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Practical Enzymology, 3rd Edition

Hans Bisswanger

ISBN: 978-3-527-34604-2 August 2019 416 Pages

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€79.10

Description

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.

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