Practical Enzymology (3rd Edition)

Rs. 26,275
  • Author: Hans Bisswanger
  • ISBN: 9783527346042
  • Publisher: Wiley Publishing
  • Edition: 3rd
  • Publication Date: October 21, 2019
  • Format: Paperback – 416 pages
  • Language: English

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A practice-oriented guide to assaying more than 100 of the most important enzymes, complete with the theoretical background and specific protocols for immediate use in the biochemical laboratory. Now expanded with a new section on metal ion determination.

Table of Contents
  1. Preface to the Third Edition xv
  2. List of Abbreviations and Symbols xvii
  3. 1 General Aspects of Enzyme Analysis 1
    1. Introduction and Essentials for Enzyme Assays 1
    2. Standard Books, Series 4
    3. Databases 4
    4. Theoretical Basis of Enzyme Assays 4
    5. Order of Enzyme Reactions 4
    6. Importance of the Reaction Order for Enzyme Reactions 6
    7. The Reaction Velocity, Significance, and Practical Aspects 10
    8. Determination of the Reaction Velocity, the Progress Curve 10
    9. Enzyme Units 15
    10. A Short Discussion About Errors in Enzyme Assays 17
    11. Practical Rules for the Preparation of Dilution Series 21
    12. Statistical Treatment of Enzyme Reactions 23
    13. Treatment of the Michaelis–Menten Equation 24
    14. General Considerations 24
    15. Linear Representations of the Michaelis–Menten Equation 26
    16. Enzyme Inhibition 28
    17. Multisubstrate Reactions 33
    18. Essential Conditions for Enzyme Assays 35
    19. Dependence on Solvents and Ionic Strength 35
    20. pH Dependency 36
    21. Isoelectric Point 38
    22. Buffers: What Must Be Regarded? 38
    23. How to Prepare Buffers? 41
    24. Temperature Dependency 42
    25. Stability of Enzymes 47
    26. Why Are Enzymes Unstable? 47
    27. How Can Enzymes Be Stabilized? 48
    28. How to Store Enzymes? 48
    29. Theory of Coupled Enzyme Reactions 51
    30. Two Coupled Reactions 51
    31. Three Coupled Reactions 54
    32. Substrate Determination 54
    33. End Point Method 55
    34. Substrate Determination by Coupled Enzyme Reactions 56
    35. Kinetic Method for Substrate Determination 57
    36. Enzymatic Cycling 57
  4. 2 Instrumental Aspects 61
    1. Spectroscopic Methods 61
    2. Absorption (UV/Vis) Photometry 61
    3. Cuvettes 72
    4. Shape 72
    5. Material 73
    6. Cleaning 73
    7. Turbidity Measurement 74
    8. Fluorescence Photometry 75
    9. Luminometry 79
    10. Polarimetry 80
    11. Electrochemical Methods 81
    12. pH Meter and Glass Electrodes 81
    13. pH Stat 82
    14. Potentiometry 83
    15. Oxygen and Carbon Dioxide Electrodes 83
    16. Radioactive Labeling 84
    17. Diverse Methods 84
  5. 3 Enzyme Assays 87
    1. Enzyme Nomenclature 88
    2. Practical Considerations for Enzyme Assays 90
  6. 4 Oxidoreductases, EC 1 95
    1. General Assay Procedures 95
    2. Optical Assay 95
    3. Fluorimetric Assay 96
    4. Alcohol Dehydrogenase (ADH), EC 1.1.1.1 96
    5. Reduction Assay 96
    6. Oxidation Assay 97
    7. Alcohol Dehydrogenase (NADP+), EC 1.1.1.2 98
    8. Homoserine Dehydrogenase, EC 1.1.1.3 99
    9. Shikimate Dehydrogenase, EC 1.1.1.25 100
    10. l-Lactate Dehydrogenase (LDH), EC 1.1.1.27 101
    11. Photometric Reduction Assay 101
    12. Fluorimetric Reduction Assay 102
    13. Oxidation Assay 103
    14. Malate Dehydrogenase (MDH), EC 1.1.1.37 104
    15. Malate Dehydrogenase (Oxaloacetate-Decarboxylating) (NAD+), EC 1.1.1.38, and Malate Dehydrogenase (Decarboxylating), EC 1.1.1.39 105
    16. Malate Dehydrogenase (Oxaloacetate-decarboxylating) (NADP+), EC 1.1.1.40 106
    17. Isocitrate Dehydrogenase (NAD+) (ICDH), EC 1.1.1.41 107
    18. Isocitrate Dehydrogenase (NADP+) (ICDH), EC 1.1.1.42 108
    19. Glucose-6-Phosphate Dehydrogenase (NADP+), EC 1.1.1.49 (G6PDH) 109
    20. Glucose Oxidase (GOD), EC 1.1.3.4 110
    21. Formate Dehydrogenase (FDH), EC 1.2.1.2 111
    22. Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH), EC 1.2.1.12 112
    23. Oxidation Assay 112
    24. Reduction Assay Coupled with 3-Phosphoglycerate Kinase (PGK) 113
    25. Long-Chain-Aldehyde Dehydrogenase, EC 1.2.1.48 114
    26. Pyruvate Dehydrogenase (Acetyl-transferring) (PDH), EC 1.2.4.1 116
    27. Ferricyanide as Electron Acceptor 116
    28. Dichlorophenolindophenol as Electron Acceptor 117
    29. Aldehyde Oxidase, EC 1.2.3.1 118
    30. Oxoglutarate Dehydrogenase (Succinyl-transferring) (OGDH), EC 1.2.4.2 119
    31. Pyruvate Ferredoxin Oxidoreductase, EC 1.2.7.1 120
    32. Assay with Cytochrome c (cyt c) as Electron Acceptor 121
    33. Alanine Dehydrogenase, EC 1.4.1.1 121
    34. Oxidation of Alanine 122
    35. Reduction of Pyruvate 122
    36. Glutamate Dehydrogenase, EC 1.4.1.3 123
    37. Leucine Dehydrogenase, EC 1.4.1.9 124
    38. l-Amino-Acid Oxidase, EC 1.4.3.2 125
    39. d-Amino-Acid Oxidase, EC 1.4.3.3 126
    40. Monoamine Oxidase, EC 1.4.3.4 126
    41. Primary Amine Oxidase, EC 1.4.3.21 127
    42. Spectrophotometric Assay 128
    43. Polarographic Assay of O2 Uptake with O2 Electrode 128
    44. Assays for Benzylamine Oxidase Activity 129
    45. Diamine Oxidase, EC 1.4.3.22 129
    46. NADH:Ubiquinone Reductase (H+-Translocating) EC 1.6.5.3 130
    47. Spectrophotometric Assay 131
    48. NADH Dehydrogenase, EC 1.6.99.3 131
    49. Factor-Independent Urate Hydroxylase, EC 1.7.3.3 132
    50. Dihydrolipoyl Dehydrogenase, EC 1.8.1.4 133
    51. Oxidation of Dihydrolipoamide 134
    52. Reduction of Lipoamide 134
    53. Glutathione Disulfide Reductase, EC 1.8.1.7 135
    54. Cytochrome-c Oxidase (COX), EC 1.9.3.1 137
    55. Spectrophotometric Assay 137
    56. Assay with Oxygen Electrode 137
    57. Catalase, EC 1.11.1.6 138
    58. Peroxidase (POD) EC 1.11.1.7 139
    59. Assay with 2,2′-Azino-bis-3-Ethylbenzothiazoline-6-Sulfonic Acid (ABTS) 140
    60. Assay with Guaiacol 140
    61. Assay with Dianisidine 141
    62. Glutathione Peroxidase, EC 1.11.1.9 142
    63. Coupled Assay with Glutathione Reductase 142
    64. Photinus luciferin 4-Monooxigenase (ATP-Hydrolyzing), EC 1.13.12.7 143
    65. Alkylglycerol Monooxygenase, EC 1.14.16.5 145
    66. Spectroscopic Assay 145
    67. Coupled Assay with HPLC Detection 145
    68. Dopamine ß-Monooxygenase, EC 1.14.17.1 147
    69. Tyrosinase, EC 1.14.18.1 148
    70. Dopa Oxidase Assay 148
    71. Dopachrome Assay 149
    72. Superoxide Dismutase (SOD), EC 1.15.1.1 149
    73. Assay with Pyrogallol 150
    74. Assay with Ferricytochrome c and Xanthine Oxidase 150
    75. Xanthine Oxidase (XOD), EC 1.17.3.2 151
  7. 5 Transferases, EC 2 153
    1. Ornithine Carbamoyltransferase (OTC), EC 2.1.3.3 153
    2. Method 1 for Color Development 154
    3. Method 2 for Color Development 154
    4. Choline O-Acetyltransferase, EC 2.3.1.6 155
    5. Carnitine O-acetyltransferase, EC 2.3.1.7 156
    6. Direct Spectroscopic Assay 156
    7. Assay with DTNB 157
    8. Dihydrolipoamide Acetyltransferase, EC 2.3.1.12 157
    9. Spectrophotometric Assay 158
    10. Stopped Assay 159
    11. Fatty Acid Synthase, EC 2.3.1.85 160
    12. 𝛾-Glutamyltransferase, EC 2.3.2.2 161
    13. Citrate Synthases, EC 2.3.3.1, EC 2.3.3.3, and EC 2.3.3.16 162
    14. ATP Citrate Lyase, EC 2.3.3.8 164
    15. Glycogen Phosphorylase, EC 2.4.1.1 165
    16. Purine-nucleoside Phosphorylase (PNP), EC 2.4.2.1 166
    17. Glutathione Transferase, EC 2.5.1.18 167
    18. Spectrophotometric Assay 168
    19. Titrimetric Assay 168
    20. Aspartate Transaminase (AAT), EC 2.6.1.1 169
    21. Alanine Transaminase, EC 2.6.1.2 170
    22. 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
    23. Tyrosine Transaminase 171
    24. Tryptophan Transaminase 171
    25. Phenylalanine (Histidine) Transaminase: 171
    26. Hexokinase (HK), EC 2.7.1.1, Glucokinase (GK), EC 2.7.1.2 173
    27. Pyruvate Kinase (PK), EC 2.7.1.40 174
    28. Acetate Kinase, EC 2.7.2.1 176
    29. Phosphoglycerate Kinase (PGK), EC 2.7.2.3 177
    30. Aspartate Kinase (AK), EC 2.7.2.4 178
    31. Creatine Kinase (CK), EC 2.7.3.2 180
    32. Coupled Assay 180
    33. pH-Colorimetric Assay 181
  8. 6 Hydrolases, EC 3 183
    1. Triacylglycerol Lipase, EC 3.1.1.3 183
    2. Assay with pH Stat (Auto-titrator) 183
    3. Fluorimetric Assay 184
    4. Phospholipase A2, EC 3.1.1.4 185
    5. Acetylcholinesterase (AChE), EC 3.1.1.7 186
    6. Cholinesterase (ButChE), EC 3.1.1.8 187
    7. pH Stat Assay 187
    8. Colorimetric Assay 188
    9. Hydroxyacylglutathione Hydrolase, EC 3.1.2.6 189
    10. Direct Assay 189
    11. Assay with DTNB 189
    12. S-Formylglutathione Hydrolase, EC 3.1.2.12 190
    13. Alkaline Phosphatase, EC 3.1.3.1 191
    14. Mammalian Alkaline Phosphatase 191
    15. Bacterial Alkaline Phosphatase 192
    16. Acid Phosphatase, EC 3.1.3.2 192
    17. Nucleotidase, EC 3.1.3.5 193
    18. Assay by Determination of Pi 194
    19. Assay by Converting Adenosine into Inosine 194
    20. Glucose-6-Phosphatase, EC 3.1.3.9 195
    21. Cyclic-Nucleotide Phosphodiesterase, EC 3.1.4.17 196
    22. Steryl-Sulfatase, EC 3.1.6.2 198
    23. Pancreatic Ribonuclease, EC 3.1.27.5 198
    24. α-Amylase, EC 3.2.1.1 199
    25. Glucan 1,4-α-Glucosidase (AMG), EC 3.2.1.3 201
    26. Coupled Assay with HK and G6PDH 201
    27. Photometric Assay with 4-Nitrophenyl-d-Glucose 202
    28. Fluorimetric Assay with 4-Methylumbelliferyl-α-d-Glucoside 202
    29. Cellulases 203
    30. β-1,4-Glucanase, EC 3.2.1.4 203
    31. β-Glucosidase, EC 3.2.1.21 203
    32. Orcinol Assay 204
    33. Activity Staining 204
    34. Lysozym, EC 3.2.1.17 206
    35. Sialidase, EC 3.2.1.18 207
    36. Fluorimetric Assay 207
    37. Activity Staining 208
    38. α-Glucosidase, EC 3.2.1.20 208
    39. Coupled Assay 208
    40. α-Glucosidase Reaction 209
    41. Glucose Determination 209
    42. Assay with 4-Nitrophenylglucopyranoside 2106.20
    43. β-Galactosidase, EC 3.2.1.23 211
    44. α-Mannosidase, EC 3.2.1.24 212
    45. Photometric Microassay 212
    46. Fluorimetric Assay 213
    47. β-Fructofuranosidase, EC 3.2.1.26 213
    48. β-Glucuronidase, EC 3.2.1.31 214
    49. Fluorimetric Assay 215
    50. β-N-Acetylhexosaminidase, EC 3.2.1.52 215
    51. Proteases, EC 3.4, General Assays 216
    52. Anson Assay 216
    53. Casein Assay 218
    54. Azocasein Assay 219
    55. Ninhydrin Assay 220
    56. Leucyl Aminopeptidase (LAP), EC 3.4.11.1, Bacterial Leucyl Aminopeptidase, EC 3.4.11.10 221
    57. Assay with Leucineamide 222
    58. Assay with Leucine-p-nitroanilide 222
    59. Peptidyl-dipeptidase A, EC 3.4.15.1 223
    60. α-Chymotrypsin, EC 3.4.21.1 224
    61. Assay with SUPHEPA 224
    62. Assay with GLUPHEPA 225
    63. Trypsin, EC 3.4.21.4 226
    64. Pancreatic Elastase, EC 3.4.21.35 227
    65. Assay with Succinyl-Ala–Ala–Ala–p-Nitroanilide 227
    66. Esterase Activity of Elastase 227
    67. Cathepsin B, EC 3.4.22.1 228
    68. Pepsin A, EC 3.4.23.1 229
    69. Asparaginase, EC 3.5.1.1 230
    70. Glutaminase, EC 3.5.1.2 232
    71. Determination of Ammonia with Nessler’s Reagent 232
    72. pH Stat Assay 233
    73. Urease, EC 3.5.1.5 233
    74. pH Stat Assay 234
    75. Photometric Assay 234
    76. Guanine Deaminase, EC 3.5.4.3 235
    77. Determination of Ammonia 236
    78. Adenosinetriphosphatase, EC 3.6.1.3 237
    79. Mg2+ Importing ATPase, EC 3.6.3.2, Na+/K+-Exchanging ATPase, EC 3.6.3.9 238
    80. Assay of Total ATPase Activity 238
    81. Assay of Mg2+-ATPase Activity 239
  9. 7 Lyases, EC 4 241
    1. Pyruvate Decarboxylase (PDC), EC 4.1.1.1 241
    2. Glutamate Decarboxylase (GAD), EC 4.1.1.15 242
    3. Fructose-bisphosphate Aldolase, EC 4.1.2.13 244
    4. Anthranilate Synthase, EC 4.1.3.27 245
    5. Carbonic Anhydrase (CA), EC 4.2.1.1 246
    6. pH Stat Assay 246
    7. Esterase Assay with 4-Nitrophenylacetate 247
    8. Fumarate Hydratase, EC 4.2.1.2 248
    9. Lactoylglutathione Lyase, EC 4.4.1.5 249
    10. Adenylate Cyclase (AC), EC 4.6.1.1 250
  10. 8 Isomerases, EC 5 253
    1. Xylose Isomerase, EC 5.3.1.5 253
    2. D-Xylose Isomerase Assay 253
    3. D-Xylose Isomerase Microplate Assay 254
    4. D-Glucose Isomerase Assay 255
    5. D-Glucose Isomerase Microplate Assay 256
    6. Glucose-6-phosphate Isomerase (G6PI), EC 5.3.1.9 256
    7. Phosphoglucomutase (PGM), EC 5.4.2.2 257
  11. 9 Ligases (Synthetases), EC 6 261
    1. Tyrosine-tRNA Ligase, EC 6.1.1.1 261
    2. Fluorimetric Assay 261
    3. ATP–32PP Exchange 262
    4. Acetate-CoA Ligase (ACL), EC 6.2.1.1 263
    5. Direct Radioactive Assay 264
    6. Coupled Spectroscopic Assay 264
    7. Glutamine Synthetase, EC 6.3.1.2 266
  12. 10 Assays for Multi-enzyme Complexes 269
    1. Pyruvate Dehydrogenase Complex (PDHC) 269
    2. Overall Activity of PDHC by NAD+ Reduction 270
    3. Overall Activity of PDHC by Dismutation Assay 270
    4. α-Oxoglutarate Dehydrogenase Complex (OGDHC) 272
    5. Overall Activity by NAD+ Reduction 273
  13. 11 Assays for Other Enzyme Relevant Parameters 275
    1. Substrate Determination 275
    2. Determination of NADP(H) by Enzymatic Cycling 275
    3. Cycling Reaction 276
    4. Determination of NAD(H) by Enzymatic Cycling 277
    5. Protein Determination 279
    6. Biuret Assay 279
    7. BCA Assay 281
    8. Assay for Soluble Proteins 281
    9. Modification for Immobilized Proteins 282
    10. Lowry Assay 282
    11. Coomassie Binding Assay (Bradford Assay) 283
    12. Assay for Soluble Proteins 284
    13. Modification for Immobilized Proteins 284
    14. Absorption Method 285
    15. Fluorimetric Assay 287
    16. Ninhydrin Assay 288
    17. Ninhydrin Assay with Hydrolysis 288
    18. Modified Ninhydrin AssayWithout Hydrolysis 289
    19. Protein Assay with 2-Hydroxy-1-naphthaldehyde 290 General Literature for Protein Assays 291
    20. Phosphate Determination 291
    21. Determination of Metal Ions 293
    22. Calcium and Magnesium 293
    23. Iron 294
    24. Determination with Ferrozine 295
    25. Determination of FeII with 1,10-Phenanthroline in the Presence of FeIII 295
    26. Copper 296
    27. Biquinoline Method 296
    28. Oxalyldihydrazide Method 297
    29. Manganese 297
    30. Colorimetric Assay 298
    31. Assay with 1-(2-Pyridylazo)-2-naphthol (PAN) 298
    32. Zinc 299
    33. Glycoprotein Assays 300
    34. Identification in Electrophoresis Gels 300
    35. Quantitative Analysis of Protein-Bound Hexoses 300
    36. Cross-linking of Proteins with Dimethylsuberimidate 301
    37. Concentrating Enzyme Solutions 302
    38. Precipitation 303
    39. Ultrafiltration and Dialysis 306
    40. Ultracentrifugation 307
    41. Lyophilization 307
    42. Other Concentration Methods 307
  14. 12 Enzyme Immunoassays 309
    1. Radioimmunoassays 309
    2. Principle of Enzyme Immunoassays 309
    3. Noncompetitive Solid-Phase Enzyme Immunoassay 311
    4. Competitive Solid-Phase Enzyme Immunoassay 312
    5. Methods for Enzyme Immunoassays and Immobilization Techniques 312
    6. Protein Coupling to Cyanogen Bromide Activated Agarose 312
    7. Coupling of Diaminohexyl Spacer 313
    8. Periodate Activation of Cellulose 314
    9. Introduction of Thiol Groups into Proteins (Antibodies) 315
    10. Conjugation of a Protein (Antibody) with an Enzyme (Peroxidase) 316
    11. Conjugation of ß-Galactosidase to Proteins (Antibodies) by MBS 316
    12. Conjugation of Alkaline Phosphatase to Antibodies by Glutaraldehyde 317
  15. 13 Binding Measurements 319
    1. Different Types of Binding 319
    2. General Considerations 319
    3. How Can Specific Reversible Binding be Identified? 320
    4. Experimental Aspects 322
    5. Binding Measurements by Size Discrimination 325
    6. Equilibrium Dialysis 325
    7. Binding of Indole to Bovine Serum Albumin 327
    8. Evaluation of Binding Experiments 329
    9. Ultrafiltration 330
    10. Gel Filtration 331
    11. Ultracentrifugation 332
    12. Spectroscopic Methods 333
    13. Difference Spectroscopy 334
    14. Difference Spectroscopic Titration of Ligands Binding to Catalase 336
    15. Evaluation of Spectroscopic Binding Curves 339
    16. Fluorescence Spectroscopy 341
    17. Binding of ANS to Bovine Serum Albumin 341
    18. Other Binding Methods 344
    19. Radioactive Labeling 344
    20. Surface Plasmon Resonance (SPR) 345
  16. 14 Enzymes in Technical Applications 347
    1. Modes of Enzyme Immobilization 347
    2. Adsorption 348
    3. Entrapment 350
    4. Encapsulation 350
    5. Cross-linking 351
    6. Covalent Immobilization to Solid Supports 351
    7. Supports 351
    8. Spacer 353
    9. Methods for Enzyme Immobilization 354
    10. Microencapsulation in Nylon Beads 355
    11. Entrapment in Polyacrylamide 355
    12. Covalent Immobilization on Glass Surfaces 356
    13. Covalent Immobilization on Controlled-Pore Glass (CPG) 358
    14. Covalent Immobilization to Polyamide 360
    15. O-Alkylation with Triethyloxonium Tetrafluoroborate 361
    16. Immobilization to Amino Groups after Partial Hydrolysis of Polyamide 363
    17. Immobilization to Carboxyl Groups After Partial Hydrolysis of Polyamide 364
    18. Immobilization to Polyester 365
    19. Immobilization by Alkaline Hydrolysis and Activation with Tosylchloride 367
    20. Alkaline Hydrolysis and Activation by Carbonyldiimidazol 368
    21. Analysis of Immobilized Enzymes 368
    22. General Principles 368
    23. Continuous Photometric Assays for Immobilized Enzymes 369
    24. Cofactors in Reactions with Immobilized Enzymes 371
    25. Enzyme Reactors 372
    26. Batch Reactor (Stirred-Tank Reactor) 373
    27. Membrane Reactor 373
    28. Solid-Bed Reactor 374
    29. Immobilized Cells 375
    30. Biosensors 375
    31. Enzyme Electrodes 375
    32. Immunoelectrodes 379
    33. Other Biosensors 379
    34. Immobilized Enzymes in Therapy 381
    35. Index 383
Author Biography

Hans Bisswanger was Professor at the Interfaculty Institute of Biochemistry at the University of Tübingen (Germany), where he has developed and taught for many years an intensive course on enzyme kinetics, enzyme technology and ligand binding. His scientific interest lies with structural and regulatory mechanisms of multi-enzyme complexes, thermophilic enzymes and the technical application of immobilized enzymes. He is the author of several well-known books on enzymology that have appeared in different languages and editions.

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