Biophysics This Page Intentionally Left Blank Biophysics Vasantha Pattabhi N. Gautham Department of Crystallography & Biophysics University of Madras, Guindy Campus Chennai, India KLUWER ACADEMIC PUBLISHERS NEW YORK, BOSTON, DORDRECHT, LONDON, MOSCOW Narosa Publishing House DELHI CHENNAI MUMBAI KOLKATA eBook ISBN: 0-306-47520-0 Print ISBN: 1-4020-0218-1 ©2002 Kluwer Academic Publishers New York, Boston, Dordrecht, London, Moscow Print ©2002 Kluwer Academic Publishers Dordrecht All rights reserved No part of this eBook may be reproduced or transmitted in any form or by any means, electronic, mechanical, recording, or otherwise, without written consent from the Publisher Created in the United States of America Visit Kluwer Online at: http://kluweronline.com and Kluwer's eBookstore at: http://ebooks.kluweronline.com This book is dedicated to Professor G.N. Ramachandran, F.R.S. who continues to inspire generations of biophysicists Wisdom grasps the truth of whatever and by whomever said. Kural 423 This Page Intentionally Left Blank Preface The initiation of biophysics in India is synonymous with the starting of the Department of Crystallography and Biophysics at the University of Madras, in 1953, under the leadership of Professor G.N. Ramachandran. Even today, nearly half-century later, there are only a handful of Universities in the country which have full-fledged biophysics departments. However, most life science curricula in the country now comprise a course in biophysics, owing to its increasing importance and relevance to the study of any biological system. This book has been written to fill a long-standing lacuna of textbooks in biophysics, suitable for students at the senior undergraduate and postgraduate level in various biological disciplines, such as biochemistry, molecular biology and even medicine. Biophysics is an interdisciplinary subject, and its treatment varies with the background of both the author and the reader. For example, some aspects of theoretical biophysics are indistinguishable from pure mathematics. On the other hand, modern biophysics has made profound contributions to subjects normally considered a part of classical biology, such as evolution. In this book we have not assumed any knowledge of higher mathematics on the part of the reader as a prerequisite. We have emphasized an understanding of the underlying physical principles of the biological phenomena. Many of the explanations may therefore appear too elementary and too verbose to sophisticated readers. We believe however that the ‘great divide’ between students of the physical sciences who are mathematically fluent, and those of the biological sciences who are less so, is too deep to be bridged with one easy leap in a single textbook. Thus the present book does not set out to make biophysicists out of all biologists. However we believe it will cater to the needs of most biologists, who stop their acquaintance with mathematics at school. In addition, chemists and physicists may also find it useful as an introduction to the subject. The teaching notes of the authors form the skeleton of the book and this was fleshed out on the basis of several years of classroom experience. Chapter 1 gives an introduction to laws of physics and chemistry and is designed to reinforce and enhance slightly the school-level understanding of the subject, which students with a background of purely biological science would have. This chapter covers quantum mechanics, molecular orbital theory, essentials of weak and strong molecular interactions, stereochemistry, fundamentals of thermodynamics and radiation biophysics. Chapters 2 and 3 deal with separation techniques and physico-chemical techniques used to study viii Preface biomolecular structure. Strictly speaking this would be counted under physical biochemistry, rather than purely biophysics. The coverage is not exhaustive and is intended to give an overview of various techniques available to unravel the properties of biomolecules. Chapters 4 to 9 describe some of the spectroscopy, microscopy, diffraction and computational techniques. These techniques are so powerful that every biophysicist must be armed with this knowledge so that it can be put to use when the need arises. These chapters aim at providing an understanding of the basic principles involved rather than thorough knowledge of the theory and practice of the techniques. The descriptions would enable most biologists to obtain an understanding of the relative merits and demerits of each technique, in order that the biochemical results, which one may come across in the general literature, become intelligible. X-ray crystallography and NMR spectroscopy have been dealt with in greater detail owing to their prominence in determining biomolecular structures. Results obtained from the application of the techniques of structural biology are reviewed in Chapter 10. This chapter is illustrated by the largest number of diagrams. Nevertheless, in a book of this nature, in which one is always trying to keep costs low, it is not possible to include every picture the authors may feel necessary. Also we would have dearly liked to include colour illustrations. The later chapters try and fill the gaps between biophysicists and biochemists. The treatment of Energy Pathways in Chapter 11 involves a lot of physical chemistry. Biomechanics in Chapter 12 and Neurophysics in Chapter 13 involve a lot of physiology. Though the advancements in the application of physical principles in these fields have been phenomenal they are probably too advanced and too detailed for the intended audience of this book. We have of course referred to several books and journal articles, review papers, etc, while writing this book. At the end of the book we have included a chapter-wise list for further reading, which would amplify and extend the contents of each chapter. This list is of course neither exhaustive nor comprehensive. We have as far as possible selected books that would be available in most University libraries. This book was written as a project sponsored and funded by the University Grants Commission, India. We take this opportunity to thank the UGC and all its officers for the invitation and support, financial and otherwise. We also thank the reviewers, whose corrections and suggestions we have tried to incorporate. Any errors that remain are, of course, ours. Our M.Sc., M.Phil and Ph.D. students have taught us (or have forced us to learn) much of the material presented in this book. We thank them all most sincerely. For actual help in preparing the book, we thank Mr A. Johnson and Mr. Prem Raj Bernard. We would like to specially thank our respective family members for their enthusiastic support throughout. Writing this book has been a rewarding experience. But as we look back over it, we feel, with Thomas Hardy, ‘the more written, the more it seems remains to be written’. VASANTHA PATTABHI N. GAUTHAM Contents Preface vii 1. Laws of Physics and Chemistry 1 1.1 Introduction 1 1.2 Quantum Mechanics 1 1.3 The Electronic Structure of Atoms 3 1.4 Molecular Orbitals andCovalent Bonds 5 1.5 Molecular Interactions 8 1.5.1 Strong interactions 9 1.5.2 Weak interactions 10 1.6 Stereochemistry andChirality 11 1.6.1 Stereochemical nomenclature 13 1.7 Thermodynamics 13 1.7.1 Entropy 16 1.7.2 Enthalpy 17 1.7.3 The free energy of a system 17 1.7.4 Chemical potential 18 1.7.5 Oxidation-reduction potential 19 1.8 Radioactivity 20 1.8.1 Rate of radioactive decay 21 1.8.2 Measurement of radioactivity 22 1.8.3 Effects of radioactivity on matter 22 1.8.4 Biological effects of radiation 23 1.8.5 Applications of radio isotopes 23 2. Separation Techniques 24 2.1 Introduction 24 2.2 Chromatography 24 2.2.1 Column chromatography 26 x Contents 2.2.2 Thin layer chromatography 26 2.2.3 Paper chromatography 26 2.2.4 Adsorption chromatography 27 2.2.5 Partition chromatography 27 2.2.6 Gas liquid chromatography (GLC) 28 2.2.7 Ion exchange chromatography 28 2.2.8 Molecular exclusion chromatography 29 2.2.9 Affinity chromatography 30 2.3 Electrophoresis 32 2.3.1 Moving boundary electrophoresis 32 2.3.2 Zone electrophoresis 33 2.3.3 Low voltage electrophoresis 33 2.3.4 High voltage electrophoresis 33 2.3.5 Gel electrophoresis 34 2.3.6 Sodium dodecyl sulphate poly acrylamide gel electrophoresis (SDS-PAGE) 2.3.7 Iso electric focussing 35 2.3.8 Continuous flow electrophoresis 35 3. Physico-Chemical Techniques to Study Biomolecules 37 3.1 Introduction 37 3.2 Hydration of Macromolecules 38 3.3 Role of Friction 38 3.4 Diffusion 39 3.5 Sedimentation 41 3.6 The Ultracentrifuge 43 3.7 Viscosity 46 3.8 Rotational Diffusion 48 3.8.1 Flow birefringence measurements 49 3.8.2 Electric birefringence 50 3.9 Light Scattering 51 3.10 Small Angle X-ray Scattering 54 4. Spectroscopy 58 4.1 Introduction 58 4.2 Ultraviolet/Visible Spectroscopy 59 4.3 Circular Dichroism (CD) and Optical Rotatory Dispersion (ORD) 61 4.4 Fluorescence Spectroscopy 65 4.5 Infrared Spectroscopy 67 4.6 Raman Spectroscopy 71 4.7 Electron Spin Resonance 74 5. Light Microscopy 77 5.1 Introduction 77 5.2 Elementary Geometrical Optics 77 5.3 The Limits of Resolution 79 5.4 Different Types of Microscopy 82 5.4.1 Bright field microscopy 82 Contents xi 5.4.2 Dark field microscopy 82 5.4.3 Phase contrast microscopy 82 5.4.4 Fluorescence microscopy 83 5.4.5 Polarising microscopy 84 6. Electron Microscopy 86 6.1 Introduction 86 6.2 Electron Optics 86 6.3 The Transmission Electron Microscope (TEM) 87 6.4 The Scanning Electron Microscope (SEM) 88 6.5 Preparation of the Specimen for Electron Microscopy 90 6.6 Image Reconstruction 91 6.7 Electron Diffraction 92 6.8 The Tunnelling Electron Microscope 92 6.9 Atomic Force Microscope 93 7.