Physical Biochemistry

Preface Acknowledgements Chapter 1 Introduction 1.1 Special chemical requirements of biomolecules 1.2 Factors affecting analyte structure and stability 1.2.1 pH effects 1.2.2 Temperature effects 1.2.3 Effects of solvent polarity 1.3 Buffering systems used in biochemistry 1.3.1 How does a buffer work? 1.3.2 Some common buffers 1.3.3 Additional components often used in buffers 1.4 Quantitation, units and data handling 1.4.1 Units used in this text 1.4.2 Quantitation of protein and biological activity 1.5 Objectives of this book Bibliography Chapter 2 Chromatography 2.1 Principles of chromatography 2.1.1 The partition coefficient 2.1.2 Phase systems used in biochemistry 2.1.3 Liquid chromatography 2.1.4 Gas chromatography 2.2 Performance parameters used in chromatography 2.2.1 Retention 2.2.2 Resolution 2.2.3 Physical basis of peak broadening 2.2.4 Plate height equation 2.2.5 Capacity factor 2.2.5 Peak symmetry 2.2.7 Significance of performance criteria in chromatography 2.3 Chromatography equipment 2.3.1 Outline of standard system used 2.3.2 Components of a chromatography system 2.3.3 Stationary phases used 2.3.4 Elution 2.4 Modes of chromatography 2.4.1 Ion exchange 2.4.2 Gel filtration 2.4.3 Reversed phase 2.4.4 Hydrophobic interaction 2.4.5 Affinity 2.4.6 Immobilised metal affinity chromatography 2.4.7 Hydroxyapatite 2.5 Open-column chromatography 2.5.1 Equipment used 2.5.2 Industrial-scale chromatography of proteins 2.6 High-performance liquid chromatography 2.6.1 Equipment used 2.6.2 Stationary phases in HPLC 2.6.3 Liquid phases in HPLC 2.7 Fast protein liquid chromatography 2.7.1 Equipment used 2.7.2 Comparison with HPLC 2.8 Perfusion chromatography 2.8.1 Theory of perfusion chromatography 2.8.2 The practice of perfusion chromatography 2.9 Membrane-based chromatography systems 2.9.1 Theoretical basis 2.9.2 Applications of membrane-based separations 2.10 Chromatography of a sample protein 2.10.1 Designing a purification protocol 2.10.2 Ion exchange chromatography of a sample protein:Glutathione S-transferases 2.10.3 HPLC of peptides from glutathione S-transferases Bibliography Chapter 3 Spectroscopic Techniques 3.1 The nature of light 3.1.1 A brief history of the theories of light 3.1.2 Wave-particle duality theory of light 3.2 The electromagnetic spectrum 3.2.1 The Electromagnetic Spectrum 3.2.2 Transitions in spectroscopy 3.3 Ultraviolet/visible absorption spectroscopy 3.3.1 Physical basis 3.3.2 Equipment used in absorption spectroscopy 3.3.3 Applications of absorption spectroscopy 3.4 Fluorescence spectroscopy 3.4.1 Physical basis of fluorescence and related phenomena 3.4.2 Measurement of fluorescence and chemiluminescence 3.4.3. External quenching of fluorescence 3.4.4 Uses of fluorescence in binding studies 3.4.5 Protein-folding studies 3.4.6 Resonance energy transfer 3.4.7 Applications of fluorescence in cell biology 3.5 Spectroscopic techniques using plane-polarised light 3.5.1 Polarised light 3.5.2 Chirality in biomolecules 3.5.3 Circular dichroism (CD) 3.5.4 Equipment used in CD 3.5.5 CD of biopolymers 3.5.6 Linear dichroism (LD) 3.5.7 LD of biomolecules 3.5.8 Plasmon resonance spectroscopy 3.6 Infrared spectroscopy 3.6.1 Physical basis of infrared spectroscopy 3.6.2 Equipment used in infrared spectroscopy 3.6.3 Uses of infrared spectroscopy in structure determination 3.6.4 Fourier transform infrared spectroscopy 3.6.5 Raman infrared spectroscopy 3.7 Nuclear magnetic resonance (NMR) spectroscopy 3.7.1 Physical basis of NMR spectroscopy 3.7.2 Effect of atomic identity on NMR 3.7.3 The chemical shift 3.7.4 Spin coupling in NMR 3.7.5 Measurement of NMR spectra 3.8 Electron spin resonance (ESR) spectroscopy 3.8.1 Physical basis of ESR spectroscopy 3.8.2 Measurement of ESR spectra 3.8.3 Uses of ESR spectroscopy in biochemistry 3.9 Lasers 3.9.1 Origin of laser beams 3.9.2 Some uses of laser beams Bibliography Chapter 4 Mass spectrometry 4.1 Principles of mass spectrometry 4.1.1 Physical basis 4.1.2 Overview of MS experiment 4.1.3 Ionisation modes 4.1.4 Equipment used in MS analysis 4.2 Mass spectrometry of proteins and peptides 4.2.1 Sample preparation 4.2.2 MS modes used in the study of proteins/peptides 4.2.3 Fragmentation of proteins/peptides in MS systems 4.3 Interfacing MS with other methods 4.3.1 MS/MS 4.3.2 LC/MS 4.3.3. GC/MS 4.4 Electrophoresis/MS 4.4 Uses of mass spectrometry in biochemistry 4.4.1 MS and microheterogeneity in proteins 4.4.2 Confirmation and analysis of peptide synthesis 4.4.3 Peptide mapping 4.4.4 Post-translational modification analysis of proteins 4.4.5 Determination of protein disulphide patterns 4.4.6 Protein sequencing by MS 4.4.7 Analysis of DNA components Bibliography Chapter 5 Electrophoresis 5.1 Principles of electrophoresis 5.1.1 Physical basis 5.1.2 Historical development of electrophoresis 5.1.3 Gel electrophoresis 5.2 Non-denaturing electrophoresis 5.2.1 Polyacrylamide non-denaturing electrophoresis 5.2.2 Protein mass determination by non-denaturing electrophoresis 5.2.3 Activity staining 5.24 Zymograms 5.3 Denaturing electrophoresis 5.3.1 SDS polyacrylamide gel electrophoresis 5.3.2 SDS polyacrylamide gel electrophoresis in reducing conditions 5.3.3 Chemical Crosslinking of Proteins - Quaternary Structure 5.3.4 Urea electrophoresis 5.4 Electrophoresis in DNA sequencing 5.4.1 Sanger dideoxynucleotide sequencing of DNA 5.4.2 Sequencing of DNA 5.4.3 Footprinting of DNA 5.4.4 Single-strand conformation polymorphism analysis of DNA 5.5 Isoelectric focusing (IEF) 5.5.1 Ampholyte structure 5.5.2 Isoelectric focusing 5.5.3 Titration curve analysis 5.5.4 Chromatofocusing 5.6 Two-dimensional SDS page 5.6.1 Basis of two-dimensional SDS Page 5.6.2 Equipment used in two-dimensional SDS Page 5.6.3 Analysis of cell proteins 5.7 Immunoelectrophoresis 5.7.1 Dot blotting and immunodiffusion tests with antibodies 5.7.2 Zone electrophoresis/immunodiffusion immunoelectrophoresis 5.7.3 Rocket immunoelectrophoresis 5.7.4 Counter-immunoelectrophoresis 5.7.5 Crossed immunoelectrophoresis (CIE) 5.8 Agarose gel electro-phoresis of nucleic acids 5.8.1 Formation of an agarose gel 5.8.2 Equipment for agarose gel electrophoresis 5.8.3 Agarose gel electrophoresis of DNA and RNA 5.8.4 Detection of DNA and RNA in gels 5.9 Pulsed field gel electrophoresis 5.9.1 Physical basis of pulsed field gel electrophoresis 5.9.2 Equipment used for pulsed field gel electrophoresis 5.9.3 Applications of pulsed field gel electrophoresis 5.10 Capillary electrophoresis 5.10.1 Physical basis of capillary electrophoresis 5.10.2 Equipment used in capillary electrophoresis 5.10.3 Variety of formats in capillary electrophoresis 5.11 Electroblotting procedures 5.11.1 Equipment used in electroblotting 5.11.2 Western blotting 5.11.3 Southern blotting of DNA 5.11.4 Northern blotting of RNA 5.11.5 Blotting as a preparative procedure for polypeptides 5.12 Electroporation of cells 5.12.1 Transformation of cells 5.12.2 Physical basis of electroporation Bibliography Chapter 6 Three-dimensional structure determination of macromolecules 6.1 The protein-folding problem 6.1.1 Proteins are only marginally stable 6.1.2 Protein folding as a two-state process 6.1.3 Protein-folding pathways 6.1.4 Chaperonins 6.2 Structure determination by NMR 6.2.1 Relaxation in one-dimensional NMR 6.2.2 The Nuclear Overhauser Effect (NOE) 6.2.3 Correlation Spectroscopy (COSY) 6.2.4 Nuclear Overhauser Effect Spectroscopy (NOESY) 6.2.5 Sequential assignment and structure elucidation 6.2.6 Multi-dimensional NMR 6.2.7 Other applications of multi-dimensional NMR 6.2.8 Limitations and advantages of multi-dimensional NMR 6.3 Crystallisation of biomacromolecules 6.3.1 What are crystals? 6.3.2 Symmetry in crystals 6.3.3 Physical basis of crystallisation 6.3.4 Crystallisation methods 6.3.5 Mounting crystals for diffraction 6.4 X-ray diffraction by crystals 6.4.1 X-rays 6.4.2 Diffraction of X-rays by crystals 6.4.3 Bragg's law 6.4.4 Reciprocal space 6.5 Calculation of electron density maps 6.5.1 Calculation of structure factors 6.5.2 Information available from the overall diffraction pattern 6.5.3 The phase problem 6.5.4 Isomorphous replacement 6.5.5 Molecular replacement 6.5.6 Anomalous scattering 6.5.7 Calculation of electron density map 6.5.8 Refinement of structure 6.5.9 Synchrotron sources 6.6 Other diffraction methods 6.6.1 Neutron diffraction 6.6.2 Electron diffraction 6.7 Comparison of X-ray crystallography with multi-dimensional NMR 6.7.1 Crystallography and NMR are complementary techniques 6.7.2 Different attributes of crystallography- and NMR-derived structures 6.8 Structural databases 6.8.1 The protein database 6.8.2 Finding a protein structure in the database Bibliography Chapter 7 Hydrodynamic methods 7.1 Viscosity 7.1.1 Definition of viscosity 7.1.2 Measurement of viscosity 7.1.3 Specific and intrinsic viscosity 7.1.4 Dependence of viscosity on characteristics of solute 7.2 Sedimentation 7.2.1 Physical basis of centrifugation 7.2.2 The Svedberg equation 7.2.3 Equipment used in centrifugation 7.2.4 Subcellular fractionation 7.2.5 Density gradient centrifugation 7.2.6 Analytical ultracentrifugation 7.2.7 Sedimentation velocity analysis 7.2.8 Sedimentation equilibrium analysis 7.3 Methods for varying buffer conditions 7.3.1 Ultrafiltration 7.3.2 Dialysis 7.3.3 Precipitation 7.4 Flow cytometry 7.4.1 Flow cytometer design 7.4.2 Cell sorting 7.4.3 Detection strategies in flow cytometry 7.4.4 Parameters measurable by flow cytometry Bibliography Chapter 8 Biocalorimetry 8.1 The main thermodynamic parameters 8.1.1 Activation energy of reactions 8.1.2 Enthalpy 8.1.3 Entropy 8.1.4 Free energy 8.2 Isothermal titration calorimetry 8.2.1 Design of an isothermal titration calorimetry experiment 8.2.2 ITC in binding experiments 8.2.3 Changes in heat capacity determined by isothermal titration calorimetry 8.3 Differential scanning calorimetry 8.3.1 Outline design of a differential scanning calorimetry experiment 8.3.2 Applications of differential scanning calorimetry 8.4 Determination of thermodynamic parameters by non-calorimetric means 8.4.1 Equilibrium constants Bibliography Appendix 1 SI units Appendix 2 The Fourier transform Index