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PHYSICAL BIOCHEMISTRY
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PHYSICAL BIOCHEMISTRY:
PRINCIPLES AND APPLICATIONS
Second Edition

David Sheehan
Department of Biochemistry
University College Cork
Ireland

A John Wiley & Sons, Ltd, Publication

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This edition first published 2009,

C 2009 John Wiley & Sons Ltd

Wiley-Blackwell is an imprint of John Wiley & Sons, formed by the merger of Wiley’s global Scientific, Technical and Medical
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Library of Congress Cataloging-in-Publication Data

Sheehan, David, 1958–

Physical biochemistry : principles and applications / David Sheehan. – 2nd ed.
p. ; cm.
Includes bibliographical references and index.
ISBN 978-0-470-85602-4 (hb) – ISBN 978-0-470-85603-1 (pb) 1. Physical biochemistry. I. Title.
[DNLM: 1. Biophysics. 2. Biochemistry. 3. Chemistry, Phisical. QT 34 S541p 2008]

QD476.2.S42 2009
572 .43–dc22 2008046672

ISBNs: 9780470856024 (HB)

9780470856031 (PB)
A catalogue record for this book is available from the British Library.
Set in 9/11pt Times by Aptara Inc., New Delhi, India
Printed in Singapore by Fabulous Printing Pte Ltd
First Impression 2009
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To the memory of my father, Patrick Sheehan (1917–2003)

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Contents
Preface xv

Chapter 1 Introduction 1

1.1 Special Chemical Requirements of Biomolecules 1

1.2 Factors Affecting Analyte Structure and Stability 2
1.2.1 pH Effects 3
1.2.2 Temperature Effects 3
1.2.3 Effects of Solvent Polarity 5
1.3 Buffering Systems Used in Biochemistry 6
1.3.1 How Does a Buffer Work? 6
1.3.2 Some Common Buffers 7
1.3.3 Additional Components Often Used in Buffers 7
1.4 Quantitation, Units and Data Handling 7
1.4.1 Units Used in the Text 7
1.4.2 Quantification of Protein and Biological Activity 8
1.5 The Worldwide Web as a Resource in Physical Biochemistry 8
1.5.1 The Worldwide Web 8
1.5.2 Web-Based Resources for Physical Biochemistry 9
1.6 Objectives of this Volume 9
References 10

Chapter 2 Chromatography 11

2.1 Principles of Chromatography 11

2.1.1 The Partition Coefficient 11
2.1.2 Phase Systems Used in Biochemistry 12
2.1.3 Liquid Chromatography 12
2.1.4 Gas Chromatography 13
2.2 Performance Parameters Used in Chromatography 14
2.2.1 Retention 14
2.2.2 Resolution 15
2.2.3 Physical Basis of Peak Broadening 15
2.2.4 Plate Height Equation 15
2.2.5 Capacity Factor 19
2.2.6 Peak Symmetry 19
2.2.7 Significance of Performance Criteria in Chromatography 20
2.3 Chromatography Equipment 20
2.3.1 Outline of Standard System Used 20
2.3.2 Components of Chromatography System 20
2.3.3 Stationary Phases Used 20
2.3.4 Elution 21
2.4 Modes of Chromatography 22
2.4.1 Ion Exchange 22
2.4.2 Gel Filtration 25
2.4.3 Reversed Phase 28
2.4.4 Hydrophobic Interaction 29
2.4.5 Affinity 31
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viii CONTENTS

2.4.6 Immobilized Metal Affinity Chromatography 35

2.4.7 Hydroxyapatite 37
2.5 Open Column Chromatography 37
2.5.1 Equipment Used 37
2.5.2 Industrial Scale Chromatography of Proteins 39
2.6 High Performance Liquid Chromatography (HPLC) 40
2.6.1 Equipment Used 40
2.6.2 Stationary Phases in HPLC 41
2.6.3 Liquid Phases in HPLC 42
2.6.4 Two Dimensional HPLC 42
2.7 Fast Protein Liquid Chromatography 43
2.7.1 Equipment Used 43
2.7.2 Comparison with HPLC 44
2.8 Perfusion Chromatography 44
2.8.1 Theory of Perfusion Chromatography 44
2.8.2 Practice of Perfusion Chromatography 45
2.9 Membrane-Based Chromatography Systems 45
2.9.1 Theoretical Basis 45
2.9.2 Applications of Membrane-Based Separations 46
2.10 Chromatography of a Sample Protein 47
2.10.1 Designing a Purification Protocol 47
2.10.2 Ion Exchange Chromatography of a Sample Protein:
Glutathione Transferases 48
2.10.3 HPLC of Peptides From Glutathione Transferases 50
References 50

Chapter 3 Spectroscopic Techniques 53

3.1 The Nature of Light 53

3.1.1 A Brief History of the Theories of Light 53
3.1.2 Wave-Particle Duality Theory of Light 55
3.2 The Electromagnetic Spectrum 55
3.2.1 The Electromagnetic Spectrum 55
3.2.2 Transitions in Spectroscopy 56
3.3 Ultraviolet/Visible Absorption Spectroscopy 58
3.3.1 Physical Basis 58
3.3.2 Equipment Used in Absorption Spectroscopy 61
3.3.3 Applications of Absorption Spectroscopy 62
3.4 Fluorescence Spectroscopy 64
3.4.1 Physical Basis of Fluorescence and Related Phenomena 64
3.4.2 Measurement of Fluorescence and Chemiluminescence 68
3.4.3 External Quenching of Fluorescence 69
3.4.4 Uses of Fluorescence in Binding Studies 72
3.4.5 Protein Folding Studies 73
3.4.6 Resonance Energy Transfer 73
3.4.7 Applications of Fluorescence in Cell Biology 75
3.5 Spectroscopic Techniques Using Plane-Polarized Light 77
3.5.1 Polarized Light 77
3.5.2 Chirality in Biomolecules 78
3.5.3 Circular Dichroism (CD) 79
3.5.4 Equipment Used in CD 80
3.5.5 CD of Biopolymers 81
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3.5.6 Linear Dichroism (LD) 83
3.5.7 LD of Biomolecules 83
3.6 Infrared Spectroscopy 84
3.6.1 Physical Basis of Infrared Spectroscopy 84
3.6.2 Equipment Used in Infrared Spectroscopy 86
3.6.3 Uses of Infrared Spectroscopy in Structure Determination 86
3.6.4 Fourier Transform Infrared Spectroscopy 87
3.6.5 Raman Infrared Spectroscopy 90
3.7 Nuclear Magnetic Resonance (NMR) Spectroscopy 91
3.7.1 Physical Basis of NMR Spectroscopy 91
3.7.2 Effect of Atomic Identity on NMR 93
3.7.3 The Chemical Shift 93
3.7.4 Spin Coupling in NMR 94
3.7.5 Measurement of NMR Spectra 95
3.8 Electron Spin Resonance (ESR) Spectroscopy 96
3.8.1 Physical Basis of ESR Spectroscopy 96
3.8.2 Measurement of ESR Spectra 98
3.8.3 Uses of ESR Spectroscopy in Biochemistry 99
3.9 Lasers 99
3.9.1 Origin of Laser Beams 100
3.9.2 Some Uses of Laser Beams 100
3.10 Surface Plasmon Resonance 103
3.10.1 Equipment Used in SPR 105
3.10.2 Use of SPR in Measurement of Adsorption Kinetics 107
References 110

Chapter 4 Mass Spectrometry 113

4.1 Principles of Mass Spectrometry 113

4.1.1 Physical Basis 113
4.1.2 Overview of MS Experiment 115
4.1.3 Ionization Modes 118
4.1.4 Equipment Used in MS Analysis 122
4.2 Mass Spectrometry of Proteins/Peptides 125
4.2.1 Sample Preparation 125
4.2.2 MS Modes Used in the Study of Proteins/Peptides 125
4.2.3 Fragmentation of Proteins/Peptides in MS Systems 125
4.3 Interfacing MS With other Methods 127
4.3.1 MS/MS 127
4.3.2 LC/MS 127
4.3.3 GC/MS 128
4.3.4 Electrophoresis/MS 129
4.4 Uses of Mass Spectrometry in Biochemistry 129
4.4.1 MS and Microheterogeneity in Proteins 130
4.4.2 Confirmation and Analysis of Peptide Synthesis 133
4.4.3 Peptide Mapping 133
4.4.4 Post-Translational Modification Analysis of Proteins 133
4.4.5 Determination of Protein Disulfide Patterns 133
4.4.6 Protein Sequencing by MS 136
4.4.7 Studies on Enzymes 139
4.4.8 Analysis of DNA Components 139
References 143
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x CONTENTS
Chapter 5 Electrophoresis 147

5.1 Principles of Electrophoresis 147

5.1.1 Physical Basis 147
5.1.2 Historical Development of Electrophoresis 148
5.1.3 Gel Electrophoresis 149
5.2 Nondenaturing Electrophoresis 153
5.2.1 Polyacrylamide Nondenaturing Electrophoresis 153
5.2.2 Protein Mass Determination by Nondenaturing Electrophoresis 153
5.2.3 Activity Staining 153
5.2.4 Zymograms 155
5.3 Denaturing Electrophoresis 155
5.3.1 SDS Polyacrylamide Gel Electrophoresis 155
5.3.2 SDS Polyacrylamide Gel Electrophoresis
in Reducing Conditions 157
5.3.3 Chemical Crosslinking of Proteins – Quaternary Structure 159
5.3.4 Urea Electrophoresis 160
5.4 Electrophoresis in DNA Sequencing 161
5.4.1 Sanger Dideoxynucleotide Sequencing of DNA 161
5.4.2 Sequencing of DNA 161
5.4.3 Footprinting of DNA 165
5.4.4 Single Strand Conformation Polymorphism Analysis
of DNA 165
5.5 Isoelectric Focusing (IEF) 166
5.5.1 Ampholyte Structure 167
5.5.2 Isoelectric Focusing 170
5.5.3 Titration Curve Analysis 170
5.5.4 Chromatofocusing 170
5.6 Immunoelectrophoresis 172
5.6.1 Dot Blotting and Immunodiffusion Tests with Antibodies 172
5.6.2 Zone Electrophoresis/Immunodiffusion Immunoelectrophoresis 174
5.6.3 Rocket Immunoelectrophoresis 174
5.6.4 Counter Immunoelectrophoresis 176
5.6.5 Crossed Immunoelectrophoresis (CIE) 176
5.7 Agarose Gel Electrophoresis of Nucleic Acids 177
5.7.1 Formation of an Agarose Gel 177
5.7.2 Equipment for Agarose Gel Electrophoresis 177
5.7.3 Agarose Gel Electrophoresis of DNA and RNA 177
5.7.4 Detection of DNA and RNA in Gels 179
5.8 Pulsed Field Gel Electrophoresis 179
5.8.1 Physical Basis of Pulsed Field Gel Electrophoresis 179
5.8.2 Equipment Used for Pulsed Field Gel Electrophoresis 181
5.8.3 Applications of Pulsed Field Gel Electrophoresis 182
5.9 Capillary Electrophoresis 183
5.9.1 Physical Basis of Capillary Electrophoresis 183
5.9.2 Equipment Used in Capillary Electrophoresis 188
5.9.3 Variety of Formats in Capillary Electrophoresis 188
5.10 Electroblotting Procedures 190
5.10.1 Equipment Used in Electroblotting 190
5.10.2 Western Blotting 190
5.10.3 Southern Blotting of DNA 192
5.10.4 Northern Blotting of RNA 194
5.10.5 Blotting as a Preparative Procedure for Polypeptides 195
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CONTENTS xi
5.11 Electroporation 196
5.11.1 Transformation of Cells 196
5.11.2 Physical Basis of Electroporation 196
References 196

Chapter 6 Three-Dimensional Structure Determination of Macromolecules 199

6.1 The Protein-Folding Problem 199

6.1.1 Proteins are only Marginally Stable 200
6.1.2 Protein Folding as a Two-State Process 203
6.1.3 Protein-Folding Pathways 204
6.1.4 Chaperones 206
6.2 Structure Determination by NMR 212
6.2.1 Relaxation in One-Dimensional NMR 212
6.2.2 The Nuclear Overhauser Effect (NOE) 214
6.2.3 Correlation Spectroscopy (COSY) 215
6.2.4 Nuclear Overhauser Effect Spectroscopy (NOESY) 217
6.2.5 Sequential Assignment and Structure Elucidation 218
6.2.6 Multi-Dimensional NMR 221
6.2.7 Other Applications of Multi-Dimensional NMR 221
6.2.8 Limitations and Advantages of Multi-Dimensional NMR 224
6.3 Crystallization of Biomacromolecules 225
6.3.1 What are Crystals? 226
6.3.2 Symmetry in Crystals 226
6.3.3 Physical Basis of Crystallization 228
6.3.4 Crystallization Methods 231
6.3.5 Mounting Crystals for Diffraction 233
6.4 X-Ray Diffraction by Crystals 235
6.4.1 X-Rays 235
6.4.2 Diffraction of X-Rays by Crystals 235
6.4.3 Bragg’s Law 236
6.4.4 Reciprocal Space 238
6.5 Calculation of Electron Density Maps 239
6.5.1 Calculation of Structure Factors 240
6.5.2 Information Available from the Overall Diffraction Pattern 241
6.5.3 The Phase Problem 241
6.5.4 Isomorphous Replacement 242
6.5.5 Molecular Replacement 244
6.5.6 Anomalous Scattering 245
6.5.7 Calculation of Electron Density Map 250
6.5.8 Refinement of Structure 251
6.5.9 Synchrotron Sources 253
6.6 Other Diffraction Methods 254
6.6.1 Neutron Diffraction 254
6.6.2 Electron Diffraction 254
6.7 Comparison of X-Ray Crystallography with Multi-Dimensional NMR 255
6.7.1 Crystallography and NMR are Complementary Techniques 255
6.7.2 Different Attributes of Crystallography- and
NMR-derived Structures 256
6.8 Structural Databases 257
6.8.1 The Protein Database 257
6.8.2 Finding a Protein Structure in the Database 257
References 259
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xii CONTENTS

Chapter 7 Hydrodynamic Methods 263

7.1 Viscosity 263

7.1.1 Definition of Viscosity 263
7.1.2 Measurement of Viscosity 264
7.1.3 Specific and Intrinsic Viscosity 265
7.1.4 Dependence of Viscosity on Characteristics of Solute 266
7.2 Sedimentation 266
7.2.1 Physical Basis of Centrifugation 266
7.2.2 The Svedberg Equation 268
7.2.3 Equipment Used in Centrifugation 269
7.2.4 Subcellular Fractionation 272
7.2.5 Density Gradient Centrifugation 273
7.2.6 Analytical Ultracentrifugation 274
7.2.7 Sedimentation Velocity Analysis 274
7.2.8 Sedimentation Equilibrium Analysis 276
7.3 Methods for Varying Buffer Conditions 279
7.3.1 Ultrafiltration 281
7.3.2 Dialysis 282
7.3.3 Precipitation 284
7.4 Flow Cytometry 286
7.4.1 Flow Cytometer Design 286
7.4.2 Cell Sorting 287
7.4.3 Detection Strategies in Flow Cytometry 288
7.4.4 Parameters Measurable by Flow Cytometry 288
References and Further Reading 290

Chapter 8 Biocalorimetry 293

8.1 The Main Thermodynamic Parameters 293

8.1.1 Activation Energy of Reactions 293
8.1.2 Enthalpy 295
8.1.3 Entropy 295
8.1.4 Free Energy 296
8.2 Isothermal Titration Calorimetry 296
8.2.1 Design of an Isothermal Titration Calorimetry Experiment 296
8.2.2 ITC in Binding Experiments 297
8.2.3 Changes in Heat Capacity Determined by Isothermal
Titration Calorimetry 297
8.3 Differential Scanning Calorimetry 300
8.3.1 Outline Design of a Differential Scanning Calorimetry Experiment 300
8.3.2 Applications of Differential Scanning Calorimetry 301
8.4 Determination of Thermodynamic Parameters by Non-Calorimetric Means 301
8.4.1 Equilibrium Constants 301
References 302

Chapter 9 Bioinformatics 305

9.1 Overview of Bioinformatics 305

9.2 Sequence Databases 309
9.2.1 Nucleotide Sequence Databases 309
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CONTENTS xiii
9.2.2 Protein Sequence Databases 309
9.2.3 Genome Databases 309
9.2.4 Expressed Sequence Tag Databases 315
9.2.5 Single Nucleotide Polymorphism (SNP) Database 315
9.3 Tools for Analysis of Primary Structures 315
9.3.1 BLAST Programs 317
9.3.2 FastA 318
9.3.3 Clustal W 318
9.3.4 Hydropathy Plots 319
9.3.5 Predicting Secondary Structure 323
9.3.6 Identifying Protein Families 325
9.4 Tertiary Structure Databases 327
9.4.1 Cambridge Database 329
9.4.2 Protein Databank (PDB) 329
9.4.3 Specialist Structural Databases 331
9.5 Programs for Analysis and Visualization of Tertiary Structure Databases 334
9.5.1 Ras Mol/Ras Top 334
9.5.2 Protein Explorer 334
9.5.3 Py MOL 334
9.5.4 Web Mol 336
9.5.5 Swiss-Pdb Viewer 336
9.6 Homology Modelling 336
9.6.1 Modelling Proteins from Known Homologous Structures 340
9.6.2 Automated Modelling 342
9.6.3 Applications of Homology Modelling to Drug Discovery 346
References 346

Chapter 10 Proteomics 349

10.1 Electrophoresis in Proteomics 349

10.1.1 Two-Dimensional SDS PAGE 350
10.1.2 Basis of 2-D SDS PAGE 350
10.1.3 Equipment Used in 2-D SDS PAGE 350
10.1.4 Analysis of Cell Proteins 351
10.1.5 Free Flow Electrophoresis 353
10.1.6 Blue Native Gel Electrophoresis 354
10.1.7 Other Electrophoresis Methods Used in Proteomics 355
10.2 Mass Spectrometry in Proteomics 355
10.2.1 Tagging Methodologies Used in MS Proteomics 355
10.2.2 Isotope-Coded Affinity Tagging (ICAT) for Cysteine-Containing Proteins 357
10.2.3 Tagging of N- and C-Termini 358
10.2.4 Tagging for Tandem MS 359
10.3 Chip Technologies in Proteomics 359
10.3.1 Microarrays 359
10.3.2 Protein Biochips 362
10.3.3 SELDI-TOF MS on Protein Chips 362
10.4 Post-Translational Modification Proteomics 366
10.4.1 Proteolysis 366
10.4.2 Glycosylation 367
10.4.3 Oxidation 372
10.4.4 Protein Disulfides 374
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xiv CONTENTS

10.4.5 The Phosphoproteome 376

Further Reading 379
References 380

Appendix 1 SI Units 381

Appendix 2 The Fourier Transform 383

Index 387
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Preface
The first edition of Physical Biochemistry: Principles and Applications set out to describe the physical basis and some
examples of applications of key physically-based techniques used in Biochemistry and other areas of molecular life science
research. In the last decade there has been a noticeable renaissance in some traditional techniques such as X-ray diffraction,
ultracentrifugation and electrophoresis in a variety of formats. In the same time-frame ‘hyphenated’ techniques (e.g. LC-
tandem MS) have become much more mainstream and some instrumentation has become available in desktop formats making
quite sophisticated analysis possible even in the nonspecialist lab. The emphasis was on a largely nonmathematical treatment
at a level appropriate to students in the penultimate year of a Biochemistry course with a view to making these techniques
comprehensible and accessible to a level intermediate between a general Biochemistry textbook and a specialist text. The
feedback I have had from many readers is that this goal was largely achieved.
My task with this second edition was to retain as much as possible of the description of physical principles whilst updating
and integrating new material into a reasonably compact volume. The first edition was strongly influenced by the effects
of the then-recent completion of the Human and other large-scale genome sequencing projects. At the time Proteomics
and other ‘-omics’ technologies were still relatively new paradigms and bioinformatics approaches largely the province of
the specialist. It is remarkable how quickly these technologies have now become embedded in many areas of biochemical
research so that they are now perceived as part of the mainstream. In the second edition I have dedicated new chapters to
proteomics and bioinformatics, respectively, to reflect this changed situation and to emphasize how interconnected physical
and computational techniques have now become.
The second edition required choices to be made and these are inevitably influenced by one’s perception of what is needed
and useful for students to know at the start of their scientific journey into molecular life science. In making these choices
I have continued to be guided by what I perceive to be the most generally-used and helpful techniques but I accept that
specialists in one or more technique may disagree.
In preparing this second edition I had help from many of the colleagues listed in the preface to the First Edition.
In addition, I must thank Dr Rebecca Green, School of Pharmaceutical Sciences, University of Nottingham, UK, for her
invaluable comments on surface plasmon resonance (Chapter 3). I am also indebted to the excellent staff at Wiley’s especially
my editors, Celia Carden and Fiona Woods for their unfailing encouragement and understanding. However, any errors in the
text are my own. I earnestly hope that the reader will find something interesting and thought-provoking in this volume and
be encouraged to explore these very powerful approaches in their work.

Prof David Sheehan

University College Cork
June 2008
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