Enzyme assays: a practical approach
Gespeichert in:
| Format: | Buch |
|---|---|
| Sprache: | English |
| Veröffentlicht: |
Oxford [u.a.]
Oxford Univ. Press
2002
|
| Ausgabe: | 2. ed. |
| Schriftenreihe: | The practical approach series
257 |
| Schlagworte: | |
| Online-Zugang: | Inhaltsverzeichnis |
| Beschreibung: | Hier auch später erschienene, unveränderte Nachdrucke Literaturangaben |
| Beschreibung: | XIX, 282 S. Ill., graph. Darst. |
| ISBN: | 0199638217 0199638209 |
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Datensatz im Suchindex
| _version_ | 1804129292337020928 |
|---|---|
| adam_text | Enzyme Assays
Second Edition
A [Praxeitoeal Approved!)
Edited by
Robert Eisenthal
and
Michael J Danson
Department of Biology and Biochemistry,
University of Bath, Bath, BA2 7AY U K
OXTORD
UNIVERSITY PRESS
Contents
List of protocols page xiii
Abbreviations xvii
1 Principles of enzyme assay and kinetic studies i
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
CONTENTS
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 Turbidimetiy 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 liquidchromatographic 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
viii
CONTENTS
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 (Rl) 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
ix
CONTENTS
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
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
x
CONTENTS
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
xi
CONTENTS
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
xii
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| series | The practical approach series |
| series2 | The practical approach series |
| spelling | Enzyme assays a practical approach ed. by Robert Eisenthal ... 2. ed. Oxford [u.a.] Oxford Univ. Press 2002 XIX, 282 S. Ill., graph. Darst. txt rdacontent n rdamedia nc rdacarrier The practical approach series 257 Hier auch später erschienene, unveränderte Nachdrucke Literaturangaben Cytochemie gtt Enzimas larpcal Enzymen gtt Enzymes - Analyse Histochemie gtt Laboratoriumonderzoek gtt Enzymes Analysis Enzymes analysis Enzym (DE-588)4014988-2 gnd rswk-swf Analyse (DE-588)4122795-5 gnd rswk-swf Bestimmung (DE-588)4144949-6 gnd rswk-swf Reinigung (DE-588)4049250-3 gnd rswk-swf Enzymatische Analyse (DE-588)4014989-4 gnd rswk-swf 1\p (DE-588)4123623-3 Lehrbuch gnd-content Enzym (DE-588)4014988-2 s Bestimmung (DE-588)4144949-6 s DE-604 Reinigung (DE-588)4049250-3 s 2\p DE-604 Analyse (DE-588)4122795-5 s 3\p DE-604 Enzymatische Analyse (DE-588)4014989-4 s 4\p DE-604 Eisenthal, Robert Sonstige oth The practical approach series 257 (DE-604)BV007921321 257 HEBIS Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=009871816&sequence=000002&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis 1\p cgwrk 20201028 DE-101 https://d-nb.info/provenance/plan#cgwrk 2\p cgwrk 20201028 DE-101 https://d-nb.info/provenance/plan#cgwrk 3\p cgwrk 20201028 DE-101 https://d-nb.info/provenance/plan#cgwrk 4\p cgwrk 20201028 DE-101 https://d-nb.info/provenance/plan#cgwrk |
| spellingShingle | Enzyme assays a practical approach The practical approach series Cytochemie gtt Enzimas larpcal Enzymen gtt Enzymes - Analyse Histochemie gtt Laboratoriumonderzoek gtt Enzymes Analysis Enzymes analysis Enzym (DE-588)4014988-2 gnd Analyse (DE-588)4122795-5 gnd Bestimmung (DE-588)4144949-6 gnd Reinigung (DE-588)4049250-3 gnd Enzymatische Analyse (DE-588)4014989-4 gnd |
| subject_GND | (DE-588)4014988-2 (DE-588)4122795-5 (DE-588)4144949-6 (DE-588)4049250-3 (DE-588)4014989-4 (DE-588)4123623-3 |
| title | Enzyme assays a practical approach |
| title_auth | Enzyme assays a practical approach |
| title_exact_search | Enzyme assays a practical approach |
| title_full | Enzyme assays a practical approach ed. by Robert Eisenthal ... |
| title_fullStr | Enzyme assays a practical approach ed. by Robert Eisenthal ... |
| title_full_unstemmed | Enzyme assays a practical approach ed. by Robert Eisenthal ... |
| title_short | Enzyme assays |
| title_sort | enzyme assays a practical approach |
| title_sub | a practical approach |
| topic | Cytochemie gtt Enzimas larpcal Enzymen gtt Enzymes - Analyse Histochemie gtt Laboratoriumonderzoek gtt Enzymes Analysis Enzymes analysis Enzym (DE-588)4014988-2 gnd Analyse (DE-588)4122795-5 gnd Bestimmung (DE-588)4144949-6 gnd Reinigung (DE-588)4049250-3 gnd Enzymatische Analyse (DE-588)4014989-4 gnd |
| topic_facet | Cytochemie Enzimas Enzymen Enzymes - Analyse Histochemie Laboratoriumonderzoek Enzymes Analysis Enzymes analysis Enzym Analyse Bestimmung Reinigung Enzymatische Analyse Lehrbuch |
| url | http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=009871816&sequence=000002&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
| volume_link | (DE-604)BV007921321 |
| work_keys_str_mv | AT eisenthalrobert enzymeassaysapracticalapproach |