cyanine dye labeling reagents containing isothiocyanate groups, Cytometry 10, 11-19, 1989; Southwick, P.L., Ernst, L.A., Tauriello, E.W., et al., Cyanine dye labeling reagents— carboxymethylindocyanine succinimidyl esters, Cytometry 11, 418-430, 1990; Mujamdar, R.B., Ernst, L.A., Mujumdar, S.R., et al., Cyanine dye labeling reagents: sulfoindocyanine succinimidyl esters, Bioconjug.Chem. 4, 105-111, 1993; Benchaib, M., Delorme, R., Pluvinage, M., et al., Evaluation of five green fluorescence-emitting streptavidin-conjugated fluorochromes for use in immunofluorescence microscopy, Histochem.Cell Biol. 106, 253-256, 1996; Mujumdar, S.R., Mujumdar, R.B., Grant, C.M., and Waggoner, A.S., Cyanine-labeling reagents: sulfobenzindocyanine succinimidyl esters, Bioconjug.Chem. 7, 356-362, 1996; Karp, N.A. and Lilley, K.S., Maximizing sensitivity for detecting changes in protein expression: experimental design using minimal CyDyes, Proteomics 5, 3105-3115, 2005; Heilmann, M., Margeat, E., Kasper, R., et al., Carbocyanine dyes as efficient reversible single-molecule optical switch, J.Am.Chem.Soc. 127, 3801-3806, 2005; Wu, T.L., Two-dimensional difference gel electrophoresis, Methods Mol.Biol.
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Cyclitols
990
328, 71-95, 2006; Boisseau, S., Mabrouk, K., Ram, N., et al., Cell penetration properties of maurocalcine, a natural venom peptide active on the intracellular ryanodine receptor, Biochim.Biophys. Acta. 1758, 308-319, 2006. There is also use of these dyes for the measurement of membrane potentials. See Miller, J.B. and Koshland, D.E., Effects of cyanine dye membrane probes on cellular properties, Nature 272, 83-84, 1978; Klausner, R.D. and Wolf, D.E., Selectivity of fluorescent lipid analogues for lipid domains, Biochemistry 19, 6199-6203, 1980; Kragh-Hansen, U., Jorgensen, K.E., and Sheikh, M.I., The use of potential-sensitive cyanine dye for studying ion-dependent electrogenic renal transport of organic solutes. Spectrophotometric measurements, Biochem.J. 208, 359368, 1982; Johnstone, R.M., Laris, P.C. and Eddy, A.A., The use of fluorescent dyes to measure membrane potentials: a critique, J.Cell Physiol. 112, 298-300, 1982; Toyomizu, M., Okamoto, K., Akiba, Y. et al,, Anacardic acid-mediated changes in membrane potential and pH gradient across liposomal membranes, Biochim.Biophys. Acta 1558, 54-62, 2002.
Cyclitols Term used to describe derivatives of hexahydroxyhexane (1,2,3,4,5,6-hexahydroxyhexane). An analogue to saccharides and serves a matrix for the development of inhibitors and activators based on saccharide structure. See Tentative rules for cyclitol nomenclature, Biochim.Biophys.Acta 165, 1-21, 1968; Orthen, B. and Popp, M., Cyclitols as cryoprotectants for spinach and chickpea thylakoids, Environ.Exp.Bot. 44, 125-132, 2000; Pelyvas, I.F., Toth, Z.G., Vereb, G., et al., Synthesis of new cyclitol compounds that influence the activity of phosphatidylinositol 4-kinase isoforms, PI4K230, J.Med.Chem. 44, 627-632, 2001; Sureshan, K.M., Shashidhar, M.S. and Varma, A.J., Cyclitol-based metal-complexing agents. Effect of the relative orientation of oxygen atoms in the ionophoric ring on the cation-binding ability of myo-inositolbased crown ethers, J.Org.Chem. 67, 6884-6888, 2002; Freeman, C., Liu, L., Banwell, M.G., et al., Use of sulfated linked cyclitols as heparin sulfate mimetics to probe the heparin/heparin sulfate binding specificity of proteins, J.Biol.Chem. 280, 8842-8849, 2005; Cochran, S., Li, C.P., and Bytheway, I., An experimental and molecular-modeling study of the binding of linked sulfated tetracyclitols to FGF-1 and FGF-2, ChemBioChem 6, 1882-1890, 2005.
Cytochrome P-450 Enzymes (CPY) A family of enzymes which have monooxygenase activity and are involved in the metabolism/catabolism of drugs. Cytochrome P450 proteins are found in high concentration in the liver. See Jung, C., Schunemann, V., and Lendzian, F. Freeze-quenched iron-oxo intermediate in cytochrome P450, Biochem.Biophys. Res.Commun. 338, 355-364, 2005; Johnson, E.F. and Stout, C.D., Structural diversity of human xenobiotic-metabolizing cytochrome P450, Biochem.Biophys.Res.Commun. 338, 331-336, 2005; Tang, W., Wang, R.W., and Lu, A.Y., Utility of recombinant cytochrome p450 enzymes: a drug metabolism perspective, Curr. Drug Metab. 6, 503-517, 2005; Krishna, D.R. and Shekar, M.S., Cytochrome P450 3A: genetic polymorphisms and inter-ethnic differences, Methods Find.Exp.Clin.Pharmacol. 27, 559-567, 2005; Sarlis, N.J. and Gourgiotis, L, Hormonal effects on drug metabolism through the CYP system: perspectives on their potential significance in the era of pharmacogenomics, Curr.Drug Targets Immune Endocr.Metabol.Disord. 5, 439-448, 2005; Grengerich, E.P., Cytochrome P405 enzymes in the generation of commercial products, Nat.Rev.Drug.Disc. 1, 359-366, 2002.
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Cytokines
Cytokeratin Cytokeratin describes intermediate filament keratins found in epithelial tissue. There are two types of cytokeratins the acidic type I cytokeratins and the basic or neutral type II cytokeratins. Cytokeratins are thought to play role in the activation of plasma prekallikrein and plasminogen. See Crewther, W.G., Fraser, R.D., Lennox, F.G., and Lindley, H., The chemistry of keratins, Adv. Protein Chem. 20, 191-346, 1965; Masri, M.S. and Friedman, M., Interactions of keratins with metal ions: uptake profiles, mode of binding, and effects on the properties of wool, Adv.Exp.Med. Biol. 48, 551-587, 1974; Fuchs, E. and Green, H., Multiple keratins of cultured human epidermal cells are translated from different mRNA molecules, Cell 17, 573-582, 1979; Fraser, R.D. and Macrae, T.P., Molecular structure and mechanical properties of keratins, Symp.Soc.Exp.Biol. 34, 211-246, 1980; Moll, R., Franke, W.W., Schiller, D.L., et al., The catalog of human cytokeratins: patterns for expression in normal epithelia, tumors and cultured cells, Cell 31, 11-24, 1982; Lazarides, E., Intermediate filaments: a chemically heterogeneous, developmentally regulated class of proteins, Annu.Rev.Biochem. 51, 219-250, 1982; Gonias, S.L., Hembrough, T.A., and Sankovic, M., Cytokeratin 8 functions as a major plasminogen receptor in select epithelial and carcinoma cells, Front. Biosci. 6, D1403-D1411, 2001; Kaplan, A.P., Joseph, K., and Silverberg, M., Pathways for bradykinin formation and inflammatory diseases, J.Allergy Clin.Immunol. 109, 195-209, 2002; Shariat-Madar, Z., Mahdi, F. and Schmaier, A.H., Assembly and activation of the plasma kallikrein/kinin system: a new interpretation, Int.Immunopharmacol. 2, 1841-1849, 2002; Langbein, L. and Schweizer, J., Keratins of the human hair follicle, Int.Rev.Cytol. 243, 1-78, 2005; Gusterson, B.A., Ross, D.T., Heath, V.J., and Stein, T., Basal cytokeratins and their relationship to the cellular origin and functional classification of breast cancer, Breast Cancer Res. 7, 143148, 2005; Skakle, J., Applications of X-ray powder diffraction in materials chemistry, Chem.Rec. 5, 252-262, 2005. See also keratin.
Cytokines Non-antibody proteins secreted by immune system cells. This is a large category and include the various interferon and interleukins as a well as other protein substances. See DeMaeyer, E.M. and Demaeyer-Guignard, J., Interferons and other Regulatory Cytokines, Wiley, New York, NY, USA, 1988; Plotnkoff, N.P., Cytokines: Stress and Immunity, CRC Press, Boca Raton, FL, USA, 1999; Cruse, J.M. and Lewis, R.E., Atlas of Immunology, CRC Press, Boca Raton, FL, 1999; Rott, I.M. and Brostoff, J., Immunology, Mosby, Edinburgh, UK, 2001; Keisari, Y. and Ofek, I., The Biology and Pathology of Innate Immunity Mechanisms, Kluwer Academic, New York, NY, 2002; Henle, W., Interference and interferon in persistent viral infections of cell cultures, J.Immunol. 91, 145-150, 1963; Isaacs, A., Interferon, Adv.Virus Res. 10, 1-38, 1963; Baron, S. and Levy, H.B., Interferon, Annu.Rev.Microbiol. 20, 291318, 1966; Silverstein, S., Macrophages and viral immunity, Semin. Hematol. 7, 185-214, 1970; Bloom, B.R., In vitro approaches to the mechanism of cell-mediated immune reactions, Adv.Immunol. 13, 101-208, 1971; Granger, G.A., Lymphokines-the mediators of cellular immunity, Ser.Hematol. 5, 8-40, 1972; Valentine, F.T., Soluble factors produced by lymphocytes, Ann.N.Y.Acad.Sci. 221, 317-323, 1974; Ward, P.A., Leukotaxis and leukotactic disorders. A review, Am.J.Pathol. 77, 520-538, 1974; Salazar-Mather, T.P. and Hokeness, K.L., Cytokine and chemokine networks: pathways to viral defense, Curr.Top.Microbiol.Immunol. 303, 29-46, 2006; Akira, S., Uematsu, S., and Takeuchi, O., Pathogen recognition
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and innate immunity, Cell 124, 783-801, 2006; Tedgui, A. and Mallat, Z., Cytokines in atherosclerosis: pathogenic and regulatory pathways, Physiol.Rev. 86, 515-581, 2006.
Cytokinesis Cell division; the division of the cytoplasm of a cell following the division of the nucleus. See Robinson, D.N. and Spudich, J.A., Mechanics and regulation of cytokinesis, Curr.Opin.Cell Biol. 16, 181-188, 2004; Mayer, U. and Jurgens, G., Cytokinesis: lines of division taking shape, Curr.Opin.Plant Biol. 7, 599-604, 2004; Albertson, R., Riggs, B., and Sullivan, W., Membrane traffic: a driving force in cytokinesis, Trends Cell Biol. 15, 92-101, 2005; Glotzer, M., The molecular requirements for cytokinesis, Science 307, 1735-1739, 2005; Burgess, D.R., Cytokinesis: new roles for myosin, Curr.Biol. 15, R310-R311, 2005; Darenfeld, H. and Mandato, C.A., Wound-induced contractile ring: a model for cytokinesis, Biochem.Cell Biol. 83, 711-720, 2005; Konopka, C.A., Scheede, J.B., Skop, A.R., and Bednarek, S.Y., Dynamin and cytokinesis, Traffic 7, 239-247, 2006.
Cytomics The molecular analysis of heterogeneous cellular systems; Davies, E., Stankovic, B., Azama, K., et al., Novel components of the plant cytoskeleton: a beginning to plant ‘cytomices,’ Plant Sci. 160, 185196, 2001; Bernas, T., Gregori, G., Asem, E.K., and Robinson, J.P., Integrating cytomics and proteomics, Mol.Cell.Proteomics 5, 2-13, 2006; Van Osta, P., Ver Donck, K., Bols, L., and Geysen, J. Cytomics and drug discovery, Cytometry A 69, 117-118, 2006; Tarnok, A., Slide-based cytometry for cytomics – a minireview, Cytometry A 69, 555-562, 2006; Herrera, G., Diaz, L., MartinezRomero, A., et al., Cytomics: a multiparametric, dynamic approach to cell research, Toxicol. In Vitro, 21, 176–182, 2006; Valet, G., Cytomics as a new potential for drug discovery, Drug Discov.Today 11, 785-791, 2006.
Cytoskeleton The internal framework of the cell; the cytoskeleton is composed largely of actin filaments and microtubules. See Wasteneys, G.O. and Yang, Z., New views on the plant cytoskeleton, Plant Physiol. 136, 3884-3891, 2004; Moller-Jensen, J. and Lowe, J., Increasing complexity of the bacterial cytoskeleton, Curr.Opin.Cell Biol. 17, 75-81, 2005; Smith, L.G. and Oppenheimer, D.G., Spatial control of cell expansion by the plant cytoskeleton, Annu.Rev.Cell Dev. Biol. 21, 271-295, 2005; Munro, E.M., PAR proteins and the cytoskeleton: a marriage of equals, Curr.Opin.Cell Biol. 18, 86-94, 2006; Boldogh, I.R. and Pon, L.A., Interactions of mitochondria with the actin cytoskeleton, Biochim.Biophys.Acta 1763, 405462, 2006; Larsson, C., Protein kinase C and the regulation of the actin cytoskeleton, Cell Signal. 18, 276-284, 2006; Logan, M.R and Mandato, C.A., Regulation of the actin cytoskeleton by PIP2 in cytokinesis, Biol.Cell. 98, 377-388, 2006; Sheetz, M.P., Sable, J.E., and Dobereiner, H.G., Continuous membrane-cytoskeleton adhesion requires continuous accommodation to lipid and cytoskeleton dynamics, Annu.Rev.Biophys.Biomol.Struct. 35, 417-434, 2006; Becker, B.E. and Gard, D.L.,Visualization of the cytoskeleton in Xenopus oocytes and eggs by confocal immunofluorescence microscropy, Methods Mol.Biol. 322, 69-86, 2006; Popowicz, G.M., Scheicher, M., Noegel, A.A., and Holak, A.A., Filamins: promiscuous organizers of the cytoskeleton, Trends Biochem.Sci. 31, 411-419, 2006.
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Desorption
Cytotoxic T-cells; cytotoxic T-lymphocytes Also known as killer cells, killer T-cells, null cells, A differentiated T cell (CD8 positive) that attacks and lyses target cells bearing specific antigens. Used in patient-specific immunotherapy with cells grown in culture. See Gillis, S., Baker, P.E., Ruscetti, F.W., and Smith, K.A., Long-term culture of human antigen-specific cytotoxic T-cell lines, J.Exptl.Med. 148, 1093-1098, 1978.
Database of Interacting Proteins (DIP) The database of interacting proteins integrates the experimental evidence available on protein interactions into a single online resource: http://dip.doe-mbi.ucla.edu See Xenarious, I., Fernandez, E., Salwinski, L., Duan, X.J. et al., DIP: the database of interacting proteins: 2001 update, Nucleic Acids Res. 29, 239-241, 2001; Deane, C.M., Salwinski, L, Xenarios, I., and Eisenberg, D., Protein interactions: two methods for assessment of the reliability of high throughput observations, Mol.Cell Proteomics 1, 349-356, 2002; Salwinski, L., Miller, C.S., Smith, A.J., et al., Nucleic Acids Res. 32, D449-451, 2004; Han, D., Kim, H.S., Seo, J., and Jang, W., A domain combination based probalistic framework for proteinprotein interaction prediction, Genome Inform.Ser.Workshop Genome Inform. 14, 250-259, 2003; Espadaler, J., Romero-Isart, O., Jackson, R.M., and Oliva, B., Prediction of protein-protein interactions using distant conservation of sequence patterns and structure relationships, Bioinformatics 21, 3360-3368, 2005.
Deconvolution An algorithm used in electrospray mass spectrometry to translate the spectra of multiply charged ions into a spectrum of molecular species.
Dendrimers A novel polymeric material cotaining a highly branched and welldefined structure. Dendrimers have been used for drug delivery, a biological matrix and for model drug distribution studies. Dedrimers are similar to dendrites which are branched crystals in which branches of crystallization proceeds at different rates. See Meldal, M. and Hilaire, P.M., Synthetic methods of glycopeptides assembly, and biological analysis of glycopeptides products, Curr. Opin.Chem.Biol. 1, 552-563, 1997; Sadler, K. and Tam, J.P., Peptide dendrimers: applications and synthesis, J.Biotechnol. 90, 195-229, 2002; Turnbull, W.B., and Stoddart, J.F., Design and synthesis of glycodendrimers, J.Biotechnol. 90, 231-255, 2002; Kobayashi, H. and Brechbiel, M.W., Dendrimer-based macromolecular MRI contrast agents: characteristics and application, Mol.Imaging 2, 1-10, 2003; Lee, C.C., MacKay, J.A., Frechet, J.M., and Szoka, F.C., Designing dendrimers for biological applications, Nat.Biotechnol. 23, 1517-1526, 2005; Qiu, L.Y. and Bae, Y.H., Polymer architechture and drug delivery, Pharm.Res.23, 1-30, 2006. Gupta, V., Agashe, H.B., Asthana, A., and Jain, N.K., Dendrimers: novel polymeric nanoarchitecture for solubility enhancement, Biomacromolecules 7, 649-658, 2006; Söntjens, S.H.M., Nettles, D.L., Carnahan, M.A., et al., Biodendrimer-based hydrogel scaffolds for cartilage tissue repair, Biomacromolecules 7, 310-316, 2006.
Desorption Process by which molecules in solid or liquid form are transformed into a gas phase.
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Detergent Perturbation
Distributed Annotation System (DAS)
Treatment of total human plasma proteins with sodium cholate and subsequent removal; resulting in “remodeling” of the lipoproteins; see Pownall, H.J., Remodeling of human plasma lipoproteins by detergent perturbation, Biochemistry 44, 9714-9722, 2005.
Myers, J.W. and Ferrell, J.E., Jr., Silencing gene expression with Dicer-generated siRNA pools, in RNA Silencing. Methods and Protocols, ed. G.G. Carmichael, Humana Press, Totowa, New Jersey, 2005; Hammond, S.M., Dicing and slicing. The core machinery of the RNA interference pathway, FEBS Lett. 579, 5822-5829, 2005.
Deterministic Series
Dictionary of Interfaces in Proteins (DIP)
A series or model which contains no random or probabilistic elements. The Cambridge Dictionary of Statistics, ed. B.S. Everitt, Cambridge University Press, Cambridge, United Kingdom, 1998.
The dictionary of interfaces in proteins is a database collecting the 3D structures of protein domains involved in interactions (patches). See Preissner, R., Goode, A., and Frommel, C., Dictionary of interfaces in proteins (DIP). Data bank of complementary molecules, J.Mol.Biol. 280, 535-550, 1998. Frommel, C., Gille, C., Goede, A., et al., Accelerating screening of 3D protein data with a graph theoretical approach, Bioinformatics 19, 2442-2447, 2003.
Diabodies An engineered non-covalent dimer of an scFv fragment which has two antigen binding sites which may be homologous or heterologous. The normal linker engineered between the V H and V L domains is 15 residues (usually glycine and serine to promote maximum flexibility) which yields as monomer; if the linker is reduced to 10 residues, a dimer (diabody) is formed while with no linker there is a trimer or higher order polymer. See Atwell, J.L., Breheney, K.A., Lawrence, L.J. et al., scFv multimers of th anti-neuraminidase antibody NC10: length of the linker between V H and V L domains dictates precisely the transition between diabodies and triabodies, Protein Eng. 12, 597-604, 1999. Todorovska, A., Roovers, R.C., Dolezal, O., et al., Design and application of diabodies, triabodies and tetrabodiese for cancer targeting, J.Immunol.Methods 248, 47-66, 2001; Holliger, P. and Hudson, P.J., Engineered antibody fragments and the rise of single domains, Nature Biotechnol. 23, 1126-1136, 2005. While diabodies are non-covalent complexes of engineered scFv constructs based on the association of the V H domain with the most available V L domain. A covalent diabody was observed with an engineered anti-carcinoembryonic antigen (CEA) diabody with cysteine residues inserted for coupling with a radiolabel. The formation of a disulfide-linked diabody was an unexpected consequence. See Olafsen, T., Cheung, C.-w., Yazaki, P.J. et al., Covalent disulfide-linked anti-CEA diabody allows site-specific conjugation and radiolabeling for tumor targeting applications, Prot.Eng.Des.Sel. 17, 21-27, 2004. Note: It has been observed that if the order of the variable regions are switch in scFv construct (V L-V H instead of V H-V L), the engineered scFv with a zero-length linker formed a dimer (diabody) instead of the expected trimer – See Arndt, M.A.E., Krauss, J., and Rybak, S.M., Antigen binding and stability properties of non-covalently linked anti-CD22 single-chain Fv dimers, FEBS Lett. 578, 257-261, 2004. See single chain Fv fragment, bibody, triabody.
Diapedesis The migration of a leukocyte through the interendothelial junction space and the extracellular matrix/basement membrane to the site of tissue inflammation; a process driven by chemotaxis.
Dicer Dicer is an RNAse III nuclease (class III) which is specific for double-stranded RNA and yields siRNAs. Structurally it consists of an amino terminal helicase domain, a PAZ domain, two RNAse III motifs, and a dsRNA binding motif. See Carmell, M.A., and Hannan, G.J., RNAse III enzymes and their initiation of gene silencing, Nat.Struct.Mol.Biol. 11, 214-218, 2004;
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Differential Scanning Calorimetry (DSC) A physical technique for the study of conformation based on measuring changes in heat capacity of a molecule under various conditions. See Zecchinon, L., Oriol, A., Netzel, U., et al., Stability domains, substrate-induced conformational changes, and hingebending motions in a psychrophilic phosphoglycerate kinase. A microcalorimetric study, J.Biol.Chem. 280, 41307-41314, 2005;
Dipolar Couplings Also residual dipolar couplings, measures the interaction between nuclei in an applied magnetic field; used for the determination of the solution structure of peptides, proteins, nucleic acids, and carbohydrates; also ligand binding, see Post, C.B., Exchangetransferred NOE spectroscopy and bound ligand structure determination, Curr.Opin.Struct.Biol. 13, 581-588, 2003; Bush, C.A., Martin-Pastor, M., and Imberty, A., Structure and conformation of complex carbohydrates of glycoproteins, glycolipids, and bacterial polysaccharides, Ann.Rev.Biophys.Biomol.Struct. 28, 269293, 1999; MacDonald, D., and Lu, P., Residual dipolar couplings in nucleic acid structure determination, Curr.Opin.Struct.Biol. 12, 337-343, 2002.
Directed Library Also focused library. A screening library of chemical compounds which may be prepared by parallel synthesis, combinatorial chemistry, phage displayed or similar multiplexed technologies. See Miller, J.L., Recent developments in focused library design: targeting gene-families, Curr.Top.Med.Chem. 6, 19-29. 2006; Xu, Y., Shi, J., Yamamoto, N., et al., A credit-card library approach for disrupting protein-protein interactions, Bioorg.Med.Chem., 14, 2660–2673, 2005; Subramanian, T., Wang, Z., Troutman, J.M., et al., Directed library of anilinogeranyl analogues of farnesyl diphosphate via mixed solid- and solution-phase synthesis, Org.Lett. 7, 2109-2112, 2005; McGregor, M.J., and Muskal, S.M., Pharmacophore fingerprinting. 1. Application to QSAR and focused library design, J.Chem.Inf.Comput.Sci. 39, 569-574, 1999.
Distributed Annotation System (DAS) The distributed annotation system is a communication protocol for the exchange of biological annotations (In genetics, the process of identifying the locations and coding regions of genes
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in a genome and determining what those genes do. An annotation is note added with comment on the function of the gene and/or coding region). See Hubbard, T., Biological information: making it accessible and integrated (and trying to make sense of it), Bioinformatics 18 (Suppl 2):S140, 2002; Olason, P.I., Integrating protein annotation resouces through the Distributed Annotation System, Nucleic Acids Res. 33, W468-470, 2005; Prlic, A., Down, T.A., and Hubbard, J.T., Adding some SPICE to DAS, Bioinformatics 21 (Suppl 2), ii40-ii41, 2005; Stamm, S., Riethovan, J.J., Le Texier, V., Gopalakrishnan, C., Kumanduri, V., Tang, Y., Barbosa-Morais, N.L., and Thanaraj, T.A., ASD: a bioinformatics resource on alternative splicing, Nucleic Acids Res. 32, D46-D55, 2006. See also http://www.cbs.dtu.dk/; http://www.cbs.dtu.dk/ cgi-bin/das
DNA Fingerprinting This procedure is also referred to as chromosomal fingerprinting, restriction enzyme analysis (REA). This is process where DNA is cleaved by a restriction endonuclease (restriction enzyme). The resulting DNA fragments are separated by gel electrophoresis ad detected by specific and non-specific probes. DNA fingerprinting is extensively used for forensic purposes. See Gazit, E. and Gazit, E., DNA fingerprinting, Isr.J.Med.Sci. 26, 158-162, 1990; Owen, R.J., Chromosomal DNA fingerprinting –a new method of species and strain identification applicable to microbial pathogens, J.Med.Microbiol. 30, 89-99, 1989; Cawood, A.H., DNA fingerprinting, Clin.Chem. 35, 1832-1837, 1989; de Gouyon, B., Julier, C., Avner, P., Georges, M, and Lathrop, M., Human variable number of tandem repeat probes as a source of polymorphic markers in experimental animals, EXS 58, 85-94, 1991; Webb, M.B. and Debenham, P.G., Cell line characterization by DNA fingerprinting: a review, Dev.Biol.Stand. 76, 39-42, 1992; Debenham, P.G., Probing identity: the changing face of DNA fingerprinting, Trends Biotechnol. 10, 96-102, 1992; McClelland, M. and Welsh, J., DNA fingerprinting by arbitrarily primed PCR, PCR Methods Appl. 4, S59-S65, 1994; Kuff, E.L. and Mietz, J.A., Analysis of DNA restriction enzyme digests by two-dimensional electrophoresis in agaraose gels, Methods Mol.Biol. 31, 177-186, 1994; Caetano-Anolles, G., Scanning of nucleic acids by in vitro amplification: new developments and applications, Nat.Biotechnol. 14, 1668-1674, 1996.
DNA Footprinting DNA is incubated with a putative binding protein and then modified with dimethyl sulfate. Methylation of DNA bases occurs at regions not protected by the protein binding. The DNA can be cleaved at guanine residues which are then cleaved by piperidine. Footprinting can also be achieved by the use of DNAse I hydrolysis, reaction with hydroxyl radicals or with metal ion-chelate complexes. With either enzymatic or chemical fragmentation, the DNA is end-labeled with 32P-phosphate to permit identification by autoradiography. This has been used to identify the sites of transcription factor binding to cis-regions on DNA. See Guille, M.J. and Kneale, G.G., Methods of the analysis of DNA-protein interactions, Mol.Biotechnol. 8, 35-52, 1997; Gao, B. and Kunos, G., DNase I Footprinting analysis of transcription factors recognizing adrenergic receptor gene promoter sequences, Methods Mol.Biol. 126, 419-429, 2000; Cappabianca, L, Thomassin, H., Pictet, R. and Grange, T., Genomic footprinting using nucleases, Methods Mol. Biol. 199, 427-442, 1999; Angelov, D., Khochbin, S., Dimitrov, S., US laser Footprinting and protein-DNA crosslinking. Application
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to chromatin, Methods Mol.Biol. 119, 481-495, 1999; Brenowitz, M., Chance, M.R., Dhavan, G., and Takamoto, K., Probing the structural dynamics of nucleic acids by quantitative time-resolved and equilibrium hydroxyl radical “Footprinting”. Curr.Opin. Struct.Biol. 12, 648-653, 2002; Knight, J.C., Functional implications of genetic variation in non-coding DNA for disease susceptibility and gene regulation, Clin.Sci. 104, 493-501, 2003.
DNA Methylation Modification (methylation) of DNA catalyzed by DNA methyltransferase enzymes. Modification occurs at cytosine and adenosine. In multicellular organisms, methylation appears to be confined to cytosine residues. See van Steensel, B. and Henikoff, S., Epigenomic profiling using microarrays, Biotechniques 35, 346-350, 2003; El-Maarri, O., Methods: DNA methylation, Adv. Exp.Med.Biol. 544, 197-204, 2003; Gut, I.G., DNA analysis by MALDI-TOF mass spectrometry, Hum.Mutat. 23, 437-441, 2004; Kapoor, A., Agius, F., and Zhu, J.K., Preventing transcriptional gene silencing by active DNA demethylation, FEBS Lett. 579, 5889-5898, 2005; Klose, R.J. and Bird, A.P., Genomic DNA methylation: the mark and its mediators, Trends in Biochem.Sci. 31, 81-97, 2006.
DNAse I Hypersenstivity site Preferred site(s) of DNA I cleavage; typically at regions where clusters of transcriptional activators bind to DNA and usually reflect a change in chromatin structure. See McGinnis, W., Shermoen, A.W., Heemskerk, J., and Beckendorf, S.K., DNA sequence changes in an upstream DNAse I-hypersensitive region are correlated with reduced gene expression, Proc.Nat.Acad. Sci.USA 80, 1063-1067, 1983; Cereghini, S., Saragosti, S., Yaniv, M. and Hamer, D.H., SV40-alpha-globulin hybrid minichromosomes. Differences in DNase I hypersensitivity of promoter and enhancer sequences, Eur.J.Biochem. 144, 545-553, 1984; Rothenberg, E.V. and Ward, S.B., A dynamic assembly of diverse transcription factors integrates activation and cell-type information for interleukin 2 gene regulation, Proc.Nat.Acad.Sci.USA 93, 9358-9365, 1996; Ishii, H., Sen, R., and Pazin, M.J., Combinatorial control of DNase I-hypersensitive site formation and erasure by immunoglobulin heavy chain enhancer-binding proteins, J.Biol. Chem. 279, 7331-7338, 2004; Hermann, B.P. and Heckert, L.L., Silencing of Fshr occurs through a conserved, hypersensitive site in the first intron, Mol.Endocrinol. 19, 2112-2131, 2005; Sun, D., Guo, K., Rusche, J.J., and Hurley, L.H., Facilitation of a structural transition in the polypurine/polypyrimidine tract within the proximal promoter region in the human VEGF gene by the presence of potassium and G-quadruplex-interactive agents, Nucleic Acids Res. 33, 6070-6080, 2005.
DNAzyme A DNA molecule that contains a catalytic motif that cleaves bound RNA in a hydrolytic reaction; also known as deoxyribozymes or DNA enzymes. See Joyce, G.F., Directed evolution of nucleic acid enzymes, Annu.Rev.Biochem. 73, 791-836, 2004; Achenbach, J.C., Chiuman, W., Cruz, R.P., and Li., Y., DNAzymes: from creation in vitro to application in vivo. Curr.Pharm.Biotechnol. 5, 321-336, 2004; Sioud, M. and Iversen, P.O., Ribozymes, DNAzymes and small interfering RNAs as therapeutics, Curr.Drug.Targets 6, 647653, 2005; Fiammengo, R. and Jaschke, A., Nucleic acid enzymes, Curr.Opin.Biotechnol. 16, 614-621, 2005.
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Domain
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Dye(s)
Domain
Drug Targeting
A contiguous (usually) series of monomer units (amino acids in proteins; nucleic acid bases in nucleic acids; monosaccharide in oligosaccharides/polysaccharides. A domain can be continuous or discontinuous and is identified by a unique function such as catalysis or binding; domains are frequently identified by homology and used to group proteins into families.
The ability to target a compound to a specific organ or cell type within an organism. The compound can be a drug/pharmaceutical or it can be a compound, such as a radioisotope, which can be used as a diagnostic. See Muzykantov, V.R., Biomedical aspects of targeted delivery of drugs to pulmonary endothelium, Expert Opin.Drug Deliv. 2, 909-926, 2005; Weissig, V., Targeted drug delivery to mammalian mitochondria in living cells, Expert Opin. Drug Deliv. 2, 89-102, 2005; Hilgenbrink, A.R. and Low, P.S., Folate-receptor-mediated drug targeting: from therapeutics to diagnostics, J.Pharm.Sci. 94, 2135-2146, 2005.
Domain Antibodies Antibodies containing a single antigen-binding domain, most ofter the VH region or the highly variable regions from the V H and VL regions. These antibodies are naturally occurring in camelids (members of the order Camelidae which includes llamas and camels). See Dick, H.M., Single domain antibodies, BMJ 300, 959, 1990; Riechman, L. and Muyldermans, S., Single domain antibodies: comparison of camel VH and camelized human VH domains, J.Immunol.Methods 231, 25-38, 1999; Stockwin, L.H. and Holmes, S., Antibodies as therapeutic agents: vive la renaissance! Expert Opin.Biol.Ther. 3, 1133-1152, 2003; Holt, L.J., Herring, C., Jespers, L.S., Woolven, B.P., and Tomlinson, I.M., Domain antibodies: proteins for therapy, Trends Biotechnol. 21, 484-490, 2003.
Drosha A member of the RNAse III family of double-stranded specific endonucleases. Drosha is a member of Class II in which each member contains tandem RNAse III catalytic motifs and one C-terminal dsRNA-binding domain. Class I proteins contain only one RNAse III catalytic domain and a dsRNA binding domain. Class III (see Dicer) contain a PAZ domain, a DUF283 domain, the tandem nuclease domains and a dsRNA-binding domain. See Carmell, M.A., and Hannan, G.J., RNAse III enzymes and their initiation of gene silencing, Nat.Struct.Mol.Biol. 11, 214-218, 2004.
Drug A drug is defined as (1) a substance recognized by an official pharmacopoeia or formulary; (2) a substance intended for use in the diagnosis, cure, mitigation, treatment, or prevention of disease; (3) a substance (other than food) intended to affect the structure or any function of the body; (4) a substance intended for use as a component of a medicine but not a device or a component, part or accessory of a device; (5) biological products are included within this definition and are generally covered by the same laws and regulations, but differences exist regarding their manufacturing processes (chemical processes vs. biological processes).
Drug Master File Drug Master Files (DMF) contain information on the processes and facilities used in drug or drug component manufacture and storage and are submitted to the FDA for examination and approval.
Drug Product The final dosage form which contains a drug substance or drug substances as well as inactive materials which are also considered as excipients. The drug product is differentiated from the drug substance but may or may not be the same as the drug substance. See http://www.fda.gov/cder/drugsatfda/glossary.htm; http://www. fda.gov/cder/ondc/Presentations/2002/01-10-19_DIA_JS.pp.
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Dye(s) A chemical compound with a structure which yields a color (a chromophore) which can be coupled either covalently or noncovalently to a substrate matrix. The ability of the compound to yield color is based on its ability to absorb light in the visible spectrum (400 – 700 nm). Dyes can be classified by various characteristics including mechanism/chemistry (e.g. basic dyes, acid dyes; acid/base indicators/redox dyes), structure (nitroso, acridine dyes, thiazole dyes) and process use (e.g. vat dyes). A dyes is a colorant (a substance which yields color) as are pigments. A dye is chemically different from a pigment which is a particle suspended in a medium as particles in paint. More recently, the term dye has expanded to include fluorescent compounds. See Venkataraman, K., The Chemistry of Synthetic Dyes, Academic Press, New York, NY, 1952; Conn, H.J., Biological Stains: A Handbook on the Nature and Uses of the Dyes Employed in the Biological; Handbook, Williams & Wilkins, Baltimore, MD, 1961; Gurr, E., Synthetic Dyes in Biology, Medicine, and Chemistry, Academic Press, London, UK, 1971; Venkataraman, K., The Chemistry of Synthetic Dyes, Academic Press, New York, NY, 1978; Egan, H. and Fishbein, L., Some Aromatic Amines and Azo Dyes in the General and Industrial Environment, International Agency for Research on Cancer, Lyon, France, 1981; Clark, G. and Koastan, F.H., History of Staining, 3rd edn., Williams & Wilkins, Baltimore, MD, 1983; Zollinger, H. Color Chemistry. Syntheses, Properties, and Applications of Organic Dyes and Pigments, 2nd edn., VCH, Weiheim, Germany, 1991; Physico-Chemical Principles of Color Chemistry, ed. A.T. Peters and H.W. Freeman, Blackie Academic and Professional, London, UK, 1996; Mason, W.T., Fluorescent and Luminescent Probes for Biological Activity: A Practical Guide to Technology for Quantitative Real-Time Analysis, Academic Press, San Diego, CA, 1999; Conn’s Biological Stains. A Handbook of Dyes, Stains, and Fluorochromes for Use in Biology and Medicine, 10th edn., ed. R.W. Horobin and J.A. Kiernan, Bios, Oxford, UK, 2002; Kasten, F.H., Cytochemical studies with acridine orange and the influence of dye contaminants in the staining of nucleic acids, Int.Rev.Cytol. 21, 141202, 1967; Meyer, M.C. and Guttman, D.E., The binding of drugs by plasma proteins, J.Pharm.Sci. 57, 895-918, 1968; Adams, C.W. Lipid histochemistry, Adv.Lipid Res. 7, 1-62, 1969; Horobin, R.W., The impurities of biological dyes: their detection, removal, occurrence and histochemical significance—a review, Histochem.J. 1, 231-265, 1969; Biswas, B.B., Basu, P.S., and Pai, M.K., Gram staining and its molecular mechanism, Int.Rev.Cytol. 29, 1-27, 1970; Anumula, K.R., Advances in fluorescence derivatization methods for high-performance liquid chromatographic analysis of glycoprotein carbohydrates, Anal.Biochem. 350, 1-23, 2006; Waggoner, A., Fluorescent labels for proteomics and genomics, Curr.Opin. Chem.Biol. 10, 62-66, 2006; Chen, H., Recent advances in azo dye degrading enzyme research, Curr.Protein Pept.Sci. 7, 101-111, 2006;
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Ectodomain Embedding 995 Mondal, K. and Gupta, M.N., The affinity concept in bioseparation: evolving paradigms and expanding range of applications, Biomol. Eng. 23, 59-76, 2006.
Ectodomain The extracellular domain of a transmembrane protein. The proteolysis of the ectodomain regions of specific proteins is described as ectodomain shedding and is catalyzed by ADAM proteases. See Rapraeger, A. and Bernfield, M., Cell surface proteoglycan of mammary epithelial cells. Protease releases a heparan sulfate-rich ectodomain from a putative membrane-anchored domain, J.Biol. Chem. 260. 4103-4109, 1985; Johnson, J.D., Wong, M.L., and Rutter, W.J., Properties of the insulin receptor ectodomain, Proc.Natl. Acad.Sci.USA 85, 7516-7520, 1988; Schaefer, E.M., Erickson, H.P., Federwisch, M., et al., Structural organization of the human insulin receptor ectodomain, J.Biol.Chem, 267. 23393-23402, 1992; Attia, J., Hicks, L., Oikawa, K.. et al., Structural properties of the myelinassociated glycoprotein ectodomain, J.Neurochem. 61, 718-726, 1993; Couet, J., Sar. S., Jolviet, A., Shedding of human thyrotropin receptor ectodomain. Involvement of a matrix metalloproteinase, J.Biol.Chem. 271, 4545-4552, 1996; Petty, H.R., Kindzelskii, A.L., Adachi, Y.. et al., Ectodomain interactions of leukocyte integrin and pro-inflammatory GPI-linked membrane proteins, J.Pharm. Biomed.Anal.15, 1405-1416, 1997; Schlondorff, J. and Blobel, C.P., Metalloprotease-disintegrins: modular proteins capable of promoting cell-cell interactions and triggering signals bv protein-ectodomain shedding, J.Cell Sci. 112, 3603-3617,1999; Dello Sbarba, P.. and Rovida, E., Transmodulation of cell surface regulatory molecules via ectodomain shedding, Biol.Chem, 383, 69-83, 2002; Arribas, J. and Borroto, A., Protein ectodomain shedding, Chem.Rev. 102, 4627-4638, 2002; l-Selectin: mechanisms and physiological significance of ectodomain cleavage,. J.Cell.Mol.Med. 9, 255-266, 2005; Higashiyama, S. and Nanba, D., ADAM-mediated ectodomain shedding of HB-EGF in receptor cross-talk, Biochim.Biophys. Acta 1751, 110-117, 2005; Garton, K.J., Gough, P.J., and Raines, E.W., Emerging roles for ectodomain shedding in the regulation of inflammatory responses, J.Leuk.Biol. 79, 1105-1116, 2006.
Electrode Potential (E°) The potential measured with a electrode in contact with a solution of its ions. Electrode potential values will predict whether a substance will be reduced or oxidized. Values are usually expressed as a reduction potential (Mn+ → M). A positive electrode potential would indicate that reduction is spontaneous. A negative potential for this reaction would suggest that the oxidation reaction (M → Mn+) would be spontaneous.
Electronegativity The tendency of an atom to pull electron towards its in a chemical bond; the difference in electronegativity between atoms in a molecule indicates polarity such that in bromoacetic acetamide permitting attack on a nucleophile such as cysteine in the protein.
Electrophoresis/MS Proteins are separated by one-dimension or more often twodimensional gel electrophoresis. The separated proteins are subjected to in situ tryptic digestion and the peptides separated by liquid chromatography and identified by mass spectrometry; Nishihara, J.C. and Champion, K.M., Quantitative evaluation of
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proteins in one- and two-dimensional polyacrylamide gels using a fluorescent stains, Electrophoresis 23, 2203-2215, 2002.
ELISA Enzyme-Linked Immunosorbent Assay – an assay based on the reaction of antibody and antigens. There are direct, indirect, direct sandwich, and indirect sandwich assay. See Maggio, E.T., Enzyme-Immunoassay, CRC Press, Boca Raton, Florida, USA, 1980; Kemeny, D.M. and Challacombe, S.J., ELISA and Other Solid Phase Immunoassays: Theoretical and Practical Aspects, Wiley, Chichester, UK, 1988; Kemeny, D.M., A Practical Guide to ELISA, Pergammon Press, Oxford, UK, 1991; Kerr, M.A. and Thorpe, R., Immunochemistry LabFax, Bios Scientific Publishers, Oxford, UK, 1994; Law, B., Immunoassay: A Practical Guide, Taylor & Francis, London, UK, 1996; Crowther, J.R., The ELISA Guidebook, Humana Press, Totowa, New Jersey, USA, 2001; Burns, R., Immunochemical Protocols, Humana Press, Totowa, NJ, USA, 2005.
Elispot The use of membranes to measure cells secreting a specific product such as an antibody or a cytokine. A membrane (nitrocellulose or PDVF) containing an antibody or other specific binding protein is placed in a microtiter plate. Cells secreting a product, such as a cytokine, are grown in this plate and the secretion of the specific product evaluated in response to stimuli. As product is secreted from an individual cell, it is captured immediately by the antibody or other specific binding protein on the membrane and subsequently detected with a probe. An individual spot then corresponds to the secretion from a single cell. There are a number of instruments designed to measure such spots. See Kalyuzhny, A., Handbook of ELISPOT: Methods and Protocols, Totowa, NJ, USA, 2005; Stot, D.I., Immunoblotting, dot-blotting, and ELISPOT assay: methods and applications, in Immunochemistry, ed. C.J. van Oss and M.H.V. van Regenmortel, Marcel Dekker, New York, New York, Chapter 35, pp. 925-948, 1994; Arvilommi, H., ELISPOT for detecting antibody-secreting cells in response to infections and vaccination, APMIS 104, 401-410, 1996; Stott, D.I., Immunoblotting, dot-blotting, and ELISPOT assays: methods and applications, J.Immunoassay 21, 273-296, 2000; Anthony, D.D. and Lehmann, P.V., T-cell epitope mapping using the ELISPOT approach, Methods 29, 260-269, 2003; Ghanekar, S.A. and Maecker, H.T., Cytokine flow cytometry: multiparametric approach to immune function analysis, Cytotherapy 5, 1-6, 2003; Letsch, A. and Scheibenbogen, C., Quantification and characterization of specific T-cells by antigen-specific cytokine production using ELISPOT assay or intracellular cytokine staining, Methods 31, 143-149, 2003; Herandez-Fuentes, M.P., Warrens, A.N., and Lechler, R.I., Immunologic monitoring, Immunol.Rev. 196, 247264, 2003; Kalyuzhny, A.E., Chemistry and biology of the ELISPOT system, Methods Mol.Biol. 302, 15-31, 2005; Periwal, S.B., Spagna, K., Shahabi, K.. et al., Statistical evaluation for detection of peptide specific interferon-gamma secreting T-cells induced by HIV vaccine determined by ELISPOT assay, J.Immunol.Methods 305, 128-134, 2005.
Embedding Infiltration of a specimen with a liquid medium (paraffin) that can be solidified/polymerized to form a matrix to support the tissue for subsequent manipulation
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Endocrine Eph 996 Receptors/Ephrin
Endocrine
Ensembl
Usually in reference to a hormone or other biological effector such as peptide growth factor or cytokine which has a systemic effect.
A data base (http://www.ensembl.org) maintained by the European Bioinformatics Institute (EMBL). This data base organizes large amounts of biological information around the sequences of large genomes. See Birney, E., Andrews, T.D., Bevan, P., et al., An overview of Ensembl, Genome Res. 14, 925-928, 2004; Baxevanis, A.D., Using genomic databases for sequence-based biological discovery, Mol.Med. 9, 185-192, 2003; Stabenau, A., McVicker, G., Melsopp, C., Procter, G., Clamp, M., and Birney, E., The Ensembl core software libraries, Genome Res. 14, 929-933, 2004; Yanai, I., Korbel, J.O., Boue, S., McWeeney, S.K., Bork, P., and Lercher, M.J., Similar gene expression profiles do not imply similar tissue functions, Trends Genet. 22, 132-138, 2006.
Endoplasmic reticulum-associated protein degradation (ERAD) A highly specific pathway for the degradation of misfolded proteins in the endoplasmic reticulum which serves as a control mechanism for protein synthesis: see Yamaski, S., Yagishita, N., Tsuchimochi, K., Nishioka, K., and Nakajima, T., Rheumatoid arthritis as a hyper-endoplasmic reticulum-associated degradation disease, Arthritis Res.Ther. 7, 181-186, 2005; Meusser, B., Hirsch, C., Jarosch, E., and Sommer, T., ERAD: the long road to destruction, Nature Cell Biol. 7, 766-772, 2005; Werner, E.D., Brodsky, J.L., and McCracken, A.A., Proteasome-dependent endoplasmic reticulum-associated protein degradation: an unconventional route to a familiar fate, Proc.Natl.Acad.Sci.USA 93, 13797-13801, 1996.
Endosome A physically distinct compartment resulting from the process of endocytosis and isolated from the rest of the cell with a permeable membrane. The endosome provides a pathway for transport of ingested materials to the lysosome. There is particular interest in this pathway for the process of antigen presentation. See Stahl, P. and Schwartz, A.L., Receptor-mediated endocytosis, J.Clin.Invest. 77, 657-662, 1986; Wagner, H., Heit, A., Schmitz, F., and Bauer, S., Targeting split vaccines to the endosome improves vaccination, Curr.Opin.Biotechnol. 15, 538-542, 2004; Boes, M., Cuvillier, A., and Ploegh, H., Membrane specializations and endosome maturation in dendritic cells and B cells, Trends Cell Biol. 14, 175-183, 2004; Karlsson, L., DM and DO shape the repertoire of peptide-like-MHC-class-II complexes, Curr.Opin.Immunol. 17, 65-70, 2005; Li, P., Gregg, J.L., Wang, N., Zhou, D., O’Donnell, P., Blum, J.S., and Crotzer, V.L., Compartmentalization of class II antigen presentation: contribution of cytoplasmic and endosomal processing, Immunol.Rev. 207, 206-217, 2005.
Enhancer Elements DNA sequences which increase transcription from a linked promoter region independent of operation and position (in contrast to proximal promoter elements). Enhancer elements are located at varying distances upstream and downstream of the linked gene. See Hankinson, O., Role of coactivators in transcriptional activation by the aryl hydrocarbon receptor, Archs. Biochem.Biophys. 433, 379-386, 2005; West. A.G. and Fraser, P., Remote control of gene transcription, Hum.Mol.Genet. 14, Spec. No. 1, R101-R111, 2005; Sipos, L. and Gyurkovics, H., Longdistance interactions between enhancers and promoters, FEBS J. 272, 3253-3259, 2005; Zhao, H. and Dean, A., Organizing the genome: enhancers and insulators, Biochem.Cell.Biol. 83, 516524, 2005.
Enlargosome An organelle resulting from a calcium-dependent, cholesterolindependent non-secretory event; Perret, E., Lakkaraju, A., Deborde, S., Schreiner, R., and Rodriguez-Boulan, E., Curr.Opin. Cell Biol. 17, 423-434, 2005.
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Ensemble Theory A proposition that several discrete compounds (proteins, nucleic, acids, carbohydrate) form a structural whole or functional whole. The term ensemble is frequently used to describe the population of discrete intermediates during the process of protein folding. See Thirumalai, D. and Hyeon, C., RNA and protein folding: common themes and variations, Biochemistry 44, 4957-4970, 2005; Dietrich, A., Buschmann, V., Muller, C., and Sauer, M., Fluorescence resonance energy transfer (FRET) and competing processes in donor-acceptor substituted DNA strands: a comparative study of ensemble and single-molecule data, J.Biotechnol. 82, 211-231, 2002; Sridevi, K., Lakshmikanth, G.S., Krishnamoorthy, G., and Udgaonkar, J.B., Increasing stability reduces conformational heterogeneity in a protein folding ensemble, J.Mol.Biol. 337, 699-711, 2004.
Enthalpy (∆H°) This is the energy change or heat of reaction for either synthetic or degradative reaction in the standard state. See standard free energy.
Entropy (s) A thermodynamic quantity that is a measure of the “disorder” or randomness in a system. For example, a crystal structure going to a liquid is associated with an increase in entropy as, for example, the melting of ice crystals to form water under standard conditions. Entropy increases for a spontaneous process. S° refers to entropy values in standard states of substances.
Eosinophil “Acid” staining leukocyte; associated with allergic inflammation. See Lee, J.J. and Lee, N.A, Eosinophil degranulation: an evolutionary vestige or a universally destructive effector function, Clin.Exp.Allergy 35, 986-994, 2005.
Eph Receptors/Ephrin Eph receptors are the largest family of receptor tyrosine kinases. The structure of Eph receptors is comprised of an extracellular domain, an intracellular domain which are linked by a transmembrane segment. Ephrin ligands bind to Eph receptors which are classified on the quality of the ephrin ligand; ephrin-A ligands bind to EphA receptors while ephrin-B ligands bind to EphB receptors. Eph receptors and ephrin ligand are integral components of cell
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Epistasis Expansins 997 surfaces and their interactions mediate growth and development. See Foo, S.S., Turner, C.J., Adams, S., et al., Ephrin-B2 controls cell motility and adhesion during blood-vessel-wall assembly, Cell 124, 161-173, 2006; Zhang, J., and Hughes, S., Role of the ephrin and Eph receptor tyrosine kinase families in angiogenesis and development of the cardiovascular system, J.Pathol. 208, 453-461, 2006; Haramis, A.P. and Perrakis, A., Selectivity and promiscuity in eph receptors, Structure 14, 169-171, 2006; Chrencik, J.E., Brooun, A., Recht, M.I., et al., Structure and thermodynamic characterization of the EphB4/Ephrin-B2 antagonist peptide complex reveals the determinants for receptor stability, Structure 14, 321-330, 2006.
Epistasis Masking of a phenotype caused by mutation of one gene by a mutation in another gene; Epistasis analysis can be used to define order of gene expression in a genetic pathway.
Epitome All epitopes present in the antigenic universe; Also defined as example, paradigm; a brief presentation or statement in most dictionaries.
Erk ½ p42/44 extracellular signal-regulated kinase, phosphorylated as a result of GPCR activation. A number of GPCR appear to converge at Erk 1/2. See Dhillon, A.S. and Kolch, W., Untying the regulation of the Raf-1 kinase, Arch.Biochem.Biophys. 404, 3-9, 2002; Chu, C.T., Levinthal, D.J., Kulich, S.M., et al., Oxidative neuronal injury. The dark side of ERK1/2, Eur.J.Biochem. 271, 2060-2066, 2004; Clark, M.J. and Traynor, J.R., Assays for G-protein-coupled receptor signaling using RGS-insensitive Galpha subunits, Methods Enzymol. 389, 155-169, 2004; Clark, A. and Sugden, P.M., Signaling through the extracellular signal-regulated kinase 1/2 cascade in cardiac myocytes, Biochem.Cell Biol. 82, 603-609, 2004.
Essential Oils A heterogeneous mixture of lipophilic substances obtained from a plant. Also referred to as absolute oils. Originally referred to as the steam distillate of the rinds of certain citrus fruits but extends for more recently used materials such as tea tree oil which is suggested to have some pharmacological use. These products are also used in aromatherapy. See Halcon, L. and Milkus, K., Staphyloccus aureus and wounds: a review of tea tree oil as a promising antimicrobial, Amer.J.Infect.Control 32, 402408, 2004; Kalemba, D. and Kunicka, A., Antibacterial and antifungal properties of essential oils, Curr.Med.Chem. 10, 813-829, 2003; Ranganna, S., Govindarajan, V.S., and Ramana, K.V., Citrus fruits—varieties, chemistry, technology, and quality evaluation. A Chemistry, Crit.Rev.Food Sci.Nutri. 18, 313-386, 1983.
EUROFAN EUROFAN (European Functional Analysis Netwood) was established to elucidate the physiological and biochemical functions of open reading frames in yeast; http://mips.gsf.de/proj/eurofan/ See Sanchez, J.C., Golaz, O., Frutiger, S., et al., The yeast SWISS-2DPAGE database, Electrophoresis 17, 556-565, 1996; Dujon, B., European Functional Analysis Netword (EUROFAN) and the functional analysis of the Saccharomyces cerevisiae
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genome, Electrophoresis 19, 617-624, 1998; Bianchi, M.M., Ngo, S., Vandenbol, M., et al., Large-scale phenotypic analysis reveals identical contributions to cell functions of known and unknown yeast genes, Yeast 18, 1397-1412, 2001; Avaro, S., Belgareh, N., Sibella-Arguelles, C., et al., Mutants defective in secretory/vacuolar pathways in the EUROFAN collection of yeast disruptants, Yeast 19, 351-371, 2002; Castrillo, J.I., Hayes, A., Mohammed, S., Gaskell, S.J., and Oliver, S.G., An optimized protocol for metabolome analysis in yeast using direct infusion electrospray mass spectrometry, Phytochemistry 62, 929-937. 2003; Davydenko, S.G., Juselius, J.K., Munder, T., Bogengruber, E., Jantti, J., and Keranen, S., Screening for novel essential genes of Saccharomyces cerevisiae involved in protein secretion, Yeast 21, 463-471, 2004.
Eutectic A mixture of components in such proportions that said mixture melts and solidifies as a single temperature lower than the melting points of the constituents or any other mixture thereof; a minimum transformation temperature between a solid solution and a mechanical mixture. This is an issue with cryobiology and therapeutic protein processing processes such as lyophilization. See Gutierrez-Merino, C., Quantitation of the Forster energy transfer for two-dimensional systems. II. Protein distribution and aggregation state in biological membranes, Biophys. Chem. 14, 259-266, 1981; Gatlin, L.A. and Nail, S.L., Protein purification process engineering. Freeze drying: a practical overview, Bioprocess Technol. 18, 317-367, 1994; Nail, S.L., Jiang, S., Chongprasert, S., and Knopp, S.A., Fundamentals of freeze-drying, Pharm. Biotechnol. 14, 281-360, 2002; Han, B., and Bischof, J.C., Thermodynamic nonequilibrium phase change behavior and thermal properties of biological solutions for cryobiology applications, J.Biomech.Eng. 126, 196-203, 2004.
Exosome A precise definition is a work in program but an exosome can be considered to be an intracellular membrane vesicle derived from fusion of endosomes with the plasma membrane. It is suggested that exosomes are involved in the intracellular transfer of molecules. See Févier, B. and Raposo, G., Exosomes: endosomal-derived vesicles shipping extracellular messages, Curr.Opin.Cell Biol. 16, 415-421, 2004; de Gassart, A., Géminard, C., Hoekstra, D., and Vidal, M., Exosome secretion: the art of reutilizing nonrecycled proteins? Traffic 5, 896-903, 2004; Chaput, N., Taïeb, Schartz, N., Flament, C., Novault, S., André, F., and Zitvogel, L. The potential of exosomes in immunotherapy of cancer, Blood Cells, Molecules, and Diseases 35, 111-115, 2005; Seaman, M.N.J., Recycle your receptors with retromer, Trends Cell Biol. 15, 68-75. 2005; Lencer, W.I. and Blumberg, R.S., A passionate kiss, then run: exocytosis and recycling of IgG by FcRn, Trends Cell Biol. 15, 5-9, 2005.
Exotoxicogenomics Study of the expression of genes important in the adaptive responses important in adaptive responses to toxic exposures.
Expansins Family of plant proteins essential for acid-induced cell wall loosening. See Cosgrove, D.J., Relaxation in a high-stress environment: The molecular basis of extensible cell walls and cell enlargement, Plant Cell 9, 1031-1041, 1997.
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Expressed Sequence Tag
998
Ferredoxin
Expressed Sequence Tag
Fenton Reaction
Usually an incomplete DAN sequence which can be “read” from either end of a gene fragment which is used as a “marker” or a “window” of gene presence in a genome; a short strand of DNA (approximately 200 base pairs long) which is usually unique to a specific cDNA and therefore can be used to identify genes and map their position in a genome. See Hartl, D.L., EST!EST!!EST!!!, Bioessays 18, 1021-1023, 1996; Gerhold, D. and Caskey, C.T., It’s the genes! EST access to human genome content, Bioessays 18, 973-981, 1996; Wilcox, A.S., Khan, A., Hopkins, J.A., and Sikela J.M., Use of 3’ untranslated sequences of human cDNA for rapid chromosome assignment and conversion to STS’s: implications for an expression map of the genome, Nucl.Acid Res. 19, 18371842, 1991; Hoffman, M., Gene expression patterns in human and mouse B cell development, Curr.Top.Microbiol.Immunol. 294, 19-29, 2005; Robson, P., The maturing of the human embryonic stem cell transcriptome profile, Trends Biotech. 22, 609-612, 2004.
Ferrous ion-dependent formation of hydroxyl radical from hydrogen peroxide; can be coupled with the oxidation of hydroxyl function to ketone/aldehydes; see Fenton, H.J.H., Oxidation of certain organic acids in the presence of ferrous salts, Proc.Chem. Soc. 15, 224-228, 1899; Goldstein, S., Meyerstein, D., and Czapski, G., The Fenton reagents, Free Rad.Biol.Med. 15, 435-445, 1993; Odyuo, M.M. and Sharan, R.N., Differential DNA strand breaking abilities of .OH and ROS generating radiomimetic chemicals and g-rays: study of plasmid dNA, pMTa4, in vitro, Free Rad.Res. 39, 499-505, 2005. Stadtman, E.R., Role of oxidized amino acids in protein breakdown and stability, Meth.Enzymol. 258, 379-393, 1995.
Expression Profiling The measurement or determination of DNA expression by the measurement RNA (transcriptomics); also used to refer to protein expression as determined by proteomic technology.
Expressional Leakage A concept where the functionally important expression of one gene can result in the ectopic expression of a neighboring gene resulting in apparent expression similarity between tissues. See de Marco, A. and de Marco, V., Bacteria co-transformed with recombinant proteins and chaperones cloned in independent plasmids are suitable for expression tuning, J.Biotechnol. 109, 45-52, 2004; Yanai, I., Korbel, J.O., Boue, S., McWeeney, S.K., Bork, P., and Lercher, M.J., Similar gene expression profiles do not imply similar tissue functions, Trends Genet. 22, 132-138, 2006.
Families of Structurally Similar Proteins (FSSP) A database based on three-dimensional comparisons of protein structures - http://ekhidna.biocenter.helsinki.fi/dali/start See Holm, L., Ouzounis, C., Sander, C., Tuparev, G., and Vriend, G., A database of protein structure families with common folding motifs, Protein Sci. 1, 1691-1698, 1992; Holm, L. and Sander, C., The FSSP database: fold classification based on structure alignment of proteins, Nucleic Acids Res. 24, 206-209, 1996; Notredame, C., Holm, L., and Higgins, D.G., COFFEE: an objective function for multiple sequence alignments, Bioinformatics 14, 407-422, 1998. Hadley, C. and Jones, D.T., A systematic comparison of protein structure classifications: SCOP, CATH and FSSP, Structure 7, 1099-1112, 1999; Getz, G., Vendruscolo, M., Sachs, D. and Domany, E., Automated assignment of SCOP and CATH protein structure classifications from FSSP scores, Proteins 46, 405-415, 2002; Edgar, R.C. and Sjolander, K., A comparison of scoring functions for protein sequence profile alignment, Bioinformatics 20, 1301-1308, 2004; Edgar, R.C. and Sjolander, K., COACH: profile-profile alignment of protein families using hidden Marikov models, Bioinformatics 20, 13091318, 2004.
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Fermentation The controlled aerobic or anaerobic process where a product is produced by yeast, molds, or bacteria from a substrate. Historically, fermentation was used to describe the action of a leavan (yeast) on a carbohydrate (saccharine) as in the production of beers and wines or a dough such as in making bread. In biotechnology manufacturing, the term fermentation is used to described the product of a biopharmaceutical by yeast or bacteria while the term cell culture is used to describe the use of animal cells or plants cells. See Wiseman, A., Principles of Biotechnology, Chapman and Hall, New York, NY, 1983; Sinclair, C.G., Kristiansen, B., and Bu’Lock, L.D., Fermentation Kinetics and Modelling, Open University Press, New York, NY, 1987; The Encyclopedia of Bioprocess Technology, ed. M.C.Flickinger and S.W. Drew, Wiley, New York, NY, 1999; Molecular Biology and Biotechnology, ed. J.M. Walker and R. Rapley, Royal Society of Chemistry, Cambridge, UK, 2000; Fermentation Biotechnology, ed. S.C. Badal, American Chemical Society, Washington, DC, 2003.
Ferredoxin A small protein which functions in the transport of electrons (reducing potential) in a variety of organisms. There are several classes of ferredoxins based on the nature of the chemistry of iron binding; Fe2S2; Fe3S4, Fe4S4. The iron is bound to cysteine residues in a cluster which also contains inorganic sulfur. See Mortenson, L.E., Nitrogen fixation: role of ferredoxin n anaerobic metabolism, Annu.Rev.Microbiol. 17, 115-138, 1963; Knaff, D.B., and Hirasawa, M., Ferredoxin-dependent chloroplast enzymes, Biochim.Biophys.Acta 1056, 93-125; Dai, S., Schwendtmayer, C., Johansson, K. et al., How does light regulate chloroplast enzymes? Structure-function studies of the ferredoxin/thioredoxin system, Q.Rev.Biophys. 33, 67-108, 2000; Schurmann, P., Redox signaling in the chloroplast: the ferredoxin/thioredoxin system, Antioxid. Redox.Signal. 5, 69-78, 2003; Carrillo, N., and Ceccarelli, E.A., Open questions in ferredoxin-NADP+ reductase catalytic mechanism, Eur.J.Biochem. 270, 1900-1915, 2003; Karplus, P.A. and Faber, H.R., Structural aspects of plant ferredoxin: NADP+ oxidoreductases, Photosynth.Res. 81, 303-315, 2004; Glastas, P., Pinotsis, N., Efthymiou, G., et al., The structure of the 2[4Fe-4S] ferredoxin from Pseudomonas aeruginosa at 1.32-A resolution: comparison with other high-resolution structures of ferredoxins and contributing structural features to reduction potential values, J.Biol.Inorg.Chem. 11, 445-458, 2006; Eckardt, N.A., Ferredoxin-thioredoxin system plays a key role in plant response to oxidative stress, Plant Cell 18, 1782, 2006.
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Ferret Diameter
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Ferret Diameter The longest chord of the project of a regular or irregular object as specific angles maximum, minimum, and average Ferret diameters can be determined by successive measurements. A value used in particle characterization; see M. Levin, Particle characterization – Tools and Methods, Laboratory Equipment, November, 2005.
Fibrillation The process of forming fibers from small, soluble polymeric materials. It is observed with amyloid fibrils in Alzheimer’s disease and with proteins during pharmaceutical processing. The term fibrillation was used in the 19th century to describe the physical changes in blood before the elucidation of fibrinogen clotting. The term fibrillation is also used to describe physical changes in structural materials with ligaments and tendons. See Arvinte, T., Cudd, A., and Drake, A.F., The structure and mechanism of formation of calcitonin fibrils, J.Biol.Chem. 268, 6415-6422, 1993; Ghosh, P. and Smith, M., The role of cartilage-derived antigens, pro-coagulant activity and fibrinolysis in the pathogenesis of osteoarthritis, Med.Hypotheses 41, 190-194, 1993; Bronfman, F.C., Garrido, J., Alvarez, A., Morgan, C. and Inestrosa, N.C., Laminin inhibits amyloid-beta-peptide fibrillation, Neurosci.Lett. 218, 201-203, 1996; Martin, J.A. and Buckwalter, J.A., Roles of articular cartilage aging and chondrocytes senesence in the pathogenesis of osteoarthritis, Iowa Orthop.J. 21, 1-7, 2001; Seyferth, S. and Lee, G., Structural studies of EDTA-induced fibrillation of salmon calcitonin, Pharm.Res. 20, 73-80, 2003; Librizzi, F. and Rischel, C., The kinetic behavior of insulin fibrillation is determined by heterogeneous nucleation pathways, Protein Sci. 14, 3129-3134, 2005; Westermark, P., Aspects on human amyloid forms and their fibril polypeptides, FEBS J. 272, 5942-5949, 2005; Pedersen, J.S., Dikov, D., Flink, J.L., et al., The changing face of glucagon fibrillation: structural polymorphism and conformational imprinting, J.Mol. Biol. 355, 501-523, 2006.
Fibroblast Growth Factor A group of peptide growth factors which regulate cell growth and proliferation.
Fibroblast Growth Factor Receptor(s) Receptor kinases which are activated by dimerization after ligand binding; Include FGFR1, FGFR2, FGFR3, FGFR4, FGFR5.
FixJ-FixL A two-component transcription regulatory system which is a global regulator of nitrogen fixation in Rhizobium meliloti, Mol. Microbiol. 5, 987-997, 1991. Sousa, F.H.S., Gonzalez, G. and Gilles-Gonazalez, M.-A., Oxygen blocks the reaction of FixL-FixJ complex with ATP but does not influence binding of FixJ or ATP to FixL, Biochemistry 44, 15359-15365, 2005.
FLAG™ FLAG™ has the sequence of AspTyrLysAspAspAsp-AspLys which includes an enterokinase cleavage site. This epitope tag can be used as a fusion partner for the expression and purification of recombinant proteins. See Einhauer, A. and Jungbauer, A.,
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The FLAG™ peptide, a versatile fusion tag for the purification of recombinant proteins, J.Biochem.Biophys.Methods 49, 455-465, 2001; Terpe, K., Overview of tag protein fusions: from molecular and biochemical fundamentals to commercial systems, Appl. Microbiol.Biotechnol. 60, 523-533, 2003; Lichty, J.J., Malecki, J.L., Agnew, H.D., Michelson-Horowitz, D.J., and Tan, S., Comparison of affinity tags for protein purification, Protein Exp.Purif. 41, 98-105, 2005.
Flap-Endonuclease (FEN; FEN-1) An enzyme with endonuclease and exonuclease activity encoded by the FEN1 gene. Cleaves branched DNA strucures including the 5’-end of Okazaki fragments. See Kunkel, T.A., Resnick, M.A., and Gordenin, D.A., Mutator specificity and disease: looking over the FENce, Cell 88, 155-158, 1997; Shen, B., Qiu, J., Hosfield, D., and Tainer, J.A., Flap endonuclease homologs in archaebacteria exist as independent proteins, Trends Biochem.Sci. 23, 171-173, 1998; Henneke, G., Freidrich-Heineken, E., and Hubscher, U., Flap endonuclease 1: a novel tumour suppressor protein, Trends Biochem.Sci. 28, 384-390, 2003; Kao, H.I. and Bambara, R.A., The protein components and mechanism of eukaryotic Okazaki fragment maturation, Crit.Rev.Biochem.Mol. Biol. 38, 433-452, 2003; Garg, P. and Burgers, P.M., DNA polymerases that propagate the eukaryotic DNA replication fork, Crit.Rev.Biochem.Mol.Biol. 40, 115-128, 2005; Olivier, M., The invader assay for SNF genotyping, Mutat.Res. 573, 103-110, 2005; Shen, B., Singh, P., Liu, R., et al., Multiple but dissectible functions of FEN-1 nucleases in nucleic acid processing, genome stability and diseases, Bioessays 27, 717729, 2005.
Flux Flux is the continuous flow of a substance. Flux can occur with electrons (Gutman, M., Electron flux through the mitochondrial ubiquinone, Biochim.Biophys.Acta 594, 53-84, 1980) and protons (Wang, J.H., Coupling of proton flux to the hydrolysis and synthesis of ATP, Annu.Rev.Biophys.Bioeng. 12, 21-34, 1983) as well and ions and other substances. Schwartz, A., Cell membrane Na+, K+ -ATPase and sarcoplasmic reticulum: possible regulators of intracellular ion activity, Fed.Proc. 35, 12791282, 1976; Meissner, G., Monovalent ion and calcium ion fluxes in sarcoplasmic reticulum, Mol.Cell.Biochem. 55, 65-82, 1983; Jones, D.P., Intracellular diffusion gradients of O2 and ATP, Am.J.Physiol. 250, C663-675, 1986; Hunter, M., Kawahara, K., and Giebisch, G., Calcium-activated epithelial potassium channels, Miner.Electrolyte Metab. 14, 48-57, 1988; , Cation Flux across Biomembranes, ed. Y. Mukohata, and L. Packer, Academic Press, New York, NY, 1979; Weir, E.K. and Hume, J.R., Ion Flux in Pulmonary Vascular Control, Plenum Press, New York, NY, 1993. Flux is defined in several ways: unidirectional influx is defined as the molar quantity of a solute passing across 1 cm 2 membrane in a unit period of time; unidirectional efflux is defined as the molar quantity of a solute crossing1 cm2 membrane outward from a cell in a unit period of time. Net flux is the difference between unidirectional influx and unidirectional efflux in a unit period of time. Understanding net flux is of importance in the design and interpretation of microdialysis studies (Schuck, V.J., Rinas, I., and Derendorf, H., In vitro microdialysis sample of docetaxel, J.Pharm.Biomed.Anal. 36, 807-813, 2004; Cano-Cebrian, M.J., Zornoza, T., Polache, A., and Granero, L., Quantitative in vivo microdialysis in pharmacokinetic studies: some reminders,
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Curr.Drug.Metab. 6, 83-90, 2005; Abrahamsson, P. and Winso, O., An assessement of calibration and performance of the microdialysis system, J.Pharm.Biomed.Anal. 39, 730-734, 2005.
Focal Adhesion A membrane area for cellular adhesion via actin filaments to the extracellular matrix/fibronectin resulting from the clustering of integrins. The interaction with fibronectin results in the formation of fibrillar adhesions considered to be more mature structures. Other intracellular proteins such as vincullin and focal adhesion kinase (FAK) are recruited to the actin cytoskeleton structure. See Otey, C.A. and Burridge, K., Patterning of the membrane cytoskeleton by the extracellular matrix, Semin. Cell Biol. 1, 391-399, 1990; Arikama, S.K., Integrins in cell adhesion and signaling, Hum.Cell 9, 181-186, 1996; Bershadsky, A.D. Balaban, N.Q., and Geiger, B., Adhesion-dependent cell mechanosensitivity, Annu.Rev.Cell Dev.Biol. 19, 677-695, 2003; Wozniak, M.A., Modzelekska, K., Kwong, L., and Keeley, P.J., Focal adhesion regulation of cell behavior, Biochim.Biophys.Acta 1692, 103119, 2004; Small, J.V. and Resch, G.P., The comings and goings of actin: coupling protrusion and retraction in cell motility, Curr. Opin.Cell Biol. 17, 517-523, 2005; Wu, M.H., Endothelial focal adhesions and barrier function, J.Physiol. 569, 359-366, 2005; Cohen, L.A. and Guan, J.L., Mechanisms of focal adhesion kinase regulation, Curr. Cancer Drug Targets 5, 629-643, 2005; Romer, L.H., Birukov, K.G., and Garcia, J.G., Focal adhesions: paradigm for a signaling nexus, Circ.Res. 98, 606-616, 2006; Legate, K.R., Montañez, O. and Fässler, R., ILK, PINCH and parvin: the tIPP of integrin signalling, Nat.Rev.Mol.Cell.Biol. 7, 20-31, 2006.
Fok1 Restriction endonuclease A type II restriction endonuclease isolated from Flavobacterium okeanokoites which has been used to identify DNA polymorphisms. There has been extensive use in the study of the vitamin D receptor gene (VDR gene). See Sugisaki, H. and Kanazawa, S., New restriction endonucleases from Flavobacterium okeanokoites (FokI) and Micrococcus luteus (MluI), Gene 16, 73-78, 1981; Kato, A., Yakura, K., and Tanifuji, S., Sequence analysis of Vicia faba repeated DNA, the FokI repet element, Nucleic Acids Res. 24, 6415-6426, 1984; Kita, K., Kotani, H., Sugisaki, H., and Tanami, M., The foci restriction-modification system. I. Organization and nucleotide sequences of the restriction and modification genes, J.Biol.Chem. 264, 5751-5756, 1989; Posfai, G. and Szybalski, W., A simple method for locating methylated based in DNA using class-IIS restriction enzymes, Gene 74, 179-181, 1988; Aggarwal, A.K. and Wah, D.A., Novel site-specific DNA endonucleases, Curr.Opin.Struct.Biol. 8, 19-25, 1998; Kovall, R.A. and Matthews, B.W., Type II restriction endonucleases: structural, functional and evolutionary relationships, Curr.Opin.Chem.Biol. 3, 578583, 1999; Akar, A., Orkunoglu, F.E., Ozata, M., Sengul, A., and Gur, A.R., Lack of association between vitamin D receptor FokI polymorphism and alopecia areata, Eur.J.Dermatol. 14, 156-158, 2004; Guy, M., Lowe, L.C., Bretherton-Watt, D., et al., Vitamin D receptor gene polymorphisms and breast cancer risk, Clin.Cancer Res. 10, 5472-5481, 2004; Claassen, M., Nouwen, J., Fang, Y., et al., Staphylococcus aureus nasal carriage is not associated with known polymorphism in the vitamin D receptor gene, FEMS Immunol.Med.Microbiol. 43, 173-176, 2005; Bolu, S.E., Orkunoglu Suer, F.E., Deniz, F., et al., The vitamin D receptor foci start codon polymorphism and bone mineral density in male hypogonadotrophic hypogonadism, J.Endocrinol.Invest. 28, 810-814, 2005.
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Foldamers Single chain polymers which can adopt secondary structure in solution and thus mimic proteins, nucleic acids, and polysaccharides; polymeric backbones which have well-defined and predictable folding properties in the solvent of choice. See Appella, D.H., Christianson, L.A., Klein, D.A., et al., Residuebased control of helix shape in beta-peptide oligomers, Nature 387, 381-384, 1997; Tanatani, A., Mio, M.J., and Moore, J.S., Chain length-dependent affinity of helical foldamers for a rodlike guest, J.Amer.Chem.Soc. 123, 1792-1793, 2001; Cubberley, M.S. and Iverson, B.L., Models of higher-order structure: foldamers and beyond, Curr.Opin.Chem.Biol. 5, 650-653, 2001; Hill, D.J., Mio, M.J., Prince, R.B., Hughes, T.S. and Moore, J.S., A field guide to foldamers, Chem.Rev. 101, 393-4012, 2001; Martinek, T.A. and Fulop, F., Side-chain control of beta-peptide secondary structures, Eur.J.Biochem. 270, 3657-3666, 2003; Sanford, A.R., Yamato, K., Yang, X., Yuan, L., Han. Y., and Gong, B., Well-defined secondary structures, Eur.J.Biochem. 271, 14161425, 2004; Cheng, R.P., Beyond de novo protein design—de novo design of non-natural folded oligomers, Curr.Opin.Struct. Biol. 14, 512-520, 2004; Stone, M.T., Heemstra, J.M., and Moore, J.S., The chain-length dependence test, Acc.Chem.Res. 39, 11-20, 2006; Schmitt, M.A., Choi, S.H., Guzei, I.A., and Gellman, S.H., New helical foldamers: heterogeneous backbones with 1:2 and 2:1 alpha:beta-amino acid residue patterns, J.Am.Chem.Soc. 128, 4538-4539, 2006.
Fragnomics The use of smaller molecules in the drug discovery process; Zartler, E.R. and Shapiro, M.J., Fragnomics: fragment-based drug discovery, Curr.Opin.Chem.Biol. 9, 366-370, 2005.
Frass Frass is debris or excrement produced by insects. This material is thought be involved with role of cockroaches in the development of asthma (See Page, K., Hughes, V.S., Bennett, G.W., and Wong, H.R., German cockroach proteases regulate matrix metalloproteinase-9 in human bronchial epithelial cells, Allergy 61, 988-995, 2006.
Free Radical/radical A molecule containing an unpaired electron can be electrically neutral. Free radicals may be created by the hemolytic cleavage of a precursor molecule free radicals can be formed by thermolytic cleavage, photolysis (ultraviolet light photolysis of hydrogen peroxide to form hydroxyl radical), radiolysis (ionizing radiation of water to form hydroxyl radical) or by homolytic cleavage with the participation of another molecule (i.e. Fenton Reaction). Perkins, J., Radical Chemistry: The Fundamentals, Oxford University Press, Oxford, United Kingdom, 2000.
fret (Fluorescence Resonance Energy Transfer) A technique for assaying the proximity of region by observed energy transfer between fluorophores. A concept similar to fluorescence quenching. With two-photon excitation, studies can be extended to the study of in vivo interactions with microscopy. See also BRET. See Milligan, G. and Bouvier, M., Methods to monitor the quaternary structure of G protein-coupled receptors,
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Freund’s Adjuvant
1001
FASEB J. 272, 2914-2925, 2005; Rasnik, I., McKinney, S.A., and Ha, T., Surfaces and orientations: much to FRET about? Acc. Chem.Res. 38, 542-548, 2005; Gertler, A., Biener, E., Ramamujan, K.V., Dijiane, J., and Herman, B., Fluorescence resonance energy transfer (FRET) microscopy in living cells as a novel tool for the study of cytokine action, J.Dairy Res. 72, Spec. No., 14-19, 2005; Cudakov, D.M., Lukyanov, S. and Lukyanov, K.A., Fluorescent proteins as a toolkit for in vivo imaging, Trends Biotechnol. 23, 605-613, 2005; Zal, T. and Gascoigne, N.R., Using live FRET imaging to reveal early protein-protein interactions during T cell activation, Curr.Opin.Immunol. 16, 674-683, 2004.
Freund’s Adjuvant A mixture of killed/lyophilized Mycobacterium bovis or Mycobacterium tuberculosis cells and oil resulting in an emulsion (referred to as Complete Freund’s adjuvant) used with an antigen to improve the immune response (antibody formation secondary to B-cell activation). Incomplete Freund’s adjuvant does not contain the bacterial cells and is used to avoid an inflammatory response. See White, R.G., Factor affecting the antibody response, Br.Med.Bull. 19, 207-213, 1963; White, R.G., Antigen adjuvants, Mod.Trends Immunol. 2, 28-52, 1967; Myrvik, Q.N., Adjuvants, Ann.N.Y.Acad.Sci. 221, 324-330, 1974; Osebold, J.W., Mechanisms for action by immunologic adjuvants, J.Am.Vet. Med.Assoc. 181, 983-987, 1982; Warren, H.S., Vogel, F.R., and Chedid, L.A., Current status of immunological adjuvants, Annu. Rev.Immunol. 4, 369-388, 1986; Claassen, E., de Leeuw, W., de Greeve, P., Hendriksen, C., and Boersma, W., Freund’s complete adjuvant: an effective but disagreeable formula, Res.Immunol. 143, 478-483, 1992; Billiau, A. and Matthys, P., Modes of action of Freund’s adjuvants in experimental models of autoimmune diseases, J.Leukoc.Biol. 70, 849-860, 2001; Cachia, P.J., Kao, D.J., and Hodges, R.S., Synthetic peptide vaccine development: measurement of polyclonal antibody affinity and cross-reactivity using a new peptide capture and release system for surface plasmon resonance spectroscopy, J.Mol.Recog. 17, 540-557, 2004; Stills, H.F., Jr., Adjuvants and antibody production: dispelling the myths associated with Freund’s complete and other adjuvants, ILAR J. 46, 280-293, 2005; Miller, L.H., Saul, A., and Mahanty, S., Revisiting Freund’s incomplete adjuvant for vaccines in the developing world, Trends Paristol. 21, 412-414, 2005.
Functional Genomics Functional genomics refers to establishing a verifiable link between gene expression and cell/organ/tissue function/dysfunction. Functional genomics refers to establishing a verifiable link between gene expression and cell/organ/tissue function/ dysfunction. See Evans, M.J., Carlton, M.B., and Russ, A.P., Gene trapping and functional genomics, Trends Genet. 13, 370-374, 1997; Schena, M., Heller, R.A., Theriault, T.P., et al., Microarrays: biotechnology’s discovery platform for functional genomics, Trends Biotechnol. 16, 301-306, 1998; Holtorf, H., Guitton, M.C., and Reski, R., Plant functional genomics, Naturewissenschaften 89, 235-249, 2002; Bader, G.D., Heilbut, A., Andrews, B., et al., Functional genomics and proteomics: charting a multidimensional map of the yeast cell, Trends Cell Sci.Biol. 13, 344-356, 2003; Kemmeren, P. and Holstege, F.C., Integrating functional genomics data, Biochem.Soc.Trans. 31, 1484-1487, 2003; Werner, T., Proteomics and regulomics: the yin and yang of functional genomics, Mass Spectrom.Rev. 23, 25-33, 2004; Brunner, A.M., Busov, V.B., and Strauss, S.H., Poplar genome sequence: functional
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genomics in an ecologically dominant plant species, Trends Plant Sci. 9, 49-56, 2004; Hughes, T.R., Robinson, M.D., Mitsakakis, N., and Johnston, M., The promise of functional genomics: completing the encyclopedia of a cell, Curr.Opin.Microbiol. 7, 546-554, 2004; Kramer, R. and Cohen, D., Functional genomics to new drug targets, Nat.Rev.Drug Discov. 3, 965-972, 2004; Vanhecke, D. and Janitz, M., Functional genomics using high-throughput RNA interference, Drug Discov.Today 10, 205-212, 2005; Sauer, S., Lange, B.M., Gobom, J., et al., Miniaturization in functional genomics and proteomics, Nat.Rev.Genet. 6, 465-476, 2005; Stoeckert, C.J., Jr., Functional genomic databases on the web, Cell Microbiol. 7, 1053-1059, 2005; Foti, M., Grannuci, F., Pelizzola, M., et al., Dendritic cells in pathogen recognition and induction of immune response: a functional genomics approach, J.Leukoc. Biol. 79, 913-916, 2006; Hunt, S.P. and Livesey, R., Functional Genomics: A Practical Approach, Oxford University Press, Oxford, UK, 2000; Functional Genomics Methods and Protocols, ed. M.J.Brownstein and A.B. Khodursky, Humana Press, Totowa, NJ, 2003; Grotewold, E., Plant Functional Genomics, Humana Press, Totowa, NJ, 2003; Zhou, J., Microbial Functional Genomics, Wiley-Liss, Hoboken, NJ, 2004.
Functional Proteomics A broad area of enquiry encompassing the study of the function of proteins in the proteome, study of changes in protein expression within the proteome, and the use of reactive chemical probes to identify enzymes in the proteome. This short list is not meant to be wholly inclusive. See Lawrence, D.S., Functional proteomics: large-scale analysis of protein kinase activity, Genome Biol. 2, REVIEWS1007, 2001; Famulok, M., Blind, M., and Mayer, G., Intramers as promising new tools in functional proteomics, Chem.Biol. 8, 931-939, 2001; Guengerich, F.P., Functional genomics and proteomics applied to the study of nutritional metabolism, Nutr.Rev. 59, 259-263, 2001; Strosberg, A.D., Functional proteomics to exploit genome sequences, Cell.Mol.Biol. 47, 1295-1299, 2001; Yanagida, M., Functional proteomics: current achievements, J.Chromatog.B.Anal.Technol.Biomed.Life Sci. 771, 89-106, 2002; Hunter, T.C., Andon, N.L., Koller, A., Yates, J.R., and Haynes, P.A., J.Chromatogr.B.Analyt.Technol.Biomed.Life Sci. 782, 165-181, 2002; Graves, P.R. and Haystead, T.A., A functional proteomics approach to signal transduction, Recent Prog. Horm.Res. 58, 1-14, 2003; Ilag, L.L., Functional proteomic screens in therapeutic protein drug discovery, Curr.Opin.Mol.Ther. 7, 538542, 2005; Wagner, V., Gessner, G., and Mittag, M., Functional proteomics: a promising approach to find novel components of the circadian system, Chronobiol.Int. 22, 403-415, 2005; Monti, M., Orru, S., Pagnozzi, D., and Pucci, P., Functional proteomics, Clin.Chim.Acta 357, 140-150, 2005.
Furin Furin is a subtilisin-like regulatory protease (subtilisn-like proprotein convertases located in the trans-Golgi network which functions in processing precursor proteins in the secretory pathway. See Molloy, S.S., Bresnahan, P.A., Leppla, S.H. et al., Human furin is a calcium-dependent serine endoprotease that recognizes the sequence Arg-X-X-Arg and efficiently cleaves anthrax protective antigen, J.Biol.Chem. 267, 16396-16402, 1992; Yanagita, M., Hoshimo, H., Nakayama, K., and Takeuchi, T., Processing of mutated proteins with tetrabasic cleavage sites to mature insulin reflects the expression of furin in nonendocrine cell lines, Endocrinology 133, 639-644, 1993; Brennan, S.O. and
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Gamma(g)-secretase
1002
Nakayama, K., Furin has the proalbumin substrate specificity and serpin inhibitor properties of an in situ convertase, FEBS Lett. 338, 147-151, 1994; Roebroek, A.J., Creemers, J.W., Ayoubi, T.A., and Van de Ven, W.J., Furin-mediated proprotein processing activity: involvement of negatively charged amino acid residues in the substrate binding site, Biochemie 76, 210-216, 1994; Denault, J.B. and Leduc, R., Furin/PACE/SPC1: a convertase involved in exocytic and endocytic processing of precursor proteins, FEBS Lett. 379, 113-116, 1996; Nakayama, K., Furin: a mammalian subtilisin/Kex2p-like endoprotease involved in processing of a wide variety of precursor proteins, Biochem.J. 327, 625-635, 1997; Rockwell, N.C., Krysan, D.J., Komiyama, T., and Fuller, B.S., Precursor processing by kex2/furin proteases, Chem. Rev. 102, 4525-4548, 2002; Fugere, M., Limperis, P.C., BeaulieuAudy, V., Gagnon, F., et al., Inhibitory potency and specificity of subtilisin-like pro-protein convertase (SPC) prodomains, J.Biol.Chem. 277, 7648-7656, 2002; Rockwell, N.C. and Thorner, J.W., The kindest cuts of all: crystal structures of kex2 and furin reveal secrets of precursor processing, Trends Biochem.Sci. 29, 80-87, 2004; The first proprotein processing proteins was described as Kex2 protease(kexin) in Saccharomyces cerevesiae (Leibowtiz, M.J. and Wickner, R.B., A chromosomal gene required for killer plasmid expression, mating, and spore maturation in Saccharomyces cerevisiae, Proc.Natl.Acad.Sci.USA 73, 2061-2065, 1976; Rogers, D.T., Saville, D., and Bussey, H., Saccharomyces cerevisiae expression mutant kex2 has altered secretory proteins and glycoproteins, Biochem.Biophys.Res. Commun. 90, 187-193, 1979; Julius, D. Brake, A., Blair, L., et al., Isolation of the putative structural gene for the lysine-argininecleaving endopeptidase required for processing of yeast preproalpha-factor, Cell 37, 1075-1089, 1984). Furin is important for the secretion of recombinant proteins in mammalian cell lines (Mark, M.R., Lokker, N.A., Zioncheck, T.F., et al., Expression and characterization of heptocyte growth factor receptor-IgG fusion proteins: effects of mutations in the potential proteolytic cleavage site on processing and ligand binding, J.Biol.Chem. 267, 26166-26171, 1992; Bristol, J.A., Freedman, S.J., Furie, B.C., and Furie, B., Profactor IX: the propeptide inhibits binding to membrane surfaces and activation by factor IXa., Biochemistry 33, 14136-14143, 1994; Groskreutz, D.J., Sliwkowski, M.X., and Gorman, C.M., Genetically engineering proinsulin constitutively processed and secreted as mature, active insulin, J.Biol. Chem. 269, 6241-6245, 1994; Lind, P., Larsson, K., Spira,J., et al., Novel forms of B-domain deleted recombinant factor VIII molecules. Construction and biochemical characterization, Eur.J.Biochem. 232, 19-27, 1995; Ayoubi, T.A., Meulemans, S.M. Roebroek, A.J., and Van de Ven, W.J., Production of recombinant proteins in Chinese hamster ovary cells overexpressing the subtilisin-like proprotein converting enzyme furin, Mol.Biol. Rep. 23, 87-95, 1996; Chiron, M.F., Fryling, C.M., and Fitzgerald, D., Furin-mediated cleavage of Pseudomonas exotoxin-derived chimeric toxins, J.Biol.Chem. 272, 31707-31711, 1997. Furins are functionally related to secretases in being protein precursor processing enzymes (Anders, L., Mertins, P., Lammich, S., et al., Furin-, ADAM 10-, and g-secretase-mediated cleavage of a receptor tyrosine phosphatase and regulation of b-cateinin’s transcriptional activity, Mol.Cell.Biol. 26, 3917-3924, 2006). There has been some work on the possible role of furin in the processing of b-secretase (Bennett, B.D., Denis, P., Haniu, M., et al., A furin-like convertase mediates propeptide cleavage of BACE, the Alzheimer’s b-secretase, J.Biol.Chem. 275, 37712-37717, 2000; Creemers, J.W.M., Dominguez, D.I., Plets, E., et al., Processing of b-secretase by furin and others members of the proprotein
9168_Book.indb 1002
G protein
convertase family, J.Biol.Chem. 276, 4211-4217, 2001; Pinnix, I., Council, J.E., Roseberry, B., et al., Convertases other than fuin cleave b-secretase to its mature form, FASEB J. 15, 1810-1812, 2001.
Gamma(g)-secretase A membrane-associated regulatory protease responsible for the cleavage of amyloid precursor protein and notch protein. Gamma(g)-secretase is composed of four subunits, presenilin, nicastrin, Aph-1 and Pen-2. Presenilin is responsible for the catalytic gamma(g)-secretase activity and nicastrin and Aph-2 have a function in substrate recognition and complex stabilization while Pen-2 assists in catalytic function. Gamma(g)-secretase is a therapeutic treatment for Alzheimer’s disease. See Mundy, D.L., Identification of the multicatalytic enzyme as a possible g-secretase for the amyloid precursor protein, Biochem.Biophys.Res. Commun. 204, 333-341. 1994; Wolfe, M.S. and Haass, C., The role of presenilins in gamma-secretase activity, J.Biol.Chem. 276, 54135416, 2001; Sisodia, S.S. and St. George-Hyslop, P.H., g-Secretase, Notch, Ab and Alzheimer’s disease: Where do the presenilins fit in?, Nat.Rev.Neurosci. 3, 281-290, 2002; Kimberly, W.T. and Wolfe, M.S., Identify and function of gamma-secretases, J.Neurosci.Res. 74, 353-360, 2003; Iwatsubo, T., The gamma secretase complex: machinery for intramembrane proteolysis, Curr.Opin.Neurobiol. 14, 379-383, 2004; Raemakers, T., Esselens, C., and Annaert, W., Presenilin 1: more than just gamma-secretase, Biochem.Soc.Trans. 33, 559-562, 2005; De Strooper, B., Nicastrin: gatekeeper of the gamma-secretase complex, Cell 122, 318-320, 2005; Churcher, I., and Beher, D., Gamma-secretase as a therapeutic target for the treatment of Alzheimer’s disease, Curr.Pharm.Des. 11, 33633382, 2005; Barten, D.M., Meredith, J.E.,Jr., Zaczek, R., et al., Gamma-secretase inhibitors for Alzheimer’s disease: balancing efficacy and toxicity, Drugs R & D 7, 87-97, 2006. Wolfe, M.S., The g-secretase complex: membrane-embedded proteolytic ensemble, Biochemistry 45, 7931-7939, 2006.
G protein A heteromeric protein which functions in signal transduction via modulation by G protein-coupled receptors (GPCRs). See Spiegel, A.M. and Downs, R.W., Jr., Guanine nucleotides: key regulators of hormone receptor-adenylate cyclase, Endocr.Rev. 2, 275-305, 1981; Cooper, D.M. and Londos, C., GTP-stimulation and inhibition of adenylate cyclase, Horiz.Biochem.Biophys. 6, 309-333, 1982; Poste, G., New insights into receptor regulation, J.Appl. Physiol. 57, 1297-1305, 1984; Cuatrecasas, P., Hormone receptors, membrane phospholipids, and protein kinases, Harvey Lect. 80, 89-128, 1984-1985; Neer, E.J., Guanine nucleotide-binding proteins involved in transmembrane signaling, Symp.Fundam. Cancer Res. 39, 123-136, 1986; Spiegel, A.M., Signal transduction by guanine nucleotide binding proteins, Mol.Cell.Endocrinol. 49, 1-16, 1987; Bockaert, J., Homburger, V., and Rouot, B., GTP binding proteins: a key role in cellular communication, Biochimie 69, 329-338, 1987; Zhang, Z., Melia, T.J., He, F., et al., How a G protein binds a membrane, J.Biol.Chem. 279, 33937-33945, 2004; Gavi, S., Shumay, E., Wang, H.Y., and Malbon, C.C., G-proteincoupled receptors and tyrosine kinases: crossroads in cell signaling and regulation, Trends Endocrinol.Metab. 17, 48-54, 2006; Sato, M., Blumer, J.B., Simon, V., and Lanier, S.M., Accessory proteins for G proteins: partners in signaling, Annu.Rev.Pharmcol. Toxicol. 46, 151-187, 2006; Houslay, M.D. and Milligan, G., G-Proteins as Mediators of Cellular Signaling Processes, Wiley, Chichester, UK, 1990; Naccache, P.H., G Proteins and Calcium
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G-Protein Coupled Receptor (GPCR)
1003
Signaling, CRC Press, Boca Raton, FL, 1990; Johnson, R.A. and Corbin, J.D., Adenyl Cyclase, G Proteins, and Guanylyl Cyclase, Academic Press, San Diego, CA, 1991; Ravi, I., Heterotrimeric G Proteins, Academic Press, San Diego, CA, 1994; Watson, S.P., and Arkinstall, S., The G-Protein Linked Receptor Factbooks, Academic Press, London, UK, 1994; Siderovski, D.P., G Proteins and Calcium Signaling, Elsevier Academic Press, Amsterdam, Netherlands, 2004.
G-Protein Coupled Receptor (GPCR) A membrane receptor which is functional linked to the activation of a trimeric G protein complex characterized by the presence of seven transmembrane segments.
Ga Protein The alpha-subunit of the heterotrimeric G protein which separates into a Ga-protein-GTP complex when GTP replaces GDP. See Albert, P.R. and Robillard, L., G protein specificity: traffic direction required, Cell Signalling 14, 407-418, 2002; Kurose, H., Ga12 and Ga13 as key regulatory mediator in signal transduction, Life Sci. 74, 155-161, 2003; Kostenis, E., Waelbroeck, M., and Milligan, G., Techniques: promiscuous Ga proteins in basic research and drug discovery, Trends Pharmacol.Sci. 26, 595-602, 2005; Herrman, R., Heck, M., Henklein, P., et al., Signal transfere from GPCRs to G proteins: role of the Ga N-terminal region in rhodopsin-transducin coupling, J.Biol.Chem, 281, 30234–30241, 2006.
Gelsolin; Gelsolin-like domains Gelsolin is signature protein for a family of protein which interact with actin and influence the structure of the cytoskeleton. Gelsolin is a calcium-dependent actin-binding protein that modulates actin filament length. See Yin, H.L., Hartwig, J.H., Maruyama, K., and Stossel, T.P., Ca 2+ control of actin filament length. Effects of macrophage gelsolin on actin polymerization, J.Biol.Chem. 256, 9693-9697, 1981; Matasudaira, P., Jakes, R., and Walker, J.E., A gelsolin-like Ca 2+ -dependent actin-binding domain in villin, Nature 315, 248-250, 1985; Dixon, R.A.F., Kobilka, B.K., Strader, D.J., Cloning of the gene and cDNA for mammalian b-adrenergic receptor and homology with rhodopsin, Nature 321, 75-79, 1986; Libert, F., Parmentier, M., Lefort, A., et al., Selective amplification and cloning of four new members of the G protein-coupled receptor family, Science 244, 569572, 1989; Yu, F.X., Zhou, D.M., and Yin, H.L., Chimeric and truncated gCap39 elucidate the requirements for actin filament severing and end capping by the gelsolin family of proteins, J.Biol.Chem. 266, 19269-19275, 1991; Wen, D., Corina, K., Chow, E.P., et al., The plasma and cytoplasmic forms of human gelsolin differ in disulfide structure, Biochemistry 35, 9700-9709, 1996; Isaacson, R.L., Weeds, A.G., and Fersht, A.R., Equilibria and kinetics of folding of gelsolin domain 2 and mutants involved in familial amyloidosis-Finnish type, Proc.Natl.Acad.Sci.USA 96, 11247-11252, 1996; Liu, Y.T. and Yin, H.L., Identification of the binding partners for flightless I, A novel protein bridging the leucine-rich repeat and the gelsolin superfamilies, J.Biol. Chem. 273, 7920-7927, 1998; Benyamini, H., Gunasekaran, K., Wolfson, H., and Nussinov, R., Conservation and amyloid formation: a study of the gelsolin-like family, Proteins 51, 266-282, 2003; Uruno, T., Remmert, K., and Hammer, J.A., 3rd., CARMIL is a potent capping protein antagonist: identification of a conserved CARMIL domain that inhibits the activity of capping
9168_Book.indb 1003
General Transcription Factors
protein and uncaps capped actin filaments, J.Biol.Chem. 281, 10635-10650, 2006.
General Transcription Factors These are protein cofactors for RNA polymerase II which are required for the function of the basal transcription apparatus. The basal apparatus can be described as the functional unit required for the accurate transcription of DNA and is directed to the 5’-end of a transcriptional unit by the core promoter. See Zheng, X.M., Moncollin, V., Egly, J.M., and Chambon, P., A general transcription factor forms a stable complex with RNA polymerase B(II), Cell 50, 361-368, 1987; DeJong, J., Bernstein, R. and Roeder, R.G., Human general transcription factor TFIIA: characterization of a cDNA encoding the small subunit and requirement for basal and activated transcription, Proc.Natl.Acad.Sci. USA 92, 3313-3317, 1995; Zaid, A., Li, R., Luciakova, K., et al., On the role of the general transcription factor Sp1 in the activation and repression of diverse mammalian oxidative phosphorylation genes, J.Bioenerg.Biomembr. 31, 129-135, 1999: Smale, J.T., Core promoter architecture for eukaryotic protein-coding genes, in Transcription: Mechanism and Regulation, ed. R.C. Conoway and J.W. Conoway, Raven Press, New York, NY, Chapter 5, pps. 63-81, 1999; Serizawa, H., Conoway, J.W. and Conaway, R.C., Transcriptional initiation by mammalian RNA polymerase II, in Transcription: Mechanism and Regulation, ed. R.C. Conaway and J.W.Conaway, Raven Press, New York, NY, Chapter 3, pps. 27-43, 1999; Pugh, B.R., RNA polymerase II transcription machinery, in Transcription Factors, ed. J. Locker, Bios/Academic Press, Oxford, UK, Chapter 1, pp. 1-16, 2001; Reid, J., Murray, I., Watt, K., et al., The androgen receptor interacts with multiple regions of the large subunit of general transcription factor TFIIF, J.Biol. Chem. 277, 41247-41253, 2002; Vullhorst, D. and Buonanno, A., Characterization of general transcription factor 3, a transcription factor involved in slow muscle-specific gene expression, J.Biol.Chem. 278, 8370-8379, 2003; Takagi, Y. and Kornberg, R.D., Mediator as a general transcription factor, J.Biol.Chem. 281, 80-89, 2006; Choudhry, M.A., Ball, A., and McEwan, I.J., The role of the general transcription factor TFIID in androgen receptordependent transcription, Mol.Endocrinol., 20, 2052–2061, 2006.
Generic Drug A generic drug is the same as a brand name drug is dosage, safety, and efficacy. Prior to approval, a generic drug must demonstrate bioequivalence or therapeutic equivalence.
Gene Expression Domain A genomic region that contains a gene and all of the cis-acting elements that are required to obtain the homeostatic level and timing of gene expression in vivo. Gene expression domains are generally defined by their ability to function independently of the site of integration into a transgene.
General Transcription Factors A group of trans-acting factors which have a central role in the initiation of transcription by RNA polymerase II (pol II). The components are likely similar to the earlier described basal transcription factors. Greenblatt, J., RNA polymerase-associated transcription factors, Trends Biochem.Sci. 16, 408-411, 1991; Corden, J.L, RNA polymerase II transcription cycles, Curr.Opin.
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Generic Drug
1004
Genet.Dev. 3, 213-218, 1993; Travers, A., Transcription: building an initiation machine, Curr.Biol. 6, 401-403, 1996; Reese, J.C., Basal transcription factors, Curr.Opin.Genet.Dev. 13, 114-118, 2003; Asturias, F.J., RNA polymerase II structure, and organization of the preinitiation complex, Curr.Opin.Struct.Biol. 14, 121129, 2004; Boeger, H., Bushnell, D.A., Davis, R., et al., Structural basis of eukaryotic gene transcription, FEBS Lett. 579, 899-903, 2005; Szutarisz, H., Dillon, N., and Tora, L., The role of enhancers as centres for general transcription factor recruitment, Trends Biochem.Sci. 30, 593-599, 2005; Gross, P. and Oelgeschlager, T., Core promoter-selective RNA polymerase II transcription, Biochem. Soc.Symp. (73), 225-236, 2006.
Generic Drug A generic drug is the same as a brand name drug is dosage, safety, strength, administration, quality, and intended use. The suitability of a generic drug is based on “therapeutic equivilance.” By law, a generic product must contain the identical amounts of the same active ingredient(s) as the brand name product. See Verbeeck, R.K., Kanfer, I., and Walker, R.B., Generic substitution: the use of medicinal products containings different salts and implications for safety and efficacy, Eur.J.Pharm.Sci. 28, 1-6, 2006; Devine, J.W., Cline, R.R., and Farley, J.F., Follow-on biologics: competition in the biopharmaceutical marketplace, J.Am.Pharm.Assoc. 46, 193201, 2006.
Genome The complete gene complement of any organism, contained in a set of chromosomes in eukaryotes, a single chromosome in bacteria, or a DNA or RNA molecule in viruses; the complete set of genes inside the cell or virus. Singer, M. and Berg, P., Genes & Genomes: A Changing Perspective, University Science Book, Mill Valley, CA, USA, 1991; Murray, T.H. and Rothstein, R.A., The Human Genome Project and the Future of Health Care, Indiana University Press, Bloomington, Indiana, USA, 1996; Brown, T.A., Genome, Bios Scientific Publishers/Wiley-Liss, New York, NY, 1999; Ridley, M., Genome:The Autobiography of a Species of 23 Chapters, HarperCollins, New York, NY, 1999.
Genome-Based Proteomics Gene-based analysis of the proteome; analytical strategies based on the knowledge of the genome. See Rosamond, J. and Allsop, A., Harnessing the power of the genome in the search for new antibiotics, Science 287, 1973-1976, 2000; Agaton, C., Uhlen, M., and Hober, S., Genome-based proteomics, Electrophoresis 25, 1280-1288, 2004; Wisz, M.S., Suarez, M.K., Holmes, M.R., and Giddings, M.C., GFSWeb: a web tool for genome-based identification of proteins from mass spectrometric samples, J.Proteome Res. 3, 1292-1295, 2004; Romero, P., Wagg, J., Green, M.L., et al., Computational prediction of human metabolic pathways from the complete human genome, Genome Biol. 6, R2, 2005; Ek. S., Adreasson, U., Hober, S., et al., From gene expression analysis to tissue microarrays: a rational approach to identify therapeutic and diagnostic targets in lymphoid malignancies, Mol.Cell. Proteomics 5, 1072-1081, 2006.
Genomic Databases See Baxevaris, A.D., Using genomic databases for sequence-based biological discovery, Molec.Med. 9, 185-192, 2003.
9168_Book.indb 1004
Glass Transition/Glass Transition Temperature
Genomics The study of the structure and function of the genome, including information about the sequence, mapping, and expression, and how genes and their products work in the organism; the study of the genetic composition of organisms.
Genotype The internally coded, inheritable information carried by all living organisms; the genetic constitution of an organism.
Glass/Glasses A large inhomogenous class of materials with highly variable mechanical and optical properties that solidify from the molten state without crystallization. The cooling of the melt must occur without crystallization. Glasses are most frequently derived from silicates by fusing with boric oxide, aluminum oxide, or phosphorus pentoxide. Glasses are generally hard, brittle, and transparent or translucent, and are considered to be supercooled liquids rather than true solids. See Santoro, M., Gorelli, F.A., Bini, F., et al., Amorphous silica-like carbon dioxide, Nature 441, 857-860, 2006; Huang, W., Day, D.E., Kittiratanapiboon, K., and Rahaman, M.N., Kinetics and mechanisms of the conversion of silicate (45S5), borate, and borosilicate glasses to hydroxyapatite in dilute phosphate solutions, J.Mater.Sci.Mater.Med. 17, 583-596, 2006; Abraham, S., Mallia, V.A., Ratheesh, K.V., et al., Reversible thermal and photochemical switching of liquid crystalline phases and luminescence in diphenylbutadienebased mesogenic dimers, J.Am.Chem.Soc. 128, 7692-7698, 2006; Lehner, A., Corbineau, F., and Bailly, C., Changes in lipid status and glass properties in cotyledons of developing sunflower seeds, Plant Cell Physiol., 47, 818–828, 2006; Chang, R. and Yethiraj, A., Dynamics of chain molecules in disordered materials, Phys. Rev.Lett. 96, 107802, 2006; Katritzky, A.R., Singh, S., Kirichenko, K. et al., In search of ionic liquids incorporating azolate anions, Chemistry 12, 4630-4641, 2006.
Glass Transition/Glass Transition Temperature The glass transition generally refers to change of a polymer from an amorphous material to an brittle material. The glass transition of a non-crystalline material is the critical temperature at which the material changes its behavior from being a glass or brittle material to an amorphous rubber-like material. For lyophilization, it is a critical temperature during the drying cycle which is important to the final product cake. See MacKenzie, A.P., Non-equilibrium freezing behavior of aqueous systems, Philos. Trans.R.Soc.Lond.B.Biol.Sci. 278, 167-189, 1977; Schenz, T.W., Israel, B., and Rosolen, M.A., Thermal analysis of water-containing systems, Adv.Exp.Med.Biol. 302, 199-214, 1991; Craig, D.Q., Royall, P.G., Kett, V.L., and Hopton, M.L., The relevance of the amorphous state to pharmaceutical dosage forms: Glassy drugs and freeze dried systems, Int.J.Pharm. 179, 179-207, 1999; Oliver, A.E., Hincha, D.K., and Crowe, J.H., Looking beyond sugars: the role of amphiphilic solutes in preventing adventitious reactions in anhydrobiotes at low water contents, Comp.Biochem. Physiol.A Mol.Integr.Physiol. 131, 515-525, 2002; Nail, S.L., Jiang, S., Chongprasert, S., and Knopp, S.A., Fundamentals of freezedrying, Pharm.Biotechnol. 14, 281-360, 2002; Franks, F., Scientific and technological aspects of aqueous glasses, Biophys.Chem. 105, 251-261, 2003; Vranic, E., Amorphous pharmaceutical solids,
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Global Proteomics
1005
Bosn.J.Basic Med. Sci. 4, 35-39, 2004; Hilden, L.R. and Morris, K.R., Physics of amorphous solids, J.Pharm.Sci. 93, 3-12, 2004.
Global Proteomics Analysis of all proteins in a cell or a tissue or an organism. See Hancock, W.S., Wu. S.L., Stanley, R.R., and Gombocz, E.W., Publishing large proteome datasets: scientific policy meets emerging technologies, Trends Biotechnol. 20(Suppl 12), S39-S44, 2002; Godovac-Zimmermann, J. and Brown, L.R., Proteomics approaches to elucidation of signal transduction pathways, Curr. Opin.Mol.Ther. 5, 241-249, 2003; Kumar, G.K. and Klein, J.B., Analysis of expression and posttranslational modification of proteins during hypoxia, J.Appl.Physiol. 96, 1178-1186, 2004; Hoskisson, P.A. and Hobbs, G., Continuous culture – making a comeback? Microbiology 151, 3153-3159, 2005.
Globulin A classic definition of proteins which are insoluble in water and soluble in dilute salt solutions and migrate more slowly than albumin in an electrophoretic system. See Cooper, G.R., Electrophoretic and ultracentrifugal analysis of normal human serum, in The Plasma Proteins, ed. F.W. Putnam, Academic Press, New York, NY, Chapter 3, pp. 51-103, 1960; . The globulins were separated into several fractions including the the g-globulins which contain the various immunoglobulin fractions and were defined as the most slowly moving protein fraction on electrophoresis at pH 8.6 (Porter, H.R., g-Globulins and antibodies, in The Plasma Proteins, ed. F.W. Putnam, Academic Press, New York, NY, Chapter 7, pp. 241-277, 1960). See Gehrke, C.W., Oh, Y.H., and Freeark, C.W., Chemical fractionation and starch gelurea electrophoretic characterization of albumins, globulins, gliadins, and glutenins in soft wheat, Anal.Biochem. 7, 439-460, 1964; Nilsson, U.R. and Mueller-Eberhard, H.J., Isolation of beta IF-globulin form human serum and its characterization as the fifth component of complement, J.Exp.Med. 122, 277-298, 1965; Sun, S.M. and Hall, T.C., Solubility characteristics of globulins from Phaseolus sees in regard to their isolation and characterization, J.Agric. Food Chem. 23, 184-189, 1975; Hauptman, S.P., Macromolecular insoluble cold globulin (MICG): a novel protein form mouse lymphocytes—I. Isolation and characterization, Immunochemistry 15, 415-422, 1978.
Glucose Oxidase An flavoprotein(FAD) enzyme(EC 1.1.3.4; b-D-glucose:oxygen 1-oxidoreductase) which catalyzes the oxidation of b-D-glucose to glucolactone/gluconic acid and hydrogen peroxide. The enzyme is highly specific for this form of glucose (Keilin, D. and Hartree, E.F., The use of glucose oxidase (Notatin) for the determination of glucose in biological material and for the study of glucose-producing systems by mannometric methods, Biochem.J. 42, 230-238, 1942; Sols, A. and de la Fuente, G., On the substrate specificity of glucose oxidase, Biochim.Biophys.Acta 24, 206-207, 1957; Wurster, B. and Hess, B., Anomeric specificity of enzymes for D-glucose metabolism, FEBS Lett. 40(Suppl), S112-S118, 1974) and is the basis of most of the assays for glucose in blood and bioreactors. The vast majority of assays measure the hydrogen peroxide released in the reaction (Kiang, S.W., Kuan, J.W., Kuan, S.S., and Guilbault, G.G., Measurment of glucose in plasma, with use of immobililized glucose oxidase and peroxidase, Clin.Chem. 22, 1378-1382, 1976; Chua, K.S. and Tan, I.K., Plasma glucose measurement with the
9168_Book.indb 1005
Glucose regulated protein, 78kD
Yellow Springs glucose analyzer, Clin.Chem. 24, 150-152, 1978; Artiss, J.D., Strandbergh, D.R., and Zak, B., On the use of a sensitive indicator reaction for the automated glucose oxidase-peroxidase coupled reaction, Clin.Biochem. 1, 334-337, 1983; Tietz Textbook of Clinical Chemistry and Molecular Diagnostics, 4th Edn., ed. C.A. Burtis, E.R. Ashwood, and D.F. Bruns, Elsevier-Saunders, St. Louis, MO, USA, 2006). Glucose oxidase was discovered the early 1900’s and originally described an antibacterial factor derived from moulds such as Pencilliium notatum and Aspergillus niger (Coulthard, C.E., Michaealis, R., Short, W.F. et al., Notatin: an antibacterial glucose aerodehydrogenase from Penicillium notatum and Penicillium resitculosum sp. nov, Biochem.J. 39, 24-36, 1945). Glucose oxidase has subsequently been identified as the antibacterial/antibiotic activity in honey (White, J.W., Jr., Subers, M.H., and Schepartz, A.I., The identification of inhibine, the antibacterial factor in honey, as hydrogen peroxide and its origin in a honey glucose-oxidase system, Biochim.Biophys.Acta 73, 57-70, 1963; Schepartz, A.T. and Subers, M.H., The glucose oxidase of honey. I. Purification and some general properties of the enzyme, Biochim.Biophys.Acta 85, 228-237, 1964; Bang, L.M., Bunting, C., and Molan, P., The effect of dilution on the rate of hydrogen peroxide production in honey and its implications for wound healing, J.Alternative Complementary Med. 9, 267-273, 2003; Badawy, O.F., Shafil, S.S., Tharwat, E.E., and Kamal, A.M., Rev.Sci.Tech. 23, 10111022, 2004) and a critical component of the honey bee invertebrate immune system (Xang, X. and Cox-Foster, D.L., Impact of an ectoparasite on the immunity and pathology of an invertebrate: evidence for host immunosuppression and viral amplification, Proc. Natl.Acad.Sci.USA 102, 7470-7475, 2005. Glucose oxidase is also involved in herbivore offense in plants (Musser, R.O., Cipollini, D.F., Hum-Musser, S.M. et al., Evidence that the caterpillar salivary enzyme glucose oxidase provides herbivore offense in solanaceous plants, Archs.Insect Biochem.Physiol. 58, 128-137, 2005.
Glucose regulated protein, 78kD Grp78; glucose regulated protein, identical with BiP, a chaperone-like protein which was also described as the immunoglobulin heavy-chain-binding protein. See Munro, S. and Pelham, H.R., An Hsp70-like protein in the ER: identity with the 78 kd glucose-regulated protein and immunoglobulin heavy chain binding protein, Cell 46, 291-300, 1986; Hendershot, L.M., Ting, J., and Lee, A.S., Identity of the immunoglobulin heavychain-binding protein with the 78,000 dalton glucose-regulated protein and the role of posttranslational modifications in its binding function, Mol.Cell Biol. 8, 4250-4256, 1988; Haas, I.G., BiP (Grp78), an essential hsp70 resident protein in the endoplasmic reticulum, Experientia 50, 1012-1020, 1994; Kleizen, B. and Braakman, I., Protein folding and quality control in the endoplasmic reticulum, Curr.Opin.Cell Biol. 16, 343-349, 2004; Okudo, H., Kato, H., Arakaki, Y. and Urade, R., Cooperation of ER-60 and BiP in the oxidative refolding of denatured proteins in vitro, J.Biochem. 138, 773-780, 2005; Sorgjerd, K., Ghafouri, B., Jonsson, B.H. et al., Retention of misfolded mutant transthyretin by the chaperone BiP/GRP78 mitigates amyloidogenesis, J.Mol.Biol. 356, 469-482, 2006; Panayi, G.S., and Corrigall, V.M., BiP regulates autoimmune inflammation and tissue damage, Autoimmune Rev. 5, 140-142, 2006; Li, J. and Lee, A.S., Stress induction of GRP78/BiP and its roles in cancer, Curr.Mol. Med. 6, 45-54, 2006; Tajima, H., and Koizumi, N., Induction of BiP by sugar independent of a cis-element for the unfolded protein response in Arabidopsis thaliana, Biochem.Biophys.Res. Commun. 346, 926-930, 2006.
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Glucosyltransferase
1006
Glucosyltransferase A glycosyltransferase specific for the transfer of glucosides. See Doyle, R.J. and Ciardi, J.E., Glucosyltransferases, Glucans, Sucrose and Dental Caries, IRL Press, Washington, DC, USA, 1983; Bleicher, R.J. and Cabot, M.C., Glucosylceramide synthesis and apoptosis, Biochim.Biophys.Acta 1585, 172-178, 2002; Yang, J., Hoffmeister, D., Liu, L., et al., Natural product glycorandomization, Bioorg.Med.Chem. 12, 1577-1584, 2004; Lorenc-Kukula, K., Korobczak, A., Aksamit-Stachurska, A., et al., Glucosyltransferase: the gene arrangement and enzyme function, Cell Mol.Biol.Lett. 9, 935-946, 2004; Trombetta, E.S. and Parodi, A.J., Glycoprotein reglucosylation, Methods 35, 328-337, 2005.
GLUT A family of membrane transporters which mediate the uptake of hexoses in mammalian cells. See Gould, G.W. and Holman G.D., The glucose transporter family—structure, function and tissue-specific expression, Biochem. J. 295, 329-341, 1993; Yang, J., Dowden, J., Tatibouet, A., Hatanaka, Y., Holman, G.D., Development of high-affinity ligands and photoaffinity labels for the d-fructose transporter GLUT5, Biochem.J. 367, 533-539, 2002;
Glycome The total carbohydrates within an organism. See Feizi, T., Progress in deciphering the information content of the ‘glycome’—a crescendo in the closing years of the millennium, Glycoconj.J. 17, 553-565, 2001; Hirabayashi, J., Arata, Y., and Kasai, K., Glycome project: concept, strategy, and preliminary application to Caenorhabditis elegans, Proteomics 1, 295-303, 2001; Loel, A., Glycome: a medical paradigm, Adv.Exp.Biol.Med. 546, 445-451, 2004; Hsu, K.L., Pilobello, K.T., and Mahal, L.K., Analyzing the dynamic bacterial glycome with a lectin microarray approach, Nat.Chem.Biol. 2, 125-126, 2006; Freeze, H.H., Genetic defects in the human glycome, Nat.Rev.Genet. 7, 537-551, 2006.
Glycomics The study of the structure, function, and interactions of carbohydrates within the gycome. See Drickhamer, K. and Taylor, M.E., Glycan arrays for functional glycomics, Genome Biol. 3, REVIEWS1034, 2002; Love, K.R. and Seeberger, P.H., Carbohydrate arrays as tools for glycomics, Angew.Chem.Int.Ed.Engl. 41, 35833586, 2002; Hirabayashi, J., Oligosaccharide microarrays for glycomics, Trends Biotechnol. 21, 141-143, 2003; Feizi, T., Fazio, F., Chai, W. and Wong, C.H., Carbohydrate microarrays - a new set of technologies at the frontiers of glycomics, Curr.Opin.Struct.Biol. 13, 637-645, 2003; Morelle, W. and Michalski, J.C., Glycomics and mass spectrometry, Curr.Pharm.Des. 11, 2615-2645, 2005; Raman, R., Raguram, S., Venkataraman, G., et al., Glycomics: an integrated systems approach to structure-function relationships of glycans, Nat.Methods 2, 817-824, 2005.
Glycosidase An enzyme that hydrolyzes glycosidic bonds; most often in oligosaccharides and polysaccharides. See Allen, H.J. and Kisailus, E.C., Glycoconjugates: Composition, Structure, and Function, Dekker, New York, New York, 1992; Guide to Techniques in Glycobiology, ed. W.J. Lennarz and G.W. Hart, Academic Press, San Diego, CA,
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Golgi Apparatus
1994; Bucke, C., Carbohydrate Biotechnology Protocols, Humana Press, Totowa, NJ, 1999; Himmel, M.E. and Baker, J.O., Glycosyl Hydrolases for Biomass Conversion, American Chemical Society, Washington, DC, 2001.
Glycosyltransferase An enzyme which synthesizes compounds with glycosidic bonds by catalyzing the transfer of glycosyl groups. See Carib, E., Carbohydrate metabolism, Annu.Rev.Biochem. 32, 321354, 1963; Heath, E.C., Complex polysaccharides, Annu.Rev. Biochem. 40, 29-56, 1971; Honjo, T. and Hayashi, O., Enzymatic ADP-ribosylation of proteins and regulation of cellular activity, Curr.Top.Cell Regul. 7, 87-127, 1973; Glycoimmunology, ed. Alavi, A. and Axford, J.S., Plenum Press, New York, New York, 1995; Molecular Glycobiology, ed. M. Fukuda and O. Hindsgaul, Oxford University Press, Oxford, UK, 1994; Endo, T., Aberrant glycosylation of alpha-dystroglycan and congenital muscular dystrophies, Acta Myol. 24, 64-69, 2005; SerafiniCessi, F., Monti, A., and Cavallone, D., N-Glycans carried by Tamm-Horsfall glycoprotein have a crucial role in the defense against urinary tract diseases, Glycoconj.J. 22, 383-394, 2005; Milewski, S., Gabriel, I., and Olchowy, J., Enzymes of UDPGlcNAc in yeast, Yeast 23, 1-14, 2006; Millar, C.M. and Brown, S.A., Oligosaccharide structures of von Willebrand factor and their potential role in von Willebrand disease, Blood Rev. 20, 83-92, 2006; Koch-Nolte, F., Adriouch, S., Bannas, P., et al., ADP-ribosylation of membrane proteins: unveiling the secrets of a crucial regulatory mechanism in mammalian cells, Ann. Med. 38, 189-199, 2006.
Goblet Cell A type of cell found in the epithelium with high occurrence in respiratory/digestive tracts which secrete mucus. See Rogers, D.F., Motor control of airway goblet cells and glands, Respir.Physiol. 125, 129-144, 2001; Jeffery, P. and Zhu, J., Mucin-producing elements and inflammatory cells, Novartis Found.Symp. 248, 51-68, 2002; Rogers, D.F., The airway goblet cell, Int.J.Biochem.Cell. Biol. 35, 1-6, 2003; Kim, S. and Nadel, J.A., Role of neutrophils in mucus hypersecretion in COPD and implications for therapy, Treat.Respir.Med. 3, 147-159, 2004; Bai, T.R. and Knight, D.A., Structural changes in the airways in asthma: observations and consequences, Clin.Sci. 108, 463-477, 2005; Rose, M.C., and Voynow, J.A., Respiratory tract mucin genes and mucin glycoproteins in health and disease, Physiol.Rev. 86, 245-278, 2006: Lievin-Le Moal, V. and Servin, A.L., The front line of enteric host defense against unwelcome intrusion of harmful microorganisms: mucins, antimicrobial peptides, and microbiota, Clin.Microbiol. Rev. 19, 315-337, 2006.
Golgi Apparatus A subcellular organelle consisting of a series of membrane structures; the Golgi apparatus can be considered as a single membrane structure containing a number of membrane-bound vesicles. The Golgi apparatus functions in the protein secretory pathway by transporting and packing of proteins for distribution elsewhere in the cell. The Golgi has a cis-side facing the endoplasmic reticulum and a trans-side which interfaces with the plasma membrane and components of the endocytotic pathway. See Whaley, W.B., The Golgi Apparatus, Springer-Verlag, New York, NY, 1975; Pavelka, M., Functional Morphology of the
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Golgins
1007
Golgi Apparatus, Springer-Verlag, Berlin, Germany, 1987; Loh, Y.P., Mechanisms of Intracellular Trafficking and Processing of Preproteins, CRC Press, Boca Raton, FL, 1993; Guidebook to the Secretory Pathway, ed J. Rothblatt and Novak, P., Oxford University Press, Oxford, UK, 1997; The Golgi Apparatus, ed. Berger, E.G. and Roth, J., Birkhäuser Verlag, Basel, 1997; Robinson, D.G., The Golgi Apparatus and the Plant Secretory Pathway,, CRC Press, Boca Raton, FL, 2003; Northcote, D.H., The Golgi apparatus, Endeavor 30, 26-33, 1971; Shnitka, T.K. and Seligman, A.M., Ultrastructural localization of enzymes, Annu.Rev.Biochem. 40, 375-396, 1971; Schachter, H., The subcellular sites of glycosylation, Biochem.Soc.Trans. 40, 47-71, 1974; Novikoff, A.B., The endoplasmic reticulum: a cytochemist’s view, Proc.Nat.Acad.Sci.USA 73, 2781-2787, 1976; Hawes, C. and Satiat-Jeunemailtre, B., The plant Golgi apparatus – going with the flow, Biochim.Biophys.Acta 1744, 466-480, 2005; Meyer, H.H., Golgi reassembly after mitosis: the AAA family meets the ubiquitin family, Biochim.Biophys.Acta 1744, 481-492, 2005; Toivola, D.M., Tao, G.Z., Hbtezion, A., Liao, J., and Omary, M.B., Cellular integrity plus: organelle—related and proteintargeting functions of intermediate filaments, Trends Cell Biol. 15, 608-617, 2005; Jolliffe, N.A., Craddock, C.P., and Frigerio, L., Pathways for protein transport to see storage granules, Biochem. Soc.Trans. 33, 1016-1018, 2005; Ungar, D., Oka, T., Kreiger, M., and Hughson, F.M., Retrograde transport on the COG railway, Trends Cell Biol. 16, 113-120, 2006; Quatela, S.E. and Phillips, M.R., Ras signaling on the Golgi, Curr.Opin.Cell Biol. 18, 162167, 2006; D’Souza-Schorey, C. and Chavrier, P., ARF proteins: roles in membrane traffic and beyond, Nat.Rev.Mol.Cell Biol. 7, 347-358, 2006.
Golgins A family of proteins found in the Golgi apparatus. The members of this protein family are characterized by the presence of a long region of coiled-coil segments thus having a tendency to form long rod-like structures. See Fritzler, M.J., Hamel, J.C., Ocha, R.L., and Chan, E.K., Molecular characterization of two human autoantigens: unique cDNAs encoding 95- and 160-kD proteins of a putative family in the Golgi complex, J.Exp.Med. 178, 49-62, 1993; Kjer-Nielsen, L., Teasdale, R.D., van Vliet, C., and Gleeson, P.A., A novel Golgi-localization domain shared by a class of coiled-coil peripheral membrane proteins, Curr.Biol. 9, 385-388, 1999; Munro, S. and Nichols, B.J., The GRIP domain – a novel Golgi-targeting domain found in several coiled-coil proteins, Curr.Biol. 9, 377-380, 1999; Pfeffer, S.R., Constructing a Golgi complex, J.Cell Biol. 155, 873-883, 2001; Barr, F.A. and Short, B., Golgins in the structure and dynamics of the Golgi apparatus, Curr.Opin.Cell Biol. 15, 405-413, 2003; Darby, M.C., vanVliet, C., Brown, D. et al., Mammalian GRIP domain proteins differ in their membrane binding properties and are recruited to distinct domains of the TGN, J.Cell Biol. 177, 5865-5874, 2004; Fridmann0Sirkis, Y., Siniossoglou, S., and Pelham, H.R., TMF is a golgin that binds Rab6 and influences Golgi morphology, BMC Cell Biol. 5, 18, 2004; Malsam, J., Satch, A., Pelletier, L., and Warren, G., Golgin tethers define subpopulations of COPI vesicles, Science 307, 1095-1098, 2005; Short, B., Haas, A., and Barr, F.A., Golgins and GTPases, giving identity and structure to the Golgi apparatus, Biochim.Biophys.Acta 1744, 383-395, 2005; Satoh, A., Beard, M., and Warren, G., Preparation and characterization of recombinant golgin tethers, Methods Enzymol. 404, 279-296, 2005.
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GTP-Binding Protein
Granzyme Granzymes are exogenous serine proteases that are contained in cytoplasmic granules in cytotoxic T cells and natural killer cells. Granzyme enter the target cell through pores created by perforin and induce apoptosis through a variety of mechanisms including caspace-dependent and caspace-independent pathways. See Jenne, D.E. and Tchopp, J., Granzymes, a family of serine proteases released from granules of cytolytic T lymphocytes upon T cell receptor stimulation, Immunol.Rev. 103, 53-71, 1988; Smyth, M.J. and Trapani, J.A., Granzymes: exogenous proteinases that induce target cell apoptosis, Immunol.Today 16, 202-206, 1995; Lieberman, J. and Fan, Z., Nuclear war: the granzyme A-bomb, Curr.Opin.Immunol. 15, 553-559, 2003; Andrade, F., CasciolaRosen, L.A., and Rosen, A., Granzyme B-induced cell death, Acta Haematol. 111, 28-41, 2004; Waterhouse, N.J., Clarke, C.J., Sedelies, K.A., Teng, M.W., and Trapani, J.A., Cytotoxic lymphocytes; instigators of dramatic target cell death, Biochem. Pharmacol. 68, 1033-1040, 2004; Ashton-Rickardt, P.G., The granule pathway of programmed cell death, Crit.Rev.Immunol. 25, 161-182, 2005; Bleackely, R.C., A molecular view of cytotoxic T lymphocyte induced killing, Biochem.Cell Biol. 83, 747-751, 2005.
Growth Can be defined as weight or mass increases with age in a multiplicative way from Medawar, P., Size, shape and Age; Essays in Growth and Form presented to D’Arcy Wentworth Thompson, Clarendon Press, Oxford, United Kingdom, p. 708, 1945 as cited by Smith, R.W. and Ottema, C., Growth, oxygen consumption and protein and RNA synthesis rates in the yolk sac larvae of the African catfish (Clarias gariepinos), Comp.Biochem.Physiol. Part A 143, 315-325, 2006.
GTP-Binding Protein Intracellular proteins which bind GTP and have a wide variety of functions including signal transduction and in turn protein synthesis and cell proliferation. These proteins are “active” when GTP is bound; on hydrolysis of the GTP to GDP, “activity” is lost. See Rouot, B., Brabet, P., Homberger, V., et al., Go, a major brain GTP binding protein in search of a function: purification, immunological and biochemical characterization, Biochimie 69, 339349, 1987; Obar, P.A., Shpetner, H.S., and Vallee, R.B., Dynamin: a microtubule-associated GTP-binding protein, J.Cell Sci.(Suppl) 14, 143-145, 1991; Lillie, T.H. and Gomperts, B.D., A cell-physiological description of GE, a GTP-binding protein that mediates exocytosis, Ciba Found.Symp. 176, 164-179, 1993; Kjeldgaard, M., Nyborg, J. and Clark, B.G., The GTP binding motif: variations on a theme, FASEB J. 10, 1347-1386, 1996; Im, M.J., Russell, M.A., and Feng, J.F., Transglutaminase II: a new class of GTP-binding protein with new biological functions, Cell Signal. 9, 477-482, 1997; Ridley, A.J., The GTP-binding protein Rho, Int.J.Biochem. Cell Biol. 29, 1225-1229, 1997; Sugden, P.H. and Clerk, A., Activation of the small GTP-binding protein Ras in the heart by hypertrophic agonists, Trends Cardiovasc.Med. 10, 1-8, 2000; Caron, E., Cellular functions of the Rap1 GTP-binding protein: a pattern emerges, J. Cell Sci. 116, 435-440, 2003; Gasper, R., Scrima, A., and Wittinghofer, A., Structural insights into HypB, a GTPbinding protein that regulates metal binding, J.Biol.Chem., 281, 27492–27502, 2006.
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Haber-Weiss Reaction Heterochromatin 1008
Haber-Weiss Reaction A cycle consisting of the reaction of hydroxyl radicals with hydrogen peroxide generating the superoxide with the subsequent reaction of superoxide with peroxide generating hydroxyl anion and hydroxyl radical; it is possible that this second reaction is catalyzed by ferric ion: see Kehrer, J.P., The Haber-Weiss reaction and mechanisms of toxicity, Toxicology 149, 43-50, 2000; Koppenol, W.H., The Haber-Weiss cycle – 70 years later, Redox Rep. 6, 229234, 2001.
Heat Capacity (Cr) The quantity of thermal energy needed to raise the temperature of an object by 1°C; Cr = mass x specific heat; see specific heat. Heat capacity in proteins is measured with techniques such as differential scanning calorimetry and isothermal titration calorimetry. An understanding of heat capacity is important in understanding the glass transition in the lyophilization of proteins. See Cooper, A., Heat capacity effects in protein folding anad ligand binding: a re-evaluation of the role of water in biomolecular thermodynamics, Biophys.Chem. 115, 89-97, 2005; Prabhu, N.V. and Sharp, K.A., Heat capacity I proteins, Annu.Rev.Phys.Chem. 56, 521-548, 2005; Lemaster, D.M., Heat capacity-independent determination of differential free energy of stability between structurally homologous proteins, Biophys. Chem. 119, 94-100, 2006; van Teeffelen, A.M., Melinders, M.B., and de Jongh, H.H., Identification of pitfalls in the analysis of heat capacity changes of beta-lactoglobulin A, Int.J.Biol.Macromol. 30, 28-34, 2005; Kozlov, A.G. and Lohman, T.M., Effects of monovalent anions on a temperature-dependent heat capacity change for Escherichia coli SSB tetramer binding to single-stranded DNA, Biochemistry 45, 5190-5205, 2006; Gribenko, A.V., Keiffer, T.R., and Makhatadze, G.I., Amino acid substitutions affecting protein dynamics in eglin C do not affect heat capacity change upon unfolding, Proteins 64, 295-300, 2006.
Heat Shock Proteins Heat shock proteins (HSP) are a family of proteins with chaperone activity. Heat shock proteins are involved in protein synthesis and folding, vesicular trafficking, and antigen presentation. Glucoseregulated protein 78 kDA (GRP78) which is also known as immunoglobulin heavy chain binding protein (BiP) is one of the better known members of this family and is constitutively expressed in the endoplasmic reticulum (ER) in a wide variety of cell types. Heat shock proteins were first described as part of the response of the cell to heat shock and other stress situations such as hypoxia. See Tissières, A., Mitchell, H.K., and Tracy, U.M., Protein synthesis in salivary glands of Drosophila melanogaster: relation to chromosome puffs, J.Mol.Biol. 84, 389-398, 1974; Schedl, P., ArtavanisTsakonas, S., Steward, R., et al,, Two hybrid plasmids with D. melanogaster DNA sequences complementary to mRNA coding for the major heat shock protein, Cell 14, 921-929, 1978; ArtavanisTsakonas, S., Schedl, P., Mirault, M.E., et al., Genes for the 70,000 dalton heat shock protein in two cloned D. melanogaster DNA segments, Cell 17, 9-18, 1979; McAlister, L. and Finklestein, D.B., Heat shock proteins and thermal resistance in yeast, Biochem. Biophys.Res.Commun. 93, 819-824, 1980; Wang, C., Gomer, R.H., and Lazarides, E., Heat shock proteins are methylated in avian and mammalian cells, Proc.Natl.Acad. Sci. USA 78, 3531-3535, 1981; Roccheri, M.C., Di Bernardo, M.G., and Giudice, G., Synthesis of heat-shock proteins in developing sea urchins, Dev.Biol. 83, 173177, 1981; Lindquist, S., Regulation of protein synthesis during
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heat shock, Nature 283, 311-314, 1981; Loomis, W.F., Wheeler, S., and Schmidt, J.A., Phosphorylation of the major heat shock protein of Dictyostelium discoideum, Mol.Cell Biol. 2, 484-489, 1982; Neidhardt, F.C., VanBogelen, R.A., and Vaughn, V., The genetics and regulation of heat-shock proteins, Annu.Rev.Genet. 18, 295329, 1984; Schlesinger, M.J., Heat shock proteins: the search for functions, J.Cell Biol. 103, 321-325, 1986; Lanks, K.W., Modulators of the eukaryotic heat shock response, Exp.Cell Res. 165, 1-10, 1986; Lindquist, S. and Craig, E.A., The heat-shock proteins, Annu. Rev. Genet. 22, 631-677, 1988; Tanguay, R.M., Transcriptional activation of heat-shock genes in eukaryotes, Biochem.Cell Biol. 66, 584-593, 1988; Pelham, H.R., Control of protein exit from the endoplasmic reticulum, Annu.Rev.Cell Biol.5, 1-23, 1989; Bukau, B., Weissman, J., and Horwich, A., Molecular chaperones and protein quality control, Cell 125, 443-451, 2006; Panyai, G.S. and Corrigal, V.W., BiP regulates autoimmune inflammation and tissue damage, Autoimmun.Rev. 5, 140-142, 2006. More recently, there has been interest in heat shock proteins as therapeutic targets in oncology. See Dai, C. and Whitesell, L., HSP90: a rising star on the horizon of anticancer targets, Future Oncol. 1, 529-540, 2005; Li, J. and Lee, A.S., Stress induction of GRP78/BiP and its role in cancer, Curr.Mol.Med. 6, 45-54, 2006; Kim, Y., Lillo, A.M., Steiniger, S.C.J., et al., Targeting heat shock proteins on cancer cells: selection, characterization, and cell-penetrating properties of a peptidic GRP78 ligand, Biochemistry 45, 9434-9444, 2006.
Hedgehog A family of proteins important in tissue formation during embryonic development; generally expressed on exterior of cell and bind to receptor on adjacent cells. Sonic hedgehog is a glycoprotein important as signal molecule during differentiation. See Lum, L. and Beachy, P.A., The Hedgehog response network: sensors, switches, and routers, Science 304, 1755-1759, 2004; Ishibashi, M., Saitsu, H., Komada, M., and Shiota, K., Signaling cascade coordinating growth of dorsal and ventral tissues of the vertebrate brain, with special reference to the involvement of Sonic Hedgehog signaling, Anat.Sci.Int. 80, 30-36, 2005; Hooper, J.A. and Scott, M.P., Communicating with Hedgehogs, Nat.Rev. Mol.Cell Biol. 6, 206-317, 2005; Kalderon, D., The mechanism of hedgehog signal transduction, Biochem.Soc.Trans. 33, 1509512, 2005; Echelard, Y., Epstein, D.J., St-Jacques, B., et al., Sonic hedgehog, a member of a family of putative signaling molecules, is implicated in the regulation of CNS polarity, Cell 75, 1417-1430, 1993; Bumcrot, D.A. and McMahon, A.P., Somite differentiation. Sonic signals somites, Curr.Biol. 5, 612-614, 1995; Johnson, R.L., Riddle, R.D., Laufer, E., and Tabin, C., Sonic hedgehog: a key mediator of anterior-posterior patterning of the limb and dorsoventral patterning of axial embryonic structures, Biochem.Soc. Trans. 22, 59-574, 1994. Davy-Grosjean, L. and Couve-Privat, S., Sonic hedgehog signaling in basel cell carcinomas, Cancer Lett. 225, 181-192, 2005.
Heterochromatin “Condensed” or modified chromatin not conducive to gene transcription. See Hyde, B.B., Ultrastructure in chromatin, Prog.Biophys.Mol.Biol. 15, 129-148, 1965; Brown, S.W., Heterochromatin, Science 151, 417-425, 1966; Back, F., The variable condition of h euchromatin and heterochromatin, Int.Rev. Cytol. 45, 25-64, 1976; Lewis, J. and Bird, A., DNA methylation and chromatin structure, FEBS Lett. 285, 155-159, 1991; Wu, C.T., Transvection, nuclear structure, and chromatin proteins,
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Heterolytic Cleavage, Heterolysis Holoenzyme 1009 J.Cell.Biol. 120, 587-590, 1993; Karpen, G.H., Position-effect variegation and the new biology of heterochromatin, Curr.Opin.Genet. Dev. 4, 281-291, 1994; Kornberg, R.D. and Lorch, Y., Interplay between chromatin structure and transcription, Curr.Opin.Cell Biol. 7, 371-375, 1995; Zhimulev, I.F., Polytene chromosomes, heterochromatin, and position effect variegation. Adv.Genet. 37, 1-566, 1998; Martin, C. and Zhang, Y., The diverse functions of histone, lysine methylation, Nat.Rev.Mol.Cell Biol. 6, 838-849, 2005 Wallace, J.A., and Orr-Weaver, T.L., Replication of heterochromatin: insights into mechanisms of epigenetic inheritance, Chromosoma 114, 389-402, 2005; Hiragami, K. and Festenstein, R., Heterochromatin protein 1: a pervasive controlling influence, Cell.Mol.Life Sci. 62, 2711-2726, 2005.
Heterolytic Cleavage, Heterolysis An uneven division of a molecule such as HCl → H+ + Cl- which usually generates ions. The hydrogenase reaction and the oxygen radical oxidation of fatty acids are example of heterolytic cleavages. See Gardner, H.W., Oxygen radical chemistry of polyunsaturated fatty acids, Free Radic.Biol.Med. 7, 65-86, 1989; Fontecilla-Camps, J.C., Frey, M., Garcin, E., et al., Hydrogenase: a hydrogen-metabolizing enzyme. What do the crystal structures tell us about its mode of action? Biochimie 79, 661-666, 1997; Richard, J.P. and Amyes, T.L., Proton transfer at carbon, Curr. Opin.Chem.Biol. 5, 626-633, 2001; Solomon, E.I., Decker, A., and Lehnert, N., Non-heme iron enzymes: contrasts to heme catalysis, Proc.Natl.Acad.Sci.USA 100, 3589-3594, 2003; Zampella,G., Bruschi, M., Fantucci, P., and De Gioia, L., Investigation of H 2 activation by [M(NHPnPr3)(‘S3’)] (M = Ni, Pd). Insight into key factors relevant to the design of hydrogenase functional models, J.Amer.Chem.Soc. 127, 13180-13189, 2005.
His-Tag Generally a hexahistidine sequence which can be attached to the carboxyl-terminal or amino-terminal end of an expressed protein. This tag can be used for the affinity purification or separation of a protein by binding to an IMAC (immobilized metal affinity chromatography) column. The tag can also be used to provide an affinity site for interaction with another molecule in solution. See Sigal, G.B., Bamdad, C., Barberis, A., Strominger, J., and Whitesides, G.M., A self-assembled monolayer for the binding and study histidine-tagged proteins by surface plasmon resonance, Anal.Chem. 68, 490-497, 1996; Hengen, P., Purification of His-Tag fusion proteins from Escherichia coli, Trends Biochem. Sci. 20, 285-286, 1995; Müller. K.M., Arndt, K.M., Bauer, K., and Plückthun, A., Tandem immobilized metal-ion affinity chromatography/immunoaffinity purification of His-tagged proteinsevaluation of two anti-his-tag monoclonal antibodies, Anal. Biochem. 259, 54-61, 1998; Altendorf, K., Stalz, W., Greie, J., Deckers-Hebestreit, G., Structure and function of the F(o) complex of the ATP synthase from Escherichia coli, J.Exptl.Biol. 203, 19-28, 2000; Terpe, K., Overview of tag protein fusions: from molecular and biochemical fundamentals to commercial systems, Appl.Microbiol.Biotechnol. 60, 523-533, 2003; Jenny, R.J., Mann, K.G., and Lundblad, R.L., A critical review of the methods for cleavage of fusion proteins with thrombin and factor Xa, Protein Expr.Purif. 31, 1-11, 2003; Meredith, G.D., Wu, H.Y., and Albritton, N.L. Targeted protein functionalization using his tags, Bioconjugate Chem. 15, 969-982, 2004; Zhao, Y., Benita, Y., Lok, M., et al., Multi-antigen immunization using IgG binding domain ZZ as carrier, Vaccine 23, 5082-5090, 2005.
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Hofmeister Series Also known as the lyotropic; the order of certain ions to “salt out” or precipitate certain hydrophilic materials from aqueous solution. Polyvalent anions such as citrate and sulfate tend to precipitate while monovalent anions such as chloride and thiocyanate tend to solubilize. A similar series exists for cations. It is thought that this phenomena is related to the ability of the various ions to bind water – hence the term “salting out.” See Cacace, M.G., Landau, E.M., and Ramsden, J.J., The Hofmeister series: salt and solvent effects on interfacial phenomena, Quart.J.Biophys. 30, 241-277, 1997; Boström, M., Tavares, F.W., Finet, S., Skouri-Panet, F., Tardieu, A., and Ninham, B.W., Why forces between proteins follow different Hofmeister series for pH above and below PI, Biophys.Chem. 117, 217-224. 2005.
Holliday Junction A transient structure formed between two adjoining DNA molecules during homologous recombination which provides for the transfer of DNA sequence between the adjacent strands. See Symington, L.S. and Kolodner, R., Partial purification of an enzyme from Saccharomyces cerevisiae that cleaves Holliday junctions, Proc.Natl.Acad.Sci.USA 82, 7247-7251, 1985; Churchill, M.E., Tullius, T.D., Kallenbach, N.R., and Seeman, N.C., Holliday recombination intermediate is twofold symmetric, Proc.Natl. Acad.Sci.USA 85, 4653-4656, 1988; Dukett, D.R., Murchie, A.I., Diekmann, S., et al., The structure of the Holliday junction, and its resolution, Cell 55, 79-89, 1988; Jeyaseelan, R. and Shanmugam, G., Human placental endonuclease cleaves Holliday junctions, Biochem.Biophys.Res. Commun. 156, 1054-1060, 1988; Sharples, G.J., Ingleston, S.M., and Lloyd, R.G., Holliday junction processing in bacteria: insights from the evolutionary conservation of RuvABC, RecG, and RusA, J.Bacteriol. 181, 5543-5550, 1999; Sharples, G.J., The X philes: structure-specific endonuclease that resolve Holliday junctions, Mol.Microbiol. 39, 823-834, 2001; Ho, P.S. and Eichman, B.F., The crystal structures of DNA Holliday junctions, Curr.Opin.Struct.Biol. 11, 302-308, 2001; Heyer, W.D., Ehmsen, K.T., and Solinger, J.A., Holliday junctions in the eukaryotic nucleus: resolution in sight? Trends Biochem. Sci. 28, 548-557, 2003; Heyer, W.D., Recombination: Holliday junction resolution and crossover formation, Curr.Biol. 14, R56R58, 2004; Khuu, P.A., Voth, A.R., Hays, F.A., and Ho, P.S., The stacked-X DNA Holliday junction and protein recognition, J.Mol. Recognit. 19, 234-242, 2006.
Holoenzyme The intact function enzyme unit which could consist of a protein, metal ions, coenzymes and other protein components. This term was originally used to describe the combination of a coenzyme or other low-molecular weight cofactor such as metal ion with a protein component designated as the apoenzyme to form the holoenzyme. More recently, the term holoenzyme has been used to describe DNA and RNA polymerases. See Hokin, L.E., Purification and molecular properties of the (sodium + potassium)adenosinetriphosphatase and reconstitution of coupled sodium and potassium transport in phospholipid vesicles containing purified enzyme, J.Exp.Zool. 194, 197-205, 1975; Dalziel, K., McFerran, N.V., and Wonacott, A.J., Glyceraldehyde-3-phosphate dehydrogenase, Philos.Trans.R.Soc.Lond.B Biol.Sci. 293, 105-118, 1981; McHenry, C.S., DNA polymerase III holoenzyme. Components, structure, and mechanism of a true replicative complex,
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Holotype Homotype 1010 J.Biol.Chem. 266, 19127-19130, 1991; Ishihama, A., A multi-functional enzyme with RNA polymerase and RNase activities: molecular anatomy of influenza virus RNA polymerase, Biochimie 78, 1097-1102, 1996; Greenblatt, J., RNA polymerase II holoenzyme and transcriptional regulation, Curr.Opin.Cell Biol. 9, 310-319, 1997; Amieux, P.S. and McKnight, G.S., The essential role of RI alpha in the maintenance of regulated PKA activity, Ann.N.Y.Acad.Sci. 968, 75-95, 2002; Taggart, A.K. and Zakian, V.A., Curr.Opin.Cell Biol. 15, 275-280, 2003; Borukhov, S. and Nudler, E., RNA polymerase holoenyzme: structure, function and biological significance, Curr. Opin.Microbiol. 6, 93-100, 2003; McHenry, C.S., Chromosomal replicases as asymmetric dimers: studies of subunit arrangement and functional consequences, Mol.Microbiol.49, 1157-1165, 2003.
Holotype The single specimen or illustration designated as the type for naming a species or subspecies when no type was specified. See Crickmore, N., Zeigler, D.R., Feitelson, J. et al., Revision of the nomenclature for the Bacillus thuringiensis pesticidal crystal proteins, Microbiol.Mol.Biol.Rev. 62, 807-813, 1998; Pecher, W.T., Robledo, J.A., and Vasta, G.R., Identification of a second rRNA gene unit in the Parkinsus andrewsi genome, J.Eukaryot. Microbiol. 51, 234-245, 2004.
Homeobox A brief sequence of nucleotides whose base sequence is virtually identical in all the genes that contain said sequence. Originally described in Drosphilia, it has now been found in many organisms including Homo sapiens. In the fruit fly, a homeobox appears to determine when particular groups of genes are expressed during development. Homobox regions encode proteins containing homeodomain regions. See Gehring, W.J. and Hiromi, Y., Homeotic genes and the homeobox, Annu.Rev. Genet. 20, 147-173, 1986; Stern, C.D. and Keynes, R.J., Spatial patterns of homeobox gene expression in the developing mammalian CNS, Trends Neurosci. 11, 190-192, 1988; Kappen, C., Schughart, K., and Ruddle, F.H., Organization and expression of homeobox genes in mouse and man, Ann.N.Y.Acad.Sci. 567, 243-252, 1989; Wray, G.A., Transcriptional regulation and the evolution of development, Int.J.Dev.Biol. 47, 675-684, 2003; Del Bene, F. and Wittbrodt, J., Cell cycle control by homeobox genes in development and disease, Semin.Cell Dev. Biol. 16, 449-460, 2005; Samuel, S. and Naora, H., Homeobox gene expression in cancer: insights from developmental regulation and deregulation, Eur.J.Cancer 41, 2428-2437, 2005.
Homeodomain A domain in a protein that is encoded for by a homeobox; these proteins are transcription factors; the homeodomains are approximately 60 amino acids in length and are composed of three a-helices and bind DNA. See Scott, M.P., Tamkun, J.W., and Hartzell, G.W., 3rd, The structure and function of the homeodomain, Biochim.Biophys.Acta. 989, 25-48, 1989; Affolter, M., Schier, A., and Gehring, W.J., Homeodomain proteins and the regulation of gene expression, Curr.Opin.Cell Biol. 2, 485-495, 1990; Izpisua-Belmonte, J.C. and Deboule, D., Homeobox genes and pattern formation in the vertebrate limb, Dev.Biol. 152, 26-36, 1992; Yates, A. and Chambers, I., The homeodomain protein Nanog and pluripotency in mouse embryonic stem cells, Biochem.Soc.Trans. 33, 1518-1521, 2005; Towle, H.C., Glucose as
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regulator of eukaryotic gene transcription, Trends Endocrinol. Metab. 16, 489-494, 2005.
Homeotic A shift in structural development as in a major shift in the developmental fate of an organ or body. See Dessain, S. and McGinnis, W., Regulating the expression and function of homeotic genes, Curr.Opin.Genet.Dev. 1, 275-282, 1991; Morata, G., Homeotic genes of Drosophila, Curr.Opin.Genet.Dev. 3, 606-614, 1993; Doboule, D. and Morata, G., Colinearity and functional hierarchy among genes of the homeotic complexes, Trends Genet. 10, 358-364, 1994; Mann, R.S., The specificity of homeotic gene function, Bioessays 17, 855-863, 1995; Duncan, I., How do single homeotic genes control multiple segment identities? Bioessays 18, 91-94, 1996; Graba, Y., Aragnol, D., and Pradel, J., Drosophila Hox complex downstream targets and the function of homeotic genes, Bioessays 19, 379-388, 1997; Reichert, H. and Simone, A., Conserved usage of gap and homeotic genes in patterning the CNS, Curr.Opin.Neurobiol. 9, 589-595, 1999; Irish, V.F., The evolution of floral homeotic gene function, Bioessays 25, 637-646, 2003; Zubko, M.K., Mitochondrial tuning fork in nuclear homeotic functions, Trends Plant Sci. 9, 61-64, 2004. See also HOX genes.
Homolytic Cleavage, Homolysis An even division of a molecule such as HCl → H. + Cl. which generates free radicals. The decomposition of a precursor molecules can proceed via either a homolytic pathway, a heterolytic pathway, or both. See White, R.E., Sligar, S.G., and Coon, M.J., Evidence for a homolytic mechanism of peroxide oxygen—oxygen bond cleavage during substrate hydroxylation by cytochrome P-450, J.Biol. Chem. 255, 11108-11011, 1980; Yang, G., Candy, T.E., Boaro, M. et al., Free radical yields from the homolysis of peroxynitrous acid, Free RadicBiol.Med. 12, 327-330, 1992; Correia, M.A., Yao, K., Allentoff, A.J. et al., Interactions of peroxy quinols with cytochromes P450 2B1, 3A1, and 3A5: influence of the apoprotein on heterocyclic versus hemolytic O-O bond cleavage, Arch.Biochem. Biophys. 317, 471-478, 1995; Barr, D.P., Martin, M.V., Guengerich, F.P., and Mason, R.P., Reaction of cytochrome P450 with cumene hydroperoxide: ESR spin-trapping evidence for the homolytic scission of the peroxide O-O bond by ferric cytochrome P450 1A2, Chem.Res.Toxicol. 9, 318-325, 1996; Marsh, E.N. and Ballou, D.P., Coupling of cobalt-carbon bond homolysis and hydrogen atom abstraction in adenosylcobalamin-dependent glutamate mutase, Biochemistry 37, 11864-11872, 1998; Licht, S.S., Booker, S., and Stubbe, J., Studies on the catalysis of carbon-cobalt bond homolysis by ribonucleoside triphosphate reductase: evidence for concerted carbon-cobalt bond homolysis and thiyl radical formation, Biochemistry 38, 1221-1233, Vlasie, M.D. and Banerjee, R., Tyrosine 89 accelerates Co-carbon bond homolysis in methylmalonyl-CoA mutase, J.Am.Chem.Soc. 125, 5431-5435, 2003; Lymar, S.V., Khairutdinov, R.F., and Hurst, J.K., Hydroxyl radical formation by O-O bond homolysis in peroxynitrous acid, Inorg.Chem. 42, 5259-5266, 2003; Rees, M.D. and Davies, M.J., Heparan sulfate degradation via reductive homolysis of its N-chloro derivatives, J.Am.Chem.Soc. 128, 3085-3097, 2006.
Homotype A structure having the same general/function as another which may or may not be opposing. For example, the left arm is a homotype of the right arm. A selectin can be a homotype of another
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Hoogsteen Bond Hydrophobins 1011 selectin. Homotypic is a descriptor referring to homotype. See Rouhandeh, H., Yau, T., and Lang, P.A., Homotypic and heterotypic interference among picornovirus ribonucleic acids, Arch. Gesamte Virusforsch. 27, 236-243, 1969; Bendini, C., Lanfranchi, A., Nobili, R., and Miyake, A., Ultrastructure of meiosis-inducing (heterotypic) and non-inducing (homotypic) cell unions in conjugation of Blepharisma, J.Cell Sci. 32, 31-43, 1978; Daunter, B., Immune response: tissue specific T-lymphocytes, Med.Hypotheses 37, 76-84, 1992; Wagner, M.C., Molnar, E.E., Molitoris, B.A., and Goebl, M.G., Loss of the homotypic fusion and vacuole protein sorting or golgi-associated retrograde protein vesicle tethering complexes results in gentamicin sensitivity in the yeast Saccharomyces cerevisiae, Antimicrob.Agents Chemother. 50, 587-595, 2006; Karaulanov, E.E., Bottcher, R.T., and Niehrs, C., A role for fibronectin-leucinerich transmembrane cell-surface proteins in homotypic cell adhesion, EMBO Rep. 7, 283-290, 2006; Brandhorst, D., Zwilling, D., Rizzoli, S.O., et al., Homotypic fusion of early endosomes: SNAREs do not determine fusion specificity, Proc.Natl.Acad.Sci.USA 103, 2701-2706, 2006; Decker, B.L. and Wickner, W.T., Enolase activates homotypic vacuole fusion and protein transport to the vacuole in yeast, J.Biol.Chem., 281, 14523–14528, 2006; Stroupe, C., Collins, K.M., Fratti, R.A., and Wickner, W., Purification of active HOPS complex reveals its affinities for phosphoinositides and the SNARE Vam7p, EMBO J. 25, 1579-1589, 2006; Brereton, H.C., Carvell, M.J., Asare-Anane, H., et al,, Homotypic cell contact enhances insulin but not glucagon secretion, Biochem.Biophys.Res.Commun. 344, 995-1000, 2006.
Hoogsteen Bond The hydrogen bonds formed in the hybridization of DNA chains to form a triple helix. See Searle, M.S. and Wickham, G., Hoogsteen versus Watson-Crick A-T basepairing in DNA complexes of a new group of ‘quinomycin-like’ antibiotics, FEBS Lett. 272, 171-174, 1990; Raghunathan, G., Miles, H.T., and Sasisekharan, V., Symmetry and structure of RNA and DNA triple helices, Biopolymers 36, 333-343, 1995; Soliva, R., Luque, F.J., and Orozco, M., Can G-C Hoogsteen-wobble pairs contribute to the stability of d(G, C-C) triplexes, Nucleic Acids Res. 27, 2248-2255, 1999; Li, J.S., Shikiya, R., Marky, L.A., and Gold, B., Triple helix forming TRIPside molecules that target mixed purine/pyrimidine DNA sequences, Biochemistry 43, 1440-1448, 2004.
Hormonology The term hormonology is used to describe the study of hormones and has been proposed as a substitute for endocrinology. See Ross, J.W., Hormonology in obstetrics, J.Natl.Med.Assoc. 46, 19-21, 1954; Swain, C.T., Hormonology, N.Engl.J.Med. 280, 388-389, 1969; Kulinskii, V.I. and Kolesnichenko, L.S., Current aspects of hormonology, Biochemistry(Mosc.) 62, 1171-1173, 1997; Holland, M.A., Occam’s razor applied to hormonology (Are cytokines produced by plants?), Plant Physiol. 115, 865-868, 1997; Hadden, D.R., 100 years of hormonology: a view from No. 1 Wimpole Street, J.R.Soc.Med. 98, 325-326, 2005; Hsueh, A.J.W., Bouchard, P. and Ben-Shlomo, I., Hormonology: a genomic perspective on hormonal research, J.Endocrinol. 187, 333-338, 2005.
HOX genes Encodes a family of transcription factors, Hox proteins. See Morgan, S., HOX genes: a continuation of embryonic patterning? Trends in Genetics 22, 67-69, 2006; Hoegg, S. and Meyer, A., HOX
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clusters as models for vertebrate genome evolution, Trends in Genetics 21, 421-424, 2005; Sekimoto, T., Yoshinobu, K., Yoshida, M., et al., Region-specific expression of murine Hox genes implies the HOX code-mediated patterning of the digestive tract, Genes to Cells 3, 51-64, 1998.
Hydrogels An easily deformed pseudo-solid mass formed from largely hydrophilic colloids dispersed in an aqueous medium (dispersion medium or continuous phase). There is considerable interest in the use of hydrogels for drug delivery. See Jhon, M.S. and Andrade, J.D., Water and hydrogels, J.Biomed.Mat.Res. 7, 509-522, 1973; Roorda, W., Do hydrogels contain different classes of water, J.Biomater.Sci. Polym.Ed. 5, 383-395, 1994; Omidian, H., Rocca, J.G., and Park, K., Advances in superporous hydrogels, J.Control.Release 102, 3-12, 2005; Frokjaer, S. and Otzen, D.E., Protein drug stability: a formulation challenge, Nat.Rev.Drug Discov. 4, 298-306, 2005; Kashyap, N., Kumar, N., and Kumar, K.N., Hydrogels for pharmaceutical and biomedical applications, Crit.Rev.Ther.Drug Carrier Syst. 22, 107-149, 2005; Fairman, R. and Akerfeldt, K.S., Peptides as novel smart materials, Curr.Opin.Struct.Biol. 15, 453-463, 2005; Young, S., Wong, M., Tabata, Y., and Mikos, A.G., Gelatin as a delivery vehicle for the controlled release of bioactive molecules, J.Control.Release 109, 256-274, 2005; Dusek, K., Reponsive Gels: Volume Transitions, Springer-Verlag, Berlin, 1993; Dumitriu, S., Polymeric Biomaterials, Marcel Dekker, New York, 1994; Zrinyl, N., Gels, Springer, Darmstadt, 1996; McCormick, C.L., SimuliResponsive Water Soluble and Amphiphilic Polymers, American Chemical Society, Washington, DC, 2001; Dumitriu, S., Polymeric Biomaterials, Marcel Dekker, New York, 2002.
Hydrophobic, Hydrophobic Effect, Hydrophobic Forces Literally, the tendency of a molecular structure to avoid water which results in an association or clustering of hydrophobic groups. The term nonpolar is frequently used to describe such groups or molecules. Polar and nonpolar groups or functions can exist in the same molecule; for example, the ε-amino group of lysine is polar but the methylene carbon chain between the ε-amino group and the a-carbon is nonpolar. See Chander, D., Interfaces and the driving force of hydrophobic assembly, Nature 437, 640-647, 2005; Kauzmann, W., Some forces in the interpretation of protein denaturation, Adv.Prot.Chem. 14, 1-63, 1959; Tanford, C., The hydrophobic effect and the organization of living matter, Science 200, 1012-1018, 1978; Kumar, S. and Nussinov, R., Close-range electrostatic interactions in proteins, ChemBioChem 3, 604-617, 2002; Kyte, J., The basis of the hydrophobic effect, Biophys.Chem. 100, 193-203, 2003; Lesk, A.M., Hydrophobicitygetting into hot water, Biophys.Chem. 105, 179-182, 2003; Seelig, J., Thermodynamics of lipid-peptide interactions, Biochim. Biophys.Acta 1666, 40-50, 2004; Hofinger, S. and Zerbetto, F., Simple models for hydrophobic hydration, Chem.Soc.Rev. 34, 1012-1020, 2005.
Hydrophobins Hydrophobins are secreted proteins functioning in fungal growth and development. Hydrophobins self-assemble at hydrophilic/ hydrophobic interfaces forming amphipathic membranes. See Wessels, J., De Vries, O., Asgeirsdottir, S.A., and Schuren, F., Hydrophobin genes involved in formation of aerial hyphae
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Hypsochromic Immunoblotting 1012 and fruit bodies in Schizophyllum, Plant.Cell, 3, 793-799, 1991; Wessels, J.G., Hydrophobins: proteins that change the nature of the fungal surface, Adv.Microb.Physiol. 38, 1-45, 1997; Ebbole, D.J., Hydrophobins and fungal infection of plants and animals, Trends Microbiol. 5, 405-408, 1997; Wosten, H.A., Hydrophobins: multipurpose proteins, Annu.Rev.Microbiol. 55, 625-646, 2001; Linder, M.B., Szilvay, G.R., Nakari-Setala, T., and Penttila, M.E., Hydrphobins: the protein-amphiphiles of filamentous fungi, FEMS Microbiol.Rev. 29, 877-896, 2005.
Hypsochromic
metal ion affinity adsorption and immobilized metal ion affinity chromatography of biomaterials. Serum protein affinities for gel-immobilized iron and nickel ions, Biochemistry 23, 1621-1630, 1982; Porath, J., Immobilized metal ion affinity chromatography, Protein Expr.Purif. 3, 263-281, 1992; Skerra, A., Engineered protein scaffolds for molecular recognition, J.Mol.Recongnit. 13, 167-187, 2000; Gaberc-Proekar, V. and Menart, V., Perspectives of immobilized-metal affinity chromatography, J.Biochem.Biophys. Methods 49, 335-360, 2001; Ueda, E.K., Gout, P.W., and Morganti, L., Current and prospective applications of metal ion-protein binding, J.Chromatog.A. 988, 1-23, 2003.
A shift of light absorption or emission to a shorter wavelength (l