Practical Enzymology

Transcription

1 Practical Enzymology Bearbeitet von Hans Bisswanger 2., vollst. überarb. Aufl Taschenbuch. XVI, 360 S. Paperback ISBN Format (B x L): 17 x 24 cm Gewicht: 720 g Weitere Fachgebiete > Chemie, Biowissenschaften, Agrarwissenschaften > Biochemie > Enzymologie Zu Leseprobe schnell und portofrei erhältlich bei Die Online-Fachbuchhandlung beck-shop.de ist spezialisiert auf Fachbücher, insbesondere Recht, Steuern und Wirtschaft. Im Sortiment finden Sie alle Medien (Bücher, Zeitschriften, CDs, ebooks, etc.) aller Verlage. Ergänzt wird das Programm durch Services wie Neuerscheinungsdienst oder Zusammenstellungen von Büchern zu Sonderpreisen. Der Shop führt mehr als 8 Millionen Produkte.

2 V Preface to the Second Edition Note to the Reader XIV Abbreviations XV XIII 1 Introduction 1 Standard Books, Series, and Databases 3 2 General Aspects of Enzyme Analysis Basic Requirements for Enzyme Assays What Must Be Observed for an Enzyme Assay? Order of Reactions Significance of the Reaction Order for Enzyme Reactions Determination of the Velocity of Enzyme Reactions Enzyme Units Progress Curves Difficulties in Determination of Initial Velocities A Short Discussion on Error Preparation of Dilution Series Considerations about Statistical Treatments Michaelis Menten Equation General Considerations Linear Representations of the Michaelis Menten Equation Enzyme Inhibition Multisubstrate Reactions Essential Conditions for Enzyme Assays Dependence on Solvents and Ionic Strength ph Dependency Isoelectric Point Buffers: What Must Be Regarded? How to Prepare Buffers? Temperature Dependency 49

3 VI Why Are Enzymes Unstable? How Can Enzymes Be Stabilized? How to Store Enzymes? Instrumental Aspects Spectroscopic Methods Absorption (UV/Vis) Photometry Cuvettes Turbidity Measurements Fluorescence Photometry Luminometry Polarimetry Electrochemical Methods ph Meter and Glass Electrodes ph Stat Potentiometry Oxygen and Carbon Dioxide Electrodes Radioactive Labeling Diverse Methods Theory of Coupled Enzyme Reactions Two Coupled Reactions Three Coupled Reactions Substrate Determination End Point Method Substrate Determination by Coupled Enzyme Reactions Kinetic Method for Substrate Determination Enzymatic Cycling 90 3 Enzyme Assays Enzyme Nomenclature Practical Considerations for Enzyme Assays Special Enzyme Assays Oxidoreductases, EC Optical Assay Fluorimetric Assay Alcohol Dehydrogenase, EC A. Reduction Assay 101 B. Oxidation Assay Alcohol Dehydrogenase (NADP + ), EC Homoserine Dehydrogenase, EC Shikimate Dehydrogenase, EC L-Lactate Dehydrogenase, EC A. Spectrophotometric Reduction Assay 106 B. Fluorimetric Reduction Assay 107 C. Oxidation Assay Malate Dehydrogenase, EC

4 VII Malate Dehydrogenase (Oxaloacetate-Decarboxylating) (NAD + ), EC , and Malate Dehydrogenase (Decarboxylating), EC Malate Dehydrogenase (Oxaloacetate-Decarboxylating) (NADP + ), EC Isocitrate Dehydrogenase (NAD + ), EC Isocitrate Dehydrogenase (NADP + ), EC Glucose-6-phosphate Dehydrogenase, EC Glucose Oxidase, EC Formate Dehydrogenase, EC Glyceraldehyde-3-phosphate Dehydrogenase, EC A. Oxidation Assay 117 B. Reduction Assay Coupled with 3-Phosphoglycerate Kinase (PGK) Pyruvate Dehydrogenase (Acetyl-Transferring), EC A. Ferricyanide as Electron Acceptor 120 B. Dichlorophenolindophenol as Electron Acceptor Oxoglutarate Dehydrogenase (Succinyl-Transferring), EC Pyruvate Ferredoxin Oxidoreductase, EC Assay with Cytochrome c as Electron Acceptor Alanine Dehydrogenase, EC A. Oxidation of Alanine 124 B. Reduction of Pyruvate Glutamate Dehydrogenase, EC Leucine Dehydrogenase, EC L-Amino Acid Oxidase, EC D-Amino Acid Oxidase, EC Monoamine Oxidase, EC Primary Amine Oxidase, EC A. Spectrophotometric Assay 131 B.PolarographicAssayofO 2 uptake with O 2 Electrode 131 C. Assays for Benzylamine Oxidase Activity Diamine Oxidase, EC Urate Oxidase, EC Dihydrolipoamide Dehydrogenase, EC A. Oxidation of Dihydrolipoamide 135 B. Reduction of Lipoamide Glutathione Disulfide Reductase, EC Catalase, EC Peroxidase, EC A. Assays with 2,2 -Azino-bis-3-ethylbenzothiazoline-6-sulfonic Acid (ABTS) 139 B. Assay with Guaiacol 140 C. Assay with Dianisidine Luciferase, EC

5 VIII Transferases, EC Dihydrolipoamide Acetyltransferase, EC A. Spectrophotometric Assay 143 B. Stopped Assay Fatty Acid Synthase, EC Phosphorylase a, EC Aspartate Transaminase, EC Alanine Transaminase, EC Tyrosine Transaminase, EC , Tryptophan Transaminase, EC , Phenylalanine Transaminase, EC Hexokinase, EC Pyruvate Kinase, EC Acetate Kinase, EC Phosphoglycerate Kinase, EC Aspartokinase, EC Hydrolases, EC Lipase, EC A. Assay with ph-stat (Auto titrator) 161 B. Fluorimetric Assay Phospholipase A 2, EC Acetylcholine-Esterase, EC Choline Esterase, EC A. ph-stat Assay 165 B. Colorimetric Assay S-Formylglutathione Hydrolase, EC Alkaline Phosphatase, EC A. Mammalian Alkaline Phosphatase 167 B. Bacterial Alkaline Phosphatase Acid Phosphatase, EC Ribonuclease (Pancreatic), EC α-amylase, EC Amyloglucosidase, EC A. Coupled Assay with HK and G6PDH 172 B. Photometric Assay with 4-Nitrophenyl-D-Glucose 172 C. Fluorimetric Assay with 4-Methylumbelliferyl-α-D-Glucoside Cellulases, β-1,4-glucanase, EC , and β-glucosidase, EC A. Orcinol Assay 174 B. Activity Staining Lysozyme, EC Sialidase, EC A. Fluorimetric Assay 178 B. Activity Staining α-glucosidase, EC A. α-glucosidase Assay 180

6 IX B. Glucose Determination 180 C. Assay with 4-Nitrophenylglucopyranoside β-galactosidase, EC β-fructosidase, EC β-glucuronidase EC A. Fluorimeteric Assay Proteases, EC 3.4, General Assays 185 A. Anson Assay 185 B. Casein-Assay 187 C. Azocasein Assay 188 D. Ninhydrin Assay Leucine Aminopeptidase, EC A. Assay with Leucineamide 191 B. Assay with Leucine-p-nitroanilide α-chymotrypsin, EC A. Assay with SUPHEPA 192 B. Assay with GLUPHEPA Pancreatic Elastase, EC (Previous EC ) 194 A. Assay with Succinyl-Ala-Ala-Ala-p-Nitronilide 194 B. Esterase Activity of Elastase Pepsin, EC Trypsin, EC Asparaginase, EC Glutaminase, EC A. Determination of Ammonia with Nessler s Reagent 199 B. ph-stat Assay Urease, EC A. ph Stat Assay 201 B. Photometric Assay Adenosinetriphosphatase, EC Lyases, EC Pyruvate Decarboxylase, EC Glutamate Decarboxylase, EC Aldolase, EC Anthranilate Synthase, EC Carbonic Anhydrase, EC A. ph-stat Assay 208 B. Esterase Assay with 4-Nitrophenylacetate Fumarase, EC Isomerases, EC Glucose/Xylose Isomerase EC A. D-Xylose Isomerase Assay 211 B. D-Xylose Isomerase Microplate Assay 212 C. D-Glucose Isomerase Assay 213 D. D-Glucose Isomerase Microplate Assay 214

7 X Phosphoglucomutase, EC Ligases (Synthetases) EC Tyrosine-tRNA Ligase, EC A. Fluorimetric Assay 216 B. ATP 32 PP-Exchange Glutamine Synthetase EC Assays for Multienzyme Complexes Pyruvate Dehydrogenase Complex (PDHC) 220 A. Overall Activity of PDHC by NAD + Reduction 221 B. Overall Activity of PDHC by by Dismutation Assay α-oxoglutarate Dehydrogenase Complex (OGDHC) 224 Overall Activity by NAD + Reduction Substrate Determination Determination of NADP(H) by Enzymatic Cycling Determination of NAD(H) Assays for Enzyme Characterization Protein Determination Biuret Assay BCA Assay 232 A. Assay for Soluble Proteins 232 B. Modification for Immobilized Proteins Lowry Assay Coomassie Binding Assay (Bradford Assay) Absorption Method Fluorimetric Assay Ninhydrin Assay Modified Ninhydrin Assay without Hydrolysis Protein Assay with 2-Hydroxy-1-naphthaldehyde Phosphate Determination Glycoprotein Assays 245 A. Detection in Electrophoresis Gels 245 B. Determination of Protein-Bound Hexoses Cross-Linking of Proteins with Dimethylsuberimidate Concentration of Enzyme Solutions Precipitation Ultrafiltration and Dialysis Ultracentrifugation Lyophilization Other Concentration Methods Enzyme Immunoassays Radioimmunoassays Noncompetitive Solid-Phase Enzyme Immunoassay Competitive Solid-Phase Enzyme Immunoassay Methods for Enzyme Immunoassays and Immobilization Techniques 257

8 XI Protein Coupling to Cyanogen Bromide Activated Agarose Coupling of Diaminohexyl Spacer Periodate Activation of Cellulose Introduction of Thiol Groups into Proteins (Antibodies) Conjugation of a Protein (Antibody) with an Enzyme (Peroxidase) Conjugation of β-galactosidase to Proteins (Antibodies) by MBS Conjugation of Alkaline Phosphatase to Antibodies by Glutaraldehyde Binding Measurements Different Types of Binding General Considerations How to Recognize Specific Reversible Binding? Experimental Aspects Binding Measurements by Size Discrimination Equilibrium Dialysis Binding of Indole to Bovine Serum Albumin Evaluation of Binding Experiments Ultrafiltration Gel Filtration Ultracentrifugation Spectroscopic Methods Difference Spectroscopy Difference Spectroscopic Titration of Ligands Binding to Catalase Evaluation of Spectroscopic Binding Curves Fluorescence Spectroscopy Binding of ANS to Bovine Serum Albumin Other Binding Methods Radioactive Labeling Surface Plasmon Resonance Enzymes in Technical Applications Modes of Enzyme Immobilization Adsorption Entrapment Encapsulation Cross Linking Covalent Immobilization to Solid Supports Supports Spacer Methods for Enzyme Immobilization Microencapsulation in Nylon Beads Entrapment in Polyacrylamide Covalent Immobilization on Glass Surfaces Covalent Immobilization on Controlled-Pore Glass 309

9 XII Covalent Immobilization to Polyamide o-alkylation with Triethyloxonium Tetrafluoroborate Immobilization to Amino Groups after Partial Hydrolysis of Polyamide Immobilization to Carboxyl Groups after Partial Hydrolysis of Polyamide Immobilization to Polyester Immobilization by Alkaline Hydrolysis and Activation with Tosylchloride Alkaline Hydrolysis and Activation by Carbonyldiimidazol Analysis of Immobilized Enzymes General Principles Continuous Photometric Assays for Immobilized Enzymes Cofactors in Reactions with Immobilized Enzymes Enzyme Reactors Batch Reactor (Stirred-Tank Reactor) Membrane Reactor Solid Bed Reactor Immobilized Cells Biosensors Enzyme Electrodes Immunoelectrodes Other Biosensors Immobilized Enzymes in Therapy 335 Appendix 337 List of enzymes according to the EC numbers 337 Index 353