Biochemistry of Antibodies

394 Pages·1970·9.32 MB·English

by  Roal’d S. Nezlin (auth.)

BIOCHEMISTRY OF
ANTIBODIES
BIOCHEMISTRY OF
ANTIBODIES
Roal'd S. Nezlin
Institute of Molecular Biology
Academy of Sciences of the USSR, Moscow
Translated from Russian by
Michel C. Vale
Translation edited by
Fred Karush
Department of Microbiology
School of Medicine
University of Pennsylvania
Philadelphia, Pennsylvania
~
PLENUM PRESS • NEW YORK-LONDON • 1970
Library of Congress Catalog Card Number 69-12534
SBN 306-30430-9
The original Russian text, first published by Nauka
Press in Moscow in 1966, has been corrected by the
author for this edition. The present translation is pub
lished under an agreement with Mezhdunarodnaya
Kniga, the Soviet book export agency.
Poa.ab8 CO.!WMOH06U1{ Ife a.aUH
BBOXHMHJI aHTBTeJI
BIOKHIMIYA ANTITEL
ISBN 978-1-4684-1793-7 ISBN 978-1-4684-1791-3 (eBook)
DOI 10.1007/978-1-4684-1791-3
© 1970 Plenum Press, New York
Softcover reprint of the hardcover 1s t edition 1970
A Division of Plenum Publishing Corporation
227 West 17th Street, New York, N.Y. 1O01l
United Kingdom edition published by Plenum Press, London
A Division of Plenum Publishing Corporation, Ltd.
Donington House, 30 Norfolk Street, London W.e. 2, England
All rights reserved
No part of this publication may be reproduced in any form
without written permission from the publisher.
Printed in the United States of America
Preface
When the history of immunology in the twentieth century is
written, the decade of the 1960's will, in all probability, stand out
as the period of greatest advance in the development of molecular
immunology. It is appropriate and useful, therefore, that a schol
arly and integrated presentation of this progress should be made
available in English. The translation of Dr. Nezlin's "Biochem
istry of Antibodies" from Russian admirably fulfills this need in
the form of a scientific monograph directed to medical and biolog
ical scientists.
The appearance of this monograph also serves to emphasize
the conceptual unification of diverse immunological phenomena
which has emerged from progress in molecular immunology. This
unity is a consequence of the key role played by the antibody mol
ecule (either in solution or cell-bound) in every biological process
properly described as immunological. Indeed, immunology as an
independent natural science can be described as the study of the
structure, interactions, and biosynthes is of the antibody molecule.
Because of this central role of the antibody molecule the "Biochem
istry of Antibodies," through its exposition of molecular immunol
ogy, provides the essential foundation for the fruitful exploration
of immunobiological phenomena. There is little doubt that the knowl
edge presented in this monograph will be instrumental in the elab
oration of the functional activities of the varieties of cell types in
volved in the immune response and in such potential medical ap
plications of immunology as the prevention of the rejection of or
gan transplants, the immunotherapy of malignancy, and the control
of fertility.
The translation of Dr. Nezlin's book has been an international
v
vi PREFACE
enterprise in which my participation, however minor, has been a
privilege and a source of personal satisfaction.
Fred Karush
Philadelphia, Pennsylvania
1970
Foreword
About 75 years have passed since it was discovered that after
addition of bacterial toxins animal serum acquires the capacity to
neutralize the effect of these toxins. It was found that serum de
rives its antitoxic properties from newly synthesized specific pro
tein components - antibodies. Not only toxins, but also almost all
other proteins, as well as certain other substances, including syn
thetic preparations, are able to stimulate synthesis of specific anti
bodies. According to the current definition, antibodies are pro
teins belonging to the class of immunoglobulins, which are able to
conjugate with the antigens responsible for their formation.
Antibody synthesis is one of the most important protective
mechanisms against infections. Antibodies also account for re
actions of hypersensitivity, which playa leading role in the de
velopment of pathological processes. Hence, it is not surprising
that for a long time antibodies, as one of the chief participants in
immunological reactions, have been of interest not only to micro
biologists and immunologists, but also to physicians, who found in
these proteins an extremely powerful and specific therapeutic
agent.
In recent years, scientists in various fields of molecular biol
ogy, such as chemists, studying the structure and properties of
proteins, biochemists, studying protein biosynthesis, and finally,
geneticists, have devoted an ever increasing amount of research to
antibodies. This interest is accounted for first of all by the fact
that antibodies serve as a convenient model for the study of many
general biological problems. Antibodies are highly specific pro
teins which form complexes only with the antigens causing their
formation. They owe this property to a very small area of the
molecular surface - the binding site. Hence, an elucidation of the
structure of the binding site would be a definite contribution to the
vii
viii FOREWORD
solution of the more general problem, i.e., the relationship of the
specific properties of the protein to its structure. Let us take yet
another example. Antibody formation begins only after addition of
an antigen. It is clear that a clarification of the question of the
site of action of the antigen would represent a definite step toward
an explanation of the regulator mechanisms of protein biosynthesis.
Of considerable importance is the fact that quite simple methods
for the isolation and accurate quantitative determination of pure
antibodies have been developed. Finally, the elusive and puzzling
aspects of the problem will always be a serious psychological
enticement for investigators.
A large quantity of data concerning the properties, struc
ture, and biosynthesis of antibodies has been accumulated from
the assiduous efforts of many research laboratories. The in
formation available requires systematization and discussion, and
it is to this end that the present monograph is devoted. In the first
two chapters two important methodological issues are discussed:
quantitative determination and isolation of antibodies in pure form.
Special attention is given to the most efficient methods - those em
ploying immunoadsorbents. The properties of antibodies are
treated in the third chapter. Recent striking achievements in re
search on the structure of antibodies are the topic of the fourth
chapter. The fifth chapter presents a summary and discussion of
a large amount of data collected in research on the biosynthesis of
antibodies, the sixth chapter explores the genetic aspects of anti
body formation, in particular, the rapidly advancing research on
aUotypes, and finally, the last chapter is devoted to a discussion
of contemporary theories on antibody synthesis. The author ex
presses his appreciation to Doctor of Biological Sciences, N. V.
Kholchev, Professor V. I. Tovarnitskiy, and Professor L. A.
Tumerman for their valuable comments, and to L. M. Kul'pin for
his assistance in planning the book.
Contents
CHAPTER I. Qua n tit at i v e Met hod s for
Determination of Antibodies 1
Heidelberger's Quantitative Precipitation Method • • • 3
Quantitative Determination of Antibodies with the
Aid of Irnmunoadsorbents 9
0 •• 0 • 0 0 0 0 0
Equilibrium Dialysis for the Quantitative
Measurement of the Hapten - Antibody
Reaction •.•.••• 15
0 • • • 0 • 0 • • • • 0 0 •
Fluorescence Methods for Antibody Determination .• 18
0
Literature Cited ... 21
0 .......... 0 0 ••• It • • • • • • •
CHAPTER II. Isolation of Pure Antibodies •.. 27
0
Nonspecific Methods ••• 27
0 • 0 ••••• 0 0 •••••• 0 • • •
Specific Methods • 31
0 0 •• 0 •••••••• 0 •• 0 •••• 0 • •
Isolation of the Antigen-Antibody Complex •••• 31
0
Dissociation of the Antigen-Antibody Complex.. 32
Temperature Change ••• 32
0 • • • • • • • • • • • •
Change in Ionic Strength ••.••• 32
0 • 0 •• 0 0 •
Changes in pH of Medium • • • 33
0 • • • • • 0 0 • •
Other Methods for Dissociation of a
Specific Complex. . • • • . • • • • • • . • . • 34
Separation of Antibody from Antigen after
Dissociation of a Specific Complex • • • • 36
Separation of Antibody from Soluble
Antigen . • . . • 36
0 • • • • • • • • • • • • 0 • • •
Separation of Antibody from Insoluble
Antigens ...••••••••• 40
0 ••• 0 0 0 0 • •
Isolation of Antibodies with the Aid of Fixed
Antigens (Synthetic Irnmunoadsorbents) • 42
Antigens Adsorbed on an Insoluble Support 42
0 • • • •
ix
x CONTENTS
Antigens Linked to an Insoluble Support via a
Chemical Bond . • • • • • • • • • 43
0 • • • • • •
Cellulose-Based Immunoadsorbents • • • • • . 45
Immunoadsorbents Based on Synthetic
Polymers .............. 51
It 0 • D ••
Literature Cited 54
CHAPTER Ill. Pro per tie s 0 fAn t i bod i e s •••.•• 65
The Chief Types of Immunoglobulins •••••••••••• 65
Other Serum Immunoglobulins • • • . . . . • • • • • • 69
Microglobulins in Plasma and Urine .•••.•• 69
0 •
Size and Shape of Molecules •••••.••.••••••••• 71
Molecular Weight. • • • • . • 75
0 • • • • • • • • • 0 • • • • • • •
Electrophoretic Properties 77
0 • • • • • • • • • • • • • • • • •
Optical Properties. 82
0 ••••••••••••••••••••••
Ultraviolet Absorption .• 82
0 •••••••••••••••
Optical Rotatory Dispersion ..•••.• 82
0 •••••••
Fluorescence Polarization. 84
0 ••••••••••••••
Chemical Properties ••• 85
0 ••• 0 ••••••••••••••
Amino Acid Composition. 85
0 •••••••••••••••
Terminal Amino Acid Residues . • . • • • • 88
0 • • • 0
Carbohydrate Component of Immunoglobulins ••• 91
Valence of Antibodies • • 91
0 • • • • • • • • • • • • • • • • • • •
Antigen Properties ••••••••••••.•• 93
0 ••••••••
Determinants Common to All Immunoglobulins .• 93
Determinants Characteristic of the Principal
Types of Immunoglobulins .••••••••• 93
Determinants of Subgroups of the Principal
Types of Immunoglobulins ••.•• 94
0 •• 0 •
Allotypical Determinants 94
0 •• 0 • 0 0 • 0 ••• 0 ••••
Determinants Exposed by Recombination of
HeavY and Light Chains •• 94
0 •••••••••
Internal Determinants .•••• 94
0 ••••••••• 0 0 • 0
Individual Determinants of Immunoglobulins and
Antibodies 95
o •• 0 0 0 • 0 • 0 0 ••• 0 •• 0 • •
Heterogeneity of Antibodies ••••.• 96
0 • • • • • • • • • • •
Literature Cited •••...•..•••••• 111
0 •• 0 • • • • • •
CONTENTS xi
CHAPTER IV. The Structure of Antibodies.. 127
Fragments Obtained by Proteolytic Cleavage
of Immunoglobulins . • • • • • • • • • • • 127
Papain-Cleaved Fragments • • • . • • . • • • • • • 128
Fragments Obtained by Hydrolysis with
Pepsin and Other Proteases. • . • • • • 134
Classification of Proteolytic Fragments. • • • • 139
Subunits and Peptide Chains Obtained by
Chemical Treatment of Molecules
of Immunoglobulins and Antibodies 139
Splitting of Disulfide Bonds. • • . . • . . • • • • • 139
Separation of Peptide Chains • • • . • . • • • • • . 143
Physicochemical Properties of Peptide
Chains 146
g • QQ •••••••••••••• 9 • 0
Molecular Weight. • • . . • • • • • . . . . • • • 146
Absorption at 280 mf.1 • • • • • • • • • • • • • • 148
Solubility • . . • . • • • • • • . • • • • • • • • • • 149
Chemical Composition of Peptide Chains
of Immunoglobulins • • • • . • • • • . • • 149
Antigenic Properties of Peptide Chains . • • • • 152
Peptide Chains of Pathological
Immunoglobulins • • • • • • • • • • . • • • 155
Role of Peptide Chains of Antibodies in the
Antigen Reaction. Molecular
Antibody Hybrids. • • • • • • • • • • • • • 156
yG-Globulin Subunits Containing Both Types
of Chains ••.••••••..••.•••• 166
Cyanogen Bromide Fragments. • • • • • • • • • • 168
Peptide Chains and Subunits of y A- and y M-
Globulins • • • • • . • • • . . . • • • • • • • 169
Classification of Peptide Chains. . • • • • • • • • 170
Structural Model of yG-Globulin Molecules • • 171
Interchain Disulfide Bonds. • • • . • • • . • • 174
The Antibody Binding Site • • • . • • • • • . • • . • • • . 175
Chemical Structure • • • • • • • . • • • • • • • • • • 175
Size of Antibody Binding Sites . • • • • • • • • • • 179
Primary Structure of Antibodies and
Immunoglobulins • • • . • • • . . • • • • • 184