Third Edition

PROTEIN
MODIFICATION
Chemical Reagents for
Copyright 2005 by CRC Press
CRC PRESS
Boca Raton London New York Washington, D.C.
Third Edition
PROTEIN
MODIFICATION
Chemical Reagents for
Roger L. Lundblad
Copyright 2005 by CRC Press

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2005 by CRC Press
No claim to original U.S. Government works
International Standard Book Number 0-8493-1983-8
Library of Congress Card Number 2004051973
Printed in the United States of America 1 2 3 4 5 6 7 8 9 0
Printed on acid-free paper

Library of Congress Cataloging-in-Publication Data

Lundblad, Roger L.
Chemical reagents for protein modication / by Roger L. Lundblad.3rd ed.
p. cm.
Includes bibliographical references and index.
ISBN 0-8493-1983-8 (alk. paper)
1. ProteinsChemical modication. I. Title.
QP551.L883 2004
572

.6dc22 2004051973

1983_C00.fm Page iv Thursday, October 7, 2004 11:53 AM
Copyright 2005 by CRC Press

Preface

This material rst appeared in 1983 as

Chemical Reagents for Protein Modication

.
The rst version was prepared in collaboration with Dr. Claudia Noyes, who moved
on to greener pastures, leaving me with sole responsibility for the later versions that
appeared in 1991 and 1994. The current edition represents a major revision from
previous editions. The chapters on amino acid analysis, peptide separation by HPLC,
and amino acid sequence determination have been removed. The material on amino
acid analysis has been set as Appendix II (determination of protein concentration),
as amino acid analysis is increasingly regarded as a gold standard in the determi-
nation of protein concentration.
In the preface of the 1994 edition I stated, While a consideration of major
biochemical journals would suggest that the use of chemical modication to study
the relationship between structure and function in proteins is no longer an active
area I retract this statement for the present edition with the caveat that it is
increasingly difcult to know whether chemical modication has been used in a
given study without a thorough consideration of the manuscript. This is most notable
in proteomics, wherein chemical modication is extensively used but never really
discussed. The present material will be of signicant value to investigators working
in proteomics. Another area is the biochemistry of aging, wherein oxidation and the
various reactions of nitric oxide are of great importance. The study of these phe-
nomena would not be possible without considering previous work on the chemical
modication of proteins.
I hope that this book will be of value to investigators with training in different
disciplines. I strongly encourage the rigorous reporting of laboratory techniques. It
is discouraging to read an otherwise useful paper in which temperature is referred
to as room temperature, reaction time is not given, buffer details are not presented,
and so on. If investigators wish to have their work seriously considered for thera-
peutic or diagnostic development, information such as precise temperature and
reaction time is required.
Finally, I strongly encourage the careful consideration of Chapter 1 before
embarking on the design of an experiment by using chemical reagents for the
modication of proteins.

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Copyright 2005 by CRC Press

Acknowledgments

In the previous version of this material,

Techniques in Protein Modication

, I com-
mented that major portions of the book had been written in various airports and at
various altitudes. I am not sure whether this is a good way to write a book, and
hence the present version has been written in Chapel Hill, NC. Thus, my rst
acknowledgment is to the libraries of the University of North Carolina at Chapel
Hill. The staff and facilities of the Health Sciences Library, the Kenan Library in
the Department of Chemistry, and the Brauer Library (Physics, Mathematics) were
very helpful. I acknowledge Professor Bryce Plapp of the University of Iowa for his
encouragement to the thermodynamically challenged and Professor Nicholas Price
of the University of Glasgow for guidance regarding content. Other individuals
making unique contributions include Professor William McClure of the University
of Southern California and Professors Charles Craik and Robert Fletterick of the
University of California at San Francisco. I am indebted to Professor Ralph Bradshaw
of the University of California at Irvine for his guidance in both the murky world
of proteomics and the continuing dominance of Duke basketball. Finally, I acknowl-
edge the continuing force of the fth oor of Flexner Hall at the Rockefeller
University.

Roger L. Lundblad

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Copyright 2005 by CRC Press

Table of Contents

Chapter 1

The Site-Specic Chemical Modication of Proteins

Chapter 2

The Modication of Amino Groups

Chapter 3

The Modication of Histidine Residues

Chapter 4

The Modication of Arginine

Chapter 5

The Modication of Carboxyl Groups

Chapter 6

The Modication of Cysteine

Chapter 7

The Modication of Cystine
Appendix I: Microprocedure for the Reduction and
Carboxymethylation of Proteins
General Comments
Materials
Procedure
Notes

Chapter 8

The Modication of Methionine

Chapter 9

The Modication of Tyrosine

Chapter 10

The Chemical Modication of Tryptophan

Chapter 11

The Chemical Cleavage of Peptide Bonds

Chapter 12

The Chemical Cross-Linking of Peptide Chains

Appendix I

Some Reagents Used in the Modication of Proteins

Appendix II

Assay of Protein Concentration in Solution
Microplate Biuret Assay
Reagent Preparation
Standard

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Copyright 2005 by CRC Press

Blank
Procedure
Comments
BCA (Bicinchoninic Acid) Assay for Protein
Reagent Preparation
Standard
Blank
Procedure
Comments
Dye-Binding Assay for Protein Using Coomassie Brilliant
Blue G-250
Reagent Preparation
Standard
Blank
Procedure
Comments
Amino Acid Analysis

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Copyright 2005 by CRC Press

Author

Dr. Roger Lundblad is a native of San Francisco, CA, and resident of Chapel Hill,
NC, where he is an independent consultant in biotechnology. He is the immediate
past editor-in-chief of

Biotechnology and Applied Biochemistry

and at present a
member of the editorial board. He is also an adjunct professor of pathology at the
University of North Carolina at Chapel Hill. Dr. Lundblad received his B.S. in
chemistry from Pacic Lutheran University in Tacoma, WA, and his Ph. D. degree
from the University of Washington in Seattle. After several years of post-doctoral
study at the Rockefeller Institute in New York, with Nobel laureates Stanford Moore
and William Stein, he joined the faculty of the University of North Carolina at Chapel
Hill in 1968. He rose quickly through the ranks and was promoted to professor of
pathology, biochemistry and periodontics in 1977. In 1991, he was recruited by
Baxter Healthcare as director of technology development at the Hyland Division
Facility in Hayward, CA. Dr. Lundblad moved to southern California in 1992 to
become director of science and technology development at Baxter Hyland in Duarte,
CA. During his time at Baxter, he became a member of the Senior Leadership Group
in the Baxter Technical Council and was chair of the committee on technical and
organizational knowledge. Dr. Lundblad left Baxter in 2000 to become an indepen-
dent consultant in biotechnology.
Dr. Lundblad is the author of more than 120 publications and is also the author
of best-selling books on protein chemistry. He has edited several books in the area of
biotechnology. Dr. Lundblad is recognized as an expert in the area of protein
chemistry, thrombosis and hemostasis, biotechnology manufacturing, process vali-
dation, assay validation, GLP laboratory compliance, product development, and
cGMP issues.

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Copyright 2005 by CRC Press