Table of Contents
Preface to the Third Edition xv
List of Abbreviations and Symbols xvii
1 General Aspects of Enzyme Analysis 1
1.1 Introduction and Essentials for Enzyme Assays 1
Standard Books, Series 4
Databases 4
1.2 Theoretical Basis of Enzyme Assays 4
1.2.1 Order of Enzyme Reactions 4
1.2.2 Importance of the Reaction Order for Enzyme Reactions 6
1.2.3 The Reaction Velocity, Significance, and Practical Aspects 10
1.2.3.1 Determination of the Reaction Velocity, the Progress Curve 10
1.2.3.2 Enzyme Units 15
1.2.3.3 A Short Discussion About Errors in Enzyme Assays 17
1.2.3.4 Practical Rules for the Preparation of Dilution Series 21
1.2.3.5 Statistical Treatment of Enzyme Reactions 23
1.2.4 Treatment of the Michaelis-Menten Equation 24
1.2.4.1 General Considerations 24
1.2.4.2 Linear Representations of the Michaelis-Menten Equation 26
1.2.5 Enzyme Inhibition 28
1.2.6 Multisubstrate Reactions 33
1.3 Essential Conditions for Enzyme Assays 35
1.3.1 Dependence on Solvents and Ionic Strength 35
1.3.2 pH Dependency 36
1.3.2.1 Isoelectric Point 38
1.3.2.2 Buffers: What Must Be Regarded? 38
1.3.2.3 How to Prepare Buffers? 41
1.3.3 Temperature Dependency 42
1.3.4 Stability of Enzymes 47
1.3.4.1 Why Are Enzymes Unstable? 47
1.3.4.2 How Can Enzymes Be Stabilized? 48
1.3.4.3 How to Store Enzymes? 48
1.4 Theory of Coupled Enzyme Reactions 51
1.4.1 Two Coupled Reactions 51
1.4.2 Three Coupled Reactions 54
1.5 Substrate Determination 54
1.5.1 End Point Method 55
1.5.2 Substrate Determination by Coupled Enzyme Reactions 56
1.5.3 Kinetic Method for Substrate Determination 57
1.5.4 Enzymatic Cycling 57
2 Instrumental Aspects 61
2.1 Spectroscopic Methods 61
2.1.1 Absorption (UV/Vis) Photometry 61
2.1.2 Cuvettes 72
2.1.2.1 Shape 72
2.1.2.2 Material 73
2.1.2.3 Cleaning 73
2.1.3 Turbidity Measurement 74
2.1.4 Fluorescence Photometry 75
2.1.5 Luminometry 79
2.1.6 Polarimetry 80
2.2 Electrochemical Methods 81
2.2.1 pH Meter and Glass Electrodes 81
2.2.2 pH Stat 82
2.2.3 Potentiometry 83
2.2.4 Oxygen and Carbon Dioxide Electrodes 83
2.3 Radioactive Labeling 84
2.4 Diverse Methods 84
3 Enzyme Assays 87
3.1 Enzyme Nomenclature 88
3.2 Practical Considerations for Enzyme Assays 90
4 Oxidoreductases, EC 1 95
4.1 General Assay Procedures 95
4.1.1 Optical Assay 95
4.1.2 Fluorimetric Assay 96
4.2 Alcohol Dehydrogenase (ADH), EC 1.1.1.1 96
4.2.1 Reduction Assay 96
4.2.2 Oxidation Assay 97
4.3 Alcohol Dehydrogenase (NADP+), EC 1.1.1.2 98
4.4 Homoserine Dehydrogenase, EC 1.1.1.3 99
4.5 Shikimate Dehydrogenase, EC 1.1.1.25 100
4.6 l-Lactate Dehydrogenase (LDH), EC 1.1.1.27 101
4.6.1 Photometric Reduction Assay 101
4.6.2 Fluorimetric Reduction Assay 102
4.6.3 Oxidation Assay 103
4.7 Malate Dehydrogenase (MDH), EC 1.1.1.37 104
4.8 Malate Dehydrogenase (Oxaloacetate-Decarboxylating) (NAD+), EC 1.1.1.38, and Malate Dehydrogenase (Decarboxylating), EC 1.1.1.39 105
4.9 Malate Dehydrogenase (Oxaloacetate-decarboxylating) (NADP+), EC 1.1.1.40 106
4.10 Isocitrate Dehydrogenase (NAD+) (ICDH), EC 1.1.1.41 107
4.11 Isocitrate Dehydrogenase (NADP+) (ICDH), EC 1.1.1.42 108
4.12 Glucose-6-Phosphate Dehydrogenase (NADP+), EC 1.1.1.49 (G6PDH) 109
4.13 Glucose Oxidase (GOD), EC 1.1.3.4 110
4.14 Formate Dehydrogenase (FDH), EC 1.2.1.2 111
4.15 Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH), EC 1.2.1.12 112
4.15.1 Oxidation Assay 112
4.15.2 Reduction Assay Coupled with 3-Phosphoglycerate Kinase (PGK) 113
4.16 Long-Chain-Aldehyde Dehydrogenase, EC 1.2.1.48 114
4.17 Pyruvate Dehydrogenase (Acetyl-transferring) (PDH), EC 1.2.4.1 116
4.17.1 Ferricyanide as Electron Acceptor 116
4.17.2 Dichlorophenolindophenol as Electron Acceptor 117
4.18 Aldehyde Oxidase, EC 1.2.3.1 118
4.19 Oxoglutarate Dehydrogenase (Succinyl-transferring) (OGDH), EC 1.2.4.2 119
4.20 Pyruvate Ferredoxin Oxidoreductase, EC 1.2.7.1 120
4.20.1 Assay with Cytochrome c (cyt c) as Electron Acceptor 121
4.21 Alanine Dehydrogenase, EC 1.4.1.1 121
4.21.1 Oxidation of Alanine 122
4.21.2 Reduction of Pyruvate 122
4.22 Glutamate Dehydrogenase, EC 1.4.1.3 123
4.23 Leucine Dehydrogenase, EC 1.4.1.9 124
4.24 l-Amino-Acid Oxidase, EC 1.4.3.2 125
4.25 d-Amino-Acid Oxidase, EC 1.4.3.3 126
4.26 Monoamine Oxidase, EC 1.4.3.4 126
4.27 Primary Amine Oxidase, EC 1.4.3.21 127
4.27.1 Spectrophotometric Assay 128
4.27.2 Polarographic Assay of O2 Uptake with O2 Electrode 128
4.27.3 Assays for Benzylamine Oxidase Activity 129
4.28 Diamine Oxidase, EC 1.4.3.22 129
4.29 NADH:Ubiquinone Reductase (H+-Translocating) EC 1.6.5.3 130
4.29.1 Spectrophotometric Assay 131
4.30 NADH Dehydrogenase, EC 1.6.99.3 131
4.31 Factor-Independent Urate Hydroxylase, EC 1.7.3.3 132
4.32 Dihydrolipoyl Dehydrogenase, EC 1.8.1.4 133
4.32.1 Oxidation of Dihydrolipoamide 134
4.32.2 Reduction of Lipoamide 134
4.33 Glutathione Disulfide Reductase, EC 1.8.1.7 135
4.34 Cytochrome-c Oxidase (COX), EC 1.9.3.1 137
4.34.1 Spectrophotometric Assay 137
4.34.2 Assay with Oxygen Electrode 137
4.35 Catalase, EC 1.11.1.6 138
4.36 Peroxidase (POD) EC 1.11.1.7 139
4.36.1 Assay with 2,2′-Azino-bis-3-Ethylbenzothiazoline-6-Sulfonic Acid (ABTS) 140
4.36.2 Assay with Guaiacol 140
4.36.3 Assay with Dianisidine 141
4.37 Glutathione Peroxidase, EC 1.11.1.9 142
4.37.1 Coupled Assay with Glutathione Reductase 142
4.38 Photinus luciferin 4-Monooxigenase (ATP-Hydrolyzing), EC 1.13.12.7 143
4.39 Alkylglycerol Monooxygenase, EC 1.14.16.5 145
4.39.1 Spectroscopic Assay 145
4.39.2 Coupled Assay with HPLC Detection 145
4.40 Dopamine ß-Monooxygenase, EC 1.14.17.1 147
4.41 Tyrosinase, EC 1.14.18.1 148
4.41.1 Dopa Oxidase Assay 148
4.41.2 Dopachrome Assay 149
4.42 Superoxide Dismutase (SOD), EC 1.15.1.1 149
4.42.1 Assay with Pyrogallol 150
4.42.2 Assay with Ferricytochrome c and Xanthine Oxidase 150
4.43 Xanthine Oxidase (XOD), EC 1.17.3.2 151
5 Transferases, EC 2 153
5.1 Ornithine Carbamoyltransferase (OTC), EC 2.1.3.3 153
5.1.1 Method 1 for Color Development 154
5.1.2 Method 2 for Color Development 154
5.2 Choline O-Acetyltransferase, EC 2.3.1.6 155
5.3 Carnitine O-acetyltransferase, EC 2.3.1.7 156
5.3.1 Direct Spectroscopic Assay 156
5.3.2 Assay with DTNB 157
5.4 Dihydrolipoamide Acetyltransferase, EC 2.3.1.12 157
5.4.1 Spectrophotometric Assay 158
5.4.2 Stopped Assay 159
5.5 Fatty Acid Synthase, EC 2.3.1.85 160
5.6 𝛾-Glutamyltransferase, EC 2.3.2.2 161
5.7 Citrate Synthases, EC 2.3.3.1, EC 2.3.3.3, and EC 2.3.3.16 162
5.8 ATP Citrate Lyase, EC 2.3.3.8 164
5.9 Glycogen Phosphorylase, EC 2.4.1.1 165
5.10 Purine-nucleoside Phosphorylase (PNP), EC 2.4.2.1 166
5.11 Glutathione Transferase, EC 2.5.1.18 167
5.11.1 Spectrophotometric Assay 168
5.11.2 Titrimetric Assay 168
5.12 Aspartate Transaminase (AAT), EC 2.6.1.1 169
5.13 Alanine Transaminase, EC 2.6.1.2 170
5.14 Tyrosine Transaminase (TAT), EC 2.6.1.5; Tryptophan Transaminase (Tam 1), EC 2.6.1.27; Phenylalanine (Histidine) Transaminase, EC 2.6.1.58 171
5.14.1 Tyrosine Transaminase 171
5.14.2 Tryptophan Transaminase 171
5.14.3 Phenylalanine (Histidine) Transaminase: 171
5.15 Hexokinase (HK), EC 2.7.1.1, Glucokinase (GK), EC 2.7.1.2 173
5.16 Pyruvate Kinase (PK), EC 2.7.1.40 174
5.17 Acetate Kinase, EC 2.7.2.1 176
5.18 Phosphoglycerate Kinase (PGK), EC 2.7.2.3 177
5.19 Aspartate Kinase (AK), EC 2.7.2.4 178
5.20 Creatine Kinase (CK), EC 2.7.3.2 180
5.20.1 Coupled Assay 180
5.20.2 pH-Colorimetric Assay 181
6 Hydrolases, EC 3 183
6.1 Triacylglycerol Lipase, EC 3.1.1.3 183
6.1.1 Assay with pH Stat (Auto-titrator) 183
6.1.2 Fluorimetric Assay 184
6.2 Phospholipase A2, EC 3.1.1.4 185
6.3 Acetylcholinesterase (AChE), EC 3.1.1.7 186
6.4 Cholinesterase (ButChE), EC 3.1.1.8 187
6.4.1 pH Stat Assay 187
6.4.2 Colorimetric Assay 188
6.5 Hydroxyacylglutathione Hydrolase, EC 3.1.2.6 189
6.5.1 Direct Assay 189
6.5.2 Assay with DTNB 189
6.6 S-Formylglutathione Hydrolase, EC 3.1.2.12 190
6.7 Alkaline Phosphatase, EC 3.1.3.1 191
6.7.1 Mammalian Alkaline Phosphatase 191
6.7.2 Bacterial Alkaline Phosphatase 192
6.8 Acid Phosphatase, EC 3.1.3.2 192
6.9 5′-Nucleotidase, EC 3.1.3.5 193
6.9.1 Assay by Determination of Pi 194
6.9.2 Assay by Converting Adenosine into Inosine 194
6.10 Glucose-6-Phosphatase, EC 3.1.3.9 195
6.11 3′,5′-Cyclic-Nucleotide Phosphodiesterase, EC 3.1.4.17 196
6.12 Steryl-Sulfatase, EC 3.1.6.2 198
6.13 Pancreatic Ribonuclease, EC 3.1.27.5 198
6.14 α-Amylase, EC 3.2.1.1 199
6.15 Glucan 1,4-α-Glucosidase (AMG), EC 3.2.1.3 201
6.15.1 Coupled Assay with HK and G6PDH 201
6.15.2 Photometric Assay with 4-Nitrophenyl-d-Glucose 202
6.15.3 Fluorimetric Assay with 4-Methylumbelliferyl-α-d-Glucoside 202
6.16 Cellulases 203
6.16.1 β-1,4-Glucanase, EC 3.2.1.4 203
6.16.2 β-Glucosidase, EC 3.2.1.21 203
6.16.3 Orcinol Assay 204
6.16.4 Activity Staining 204
6.17 Lysozym, EC 3.2.1.17 206
6.18 Sialidase, EC 3.2.1.18 207
6.18.1 Fluorimetric Assay 207
6.18.2 Activity Staining 208
6.19 α-Glucosidase, EC 3.2.1.20 208
6.19.1 Coupled Assay 208
6.19.1.1 α-Glucosidase Reaction 209
6.19.1.2 Glucose Determination 209
6.19.2 Assay with 4-Nitrophenylglucopyranoside 210
6.20 β-Galactosidase, EC 3.2.1.23 211
6.21 α-Mannosidase, EC 3.2.1.24 212
6.21.1 Photometric Microassay 212
6.21.2 Fluorimetric Assay 213
6.22 β-Fructofuranosidase, EC 3.2.1.26 213
6.23 β-Glucuronidase, EC 3.2.1.31 214
6.23.1 Fluorimetric Assay 215
6.24 β-N-Acetylhexosaminidase, EC 3.2.1.52 215
6.25 Proteases, EC 3.4, General Assays 216
6.25.1 Anson Assay 216
6.25.2 Casein Assay 218
6.25.3 Azocasein Assay 219
6.25.4 Ninhydrin Assay 220
6.26 Leucyl Aminopeptidase (LAP), EC 3.4.11.1, Bacterial Leucyl Aminopeptidase, EC 3.4.11.10 221
6.26.1 Assay with Leucineamide 222
6.26.2 Assay with Leucine-p-nitroanilide 222
6.27 Peptidyl-dipeptidase A, EC 3.4.15.1 223
6.28 α-Chymotrypsin, EC 3.4.21.1 224
6.28.1 Assay with SUPHEPA 224
6.28.2 Assay with GLUPHEPA 225
6.29 Trypsin, EC 3.4.21.4 226
6.30 Pancreatic Elastase, EC 3.4.21.35 227
6.30.1 Assay with Succinyl-Ala-Ala-Ala-p-Nitroanilide 227
6.30.2 Esterase Activity of Elastase 227
6.31 Cathepsin B, EC 3.4.22.1 228
6.32 Pepsin A, EC 3.4.23.1 229
6.33 Asparaginase, EC 3.5.1.1 230
6.34 Glutaminase, EC 3.5.1.2 232
6.34.1 Determination of Ammonia with Nessler’s Reagent 232
6.34.2 pH Stat Assay 233
6.35 Urease, EC 3.5.1.5 233
6.35.1 pH Stat Assay 234
6.35.2 Photometric Assay 234
6.36 Guanine Deaminase, EC 3.5.4.3 235
6.36.1 Determination of Ammonia 236
6.37 Adenosinetriphosphatase, EC 3.6.1.3 237
6.38 Mg2+ Importing ATPase, EC 3.6.3.2, Na+/K+-Exchanging ATPase, EC 3.6.3.9 238
6.38.1 Assay of Total ATPase Activity 238
6.38.2 Assay of Mg2+-ATPase Activity 239
7 Lyases, EC 4 241
7.1 Pyruvate Decarboxylase (PDC), EC 4.1.1.1 241
7.2 Glutamate Decarboxylase (GAD), EC 4.1.1.15 242
7.3 Fructose-bisphosphate Aldolase, EC 4.1.2.13 244
7.4 Anthranilate Synthase, EC 4.1.3.27 245
7.5 Carbonic Anhydrase (CA), EC 4.2.1.1 246
7.5.1 pH Stat Assay 246
7.5.2 Esterase Assay with 4-Nitrophenylacetate 247
7.6 Fumarate Hydratase, EC 4.2.1.2 248
7.7 Lactoylglutathione Lyase, EC 4.4.1.5 249
7.8 Adenylate Cyclase (AC), EC 4.6.1.1 250
8 Isomerases, EC 5 253
8.1 Xylose Isomerase, EC 5.3.1.5 253
8.1.1 D-Xylose Isomerase Assay 253
8.1.2 D-Xylose Isomerase Microplate Assay 254
8.1.3 D-Glucose Isomerase Assay 255
8.1.4 D-Glucose Isomerase Microplate Assay 256
8.2 Glucose-6-phosphate Isomerase (G6PI), EC 5.3.1.9 256
8.3 Phosphoglucomutase (PGM), EC 5.4.2.2 257
9 Ligases (Synthetases), EC 6 261
9.1 Tyrosine-tRNA Ligase, EC 6.1.1.1 261
9.1.1 Fluorimetric Assay 261
9.1.2 ATP-32PP Exchange 262
9.2 Acetate-CoA Ligase (ACL), EC 6.2.1.1 263
9.2.1 Direct Radioactive Assay 264
9.2.2 Coupled Spectroscopic Assay 264
9.3 Glutamine Synthetase, EC 6.3.1.2 266
10 Assays for Multi-enzyme Complexes 269
10.1 Pyruvate Dehydrogenase Complex (PDHC) 269
10.1.1 Overall Activity of PDHC by NAD+ Reduction 270
10.1.2 Overall Activity of PDHC by Dismutation Assay 270
10.2 α-Oxoglutarate Dehydrogenase Complex (OGDHC) 272
10.2.1 Overall Activity by NAD+ Reduction 273
11 Assays for Other Enzyme Relevant Parameters 275
11.1 Substrate Determination 275
11.1.1 Determination of NADP(H) by Enzymatic Cycling 275
11.1.1.1 Cycling Reaction 276
11.1.2 Determination of NAD(H) by Enzymatic Cycling 277
11.2 Protein Determination 279
11.2.1 Biuret Assay 279
11.2.2 BCA Assay 281
11.2.2.1 Assay for Soluble Proteins 281
11.2.2.2 Modification for Immobilized Proteins 282
11.2.3 Lowry Assay 282
11.2.4 Coomassie Binding Assay (Bradford Assay) 283
11.2.4.1 Assay for Soluble Proteins 284
11.2.4.2 Modification for Immobilized Proteins 284
11.2.5 Absorption Method 285
11.2.6 Fluorimetric Assay 287
11.2.7 Ninhydrin Assay 288
11.2.7.1 Ninhydrin Assay with Hydrolysis 288
11.2.7.2 Modified Ninhydrin AssayWithout Hydrolysis 289
11.2.8 Protein Assay with 2-Hydroxy-1-naphthaldehyde 290 General Literature for Protein Assays 291
11.3 Phosphate Determination 291
11.4 Determination of Metal Ions 293
11.4.1 Calcium and Magnesium 293
11.4.2 Iron 294
11.4.2.1 Determination with Ferrozine 295
11.4.2.2 Determination of FeII with 1,10-Phenanthroline in the Presence of FeIII 295
11.4.3 Copper 296
11.4.3.1 Biquinoline Method 296
11.4.3.2 Oxalyldihydrazide Method 297
11.4.4 Manganese 297
11.4.4.1 Colorimetric Assay 298
11.4.4.2 Assay with 1-(2-Pyridylazo)-2-naphthol (PAN) 298
11.4.5 Zinc 299
11.5 Glycoprotein Assays 300
11.5.1 Identification in Electrophoresis Gels 300
11.5.2 Quantitative Analysis of Protein-Bound Hexoses 300
11.6 Cross-linking of Proteins with Dimethylsuberimidate 301
11.7 Concentrating Enzyme Solutions 302
11.7.1 Precipitation 303
11.7.2 Ultrafiltration and Dialysis 306
11.7.3 Ultracentrifugation 307
11.7.4 Lyophilization 307
11.7.5 Other Concentration Methods 307
12 Enzyme Immunoassays 309
12.1 Radioimmunoassays 309
12.2 Principle of Enzyme Immunoassays 309
12.3 Noncompetitive Solid-Phase Enzyme Immunoassay 311
12.4 Competitive Solid-Phase Enzyme Immunoassay 312
12.5 Methods for Enzyme Immunoassays and Immobilization Techniques 312
12.5.1 Protein Coupling to Cyanogen Bromide Activated Agarose 312
12.5.2 Coupling of Diaminohexyl Spacer 313
12.5.3 Periodate Activation of Cellulose 314
12.5.4 Introduction of Thiol Groups into Proteins (Antibodies) 315
12.5.5 Conjugation of a Protein (Antibody) with an Enzyme (Peroxidase) 316
12.5.6 Conjugation of ß-Galactosidase to Proteins (Antibodies) by MBS 316
12.5.7 Conjugation of Alkaline Phosphatase to Antibodies by Glutaraldehyde 317
13 Binding Measurements 319
13.1 Different Types of Binding 319
13.1.1 General Considerations 319
13.1.2 How Can Specific Reversible Binding be Identified? 320
13.1.3 Experimental Aspects 322
13.2 Binding Measurements by Size Discrimination 325
13.2.1 Equilibrium Dialysis 325
13.2.1.1 Binding of Indole to Bovine Serum Albumin 327
13.2.2 Evaluation of Binding Experiments 329
13.2.3 Ultrafiltration 330
13.2.4 Gel Filtration 331
13.2.5 Ultracentrifugation 332
13.3 Spectroscopic Methods 333
13.3.1 Difference Spectroscopy 334
13.3.1.1 Difference Spectroscopic Titration of Ligands Binding to Catalase 336
13.3.1.2 Evaluation of Spectroscopic Binding Curves 339
13.3.2 Fluorescence Spectroscopy 341
13.3.2.1 Binding of ANS to Bovine Serum Albumin 341
13.4 Other Binding Methods 344
13.4.1 Radioactive Labeling 344
13.4.2 Surface Plasmon Resonance (SPR) 345
14 Enzymes in Technical Applications 347
14.1 Modes of Enzyme Immobilization 347
14.1.1 Adsorption 348
14.1.2 Entrapment 350
14.1.3 Encapsulation 350
14.1.4 Cross-linking 351
14.1.5 Covalent Immobilization to Solid Supports 351
14.1.5.1 Supports 351
14.1.5.2 Spacer 353
14.2 Methods for Enzyme Immobilization 354
14.2.1 Microencapsulation in Nylon Beads 355
14.2.2 Entrapment in Polyacrylamide 355
14.2.3 Covalent Immobilization on Glass Surfaces 356
14.2.4 Covalent Immobilization on Controlled-Pore Glass (CPG) 358
14.2.5 Covalent Immobilization to Polyamide 360
14.2.5.1 O-Alkylation with Triethyloxonium Tetrafluoroborate 361
14.2.5.2 Immobilization to Amino Groups after Partial Hydrolysis of Polyamide 363
14.2.5.3 Immobilization to Carboxyl Groups After Partial Hydrolysis of Polyamide 364
14.2.6 Immobilization to Polyester 365
14.2.7 Immobilization by Alkaline Hydrolysis and Activation with Tosylchloride 367
14.2.8 Alkaline Hydrolysis and Activation by Carbonyldiimidazol 368
14.3 Analysis of Immobilized Enzymes 368
14.3.1 General Principles 368
14.3.2 Continuous Photometric Assays for Immobilized Enzymes 369
14.3.3 Cofactors in Reactions with Immobilized Enzymes 371
14.4 Enzyme Reactors 372
14.4.1 Batch Reactor (Stirred-Tank Reactor) 373
14.4.2 Membrane Reactor 373
14.4.3 Solid-Bed Reactor 374
14.4.4 Immobilized Cells 375
14.5 Biosensors 375
14.5.1 Enzyme Electrodes 375
14.5.2 Immunoelectrodes 379
14.5.3 Other Biosensors 379
14.6 Immobilized Enzymes in Therapy 381
Index 383
