Cover
Preface to the Third Edition
List of Abbreviations and Symbols
1 General Aspects of Enzyme Analysis
1. 1.1 Introduction and Essentials for Enzyme Assays
2. 1.2 Theoretical Basis of Enzyme Assays
3. 1.3 Essential Conditions for Enzyme Assays
4. 1.4 Theory of Coupled Enzyme Reactions
5. 1.5 Substrate Determination
2 Instrumental Aspects
1. 2.1 Spectroscopic Methods
2. 2.2 Electrochemical Methods
3. 2.3 Radioactive Labeling
4. 2.4 Diverse Methods
3 Enzyme Assays
1. 3.1 Enzyme Nomenclature
2. 3.2 Practical Considerations for Enzyme Assays
4 Oxidoreductases, EC 1
1. 4.1 General Assay Procedures
2. 4.2 Alcohol Dehydrogenase (ADH), EC 1.1.1.1
3. 4.3 Alcohol Dehydrogenase (NADP⁺), EC 1.1.1.2
4. 4.4 Homoserine Dehydrogenase, EC 1.1.1.3
5. 4.5 Shikimate Dehydrogenase, EC 1.1.1.25
6. 4.6 L‐Lactate Dehydrogenase (LDH), EC 1.1.1.27
7. 4.7 Malate Dehydrogenase (MDH), EC 1.1.1.37
8. 4.8 Malate Dehydrogenase (Oxaloacetate‐Decarboxylating) (NAD⁺), EC 1.1.1.38, and Malate Dehydrogenase (Decarboxylating), EC 1.1.1.39
9. 4.9 Malate Dehydrogenase (Oxaloacetate‐decarboxylating) (NADP⁺), EC 1.1.1.40
10. 4.10 Isocitrate Dehydrogenase (NAD⁺) (ICDH), EC 1.1.1.41
11. 4.11 Isocitrate Dehydrogenase (NADP⁺) (ICDH), EC 1.1.1.42
12. 4.12 Glucose‐6‐Phosphate Dehydrogenase (NADP⁺), EC 1.1.1.49 (G6PDH)
13. 4.13 Glucose Oxidase (GOD), EC 1.1.3.4
14. 4.14 Formate Dehydrogenase (FDH), EC 1.2.1.2
15. 4.15 Glyceraldehyde‐3‐Phosphate Dehydrogenase (GAPDH), EC 1.2.1.12
16. 4.16 Long‐Chain‐Aldehyde Dehydrogenase, EC 1.2.1.48
17. 4.17 Pyruvate Dehydrogenase (Acetyl‐transferring) (PDH), EC 1.2.4.1
18. 4.18 Aldehyde Oxidase, EC 1.2.3.1
19. 4.19 Oxoglutarate Dehydrogenase (Succinyl‐transferring) (OGDH), EC 1.2.4.2
20. 4.20 Pyruvate Ferredoxin Oxidoreductase, EC 1.2.7.1
21. 4.21 Alanine Dehydrogenase, EC 1.4.1.1
22. 4.22 Glutamate Dehydrogenase, EC 1.4.1.3
23. 4.23 Leucine Dehydrogenase, EC 1.4.1.9
24. 4.24 L‐Amino‐Acid Oxidase, EC 1.4.3.2
25. 4.25 D‐Amino‐Acid Oxidase, EC 1.4.3.3
26. 4.26 Monoamine Oxidase, EC 1.4.3.4
27. 4.27 Primary Amine Oxidase, EC 1.4.3.21
28. 4.28 Diamine Oxidase, EC 1.4.3.22
29. 4.29 NADH:Ubiquinone Reductase (H⁺‐Translocating) EC 1.6.5.3
30. 4.30 NADH Dehydrogenase, EC 1.6.99.3
31. 4.31 Factor‐Independent Urate Hydroxylase, EC 1.7.3.3
32. 4.32 Dihydrolipoyl Dehydrogenase, EC 1.8.1.4
33. 4.33 Glutathione Disulfide Reductase, EC 1.8.1.7
34. 4.34 Cytochrome‐c Oxidase (COX), EC 1.9.3.1
35. 4.35 Catalase, EC 1.11.1.6
36. 4.36 Peroxidase (POD) EC 1.11.1.7
37. 4.37 Glutathione Peroxidase, EC 1.11.1.9
38. 4.38 Photinus luciferin 4‐Monooxigenase (ATP‐Hydrolyzing), EC 1.13.12.7
39. 4.39 Alkylglycerol Monooxygenase, EC 1.14.16.5
40. 4.40 Dopamine ß‐Monooxygenase, EC 1.14.17.1
41. 4.41 Tyrosinase, EC 1.14.18.1
42. 4.42 Superoxide Dismutase (SOD), EC 1.15.1.1
43. 4.43 Xanthine Oxidase (XOD), EC 1.17.3.2
5 Transferases, EC 2
1. 5.1 Ornithine Carbamoyltransferase (OTC), EC 2.1.3.3
2. 5.2 Choline O‐Acetyltransferase, EC 2.3.1.6
3. 5.3 Carnitine O‐acetyltransferase, EC 2.3.1.7
4. 5.4 Dihydrolipoamide Acetyltransferase, EC 2.3.1.12
5. 5.5 Fatty Acid Synthase, EC 2.3.1.85
6. 5.6 γ‐Glutamyltransferase, EC 2.3.2.2
7. 5.7 Citrate Synthases, EC 2.3.3.1, EC 2.3.3.3, and EC 2.3.3.16
8. 5.8 ATP Citrate Lyase, EC 2.3.3.8
9. 5.9 Glycogen Phosphorylase, EC 2.4.1.1
10. 5.10 Purine‐nucleoside Phosphorylase (PNP), EC 2.4.2.1
11. 5.11 Glutathione Transferase, EC 2.5.1.18
12. 5.12 Aspartate Transaminase (AAT), EC 2.6.1.1
13. 5.13 Alanine Transaminase, EC 2.6.1.2
14. 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
15. 5.15 Hexokinase (HK), EC 2.7.1.1, Glucokinase (GK), EC 2.7.1.2
16. 5.16 Pyruvate Kinase (PK), EC 2.7.1.40
17. 5.17 Acetate Kinase, EC 2.7.2.1
18. 5.18 Phosphoglycerate Kinase (PGK), EC 2.7.2.3
19. 5.19 Aspartate Kinase (AK), EC 2.7.2.4
20. 5.20 Creatine Kinase (CK), EC 2.7.3.2
6 Hydrolases, EC 3
1. 6.1 Triacylglycerol Lipase, EC 3.1.1.3
2. 6.2 Phospholipase A₂, EC 3.1.1.4
3. 6.3 Acetylcholinesterase (AChE), EC 3.1.1.7
4. 6.4 Cholinesterase (ButChE), EC 3.1.1.8
5. 6.5 Hydroxyacylglutathione Hydrolase, EC 3.1.2.6
6. 6.6 S‐Formylglutathione Hydrolase, EC 3.1.2.12
7. 6.7 Alkaline Phosphatase, EC 3.1.3.1
8. 6.8 Acid Phosphatase, EC 3.1.3.2
9. 6.9 5′‐Nucleotidase, EC 3.1.3.5
10. 6.10 Glucose‐6‐Phosphatase, EC 3.1.3.9
11. 6.11 3′,5′‐Cyclic‐Nucleotide Phosphodiesterase, EC 3.1.4.17
12. 6.12 Steryl‐Sulfatase, EC 3.1.6.2
13. 6.13 Pancreatic Ribonuclease, EC 3.1.27.5
14. 6.14 α‐Amylase, EC 3.2.1.1
15. 6.15 Glucan 1,4‐α‐Glucosidase (AMG), EC 3.2.1.3
16. 6.16 Cellulases
17. 6.17 Lysozym, EC 3.2.1.17
18. 6.18 Sialidase, EC 3.2.1.18
19. 6.19 α‐Glucosidase, EC 3.2.1.20
20. 6.20 β‐Galactosidase, EC 3.2.1.23
21. 6.21 α‐Mannosidase, EC 3.2.1.24
22. 6.22 β‐Fructofuranosidase, EC 3.2.1.26
23. 6.23 β‐Glucuronidase, EC 3.2.1.31
24. 6.24 β‐N‐Acetylhexosaminidase, EC 3.2.1.52
25. 6.25 Proteases, EC 3.4, General Assays
26. 6.26 Leucyl Aminopeptidase (LAP), EC 3.4.11.1, Bacterial Leucyl Aminopeptidase, EC 3.4.11.10
27. 6.27 Peptidyl‐dipeptidase A, EC 3.4.15.1
28. 6.28 α‐Chymotrypsin, EC 3.4.21.1
29. 6.29 Trypsin, EC 3.4.21.4
30. 6.30 Pancreatic Elastase, EC 3.4.21.35
31. 6.31 Cathepsin B, EC 3.4.22.1
32. 6.32 Pepsin A, EC 3.4.23.1
33. 6.33 Asparaginase, EC 3.5.1.1
34. 6.34 Glutaminase, EC 3.5.1.2
35. 6.35 Urease, EC 3.5.1.5
36. 6.36 Guanine Deaminase, EC 3.5.4.3
37. 6.37 Adenosinetriphosphatase, EC 3.6.1.3
38. 6.38 Mg²⁺ Importing ATPase, EC 3.6.3.2, Na⁺/K⁺‐Exchanging ATPase, EC 3.6.3.9
7 Lyases, EC 4
1. 7.1 Pyruvate Decarboxylase (PDC), EC 4.1.1.1
2. 7.2 Glutamate Decarboxylase (GAD), EC 4.1.1.15
3. 7.3 Fructose‐bisphosphate Aldolase, EC 4.1.2.13
4. 7.4 Anthranilate Synthase, EC 4.1.3.27
5. 7.5 Carbonic Anhydrase (CA), EC 4.2.1.1
6. 7.6 Fumarate Hydratase, EC 4.2.1.2
7. 7.7 Lactoylglutathione Lyase, EC 4.4.1.5
8. 7.8 Adenylate Cyclase (AC), EC 4.6.1.1
8 Isomerases, EC 5
1. 8.1 Xylose Isomerase, EC 5.3.1.5
2. 8.2 Glucose‐6‐phosphate Isomerase (G6PI), EC 5.3.1.9
3. 8.3 Phosphoglucomutase (PGM), EC 5.4.2.2
9 Ligases (Synthetases), EC 6
1. 9.1 Tyrosine‐tRNA Ligase, EC 6.1.1.1
2. 9.2 Acetate‐CoA Ligase (ACL), EC 6.2.1.1
3. 9.3 Glutamine Synthetase, EC 6.3.1.2
10 Assays for Multi‐enzyme Complexes
1. 10.1 Pyruvate Dehydrogenase Complex (PDHC)
2. 10.2 α‐Oxoglutarate Dehydrogenase Complex (OGDHC)
11 Assays for Other Enzyme Relevant Parameters
1. 11.1 Substrate Determination
2. 11.2 Protein Determination
3. General Literature for Protein Assays
4. 11.3 Phosphate Determination
5. 11.4 Determination of Metal Ions
6. 11.5 Glycoprotein Assays
7. 11.6 Cross‐linking of Proteins with Dimethylsuberimidate
8. 11.7 Concentrating Enzyme Solutions
12 Enzyme Immunoassays
1. 12.1 Radioimmunoassays
2. 12.2 Principle of Enzyme Immunoassays
3. 12.3 Noncompetitive Solid‐Phase Enzyme Immunoassay
4. 12.4 Competitive Solid‐Phase Enzyme Immunoassay
5. 12.5 Methods for Enzyme Immunoassays and Immobilization Techniques
6. General Literature
13 Binding Measurements
1. 13.1 Different Types of Binding
2. 13.2 Binding Measurements by Size Discrimination
3. General Literature
4. 13.3 Spectroscopic Methods
5. 13.4 Other Binding Methods
14 Enzymes in Technical Applications
1. 14.1 Modes of Enzyme Immobilization
2. 14.2 Methods for Enzyme Immobilization
3. 14.3 Analysis of Immobilized Enzymes
4. General Literature about Immobilization
5. 14.4 Enzyme Reactors
6. 14.5 Biosensors
7. 14.6 Immobilized Enzymes in Therapy
Index
End User License Agreement

List of Tables

Chapter 1
1. Table 1.1 pK _a values of functional groups of various amino acids.
2. Table 1.2 Biological buffers (pK _a values and pH range refer to 25 °C)...
3. Table 1.3 Frequently applied additives for enzyme assays.
4. Table 1.4 Time required for the conversion of substrate to product assu...
Chapter 3
1. Table 3.1 Frequently used stock solutions for enzyme assays.
Chapter 11
1. Table 11.1 Determination of the amount of protein and nucleic acids by ...
2. Table 11.2 Precipitation of protein with ammonium sulfate.
Chapter 14
1. Table 14.1 Cross‐linking reagents (according to Pierce Handbook 1989, P...

List of Illustrations

Chapter 1
1. Figure 1.1 Progress curves of various reaction orders. (a) Direct plotting...
2. Figure 1.2 Schematic representation of the three phases of a progress curv...
3. Figure 1.3 Determination of velocities of enzyme reactions from progress c...
4. Figure 1.4 Disturbance of velocity determination by the dead time. Progres...
5. Figure 1.5 Linearity of progress curves depends on the enzyme amount: less...
6. Figure 1.6 Stopped assay performed under nonideal conditions. The measurem...
7. Figure 1.7 Stopped assay performed under ideal conditions. A series of mea...
8. Figure 1.8 Error distributions. (a) Direct plotting of experimental data a...
9. Figure 1.9 Pipetting and mixing in assay solutions. (I–III) Pipetting of a...
10. Figure 1.10 Plunging the pipette directly into the assay solution: some so...
11. Figure 1.11 Different modes for preparation of a dilution series. (a) Dire...
12. Figure 1.12 Artificial deviations from the progress curve due to insuffici...
13. Figure 1.13 Michaelis–Menten presentation of enzyme velocities measured in...
14. Figure 1.14 Relative saturation (in percentage) of an enzyme in dependence...
15. Figure 1.15 Diagrams for linear presentation of the Michaelis–Menten data....
16. Figure 1.16 Three main mechanisms of enzyme inhibition in the linear prese...
17. Figure 1.17 Secondary plot (right), derived from the slopes (a) and the or...
18. Figure 1.18 Substrate inhibition. (a) Michaelis–Menten plot; (b) Lineweave...
19. Figure 1.19 Multisubstrate mechanisms in the Lineweaver–Burk diagram. , l...
20. Figure 1.20 pH behavior of the enzyme activity. pH optimum curve: the enzy...
21. Figure 1.21 Temperature behavior of the enzyme activity in direct plotting...
22. Figure 1.22 Temperature stability of the enzyme activity in direct and sem...
23. Figure 1.23 Progress curve of a coupled enzyme assay. For substrate determ...
Chapter 2
1. Figure 2.1 Construction schemes of absorption photometers. (a) Filter phot...
2. Figure 2.2 Determination of the velocity from a progress curve by regressi...
3. Figure 2.3 Progress curves of an enzyme catalyzed reaction. Panel (a) show...
4. Figure 2.4 Progress curves of enzyme reactions displayed on a photometric ...
5. Figure 2.5 Spectra of oxidized and reduced NAD. The wavelengths of the abs...
6. Figure 2.6 Spectral features of cuvettes made from different materials.
7. Figure 2.7 Construction scheme of a spectrofluorimeter.
8. Figure 2.8 Glass electrode with single‐rod measuring cell, connected with ...
Chapter 11
1. Figure 11.1 Structure of the BCA–Cu⁺ complex.
2. Figure 11.2 UV absorption spectra. (a) Absorption of the three aromatic am...
Chapter 12
1. Figure 12.1 Schemes of different immunoassays. (a) Competitive radioimmuno...
Chapter 13
1. Figure 13.1 Curves for specific and unspecific binding. The determination ...
2. Figure 13.2 (a) Principle of equilibrium dialysis and (b) scheme of an equ...
3. Figure 13.3 Indole reaction with Ehrlich's reagent.
4. Figure 13.4 Evaluation of a spectroscopic titration curve. For details see...
5. Figure 13.5 Schematic representation of a surface plasmon device.
Chapter 14
1. Figure 14.1 Various types of immobilization techniques.
2. Figure 14.2 Modes of immobilization of enzymes to solid supports. (a) Diff...
3. Figure 14.3 Entrapment of enzymes by polymerization of acrylamide with met...
4. Figure 14.4 Silanization of a glass surface and covalent fixation of an en...
5. Figure 14.5 Immobilization of enzymes to controlled pore glass. I, Formati...
6. Figure 14.6 Covalent immobilization of enzymes to polyamide. Polyamide is ...
7. Figure 14.7 Formation of polyester by polycondensation of terephthalate wi...
8. Figure 14.8 Immobilization of enzymes to polyester by tosylchloride activa...
9. Figure 14.9 Sequential determination of the activity of a matrix‐bound enz...
10. Figure 14.10 Enzyme reactor arrangement for continuous observation of the ...
11. Figure 14.11 Types of enzyme reactors.
12. Figure 14.12 Schematic diagram of an enzyme electrode. Adapted from Römpp ...
13. Figure 14.13 Schematic diagram of an enzyme field effect transistor

Copyright

Author

Hans Bisswanger

Eberhard‐Karls‐Universität Tübingen

Interfakultäres Institut für Biochemie

Hoppe‐Seyler‐Str. 4

72076 Tübingen

Germany

Email: bisswanger@uni‐tuebingen.de

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List of Abbreviations and Symbols

Only repeatedly used abbreviations, special abbreviations are defined at the respective section.

A: absorption
[A]: ligand concentration
[A]₀: total ligand concentration
A, B, C: specific binding ligands
ABTS: 2,2′‐azino‐bis‐3‐ethylbenzothiazoline‐6‐sulfonic acid
ADH: alcohol dehydrogenase
ANS: anilinonaphthalene sulfonate
BAPNA: N′‐benzoyl‐L‐arginine‐p‐nitroanilide
BCA: bicinchoninic acid
BSA: bovine serum albumin
CDI: carbonyldiimidazol
CoA: coenzyme A
CPG: controlled pore glass
d: density
DMSO: dimethylsulfoxide
DPIP: 2,6‐dichlorophenolindophenol
DTE: dithioerythritol
DTNB: 5,5′‐dithio‐bis(2‐nitrobenzoic acid), Ellman's reagent
DTT: dithiothreitol, Cleland's reagent
E, [E]: enzyme, enzyme concentration
[E]₀: total enzyme concentration
ε_nm: absorption (“extinction”) coefficient at the wavelength indicated
ε_r: dielectric constant
EDTA: ethylenediaminetetraacetic acid
EIA: enzyme immunoassays
ELISA: enzyme‐linked immunoadsorbent assays
FAD: flavin adenine dinucleotide
FMN: flavin mononucleotide
GOD: glucose oxidase
h: Planck's constant
HK: hexokinase
I: light intensity
IU: International enzyme unit (μmol min⁻¹)
k: rate constant
kat: Katal (mol s⁻¹)
k _cat: catalytical constant
K _d: dissociation constant
K _m: Michaelis constant
LDH: lactate dehydrogenase
M _r: relative molecule mass
MDH: malate dehydrogenase
n: number of subunits
NAD: nicotinamide adenine dinucleotide ¹
NADH: reduced nicotinamide adenine dinucleotide¹
NADP: nicotinamide adenine dinucleotide phosphate¹
NADPH: reduced nicotinamide adenine dinucleotide phosphate¹
ONPG: o‐nitrophenyl β‐D‐galactopyranoside
ORD: optical rotatory dispersion
P, Q, R: products
PAGE: polyacrylamide gel electrophoresis
PBS: phosphate buffered saline
PEG: polyethylene glycol
PLP: pyridoxal 5‐phosphate
PMSF: phenylmethylsulfonyl fluoride
POD: peroxidase
R: gas constant (8.3145 J mol⁻¹ K⁻¹)
RIA: radioimmunoassay
RN: recommended name
rpm: rotations per min
RT: room temperature
S: substrate
SA: specific enzyme activity
SDS: sodium dodecyl sulfate
SN: systematic name
ThDP: thiamine diphosphate
TCA: trichloroacetic acid
TRIS: tris(hydroxymethyl)aminomethane
ν: reaction velocity
V: maximum reaction velocity
ν_i: initial reaction velocity

Practical Enzymology

Copyright

Preface to the Third Edition

List of Abbreviations and Symbols

Note