TUTORIAL CHEM ISTRY TEXTS 16 Biophysical Chemistry A L A N C O O P E R Glasgow University RSeC ROYAL SoClEpl OF CHEMISTRY Cover images tc) Murray Robertsonjvisual elements 1998-99, taken from the 109 Visual Elements Periodic Table, available at www.chemsoc.org/viselements ISBN 0-85404-480-9 A catalogue record for this book is available from the British Library 4.) The Royal Society of Chemistry 2004 All rights rescvwcl Apurt from any fair deuling fiw the purposes qf research or privatc study, or criticism or reviews us perniitted under the trrnzs qf the UK Copyright, Designs and Patents Act, 1988, this publication rnq riot be reproduced, stored or transmitted, in urij'jorni or hip any nieuns, ~citlioutt he prior permisxion in w)ritingq f The Royul Society of Chemistry,o r in the case of reprographic reproduction only in accordance with the terms oftlie licences issued bj' the Copyright Licmsing Agency in the UK,o r in uccorcicmce with the terms of the licences issued by the uppropriate Reproduction Rights Organization outside the UK. Enquiries conccrning reproduc*tiono utside the ternis stated here should be sent to The Royul Society qf Clieniistrj- at the crddress printed on this puge. Published by The Royal Society of Chemistry, Thomas Graham House, Science Park, Milton Road, Cambridge CB4 OWF, UK Registered Charity No. 207890 For further information see our web site at www.rsc.org Typeset in Great Britain by Alden Bookset, Northampton Printed and bound by Italy by Rotolito Lombarda Preface Biology is chemistry on an impressive scale. It is a product of evolution, the outcome of countless random experiments, resulting in the exquisite complexity of the biological world of which we are a part. Setting aside any philosophical considerations, living organisms including ourselves are - - simply nothing more than wet, floppy bags of chemistry: complicated mixtures of molecules interacting in a multitude of ways. All this takes place mainly in water, a solvent that most chemists try to avoid because of its complexities. However, we can learn from this. In the course of evolution, biology has had the opportunity to perform vastly more experiments than we can ever contemplate in the laboratory. The resulting chemistry is fascinating in its own right, and we can quite rightly study it for its intellectual satisfaction alone. We can also, if we choose, apply what we learn to other areas of chemistry and to its applications in biomedical and environmental areas. This book is about the physical chemistry of biological macromolecules and how we can study it. The approach here is unashamedly experimental: this is the way science actually works, and in any case we do not yet have the rigorous theoretical understanding perhaps found in more mature areas of chemistry. This is what makes it a fun topic, and why it poses fascinating challenges for both theoretical and experimental scientists. The level adopted in this tutorial text should be suitable for early undergraduate years in chemical or physical sciences. However, since this interdisciplinary topic is often postponed to later years, the book will also act as a basis for more advanced study. Students in other areas of biological sciences might also appreciate the less intimidating approach to physical chemistry that I have attempted here. The term “biophysical chemistry” was brought to prominence by the work of John T. Edsall (1902-2002), who died just prior to his 100th birthday. Together with Jeffries Wyman, he wrote the original classic text: Biophysical Chemistry, Volume I (Academic Press, 1958), but there never was a Volume 2. This book is dedicated to him and to the many other physical scientists who have dared to enter biological territory. With thanks to my family and other animals who have tolerated me during the writing of this text, and to my students and other colleagues who have checked and corrected some of the material. I did not always follow their suggestions so just blame me. - Alan Cooper Glasgo 1.2, iii EDITOR-IN-CHIEF EXECUTIVE EDITORS EDUCAlIONAL CONSULTANT Profkssor E W Ahel Put fessor A G Du vies Mr M Berry Professor D Phillips Projessor J D Woollins This series of books consists of short. single-topic or modular texts, concentrating on the fundamental areas of chemistry taught in undergraduate science courses. Each book provides a concise account of the basic principles underlying a given subject, embodying an independent- learning philosophy and including worked examples. The one topic, one book approach ensures that the series is adaptable to chemistry courses across a variety of institutions. TITLES IN THE SERIES FO RTH CO M I N G T I T 1- E S Stereochemistry D G Morris Mechanisms in Organic Reactions Reactions and Characterization of Solids Molecular Interactions S E Dann Biology for Chemists Main Group Chemistry W Henderson Nucleic Acids d- and f-Block Chemistry C J Jones Organic Spectroscopic Analysis Structure and Bonding J Burrett Functional Group Chemistry J R Hunson Organotransition Metal Chemistry A F Hill Heterocyclic Chemistry M Sainsbury Atomic Structure and Periodicity J Burratt Thermodynamics and Statistical Mechanics J M Seddon and J D Gale Basic Atomic and Molecular Spectroscopy J M Hollas Organic Synthetic Methods J R Hunson Aromatic Chemistry J D Hepworth, D R Wuring und M J Waring Quantum Mechanics for Chemists D 0 Hayward Peptides and Proteins S Doonun Biophysical Chemistry A Cooper Natural Products: The Secondary Metabolites J R Hanson Maths for Chemists, Volume I, Numbers, Functions and Calculus M Cockett and G Doggett Maths for Chemists, Volume 11, Power Series, Complex Numbers and Linear Algebra M Cockett und G Doggett Inorganic Chemistry in Aqueous Solution J Burreti Further information about this series is uvailable at www.r.sc.orgftct Order and enquiries should he sent to: Sales and Customer Care, Royal Society of Chemistry, Thomas Graham House, Science Park, Milton Road, Cambridge CB4 OWF, UK + + Tel: 44 I223 432360; Fax: 44 1223 42601 7; Email: [email protected] Contents 1.1 Introduction 1 1.2 Proteins and Polypeptides 2 1.3 Polynucleotides 7 1.4 Polysaccharides 9 1.5 Fats, Lipids and Detergents 10 I .6 Water 11 1.7 Acids, Bases, Buffers and Polyelectroytes 14 1.8 A Note about Units 17 2.1 Electromagnetic Waves and their Interactions 21 2.2 UV/Visible Spectroscopy 28 2.3 Circular Dichroism 40 2.4 Fluorescence 43 2.5 Vibrational Spectroscopy: IR and Raman 55 2.6 NMR (Brief Overview) 59 3.1 Introduction 70 3.2 Ion Sources 71 3.3 Ionization Methods 72 3.4 Mass Analysers 73 3.5 Detection 76 3.6 Applications of MS 76 V vi Contents 4.1 Density and Molecular Volume 82 4.2 Analytical U1 t racen t ri fuga tion 86 4.3 Sedimentation Equilibrium 87 4.4 Sedimentation Rate 88 4.5 Diffusion and Brownian Motion 91 4.6 Dynamic Light Scattering 94 4.7 Viscosity 94 5.1 A Bluffer’s Guide to Molecular Thermodynamics 99 5.2 Differential Scanning Calorimetry 103 5.3 Isothermal Titration Calorimetry 107 5.4 Binding Equilibrium 108 5.5 General Methods for Determining Thermodynamic Properties 110 5.6 Equilibrium Dialysis 114 5.7 Protein Solubility and Crystallization 115 6.1 Basic Kinetics 123 6.2 Rapid Reaction Techniques 127 6.3 Relaxation Methods 130 6.4 Hydrogen Exchange 132 6.5 Surface Plasmon Resonance 134 6.6 Enzyme Kinetics 136 7.1 Chromatography 143 7.2 Electrophoresis 148 8.1 How Many Molecules Can Stand on the Head of a Pin? 155 8.2 Thermodynamic Fluctuations and the Ergodic Hypothesis 157 Contents vii 8.3 Atomic Force Microscopy 159 8.4 Optical Tweezers and Traps 162 8.5 Single-molecule Fluorescence 163 8.6 Electron Microscopy 165