Chemistry of Protein and Nucleic Acid Cross-Linking and Conjugation: 2nd Edition (Hardback) book cover

Chemistry of Protein and Nucleic Acid Cross-Linking and Conjugation

2nd Edition

By Shan S. Wong, David M. Jameson

CRC Press

622 pages | 3 Color Illus. | 198 B/W Illus.

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Since the publication of the first edition of Chemistry of Protein Conjugation and Cross-Linking in 1991, new cross-linking reagents, notably multifunctional cross-linkers, have been developed and synthesized. The completion of the human genome project has opened a new area for studying nucleic acid and protein interactions using nucleic acid cross-linking reagents, and advances have also been made in the area of biosensors and microarray biochips for the detection and analysis of genes, proteins, and carbohydrates. In addition, developments in physical techniques with unprecedented sensitivity and resolution have facilitated the analysis of cross-linked products.

Updated to reflect the advances of the 21st century, this book offers:

  • An overview of the chemical principles underlying the processes of cross-linking and conjugation
  • A thorough list of cross-linking reagents published in the literature since the first edition, covering monofunctional, homobifunctional, heterobifunctional, multifunctional, and zero-length cross-linkers
  • Reviews of the use of these reagents in studying protein tertiary structures, geometric arrangements of subunits within complex proteins and nucleic acids, near-neighbor analysis, protein-to-protein or ligand–receptor interactions, and conformational changes of biomolecules
  • Discusses the application of immunoconjugation for immunoassays, immunotoxins for targeted therapy, microarray technology for analysis of various biomolecules, and solid state chemistry for immobilizations

Table of Contents

Overview of Protein Conjugation


Review of Protein and Nucleic Acid Chemistry


Protein Composition

Protein Functional Groups

Nucleic Acid Chemistry


Reagents Targeted to Specific Functional Groups


Sulfhydryl Reagents

Amino Group–Specific Reagents

Reagents Directed toward Carboxyl Groups

Arginine-Specific Reagents.

Histidine-Selective Reagents

Methionine-Alkylating Reagents

Tryptophan-Specific Reagents

Serine-Modifying Reagents


How to Design and Choose Cross-Linking Reagents


Use of Nucleophilic Reactions

Use of Electrophilic Reactions

Incorporating Group-Directed Reagents

Incorporating Photoactivatable Nonspecific Groups

Changing the Water Solubility of Cross-Linkers

Incorporating Special Characteristics in the Bridge Spacer


Homobifunctional Cross-Linking Reagents


Amino Group–Directed Cross-Linkers

Sulfhydryl Group–Directed Cross-Linkers

Carboxyl Group–Directed Cross-Linking Agents

Phenolate and Imidazolyl Group–Directed Cross-Linking Reagents

Arginine Residue–Directed Cross-Linkers

Methionine Residue Cross-Linking Agent

Carbohydrate Moiety–Specific Reagents

Nondiscriminatory Photoactivatable Cross-linkers

Noncovalent Homobifunctional Cross-Linking Reagents

Nucleic Acid Cross-Linking Reagents


Heterobifunctional Cross-Linkers


Group-Selective Heterobifunctional Reagents for Protein Cross- Linking

Protein-Photosensitive Heterobifunctional Cross-Linking Reagents

Noncovalent Immunoglobulin Cross-Linking System

Heterobifunctional Nucleic Acid Cross-Linking Reagents


Multifunctional Cross-Linking Reagents


Trifunctional Cross-Linkers

Tetrafunctional Cross-Linkers

Multifunctional Cross-Linkers

Noncovalent Cross-Linkers


Monofunctional and Zero -Length Cross -Linking Reagents


Monofunctional Cross-Linking Reagents

Zero-Length Cross-Linking Reagents


General Approaches for Chemical Cross-Linking


Classification of Cross-Linking Procedures

General Conditions for Cross-Linking

Cross-Linking Protocols for Commonly Used Reagents

Cross-Linking Protocols Based on Biological Systems

Conditions for Cleavage of Cross-Linked Complexes

Reaction Complications


Analysis of Cross-Linked Products




Applications of Chemical Cross-Linking to the Study of Biological Macromolecules


Determination of Tertiary Structures of Proteins

Determination of Quaternary Structures of Proteins

Determination of Protein–Protein Interactions

Detection of Protein Conformational Changes

Determination of Nucleic Acid Interaction and Nucleic Acid–Protein Interaction

Effects of Cross-Linking on Structural Stability and Biological Activity


Applications of Chemical Conjugation in the Preparation of Immunoconjugates and Immunogens


Preparation of Immunoconjugates

Preparation of Immunogens

Characterization of Conjugation Methods


Application of Chemical Conjugation for the Preparation of Immunotoxins and Other Drug Conjugates for Targeting Therapeutics


Targeting Agents and Toxins

Preparation of Therapeutic Conjugates


Application of Chemical Conjugation to Solid-State Chemistry


Functionalities of Matrices

Protein Immobilization by Matrix Activation

Cross-Linking Reagents Commonly Used for Immobilization of Biomolecules

Immobilization by Cross-Linking through Carbohydrate Chains

Examples of Applications of Solid-Phase Immobilization Chemistry


Appendix A: Amino Group–Directed Homobifunctional Cross-Linkers

Appendix B: Sulfhydryl Group– Directed Homobifunctional Cross-Linkers.

Appendix C: Phenolate- and Imidazolyl-Group-Directed Reagents: Bisdiazonium Precursors

Appendix D: Group Selective Heterobifunctional Cross-Linkers

Appendix E: Photoactivatable Heterobifunctional Cross-Linking Reagents


About the Authors

Shan S. Wong, Ph.D., recently retired from the National Institutes of Health, where he served as a scientific review administrator and a program officer. In the latter capacity, he oversaw scientific programs in the area of alternative and complementary medicine. Previously, he served as director of clinical chemistry at Hermann Hospital and Lyndon B. Johnson General Hospital in Houston, Texas, and as a faculty member at the University of Texas Health Science Center at Houston. Before joining the University of Texas, Dr. Wong was a full professor of chemistry at the University of Massachusetts at Lowell. In addition to teaching at the University of Massachusetts at Lowell, he also taught chemistry courses at Denison University, Granville, Ohio, and Ohio State University, Columbus.

Dr. Wong has published extensively in various scientific journals in the area of enzymology and clinical chemistry. He has received numerous honors and awards and has been active in various professional societies.

David M. Jameson, Ph.D., joined the Department of Cell and Molecular Biology at the John A. Burns School of Medicine at the University of Hawaii in 1989, where he is presently a full professor. Before moving to Hawaii he was on the faculty of the Pharmacology Department at the University of Texas Southwestern Medical School in Dallas.

Dr. Jameson received his BS in chemistry from Ohio State University in 1971 and his PhD in biochemistry from the University of Illinois at Urbana-Champaign in 1978. His thesis advisor was Gregorio Weber, who laid the foundations of modern fluorescence spectroscopy. Dr. Jameson carried out postdoctoral research at the Université Paris-Sud at Orsay, France before returning to the University of Illinois for a postdoctoral period in Gregorio Weber’s laboratory. In 1983, he joined the Pharmacology Department at the University of Texas Southwestern Medical Center at Dallas as an assistant professor. In 1989, he moved to the University of Hawaii.

Dr. Jameson’s primary research focus has always been the development and application of fluorescence approaches for the study of biomolecular interactions, in particular protein–protein and protein–ligand interactions. He has published extensively in this area (some 130 publications to date) and has received funding from the National Science Foundation, the American Heart Association, and the National Institutes of Health. He has also received the Established Investigator Award from the American Heart Association and the 2004 Gregorio Weber Award for Excellence in Fluorescence Theory and Application. He lectures at numerous fluorescence workshops around the world and is co-organizer of the International Weber Symposium on Innovative Fluorescence Methodologies in Biochemistry and Medicine held every three years in Hawaii.

Subject Categories

BISAC Subject Codes/Headings:
MEDICAL / Biochemistry
SCIENCE / Chemistry / Organic