Physical Biochemistry : Principles and Applications,9780471986638

Physical Biochemistry : Principles and Applications

by
ISBN13: 9780471986638
ISBN10: 0471986631
Format: Paperback
Pub. Date: 2000-06-01
Publisher(s): WILEY
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Summary

This book will be invaluable to undergraduates and postgraduates studying biochemistry, molecular biology and related disciplines.

Table of Contents

Preface xv
Acknowledgements xvii
Introduction
1(11)
Special chemical requirements of biomolecules
1(2)
Factors affecting analyte structure and stability
3(4)
pH effects
3(1)
Temperature effects
4(2)
Effects of solvent polarity
6(1)
Buffering systems used in biochemistry
7(2)
How does a buffer work?
7(1)
Some common buffers
8(1)
Additional components often used in buffers
8(1)
Quantitation, units and data handling
9(1)
Units used in this text
9(1)
Quantitation of protein and biological activity
9(1)
Objectives of this book
10(2)
Bibliography
11(1)
Chromatography
12(49)
Principles of chromatography
12(4)
The partition coefficient
12(1)
Phase systems used in biochemistry
13(1)
Liquid chromatography
14(1)
Gas chromatography
15(1)
Performance parameters used in chromatography
16(7)
Retention
16(1)
Resolution
16(1)
Physical basis of peak broadening
17(1)
Plate height equation
17(6)
Capacity factor
23(1)
Peak symmetry
23(1)
Significance of performance criteria in chromatography
23(1)
Chromatography equipment
23(3)
Outline of standard system used
23(1)
Components of a chromatography system
24(1)
Stationary phases used
24(1)
Elution
24(2)
Modes of chromatography
26(18)
Ion exchange
27(3)
Gel filtration
30(3)
Reversed phase
33(1)
Hydrophobic interaction
34(3)
Affinity
37(2)
Immobilised metal affinity chromatography
39(4)
Hydroxyapatite
43(1)
Open-column chromatography
44(2)
Equipment used
44(1)
Industrial-scale chromatography of proteins
45(1)
High-performance liquid chromatography
46(4)
Equipment used
46(2)
Stationary phases in HPLC
48(1)
Liquid phases in HPLC
49(1)
Fast protein liquid chromatography
50(1)
Equipment used
50(1)
Comparison with HPLC
51(1)
Perfusion chromatography
51(3)
Theory of perfusion chromatography
51(1)
The practice of perfusion chromatography
52(2)
Membrane-based chromatography systems
54(1)
Theoretical basis
54(1)
Applications of membrane-based separations
54(1)
Chromatography of a sample protein
55(6)
Designing a purification protocol
55(1)
Ion exchange chromatography of a sample protein: Glutathione S-transferases
56(1)
HPLC of peptides from glutathione S-transferases
57(2)
Bibliography
59(2)
Spectroscopic Techniques
61(60)
The nature of light
61(4)
A brief history of the theories of light
61(3)
Wave--particle duality theory of light
64(1)
The electromagnetic spectrum
65(1)
The Electromagnetic Spectrum
65(1)
Transitions in spectroscopy
66(1)
Ultraviolet/visible absorption spectroscopy
66(8)
Physical basis
66(5)
Equipment used in absorption spectroscopy
71(1)
Applications of absorption spectroscopy
72(2)
Fluorescence spectroscopy
74(14)
Physical basis of fluorescence and related phenomena
74(4)
Measurement of fluorescence and chemiluminescence
78(2)
External quenching of fluorescence
80(3)
Uses of fluorescence in binding studies
83(1)
Protein-folding studies
84(1)
Resonance energy transfer
85(1)
Applications of fluorescence in cell biology
86(2)
Spectroscopic techniques using plane-polarised light
88(7)
Polarised light
88(1)
Chirality in biomolecules
89(1)
Circular dichroism (CD)
90(1)
Equipment used in CD
91(1)
CD of biopolymers
92(2)
Linear dichroism (LD)
94(1)
LD of biomolecules
94(1)
Plasmon resonance spectroscopy
95(1)
Infrared spectroscopy
95(8)
Physical basis of infrared spectroscopy
95(2)
Equipment used in infrared spectroscopy
97(1)
Uses of infrared spectroscopy in structure determination
98(1)
Fourier transform infrared spectroscopy
98(2)
Raman infrared spectroscopy
100(3)
Nuclear magnetic resonance (NMR) spectroscopy
103(6)
Physical basis of NMR spectroscopy
103(2)
Effect of atomic identity on NMR
105(1)
The chemical shift
106(1)
Spin coupling in NMR
107(1)
Measurement of NMR spectra
108(1)
Electron spin resonance (ESR) spectroscopy
109(4)
Physical basis of ESR spectroscopy
111(1)
Measurement of ESR spectra
111(1)
Uses of ESR spectroscopy in biochemistry
112(1)
Lasers
113(8)
Origin of laser beams
114(2)
Some uses of laser beams
116(1)
Bibliography
117(4)
Mass spectrometry
121(32)
Principles of mass spectrometry
121(13)
Physical basis
121(2)
Overview of MS experiment
123(3)
Ionisation modes
126(5)
Equipment used in MS analysis
131(3)
Mass spectrometry of proteins and peptides
134(1)
Sample preparation
134(1)
MS modes used in the study of proteins/peptides
134(1)
Fragmentation of proteins/peptides in MS systems
134(1)
Interfacing MS with other methods
135(3)
MS/MS
136(1)
LC/MS
136(1)
GC/MS
137(1)
Electrophoresis/MS
138(1)
Uses of mass spectrometry in biochemistry
138(15)
MS and microheterogeneity in proteins
139(3)
Confirmation and analysis of peptide synthesis
142(2)
Peptide mapping
144(1)
Post-translational modification analysis of proteins
145(1)
Determination of protein disulphide patterns
146(2)
Protein sequencing by MS
148(1)
Analysis of DNA components
149(1)
Bibliography
150(3)
Electrophoresis
153(62)
Principles of electrophoresis
153(7)
Physical basis
153(2)
Historical development of electrophoresis
155(1)
Gel electrophoresis
155(5)
Non-denaturing electrophoresis
160(4)
Polyacrylamide non-denaturing electrophoresis
160(1)
Protein mass determination by non-denaturing electrophoresis
160(1)
Activity staining
161(3)
Zymograms
164(1)
Denaturing electrophoresis
164(5)
SDS polyacrylamide gel electrophoresis
164(1)
SDS polyacrylamide gel electrophoresis in reducing conditions
165(1)
Chemical Crosslinking of Proteins---Quaternary Structure
165(2)
Urea electrophoresis
167(2)
Electrophoresis in DNA sequencing
169(8)
Sanger dideoxynucleotide sequencing of DNA
169(1)
Sequencing of DNA
170(3)
Footprinting of DNA
173(1)
Single-strand conformation polymorphism analysis of DNA
174(3)
Isoelectric focusing (IEF)
177(6)
Ampholyte structure
177(1)
Isoelectric focusing
178(1)
Titration curve analysis
179(2)
Chromatofocusing
181(2)
Two-dimensional SDS page
183(3)
Basis of two-dimensional SDS PAGE
183(1)
Equipment used in two-dimensional SDS PAGE
184(1)
Analysis of cell proteins
185(1)
Immunoelectrophoresis
186(4)
Dot blotting and immunodiffusion tests with antibodies
186(2)
Zone electrophoresis/immunodiffusion immunoelectrophoresis
188(1)
Rocket immunoelectrophoresis
189(1)
Counter-immunoelectrophoresis
190(1)
Crossed immunoelectrophoresis (CIE)
190(1)
Agarose gel electro-phoresis of nucleic acids
190(3)
Formation of an agarose gel
190(1)
Equipment for agarose gel electrophoresis
190(1)
Agarose gel electrophoresis of DNA and RNA
191(2)
Detection of DNA and RNA in gels
193(1)
Pulsed field gel electrophoresis
193(4)
Physical basis of pulsed field gel electrophoresis
193(2)
Equipment used for pulsed field gel electrophoresis
195(2)
Applications of pulsed field gel electrophoresis
197(1)
Capillary electrophoresis
197(7)
Physical basis of capillary electrophoresis
197(6)
Equipment used in capillary electrophoresis
203(1)
Variety of formats in capillary electrophoresis
203(1)
Electroblotting procedures
204(7)
Equipment used in electroblotting
206(1)
Western blotting
206(2)
Southern blotting of DNA
208(1)
Northern blotting of RNA
209(1)
Blotting as a preparative procedure for polypeptides
210(1)
Electroporation of cells
211(4)
Transformation of cells
211(1)
Physical basis of electroporation
211(1)
Bibliography
211(4)
Three-dimensional structure determination of macromolecules
215(72)
The protein-folding problem
216(14)
Proteins are only marginally stable
216(4)
Protein folding as a two-state process
220(1)
Protein-folding pathways
221(2)
Chaperonins
223(7)
Structure determination by NMR
230(15)
Relaxation in one-dimensional NMR
230(2)
The Nuclear Overhauser Effect (NOE)
232(2)
Correlation Spectroscopy (COSY)
234(1)
Nuclear Overhauser Effect Spectroscopy (NOESY)
235(2)
Sequential assignment and structure elucidation
237(3)
Multi-dimensional NMR
240(2)
Other applications of multi-dimensional NMR
242(1)
Limitations and advantages of multi-dimensional NMR
243(2)
Crystallisation of biomacromolecules
245(12)
What are crystals?
245(1)
Symmetry in crystals
246(2)
Physical basis of crystallisation
248(4)
Crystallisation methods
252(3)
Mounting crystals for diffraction
255(2)
X-ray diffraction by crystals
257(6)
X-rays
257(1)
Diffraction of X-rays by crystals
258(1)
Bragg's law
259(2)
Reciprocal space
261(2)
Calculation of electron density maps
263(16)
Calculation of structure factors
263(1)
Information available from the overall diffraction pattern
264(1)
The phase problem
265(1)
Isomorphous replacement
266(2)
Molecular replacement
268(1)
Anomalous scattering
269(6)
Calculation of electron density map
275(1)
Refinement of structure
276(2)
Synchrotron sources
278(1)
Other diffraction methods
279(2)
Neutron diffraction
279(1)
Electron diffraction
280(1)
Comparison of X-ray crystallography with multi-dimensional NMR
281(1)
Crystallography and NMR are complementary techniques
281(1)
Different attributes of crystallography- and NMR-derived structures
282(1)
Structural databases
282(5)
The protein database
283(1)
Finding a protein structure in the database
283(2)
Bibliography
285(2)
Hydrodynamic methods
287(30)
Viscosity
287(5)
Definition of viscosity
287(1)
Measurement of viscosity
288(1)
Specific and intrinsic viscosity
289(1)
Dependence of viscosity on characteristics of solute
290(2)
Sedimentation
292(13)
Physical basis of centrifugation
292(1)
The Svedberg equation
293(1)
Equipment used in centrifugation
294(1)
Subcellular fractionation
295(1)
Density gradient centrifugation
296(3)
Analytical ultracentrifugation
299(1)
Sedimentation velocity analysis
300(2)
Sedimentation equilibrium analysis
302(3)
Methods for varying buffer conditions
305(5)
Ultrafiltration
305(2)
Dialysis
307(2)
Precipitation
309(1)
Flow cytometry
310(7)
Flow cytometer design
311(1)
Cell sorting
312(1)
Detection strategies in flow cytometry
313(2)
Parameters measurable by flow cytometry
315(1)
Bibliography
315(2)
Biocalorimetry
317(12)
The main thermodynamic parameters
318(3)
Activation energy of reactions
318(1)
Enthalpy
318(1)
Entropy
319(1)
Free energy
320(1)
Isothermal titration calorimetry
321(2)
Design of an isothermal titration calorimetry experiment
321(1)
ITC in binding experiments
322(1)
Changes in heat capacity determined by isothermal titration calorimetry
323(1)
Differential scanning calorimetry
323(3)
Outline design of a differential scanning calorimetry experiment
325(1)
Applications of differential scanning calorimetry
325(1)
Determination of thermodynamic parameters by non-calorimetric means
326(3)
Equilibrium constants
326(1)
Bibliography
327(2)
Appendix 1 SI units 329(1)
Appendix 2 The Fourier transform 330(5)
Index 335

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