Practical Enzymology

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.

• Preface to the Third Edition xvList of Abbreviations and Symbols xvii1 General Aspects of Enzyme Analysis 11.1 Introduction and Essentials for Enzyme Assays 1Standard Books, Series 4Databases 41.2 Theoretical Basis of Enzyme Assays 41.2.1 Order of Enzyme Reactions 41.2.2 Importance of the Reaction Order for Enzyme Reactions 61.2.3 The Reaction Velocity, Significance, and Practical Aspects 101.2.3.1 Determination of the Reaction Velocity, the Progress Curve 101.2.3.2 Enzyme Units 151.2.3.3 A Short Discussion About Errors in Enzyme Assays 171.2.3.4 Practical Rules for the Preparation of Dilution Series 211.2.3.5 Statistical Treatment of Enzyme Reactions 231.2.4 Treatment of the Michaelis–Menten Equation 241.2.4.1 General Considerations 241.2.4.2 Linear Representations of the Michaelis–Menten Equation 261.2.5 Enzyme Inhibition 281.2.6 Multisubstrate Reactions 331.3 Essential Conditions for Enzyme Assays 351.3.1 Dependence on Solvents and Ionic Strength 351.3.2 pH Dependency 361.3.2.1 Isoelectric Point 381.3.2.2 Buffers: What Must Be Regarded? 381.3.2.3 How to Prepare Buffers? 411.3.3 Temperature Dependency 421.3.4 Stability of Enzymes 471.3.4.1 Why Are Enzymes Unstable? 471.3.4.2 How Can Enzymes Be Stabilized? 481.3.4.3 How to Store Enzymes? 481.4 Theory of Coupled Enzyme Reactions 511.4.1 Two Coupled Reactions 511.4.2 Three Coupled Reactions 541.5 Substrate Determination 541.5.1 End Point Method 551.5.2 Substrate Determination by Coupled Enzyme Reactions 561.5.3 Kinetic Method for Substrate Determination 571.5.4 Enzymatic Cycling 572 Instrumental Aspects 612.1 Spectroscopic Methods 612.1.1 Absorption (UV/Vis) Photometry 612.1.2 Cuvettes 722.1.2.1 Shape 722.1.2.2 Material 732.1.2.3 Cleaning 732.1.3 Turbidity Measurement 742.1.4 Fluorescence Photometry 752.1.5 Luminometry 792.1.6 Polarimetry 802.2 Electrochemical Methods 812.2.1 pH Meter and Glass Electrodes 812.2.2 pH Stat 822.2.3 Potentiometry 832.2.4 Oxygen and Carbon Dioxide Electrodes 832.3 Radioactive Labeling 842.4 Diverse Methods 843 Enzyme Assays 873.1 Enzyme Nomenclature 883.2 Practical Considerations for Enzyme Assays 904 Oxidoreductases, EC 1 954.1 General Assay Procedures 954.1.1 Optical Assay 954.1.2 Fluorimetric Assay 964.2 Alcohol Dehydrogenase (ADH), EC 1.1.1.1 964.2.1 Reduction Assay 964.2.2 Oxidation Assay 974.3 Alcohol Dehydrogenase (NADP+), EC 1.1.1.2 984.4 Homoserine Dehydrogenase, EC 1.1.1.3 994.5 Shikimate Dehydrogenase, EC 1.1.1.25 1004.6 l-Lactate Dehydrogenase (LDH), EC 1.1.1.27 1014.6.1 Photometric Reduction Assay 1014.6.2 Fluorimetric Reduction Assay 1024.6.3 Oxidation Assay 1034.7 Malate Dehydrogenase (MDH), EC 1.1.1.37 1044.8 Malate Dehydrogenase (Oxaloacetate-Decarboxylating) (NAD+), EC 1.1.1.38, and Malate Dehydrogenase (Decarboxylating), EC 1.1.1.39 1054.9 Malate Dehydrogenase (Oxaloacetate-decarboxylating) (NADP+), EC 1.1.1.40 1064.10 Isocitrate Dehydrogenase (NAD+) (ICDH), EC 1.1.1.41 1074.11 Isocitrate Dehydrogenase (NADP+) (ICDH), EC 1.1.1.42 1084.12 Glucose-6-Phosphate Dehydrogenase (NADP+), EC 1.1.1.49 (G6PDH) 1094.13 Glucose Oxidase (GOD), EC 1.1.3.4 1104.14 Formate Dehydrogenase (FDH), EC 1.2.1.2 1114.15 Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH), EC 1.2.1.12 1124.15.1 Oxidation Assay 1124.15.2 Reduction Assay Coupled with 3-Phosphoglycerate Kinase (PGK) 1134.16 Long-Chain-Aldehyde Dehydrogenase, EC 1.2.1.48 1144.17 Pyruvate Dehydrogenase (Acetyl-transferring) (PDH), EC 1.2.4.1 1164.17.1 Ferricyanide as Electron Acceptor 1164.17.2 Dichlorophenolindophenol as Electron Acceptor 1174.18 Aldehyde Oxidase, EC 1.2.3.1 1184.19 Oxoglutarate Dehydrogenase (Succinyl-transferring) (OGDH), EC 1.2.4.2 1194.20 Pyruvate Ferredoxin Oxidoreductase, EC 1.2.7.1 1204.20.1 Assay with Cytochrome c (cyt c) as Electron Acceptor 1214.21 Alanine Dehydrogenase, EC 1.4.1.1 1214.21.1 Oxidation of Alanine 1224.21.2 Reduction of Pyruvate 1224.22 Glutamate Dehydrogenase, EC 1.4.1.3 1234.23 Leucine Dehydrogenase, EC 1.4.1.9 1244.24 l-Amino-Acid Oxidase, EC 1.4.3.2 1254.25 d-Amino-Acid Oxidase, EC 1.4.3.3 1264.26 Monoamine Oxidase, EC 1.4.3.4 1264.27 Primary Amine Oxidase, EC 1.4.3.21 1274.27.1 Spectrophotometric Assay 1284.27.2 Polarographic Assay of O2 Uptake with O2 Electrode 1284.27.3 Assays for Benzylamine Oxidase Activity 1294.28 Diamine Oxidase, EC 1.4.3.22 1294.29 NADH:Ubiquinone Reductase (H+-Translocating) EC 1.6.5.3 1304.29.1 Spectrophotometric Assay 1314.30 NADH Dehydrogenase, EC 1.6.99.3 1314.31 Factor-Independent Urate Hydroxylase, EC 1.7.3.3 1324.32 Dihydrolipoyl Dehydrogenase, EC 1.8.1.4 1334.32.1 Oxidation of Dihydrolipoamide 1344.32.2 Reduction of Lipoamide 1344.33 Glutathione Disulfide Reductase, EC 1.8.1.7 1354.34 Cytochrome-c Oxidase (COX), EC 1.9.3.1 1374.34.1 Spectrophotometric Assay 1374.34.2 Assay with Oxygen Electrode 1374.35 Catalase, EC 1.11.1.6 1384.36 Peroxidase (POD) EC 1.11.1.7 1394.36.1 Assay with 2,2′-Azino-bis-3-Ethylbenzothiazoline-6-Sulfonic Acid (ABTS) 1404.36.2 Assay with Guaiacol 1404.36.3 Assay with Dianisidine 1414.37 Glutathione Peroxidase, EC 1.11.1.9 1424.37.1 Coupled Assay with Glutathione Reductase 1424.38 Photinus luciferin 4-Monooxigenase (ATP-Hydrolyzing), EC 1.13.12.7 1434.39 Alkylglycerol Monooxygenase, EC 1.14.16.5 1454.39.1 Spectroscopic Assay 1454.39.2 Coupled Assay with HPLC Detection 1454.40 Dopamine ß-Monooxygenase, EC 1.14.17.1 1474.41 Tyrosinase, EC 1.14.18.1 1484.41.1 Dopa Oxidase Assay 1484.41.2 Dopachrome Assay 1494.42 Superoxide Dismutase (SOD), EC 1.15.1.1 1494.42.1 Assay with Pyrogallol 1504.42.2 Assay with Ferricytochrome c and Xanthine Oxidase 1504.43 Xanthine Oxidase (XOD), EC 1.17.3.2 1515 Transferases, EC 2 1535.1 Ornithine Carbamoyltransferase (OTC), EC 2.1.3.3 1535.1.1 Method 1 for Color Development 1545.1.2 Method 2 for Color Development 1545.2 Choline O-Acetyltransferase, EC 2.3.1.6 1555.3 Carnitine O-acetyltransferase, EC 2.3.1.7 1565.3.1 Direct Spectroscopic Assay 1565.3.2 Assay with DTNB 1575.4 Dihydrolipoamide Acetyltransferase, EC 2.3.1.12 1575.4.1 Spectrophotometric Assay 1585.4.2 Stopped Assay 1595.5 Fatty Acid Synthase, EC 2.3.1.85 1605.6 ;;-Glutamyltransferase, EC 2.3.2.2 1615.7 Citrate Synthases, EC 2.3.3.1, EC 2.3.3.3, and EC 2.3.3.16 1625.8 ATP Citrate Lyase, EC 2.3.3.8 1645.9 Glycogen Phosphorylase, EC 2.4.1.1 1655.10 Purine-nucleoside Phosphorylase (PNP), EC 2.4.2.1 1665.11 Glutathione Transferase, EC 2.5.1.18 1675.11.1 Spectrophotometric Assay 1685.11.2 Titrimetric Assay 1685.12 Aspartate Transaminase (AAT), EC 2.6.1.1 1695.13 Alanine Transaminase, EC 2.6.1.2 1705.14 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 1715.14.1 Tyrosine Transaminase 1715.14.2 Tryptophan Transaminase 1715.14.3 Phenylalanine (Histidine) Transaminase: 1715.15 Hexokinase (HK), EC 2.7.1.1, Glucokinase (GK), EC 2.7.1.2 1735.16 Pyruvate Kinase (PK), EC 2.7.1.40 1745.17 Acetate Kinase, EC 2.7.2.1 1765.18 Phosphoglycerate Kinase (PGK), EC 2.7.2.3 1775.19 Aspartate Kinase (AK), EC 2.7.2.4 1785.20 Creatine Kinase (CK), EC 2.7.3.2 1805.20.1 Coupled Assay 1805.20.2 pH-Colorimetric Assay 1816 Hydrolases, EC 3 1836.1 Triacylglycerol Lipase, EC 3.1.1.3 1836.1.1 Assay with pH Stat (Auto-titrator) 1836.1.2 Fluorimetric Assay 1846.2 Phospholipase A2, EC 3.1.1.4 1856.3 Acetylcholinesterase (AChE), EC 3.1.1.7 1866.4 Cholinesterase (ButChE), EC 3.1.1.8 1876.4.1 pH Stat Assay 1876.4.2 Colorimetric Assay 1886.5 Hydroxyacylglutathione Hydrolase, EC 3.1.2.6 1896.5.1 Direct Assay 1896.5.2 Assay with DTNB 1896.6 S-Formylglutathione Hydrolase, EC 3.1.2.12 1906.7 Alkaline Phosphatase, EC 3.1.3.1 1916.7.1 Mammalian Alkaline Phosphatase 1916.7.2 Bacterial Alkaline Phosphatase 1926.8 Acid Phosphatase, EC 3.1.3.2 1926.9 5′-Nucleotidase, EC 3.1.3.5 1936.9.1 Assay by Determination of Pi 1946.9.2 Assay by Converting Adenosine into Inosine 1946.10 Glucose-6-Phosphatase, EC 3.1.3.9 1956.11 3′,5′-Cyclic-Nucleotide Phosphodiesterase, EC 3.1.4.17 1966.12 Steryl-Sulfatase, EC 3.1.6.2 1986.13 Pancreatic Ribonuclease, EC 3.1.27.5 1986.14 α-Amylase, EC 3.2.1.1 1996.15 Glucan 1,4-α-Glucosidase (AMG), EC 3.2.1.3 2016.15.1 Coupled Assay with HK and G6PDH 2016.15.2 Photometric Assay with 4-Nitrophenyl-d-Glucose 2026.15.3 Fluorimetric Assay with 4-Methylumbelliferyl-α-d-Glucoside 2026.16 Cellulases 2036.16.1 β-1,4-Glucanase, EC 3.2.1.4 2036.16.2 β-Glucosidase, EC 3.2.1.21 2036.16.3 Orcinol Assay 2046.16.4 Activity Staining 2046.17 Lysozym, EC 3.2.1.17 2066.18 Sialidase, EC 3.2.1.18 2076.18.1 Fluorimetric Assay 2076.18.2 Activity Staining 2086.19 α-Glucosidase, EC 3.2.1.20 2086.19.1 Coupled Assay 2086.19.1.1 α-Glucosidase Reaction 2096.19.1.2 Glucose Determination 2096.19.2 Assay with 4-Nitrophenylglucopyranoside 2106.20 β-Galactosidase, EC 3.2.1.23 2116.21 α-Mannosidase, EC 3.2.1.24 2126.21.1 Photometric Microassay 2126.21.2 Fluorimetric Assay 2136.22 β-Fructofuranosidase, EC 3.2.1.26 2136.23 β-Glucuronidase, EC 3.2.1.31 2146.23.1 Fluorimetric Assay 2156.24 β-N-Acetylhexosaminidase, EC 3.2.1.52 2156.25 Proteases, EC 3.4, General Assays 2166.25.1 Anson Assay 2166.25.2 Casein Assay 2186.25.3 Azocasein Assay 2196.25.4 Ninhydrin Assay 2206.26 Leucyl Aminopeptidase (LAP), EC 3.4.11.1, Bacterial Leucyl Aminopeptidase, EC 3.4.11.10 2216.26.1 Assay with Leucineamide 2226.26.2 Assay with Leucine-p-nitroanilide 2226.27 Peptidyl-dipeptidase A, EC 3.4.15.1 2236.28 α-Chymotrypsin, EC 3.4.21.1 2246.28.1 Assay with SUPHEPA 2246.28.2 Assay with GLUPHEPA 2256.29 Trypsin, EC 3.4.21.4 2266.30 Pancreatic Elastase, EC 3.4.21.35 2276.30.1 Assay with Succinyl-Ala–Ala–Ala–p-Nitroanilide 2276.30.2 Esterase Activity of Elastase 2276.31 Cathepsin B, EC 3.4.22.1 2286.32 Pepsin A, EC 3.4.23.1 2296.33 Asparaginase, EC 3.5.1.1 2306.34 Glutaminase, EC 3.5.1.2 2326.34.1 Determination of Ammonia with Nessler’s Reagent 2326.34.2 pH Stat Assay 2336.35 Urease, EC 3.5.1.5 2336.35.1 pH Stat Assay 2346.35.2 Photometric Assay 2346.36 Guanine Deaminase, EC 3.5.4.3 2356.36.1 Determination of Ammonia 2366.37 Adenosinetriphosphatase, EC 3.6.1.3 2376.38 Mg2+ Importing ATPase, EC 3.6.3.2, Na+/K+-Exchanging ATPase, EC 3.6.3.9 2386.38.1 Assay of Total ATPase Activity 2386.38.2 Assay of Mg2+-ATPase Activity 2397 Lyases, EC 4 2417.1 Pyruvate Decarboxylase (PDC), EC 4.1.1.1 2417.2 Glutamate Decarboxylase (GAD), EC 4.1.1.15 2427.3 Fructose-bisphosphate Aldolase, EC 4.1.2.13 2447.4 Anthranilate Synthase, EC 4.1.3.27 2457.5 Carbonic Anhydrase (CA), EC 4.2.1.1 2467.5.1 pH Stat Assay 2467.5.2 Esterase Assay with 4-Nitrophenylacetate 2477.6 Fumarate Hydratase, EC 4.2.1.2 2487.7 Lactoylglutathione Lyase, EC 4.4.1.5 2497.8 Adenylate Cyclase (AC), EC 4.6.1.1 2508 Isomerases, EC 5 2538.1 Xylose Isomerase, EC 5.3.1.5 2538.1.1 D-Xylose Isomerase Assay 2538.1.2 D-Xylose Isomerase Microplate Assay 2548.1.3 D-Glucose Isomerase Assay 2558.1.4 D-Glucose Isomerase Microplate Assay 2568.2 Glucose-6-phosphate Isomerase (G6PI), EC 5.3.1.9 2568.3 Phosphoglucomutase (PGM), EC 5.4.2.2 2579 Ligases (Synthetases), EC 6 2619.1 Tyrosine-tRNA Ligase, EC 6.1.1.1 2619.1.1 Fluorimetric Assay 2619.1.2 ATP–32PP Exchange 2629.2 Acetate-CoA Ligase (ACL), EC 6.2.1.1 2639.2.1 Direct Radioactive Assay 2649.2.2 Coupled Spectroscopic Assay 2649.3 Glutamine Synthetase, EC 6.3.1.2 26610 Assays for Multi-enzyme Complexes 26910.1 Pyruvate Dehydrogenase Complex (PDHC) 26910.1.1 Overall Activity of PDHC by NAD+ Reduction 27010.1.2 Overall Activity of PDHC by Dismutation Assay 27010.2 α-Oxoglutarate Dehydrogenase Complex (OGDHC) 27210.2.1 Overall Activity by NAD+ Reduction 27311 Assays for Other Enzyme Relevant Parameters 27511.1 Substrate Determination 27511.1.1 Determination of NADP(H) by Enzymatic Cycling 27511.1.1.1 Cycling Reaction 27611.1.2 Determination of NAD(H) by Enzymatic Cycling 27711.2 Protein Determination 27911.2.1 Biuret Assay 27911.2.2 BCA Assay 28111.2.2.1 Assay for Soluble Proteins 28111.2.2.2 Modification for Immobilized Proteins 28211.2.3 Lowry Assay 28211.2.4 Coomassie Binding Assay (Bradford Assay) 28311.2.4.1 Assay for Soluble Proteins 28411.2.4.2 Modification for Immobilized Proteins 28411.2.5 Absorption Method 28511.2.6 Fluorimetric Assay 28711.2.7 Ninhydrin Assay 28811.2.7.1 Ninhydrin Assay with Hydrolysis 28811.2.7.2 Modified Ninhydrin AssayWithout Hydrolysis 28911.2.8 Protein Assay with 2-Hydroxy-1-naphthaldehyde 290 General Literature for Protein Assays 29111.3 Phosphate Determination 29111.4 Determination of Metal Ions 29311.4.1 Calcium and Magnesium 29311.4.2 Iron 29411.4.2.1 Determination with Ferrozine 29511.4.2.2 Determination of FeII with 1,10-Phenanthroline in the Presence of FeIII 29511.4.3 Copper 29611.4.3.1 Biquinoline Method 29611.4.3.2 Oxalyldihydrazide Method 29711.4.4 Manganese 29711.4.4.1 Colorimetric Assay 29811.4.4.2 Assay with 1-(2-Pyridylazo)-2-naphthol (PAN) 29811.4.5 Zinc 29911.5 Glycoprotein Assays 30011.5.1 Identification in Electrophoresis Gels 30011.5.2 Quantitative Analysis of Protein-Bound Hexoses 30011.6 Cross-linking of Proteins with Dimethylsuberimidate 30111.7 Concentrating Enzyme Solutions 30211.7.1 Precipitation 30311.7.2 Ultrafiltration and Dialysis 30611.7.3 Ultracentrifugation 30711.7.4 Lyophilization 30711.7.5 Other Concentration Methods 30712 Enzyme Immunoassays 30912.1 Radioimmunoassays 30912.2 Principle of Enzyme Immunoassays 30912.3 Noncompetitive Solid-Phase Enzyme Immunoassay 31112.4 Competitive Solid-Phase Enzyme Immunoassay 31212.5 Methods for Enzyme Immunoassays and Immobilization Techniques 31212.5.1 Protein Coupling to Cyanogen Bromide Activated Agarose 31212.5.2 Coupling of Diaminohexyl Spacer 31312.5.3 Periodate Activation of Cellulose 31412.5.4 Introduction of Thiol Groups into Proteins (Antibodies) 31512.5.5 Conjugation of a Protein (Antibody) with an Enzyme (Peroxidase) 31612.5.6 Conjugation of ß-Galactosidase to Proteins (Antibodies) by MBS 31612.5.7 Conjugation of Alkaline Phosphatase to Antibodies by Glutaraldehyde 31713 Binding Measurements 31913.1 Different Types of Binding 31913.1.1 General Considerations 31913.1.2 How Can Specific Reversible Binding be Identified? 32013.1.3 Experimental Aspects 32213.2 Binding Measurements by Size Discrimination 32513.2.1 Equilibrium Dialysis 32513.2.1.1 Binding of Indole to Bovine Serum Albumin 32713.2.2 Evaluation of Binding Experiments 32913.2.3 Ultrafiltration 33013.2.4 Gel Filtration 33113.2.5 Ultracentrifugation 33213.3 Spectroscopic Methods 33313.3.1 Difference Spectroscopy 33413.3.1.1 Difference Spectroscopic Titration of Ligands Binding to Catalase 33613.3.1.2 Evaluation of Spectroscopic Binding Curves 33913.3.2 Fluorescence Spectroscopy 34113.3.2.1 Binding of ANS to Bovine Serum Albumin 34113.4 Other Binding Methods 34413.4.1 Radioactive Labeling 34413.4.2 Surface Plasmon Resonance (SPR) 34514 Enzymes in Technical Applications 34714.1 Modes of Enzyme Immobilization 34714.1.1 Adsorption 34814.1.2 Entrapment 35014.1.3 Encapsulation 35014.1.4 Cross-linking 35114.1.5 Covalent Immobilization to Solid Supports 35114.1.5.1 Supports 35114.1.5.2 Spacer 35314.2 Methods for Enzyme Immobilization 35414.2.1 Microencapsulation in Nylon Beads 35514.2.2 Entrapment in Polyacrylamide 35514.2.3 Covalent Immobilization on Glass Surfaces 35614.2.4 Covalent Immobilization on Controlled-Pore Glass (CPG) 35814.2.5 Covalent Immobilization to Polyamide 36014.2.5.1 O-Alkylation with Triethyloxonium Tetrafluoroborate 36114.2.5.2 Immobilization to Amino Groups after Partial Hydrolysis of Polyamide 36314.2.5.3 Immobilization to Carboxyl Groups After Partial Hydrolysis of Polyamide 36414.2.6 Immobilization to Polyester 36514.2.7 Immobilization by Alkaline Hydrolysis and Activation with Tosylchloride 36714.2.8 Alkaline Hydrolysis and Activation by Carbonyldiimidazol 36814.3 Analysis of Immobilized Enzymes 36814.3.1 General Principles 36814.3.2 Continuous Photometric Assays for Immobilized Enzymes 36914.3.3 Cofactors in Reactions with Immobilized Enzymes 37114.4 Enzyme Reactors 37214.4.1 Batch Reactor (Stirred-Tank Reactor) 37314.4.2 Membrane Reactor 37314.4.3 Solid-Bed Reactor 37414.4.4 Immobilized Cells 37514.5 Biosensors 37514.5.1 Enzyme Electrodes 37514.5.2 Immunoelectrodes 37914.5.3 Other Biosensors 37914.6 Immobilized Enzymes in Therapy 381Index 383