List of protocols page ....................................... xiii
Abbreviations ................................................ xvii
1. Principles of enzyme assay and kinetic studies ............... 1
Keith F. Tipton
1. Introduction .............................................. 1
2. Behaviour of assays ....................................... 1
Reaction progress curves .................................. 1
Initial rate measurements ................................. 5
Integrated rate equations ................................. 6
Bursts and lags in progress curves ........................ 7
Blank rates .............................................. 11
3. The effects of enzyme concentration ...................... 15
Direct proportionality ................................... 15
Upward curvature ......................................... 17
Downward curvature ....................................... 18
4. Expression of enzyme activity ............................ 18
Units and specific activity .............................. 19
The katal ................................................ 19
Stoichiometry ............................................ 19
Conditions for activity measurements ..................... 20
5. The effects of substrate concentration ................... 20
The Michaelis-Menten relationship ........................ 20
Failure to obey the Michaelis-Menten equation ............ 21
6. Experimental approaches .................................. 29
Type of assay ............................................ 29
Choice of assay method ................................... 38
The effects of pH ........................................ 39
Practical considerations ................................. 40
Conclusions .............................................. 44
References ............................................... 44
2. Photometric assays .......................................... 49
Robert A. John
1. Introduction ............................................. 49
2. Absorption ............................................... 49
Terminology .............................................. 49
Absorbance ............................................... 50
Limitations and sources of error ......................... 52
Absorbance range ......................................... 55
Measurement of low rates of absorbance change ............ 55
Use of absorbance coefficient ............................ 56
Continuous assays ........................................ 58
Discontinuous assays ..................................... 63
Examples of enzymes assayed by absorbance change ......... 64
3. Turbidimetry ............................................. 70
4. Fluorescence ............................................. 71
The fluorescence spectrometer ............................ 72
Quantitation of fluorescence ............................. 72
Causes of non-linearity - the inner filter effect ........ 73
Examples of fluorimetric enzyme assays ................... 73
References ............................................... 77
3. Radiometric assays .......................................... 79
Kelvin T. Hughes
1. Introduction ............................................. 79
2. Techniques ............................................... 80
Ion-exchange methods ..................................... 80
Precipitation of macromolecules .......................... 81
Solvent extraction methods ............................... 81
Paper and thin-layer chromatographic (TLC) methods ....... 81
Electrophoretic methods .................................. 81
Scintillation Proximity Assay (SPA) ...................... 82
3. Experimental design ...................................... 98
4. Microplate technology .................................... 99
5. Measurement of radioactivity ............................ 100
6. Automation of assays .................................... 100
Acknowledgements ........................................ 100
References .............................................. 100
4. High performance liquid chromatographic assays ............. 103
Shabih E.H. Syed
1. Introduction ............................................ 103
2. Theory of HPLC .......................................... 103
Introduction ............................................ 103
Chromatographic parameters .............................. 104
3. Retention mechanism ..................................... 106
Characteristics of silica ............................... 106
Polymeric packings ...................................... 107
Reverse phase chromatography ............................ 108
Influence of composition of mobile phase ................ 111
Effect of pH and salts .................................. 112
Influence of temperature ................................ 112
Ion-pair chromatography ................................. 113
Ion-exchange resins ..................................... 114
Size-exclusion chromatography ........................... 116
4. Instrumentation ......................................... 117
Essential components of an HPLC system .................. 117
Pumps ................................................... 117
Biocompatibility ........................................ 118
Sample injection ........................................ 118
5. Detectors ............................................... 119
UV/visible detectors .................................... 119
Fluorescent detectors ................................... 120
Refractive-index (RI) detectors ......................... 121
Electrochemical detectors ............................... 122
Radioactivity monitors .................................. 122
6. Practical considerations ................................ 123
Selection of a chromatographic mode ..................... 123
Solvent selection ....................................... 123
De-gassing and filtration of solvents ................... 124
Sample preparation ...................................... 124
Column packing .......................................... 124
Column protection ....................................... 125
Tubing .................................................. 125
7. Application of HPLC to enzymatic analysis ............... 125
Hydrolases .............................................. 125
Isomerases .............................................. 127
Lyases .................................................. 130
Ligases ................................................. 133
Oxidoreductases ......................................... 136
Transferases ............................................ 136
References .............................................. 137
5. Electrochemical assays: the oxygen electrode ............... 141
J.B. Clark
1. Introduction ............................................ 141
2. Theory and principles ................................... 141
3. Current/voltage relationships ........................... 142
4. Sensitivity ............................................. 142
5. Calibration ............................................. 142
6. Electrode systems ....................................... 144
7. Polarographic assays .................................... 145
Tissue/organelle respiration studies .................... 145
Specific enzyme studies ................................. 146
References .............................................. 148
6. Electrochemical assays: the nitric oxide electrode ......... 149
R.D. Hurst and J.B. Clark
1. Introduction ............................................ 149
2. Principles of detection ................................. 149
3. Principles of selectivity and sensitivity ............... 149
4. Environmental influences ................................ 150
Temperature ............................................. 150
Electrical interference ................................. 150
5. Membrane integrity and maintenance ...................... 151
6. Calibration ............................................. 151
Calibration for liquid measurements ..................... 151
Calibration for gas-phase measurements .................. 153
7. NO and cellular respiration studies ..................... 153
References .............................................. 155
7. Electrochemical assays: the pH-stat ........................ 157
Keith Brocklehurst
1. Introduction ............................................ 157
2. The basis of pH-stat methodology ........................ 157
Principle and general approach .......................... 157
pH-stat components and their functions .................. 158
Some limitations and sources of error ................... 159
3. Commercial and custom-made pH-stat assemblies ........... 159
The range of equipment .................................. 159
Some pH-stat systems described in the literature ........ 159
4. General pH-stat procedure and specific protocols for
some individual enzymes ................................. 162
Procedures .............................................. 162
5. A systematic error in pH-stat assays of enzymes in
haemolysates ............................................ 168
6. Concluding comment ...................................... 168
References .............................................. 168
8. Enzyme assays after gel electrophoresis .................... 171
Gunter M. Rothe
1. Introduction ............................................ 171
2. Preparation of enzyme extracts .......................... 171
Extraction of microorganisms ............................ 171
Animal soft tissues ..................................... 172
Mammalian blood ......................................... 173
Insects ................................................. 173
Plant tissues ........................................... 173
3. Principles of enzyme visualization ...................... 175
Methods to visualize oxidative enzymes .................. 175
Methods to visualize transferases ....................... 177
Methods to visualize hydrolases ......................... 178
Methods to visualize lyases, isomerases and ligases ..... 180
4. A compilation of protocols to visualize enzymes
following electrophoretic separation .................... 180
Staining protocols ...................................... 184
Buffer systems for electrophoresis ...................... 201
References .............................................. 206
9. Techniques for enzyme extraction ........................... 209
Nicholas C. Price and Lewis Stevens
1. Introduction: scope of the chapter ...................... 209
2. Disruption of tissues and cells ......................... 210
Choice of tissue ........................................ 210
Disruption of tissue and separation of cells ............ 211
Disruption of cells ..................................... 212
3. Protection of enzyme activity ........................... 215
Control of pH ........................................... 215
Control of temperature .................................. 215
Control of proteolysis .................................. 216
Protection of thiol groups .............................. 217
Protection against heavy metals ......................... 217
Control of mechanical stress ............................ 218
Effects of dilution ..................................... 218
4. Assays of enzymes in unfractionated cell-extracts ....... 219
The presence of endogenous inhibitors ................... 219
Interference from other reactions ....................... 219
Removal of substrate .................................... 219
Turbidity of extract .................................... 220
5. Concluding remarks ...................................... 220
References .............................................. 220
Appendix 1 Buffers and control of pH .................... 221
Appendix 2 The determination of protein ................. 223
References for Appendices ............................... 224
10.Determination of active site concentration ................. 225
Mark T. Martin
1. Introduction ............................................ 225
2. Areas of application .................................... 225
3. Categories of titration methods ......................... 226
Activity bursts ......................................... 226
Inhibitor titration ..................................... 228
Special techniques ...................................... 230
References .............................................. 233
11.High throughput screening - considerations for enzyme
assays ..................................................... 235
David Hayes and Geoff Mellor
1. Introduction ............................................ 235
2. The drug discovery process .............................. 235
A historical perspective ................................ 235
A model of drug discovery ............................... 236
3. High throughput screening ............................... 237
Compounds for screening ................................. 239
Considerations for high throughput assays ............... 240
4. Enzymatic considerations ................................ 245
5. Assay formats for enzymatic HTS ......................... 246
6. Automation .............................................. 246
7. Developments ............................................ 247
Higher density plates ................................... 247
References .............................................. 247
12.Statistical analysis of enzyme kinetic data ................ 249
Athel Cornish-Bowden
1. Introduction ............................................ 249
2. Derivation of relationships ............................. 250
3. Defining objectives ..................................... 250
4. Basic assumptions of least squares ...................... 251
5. Fitting the Michaelis-Menten equation ................... 252
6. Equations with more than two parameters ................. 258
7. Detecting lack of fit ................................... 258
8. Estimating pure error ................................... 260
9. Distribution-free methods ............................... 262
10.Residual plots .......................................... 264
11.A note about rounding ................................... 267
References .............................................. 268
List of suppliers ............................................. 269
Enzyme index .................................................. 273
General index ................................................. 277
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