Enzyme Assays A Practical Approach
by Eisenthal, Robert; Danson, Michael J.-
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Summary
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 |
| Table of Contents provided by Syndetics. All Rights Reserved. |
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