Sulfhydryl and Disulfide Groups of Proteins

282 Pages·1974·8.771 MB·English

by  Yu. M. Torchinskii (auth.)

SULFHYDRYL AND
DISULFIDE GROUPS
OF PROTEINS
STUDIES IN SOVIET SCIENCE
LIFE SCIENCES
1973
Motile Muscle and Cell Models
N. I. Arronet
Pathological Effects of Radio Waves
M. S. Tolgskaya and Z. V. Gordon
Central Regulation of the Pituitary-Adrenal Complex
E. V. Naumenko
1974
Sulfhydryl and Disulfide Groups of Proteins
Yu. M. Torchinskii
Mechanisms of Genetic Recombination
V.V.Kushev
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STUD~SINSOWETSC~NCE
SULFHYDRYL AND
DISULFIDE GROUPS
OF PROTEINS
Yu. M. Torchinskii
Institute of Molecular Biology
Academy of Sciences of the USSR
Moscow, USSR
Translated from Russian by
H. B. F. Dixon
Department of Biochemistry
University of Cambridge
Cambridge, England
Revised by Yu. M. Torchinskii and H. B. F. Dixon
CONSULTANTS BUREAU • NEW YORK AND LONDON
Library of Congress Cataloging in Publication Data
Torchinskrr, Writ Moiseevich.
Sulfhydryl and disulfide groups of proteins.
(Studies in Soviet science)
Translation of Sul'fgidril'nye i disul'fidnye gruppy belkov.
1. Proteins. 2. Thiols. 3. Sulphides. I. Title. II. Series. [DNLM: 1. Cystine. 2.
Sulfhydryl compounds. QU60 T676s 1974]
QP551.T6913 574.1'9245 73-83903
ISBN 978-1-4757-0129-6
The original Russian text, published by Nauka Press in Moscow in 1971,
has been corrected by the author for the present edition. This translation
is published under an agreement with the Copyright Agency of
the USSR (VAAP).
CYJlbcllrHAPHJlbHJ>le H AHcyJlhcllHJl.HblC rpynnbl lieJlKOB
/0. M. TOP'IUfICfCutl
SUL'FGIORIL'NYE I OISUL'FIONYE GRUPPY BELKOV
Yu. M. Torchinskii
ISBN 978-1-4757-0129-6 ISBN 978-1-4757-0127-2 (eBook)
DOI 10.1007/978-1-4757-0127-2
© 1974 Consultants Bureau, New York
Softcover reprint of the hardcover 1s t edition 1974
A Division of Plenum Publishing Corporation
227 West 17th Street, New York, N.Y. 10011
United Kingdom edition published by Consultants Bureau, London
A Division of Plenum Publishing Company, Ltd.
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All rights reserved
No part of this book may be reproduced, stored in a retrieval system, or transmitted,
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recording, or otherwise, without written permission from the Publisher
Contents
Preface .•................................ ix
Introduction ....... . 1
Part I
The Chemistry of SH and S-S Groups and
Methods for Their Determination
CHAPTER 1. The Chemical Properties of SH
Groups. Sulfhydryl Reagents
1. The Ionization of SH Groups. . . . . . . . . . . . . . . . 7
2. Hydrogen Bond Formation. . . . . . . . . . . . . . . . . 12
3. Acylation of SH Groups. Properties of Thioesters . 14
4. ReactionwithCyanate .•.................. 17
5. Reaction with Carbonyl Compounds. • . . . . . . . • . 20
6. Alkylation and Arylation of SH Groups. • . . . . . . . 23
a. Addition to an Activated Double Bond •.. . . . • 23
b. Reactions with Quinones. . . . . . . . . . . . . . . . 27
c. Reactions with Haloacids and Their Amides . • . 29
d. Reactions with Ethylene Oxide and
Ethylene Imine . . . . . . • . . . . . . . . . . . . . . . 35
e. Reactions with O-Methylisourea and Methyl-
p-nitrobenzenesulfonate . . • . . . . . . • . . . . . . 36
f. Arylation . . • . . . . . . . . . . . . . . . . . . . . . . . 38
g. Mixed Reactions ..................... 40
7. Reactions with Metal Ions and with Organic
Mercury Compounds . . . . . . . . . . . • . . . • . . . . . . 40
8. Reaction with Arsenic Compounds. . . . . . . . . • . . 44
9. Oxidation of SH Groups . . . . . . . . . . . . . . • • • • . 46
v
vi CONTENTS
10. The Thiol-Disulfide Exchange Reaction and
Its Significance .•....................•.. 54
11. Reaction with Sulfenyl Halides . . . . . . . . . . . . . . 59
12. Concluding Remarks. . . . . . . . . . . . . . . . . . . . . 60
CHAPTER 2. Che mi c a I Pr op e rtie s of S-S Gr oup s
and Methods for Their Scission
1. The Geometry and Optical Properties of the
Cystine Molecule . . . . . . . . . . • . . . . . . . . . . . . . 65
2. Reaction with Metal Ions . . . . . . . . . . . . . . . . . . 68
3. Reaction with Sulfite. • . . . . . . . . • . . . . . . . . . . 69
4. Reaction with Cyanide. . . . . . . . . . . . . . . . . . . . 74
5. Reaction with Monothiophosphoric Acid . . • . . . . . 76
6. Scission in Alkaline Medium ... . . . . . . . . • . . . 77
7. Reduction with Thiols . . . . . . . . . . . . . . . . . . . . 81
8. Disulfide Exchange. . . . . . . . . . . • . . . . . . . . • . 84
9. Reduction by Sodium Borohydride . . • • . . . . . . . . 86
10. Electrolytic Reduction . . . . . . . . . . . . . . . . . . . 87
11. Reduction by Tertiary Phosphines . . . . . • . . . . . • 88
12. Desulfurization with Raney Nickel. . . . . . . . . . . • 89
13. Photolysis ......•...•......•......... 90
14. Oxidation of S-S Groups. Identification of
Cystine-Containing Peptides . . . . . . . . . . . • . . . . . 94
15. Concluding Remarks. . . . . . . . . . • . . . . . . . . 97
CHAPTER 3. Methods for the Quantitative
Determination of SH and S-S
Groups in Proteins
1. The Determination of SH Groups. . . • • . • • • • • • • 99
a. Methods Based on Mercaptide Formation. . . . . 99
b. Methods Based on Alkylation. . . . . . . . . . . .. 107
c. Methods Based on Reactions with Disulfides
and Sulfenyl Halides. • • . . . . . . • . . . . . • • •. 109
d. Methods Based on Oxidation • . . . • • . • • • • •. 116
2. Determination of S-S Groups. • • . • . . . • . . • . .• 116
a. Determination as Cysteic Acid. • • • • . . • . . .• 117
b. Determination as S-sulfocysteine . . . . . . . . .. 118
c. Determination as S-,B-(4-pyridylethyl)
cysteine. • • • • • . . • . . . . . . . . . . . . . . . • .. 118
d. Determination as S-2-(2-quinolylethyl)
cysteine. . . . • . . . • . . . • . . . . . . . . . . . . .. 119
CONTENTS vii
e. Determination as S-(p-nitrophenethyl)
cysteine ....................... 119
f. Amperometric Titration ................ 120
g. Fluorometric Method .............•.... 121
h. Methods Based on Reduction of S-S Groups
and the Subsequent Use of Ellman's Reagent ... 122
i. Determination as Hydrogen Sulfide 123
• • 1:1 • • • • • •
j. Direct Spectrophotometric Measurement
of Reduction by Dithiothreitol ............. 123
CHAPTER 4. The Reactivity of SH Groups
in Proteins
1. Types of SH Groups . . . . . . . • • . . . . . . . • . . .. 125
2. Causes of Altered Reactivity of SH Groups. • . . .. 126
a. Mechanisms of Activation of SH Groups. • • . •• 127
b. Mechanisms of "Masking" SH Groups. The
Formation of Intramolecular Bonds .... 132
3. Conclusions 138
Part II
The Role of SH and S-S Groups in Enzymes
and Other Proteins
CHAPTER 5. The Essential SH Groups of
Enzymes and Methods for Their
Identification
1. The Concept of Essential Groups •... . . . . • . .. 141
2. Direct Methods of Identifying SH Groups in the
Active Sties of Enzymes ........•.......... 143
3. Indirect Methods of Identifying SH Groups
in the Active Sites of Enzymes . . . . . . . . . . . . . .. 151
CHAPTER 6. The Role of SH Groups in E;nzymes
1. The Role of SH Groups in Catalysis. . . . . . . . . .. 155
a. SH Groups of Proteins as Nucleophilic
Catalysts • . . • • . . . . . . . . . . . . . • • . . . . .. 155
b. Dithiol-Disulfide Conversions in the Active
Sites of Oxidizing Enzymes. . . . . • . . . . . . .. 168
c. Thiol Cofactors . . . . . • . . . . • . . . . . . • • . .. 172
2. The Role of SH Groups in the Binding of
Substrates and Cofactors (Metal Ions
and Coenzymes) • • • • . . . . • • • • • • • . • . • • . • • .. 185
viii CONTENTS
3. The Role of SH Groups in the Maintenance of
the Native Conformation of Enzymes . . . . . . • . • •• 198
4. Conclusion . . . . • . . . • • . . . . . • . • . . • . . . • .. 206
CHAPTER 7. The Role of S-S Groups in Proteins
1. The Role of S-S Groups in Stabilizing the
Macromolecular Structure of Proteins. • • • • . • • .• 207
2. The Formation of S-S Groups in the Renatura-
tion and Biosynthesis of Proteins •••.•..•...•. 215
3. S-S Groups in Immunoglobulins . • . • • . . . • . • •. 221
4. S-S Groups in Peptide Hormones . • . . . • • • • • •. 224
5. S-S Groups in Fibrous Proteins . . • • • • . • . • . •• 227
6. Concluding Remarks. • • . . . . . . . . . . . . . • • • •. 231
Reference s . • • . . . . . . . . . • • . • • • . . . . • . . . • . • . . .• 233
Index • • • . • . . . . . . • • . • • • . . . • . . . . . . . . . . • . • • .. 265
Preface
(Written for the original Russian edition)
Out of all the functional groups of protein molecules that be
long to the side chains of amino acid residues, the sulfhydryl or
thiol (SH) groups of cysteine residues and the disulfide (S-S)
groups of cystine have for long attracted particular attention from
chemists and biochemists. This is due, on the one hand, to the
high chemical reactivity of these groups, which enter into many,
and often very characteristic and selective, reactions with several
types of compound. On the other hand, this attention is connected
with the great significance of SH and S-S groups for the specific
functions of a number of enzymes, hormones, and other biological
ly active proteins, and consequently for the normal course of many
physiological processes.
The varied functions of sulfhydryl and disulfide groups in en
zymes are particularly interesting; their role can consist of bind
ing substrates and cofactors, in direct participation in the catalytic
act, or in maintaining (in ways not so far sufficiently studied) the
native, catalytically active conformation of the protein.
It is entirely natural that the detailed study of the chemical
behavior and biological role of these sulfur-containing groups in
proteins (and in such low-molecular cofactors as glutathione, co
enzyme A, lipoiC acid, etc.) has, in addition to its academic, gen
eral biological interest, great Significance for many applied branches
of medicine and biology (toxicology, radiobiology, diagnostics and
therapeutics, biochemical engineering, plant growing, etc.).
ix