Enzyme Assays A Practical Approach

by Eisenthal, Robert; Danson, Michael

• ISBN13: 9780199638208
• ISBN10: 0199638209
• Edition: 2nd
• Format: Paperback
• Copyright: 2002-06-20
• Publisher: Oxford University Press

Summary

Enzyme assays are among the most frequently performed procedures in biochemistry and are routinely used to estimate the amount of enzyme present in a cell or tissue, to follow the purification of an enzyme, or to determine the kinetic parameters of a system. The range of techniques used to measure the rate of an enzyme-catalysed reaction is limited only by the nature of the chemical change and the ingenuity of the investigator. This book describes the design and execution of enzyme assays, covering both general principles and specific chapters. Building upon the highly popular first edition, this book combines revised or rewritten chapters with entirely new contributions. Topics include include experimental protocols covering photometric, radiometric, HPLC, and electrochemical assays, along with methods for determining enzyme assays after gel electrophoresis. The theory underlying each method is outlined, together with a description of the instrumentation, sensitivity and sources of error. Also included are chapters on the principles of enzyme assay and kinetic studies; techniques for enzyme extraction; high- throughout screening; statistical analysis of enzyme kinetic data; and the determination of active site concentration. This second edition of Enzyme Assays will be valuable not only to biochemists, but to researchers in all areas of the life sciences.

Table of Contents

List of protocols	p. xiii
Abbreviations	p. xvii
Principles of enzyme assay and kinetic studies	p. 1
Introduction	p. 1
Behaviour of assays	p. 1
Reaction progress curves	p. 1
Initial rate measurements	p. 5
Integrated rate equations	p. 6
Bursts and lags in progress curves	p. 7
Blank rates	p. 11
The effects of enzyme concentration	p. 15
Direct proportionality	p. 15
Upward curvature	p. 17
Downward curvature	p. 18
Expression of enzyme activity	p. 18
Units and specific activity	p. 19
The katal	p. 19
Stoichiometry	p. 19
Conditions for activity measurements	p. 20
The effects of substrate concentration	p. 20
The Michaelis-Menten relationship	p. 20
Failure to obey the Michaelis-Menten equation	p. 21
Experimental approaches	p. 29
Type of assay	p. 29
Choice of assay method	p. 38
The effects of pH	p. 39
Practical considerations	p. 40
Conclusions	p. 44
References	p. 44
Photometric assays	p. 49
Introduction	p. 49
Absorption	p. 49
Terminology	p. 49
Absorbance	p. 50
Limitations and sources of error	p. 52
Absorbance range	p. 55
Measurement of low rates of absorbance change	p. 55
Use of absorbance coefficient	p. 56
Continuous assays	p. 58
Discontinuous assays	p. 63
Examples of enzymes assayed by absorbance change	p. 64
Turbidimetry	p. 70
Fluorescence	p. 71
The fluorescence spectrometer	p. 72
Quantitation of fluorescence	p. 72
Causes of non-linearity - the inner filter effect	p. 73
Examples of fluorimetric enzyme assays	p. 73
References	p. 77
Radiometric assays	p. 79
Introduction	p. 79
Techniques	p. 80
Ion-exchange methods	p. 80
Precipitation of macromolecules	p. 81
Solvent extraction methods	p. 81
Paper and thin-layer chromatographic (TLC) methods	p. 81
Electrophoretic methods	p. 81
Scintillation Proximity Assay (SPA)	p. 82
Experimental design	p. 98
Microplate technology	p. 99
Measurement of radioactivity	p. 100
Automation of assays	p. 100
Acknowledgements	p. 100
References	p. 100
High performance liquid chromatographic assays	p. 103
Introduction	p. 103
Theory of HPLC	p. 103
Introduction	p. 103
Chromatographic parameters	p. 104
Retention mechanism	p. 106
Characteristics of silica	p. 106
Polymeric packings	p. 107
Reverse phase chromatography	p. 108
Influence of composition of mobile phase	p. 111
Effect of pH and salts	p. 112
Influence of temperature	p. 112
Ion-pair chromatography	p. 113
Ion-exchange resins	p. 114
Size-exclusion chromatography	p. 116
Instrumentation	p. 117
Essential components of an HPLC system	p. 117
Pumps	p. 117
Biocompatibility	p. 118
Sample injection	p. 118
Detectors	p. 119
UV/visible detectors	p. 119
Fluorescent detectors	p. 120
Refractive-index (RI) detectors	p. 121
Electrochemical detectors	p. 122
Radioactivity monitors	p. 122
Practical considerations	p. 123
Selection of a chromatographic mode	p. 123
Solvent selection	p. 123
De-gassing and filtration of solvents	p. 124
Sample preparation	p. 124
Column packing	p. 124
Column protection	p. 125
Tubing	p. 125
Application of HPLC to enzymatic analysis	p. 125
Hydrolases	p. 125
Isomerases	p. 127
Lyases	p. 130
Ligases	p. 133
Oxidoreductases	p. 136
Transferases	p. 136
References	p. 137
Electrochemical assays: the oxygen electrode	p. 141
Introduction	p. 141
Theory and principles	p. 141
Current/voltage relationships	p. 142
Sensitivity	p. 142
Calibration	p. 142
Electrode systems	p. 144
Polarographic assays	p. 145
Tissue/organelle respiration studies	p. 145
Specific enzyme studies	p. 146
References	p. 148
Electrochemical assays: the nitric oxide electrode	p. 149
Introduction	p. 149
Principles of detection	p. 149
Principles of selectivity and sensitivity	p. 149
Environmental influences	p. 150
Temperature	p. 150
Electrical interference	p. 150
Membrane integrity and maintenance	p. 151
Calibration	p. 151
Calibration for liquid measurements	p. 151
Calibration for gas-phase measurements	p. 153
NO and cellular respiration studies	p. 153
References	p. 155
Electrochemical assays: the pH-stat	p. 157
Introduction	p. 157
The basis of pH-stat methodology	p. 157
Principle and general approach	p. 157
pH-stat components and their functions	p. 158
Some limitations and sources of error	p. 159
Commercial and custom-made pH-stat assemblies	p. 159
The range of equipment	p. 159
Some pH-stat systems described in the literature	p. 159
General pH-stat procedure and specific protocols for some individual enzymes	p. 162
Procedures	p. 162
A systematic error in pH-stat assays of enzymes in haemolysates	p. 168
Concluding comment	p. 168
References	p. 168
Enzyme assays after gel electrophoresis	p. 171
Introduction	p. 171
Preparation of enzyme extracts	p. 171
Extraction of microorganisms	p. 171
Animal soft tissues	p. 172
Mammalian blood	p. 173
Insects	p. 173
Plant tissues	p. 173
Principles of enzyme visualization	p. 175
Methods to visualize oxidative enzymes	p. 175
Methods to visualize transferases	p. 177
Methods to visualize hydrolases	p. 178
Methods to visualize lyases, isomerases and ligases	p. 180
A compliation of protocols to visualize enzymes following electrophoretic separation	p. 180
Staining protocols	p. 184
Buffer systems for electrophoresis	p. 201
References	p. 206
Techniques for enzyme extraction	p. 209
Introduction: scope of the chapter	p. 209
Disruption of tissues and cells	p. 210
Choice of tissue	p. 210
Disruption of tissue and separation of cells	p. 211
Disruption of cells	p. 212
Protection of enzyme activity	p. 215
Control of pH	p. 215
Control of temperature	p. 215
Control of proteolysis	p. 216
Protection of thiol groups	p. 217
Protection against heavy metals	p. 217
Control of mechanical stress	p. 218
Effects of dilution	p. 218
Assays of enzymes in unfractionated cell-extracts	p. 219
The presence of endogenous inhibitors	p. 219
Interference from other reactions	p. 219
Removal of substrate	p. 219
Turbidity of extract	p. 220
Concluding remarks	p. 220
References	p. 220
Buffers and control of pH	p. 221
The determination of protein	p. 223
References for Appendices	p. 224
Determination of active site concentration	p. 225
Introduction	p. 225
Areas of application	p. 225
Categories of titration methods	p. 226
Activity bursts	p. 226
Inhibitor titration	p. 228
Special techniques	p. 230
References	p. 233
High throughput screening--considerations for enzyme assays	p. 235
Introduction	p. 235
The drug discovery process	p. 235
A historical perspective	p. 235
A model of drug discovery	p. 236
High throughput screening	p. 237
Compounds for screening	p. 239
Considerations for high throughput assays	p. 240
Enzymatic considerations	p. 245
Assay formats for enzymatic HTS	p. 246
Automation	p. 246
Developments	p. 247
Higher density plates	p. 247
References	p. 247
Statistical analysis of enzyme kinetic data	p. 249
Introduction	p. 249
Derivation of relationships	p. 250
Defining objectives	p. 250
Basic assumptions of least squares	p. 251
Fitting the Michaelis-Menten equation	p. 252
Equations with more than two parameters	p. 258
Detecting lack of fit	p. 258
Estimating pure error	p. 260
Distribution-free methods	p. 262
Residual plots	p. 264
A note about rounding	p. 267
References	p. 268
List of suppliers	p. 269
Enzyme index	p. 273
General index	p. 277
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