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During the past decade, fluorescence techniques have come to occupy a position of central importance in biochemistry. Such areas as laser techniques, radiation less energy transfer, and nanosecond fluorometry have evolved from esoteric research specialties into standard procedures that are applied routinely to bio chemical problems. Indeed, discussion of the above three areas occupies the greater part of this book. Its level and approach are appropriate for the bio logical or physical scientist who is interested in applying fluorescence techniques, but is not necessarily an expert in this area. The coverage of the literature has, in general, been selective rather than exhaustive. It is likely that what is summarized here will prove resistant to the erosion of time and provide a basis for the future evolution of this rapidly developing area of science. Robert F. Steiner Catonsville, Maryland vii Contents Chapter 1 Some Principles Governing the Luminescence of Organic Molecules R. M Hochstrasser 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 2. Spontaneous Emission . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 2. 1. General Considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 2. 2. Luminescence from Nearby States . . . . . . . . . . . . . . . . . . . . . . . . . 4 2. 3. Multiple State Decay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 3. Molecular Luminescence Characteristics . . . . . . . . . . . . . . . . . . . . . . . . . 5 3. 1. The Transition Dipole Moment . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 3. 2. Determination of Transition-Moment Directions from Fluorescence . . . 8 3. 3. Polarization of Fluorescence from Crystals. . . . . . . . . . . . . . . . . . . . 9 4. Principles of Luminescence Experiments Carried Out with Lasers. . . . . . . 12 4. 1. Nonlinear Processes and Optical Pumping . . . . . . . . . . . . . . . . . . . 12 5. Coherent Interactions of Molecules and Light . . . . . . . . . . . . . . . . . . . . 16 5. 1. The Distinctions between Fluorescence and Resonance Raman Effects 17 6. Ultrafast Fluorescence Decay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
The goal of this book is to give a systematic description of the main principles of affinity modification and applications, consideration of possibilities, and restrictions of the method. Modification within specific complexes is a special case of chemical modification which is widely used in the nonaddressed version in biochemistry and related areas. Therefore, we have included in the first introductory paper chapter of the book general considerations of chemical modifications of biopolymers and the application of biopolymers.
Photonics of biopolymers discusses the processes of energy transformation in photoexcited proteins, nucleic acids, membranes and model systems. The author addresses, among other topics: Light absorption, screening and reabsorption; photometric studies of protein; energy transfer mechanics; fluorescent probes; photomodulation of enzymes, and photoactiviation. Much of the information stems from the author's own wide experience in the field.
The choice of title for this collective volume reflects the desire of the editors and authors to make clear that, while the bulk of the material is concerned with luminescence, other aspects of the excited state have not been excluded. In the five years which have elapsed since the publication of the classical monograph of Konev, a wealth of new information has ap peared on the emission properties of proteins and nucleic acids. Indeed, since new publications in this area appear to be proliferating in a geometric ratio, this may be the last opportunity to provide a comprehensive summary of the field in a book which is not of prohibitive length. This is what we have attempted to do here. While the orientation of each chapter naturally reflects the interests and point of view of the author, there has been a general effort to present .a critical assessment of existing results and interpretations, rather than a compendium of data with minimal comment. Finally, it should be stressed that the rapid evolution of the subject at the time of writing makes it inevitable that the book will age to some degree over the next few years, although this will occur at differing rates for the various chapters. We can only hope that most of the material in this interim summing-up will prove resistant to the erosion of time and provide a solid foundation for further progress.
The book is concerned with the application of physical techniques to the study of the structure and interactions of biopolymers. The treatment is confined to those procedures applicable to solutions. The material has been tested on students in actual classes, thereby permitting the elimination of ambiguities and potential points of difficulty. Stress has been placed upon lucidity of treatment, and difficult steps in derivations have been explained. The mathematical exposition has been made as clear and simple as feasible. Examples of actual data are given.
This monograph—based on the authors course lectures on radiation chemistry of biopolymers—deals with the primary stages of radiation-chemical transformation of main biopolymers. Special attention is given to radiolysis of their aqueous solutions, formation and conversion mechanisms of macroradicals, synthesized in acts of solvent radical interaction with biopolymer molecules and in their natural complexes.
Physics.
Biopolymers deals with the methods of physical characterization and the principles underlying them, with emphasis on quantitative aspects of sequence, conformation, and structure in both laboratory-synthesized and native biopolymers. The book reviews structural units of biopolymers and describes characterization of biopolymers, the available techniques, the evaluation of underlying principles, and experimental applications. Some of these methods include Raman spectroscopy, theoretical conformation analysis, electron microscopy, and morphology of laboratory-synthesized polymers. The text explains the factors controlling conformation of polypeptides, the steric maps of dipeptides, potential energy maps, and the calculation of tertiary polypeptide structure. The investigator can use X-ray diffraction to determine the structure of polymers and macromolecules, such as diffraction by a crystal, by poorly crystalline polymer systems, or by a helical chain. The book notes that materials that can be crystallized from strong solvents reveal morphology similar to that of commercial polymers, which are different from that of polypeptides or proteins in native tissue. The text explains the basis of infrared and Raman spectroscopy in probing molecular structure and conformation of biological macromolecules. The investigator can also employ nuclear magnetic resonance and dielectric relaxation for conformation in physical organic chemistry, outside of biological macromolecule applications. The book can prove helpful for researchers in ultra-trace analysis, polymer research, and analytical chemistry.
Fluorescence and phosphorescence are proving to be extremely sensitive probes for elucidating conformation of proteins and nucleic acids and for studying molecular interactions. Newer instrumentation and techniques hold forth great promise for the future of these luminescence methods in biopolymer research. It must be noted, however, that the discovery that certain amino acids, purines, and pyrimidines emit fluorescence or phosphorescence is relatively recent, occurring within the last decade. Professor Konev is one of the pioneers in the application of these procedures to biopolymers and is highly qualified to write about this subject. This book, though written largely as a monograph of the author's own contributions, is also an excellent review of the subject. Of particular interest are the references to many important Russian papers in this field which have not been recognized in the Western literature. It is apparent from this book that fluorescence and phosphorescence methods are being used about as widely in Russia as elsewhere in the world and that we must not overlook these im portant contributions. Konev's studies on protein fluorescence have been widely recognized. It is of interest to learn about these and other of his applications. The last part of the book, which deals with fluorescence as a means to probe into the structure and conforma tion of macromolecules in intact cells, is most interesting. Aside from published symposia this book is the first written specifically about luminescence of biopolymers. Sidney Udenfriend Bethesda. Maryland May, 1967 v CONTENTS Introduction . • • . . . . . . . . . • . . . . . . . . . .