Sách Bioanalytical chemistry

Preface xv

Acknowledgments xvii

1. Spectroscopic Methods for Matrix Characterization 1

1.1 Introduction 1

1.2 Total Protein 2

1.2.1 Lowry Method 3

1.2.2 Smith (BCA) Method 3

1.2.3 Bradford Method 4

1.2.4 Ninhydrin-Based Assay 5

1.2.5 Other Protein Quantitation Methods 5

1.3 Total DNA 7

1.3.1 Diaminobenzoic Acid Method 7

1.3.2 Diphenylamine Method 9

1.3.3 Other Fluorometric Methods 9

1.4 Total RNA 10

1.5 Total Carbohydrate 11

1.5.1 Ferricyanide Method 11

1.5.2 Phenol–Sulfuric Acid Method 11

1.5.3 2-Aminothiophenol Method 12

1.5.4 Purpald Assay for Bacterial Polysaccharides 12

1.6 Free Fatty Acids 13

References 14

Problems 15

2. Enzymes 16

2.1 Introduction 16

2.2 Enzyme Nomenclature 17

2.3 Enzyme Commission Numbers 18

2.4 Enzymes in Bioanalytical Chemistry 19

2.5 Enzyme Kinetics 21

2.5.1 Simple One-Substrate Enzyme Kinetics 23

2.5.2 Experimental Determination of Michaelis–Menten

Parameters 24

2.5.2.1 Eadie–Hofstee Method 25

2.5.2.2 Hanes Method 25

2.5.2.3 Lineweaver–Burk Method 26

2.5.2.4 Cornish–Bowden–Eisenthal Method 27

2.5.3 Comparison of Methods for the Determination

ofKmValues 28

2.5.4 One-Substrate, Two-Product Enzyme Kinetics 29

2.5.5 Two-Substrate Enzyme Kinetics 29

2.5.6 Examples of Enzyme-Catalyzed Reactions and

Their Treatment 31

2.6 Enzyme Activators 32

2.7 Enzyme Inhibitors 33

2.7.1 Competitive Inhibition 34

2.7.2 Noncompetitive Inhibition 35

2.7.3 Uncompetitive Inhibition 35

2.8 Enzyme Units and Concentrations 36

Suggested References 38

References 38

Problems 38

3. Quantitation of Enzymes and Their Substrates 41

3.1 Introduction 41

3.2 Substrate Depletion or Product Accumulation 42

3.3 Direct and Coupled Measurements 43

3.4 Classification of Methods 45

3.5 Instrumental Methods 47

3.5.1 Optical Detection 47

3.5.1.1 Absorbance 47

3.5.1.2 Fluorescence 49

3.5.1.3 Luminescence 51

3.5.1.4 Nephelometry 53

3.5.2 Electrochemical Detection 53

3.5.2.1 Amperometry 53

3.5.2.2 Potentiometry 54

3.5.2.3 Conductimetry 54

3.5.3 Other Detection Methods 55

3.5.3.1 Radiochemical 55

3.5.3.2 Manometry 55

3.5.3.3 Calorimetry 56

3.6 Ultra-High-Throughput Assays (HTA) 56

3.7 Practical Considerations for Enzymatic Assays 57

Suggested References 57