This book highlights the applications of coupled bioluminescence assay techniques to real-world problems in drug discovery, environmental and chemical analysis, and biodefense. It separates theoretical aspects from the applied sections in a clear and readable way. Coupled Bioluminescent Assays, explains the uses of CB technologies across drug discovery to analyze toxicity, drug receptors, and enzymes. It covers applications in environmental analysis and biodefense, including cytotoxicity, fertilizer and explosives analysis, and nerve agent and pesticide detection. This is the premier reference on coupled bioluminescent assays for chemists, biochemists, and molecular biologists.

Michael J. Corey, PHD, is a consulting biochemist and a leading authority on coupled bioluminescent assays. A former head of assay development in biotechnology, he is a founding member of the Scientific Advisory Board of Cell Technology, Inc. Dr. Corey holds key patents in the field of coupled bioluminescence.

This book enables readers to take full advantage of coupled bioluminescent (CB) assay techniques by demonstrating how they are used to solve real-world problems. It guides readers involved in drug discovery and biomedicine through a broad range of valuable CB techniques, including assays of kinases, phosphatases, and other enzymes; cytotoxicity measurements; and high-throughput screening of receptor modulators. In each case, CB methods are compared with competing technologies to enable informed choices. In addition, the book's coverage of CB methods in biomonitoring; analysis of nitrate, a critical marker of explosives residue and fertilizer runoff; and nerve agent and pesticide detection supports the work of researchers involved in biodefense as well as environmental and food sciences.

Coupled Bioluminescent Assays is divided into three parts:

• Background to coupled bioluminescent assays

• Biomedical applications of coupled bioluminescence

• Other applications of coupled bioluminescence

Highlights of coverage include:

• CB cytotoxicity assays

• CB assays of kinases and phosphatases

• CB measurements of G protein coupled receptor activity

• Assessment of nitric oxide synthase activity by CB

• CB determination of bioburden and sterility

• Emerging regulatory concerns

Each chapter clearly presents both underlying theory and applications. References facilitate further investigations by guiding readers to the primary literature.

CB techniques offer advantages in speed and sensitivity that fluorometric techniques generally cannot match. Bioluminescent reaction schemes are typically one to several orders of magnitude more sensitive than fluorometric assays, yet these techniques have been vastly underutilized. With this book as their guide, chemists, biochemists, and molecular biologists can take full advantage of a broad range of available CB assays to achieve their research goals.

Preface xiii

Part I Background to Coupled Bioluminescent Assays 1

1 Introduction 3

1.1 Introduction to Coupled Bioluminescent Assays, 3

1.2 Luminescent Technologies of the Life Sciences, 4

1.3 Varieties of Fluorometric Assays, 7

1.3.1 Time-Resolved Fluorescence, 8

1.3.2 Fluorescence Resonance Energy Transfer, 8

1.3.3 Fluorescence Polarization, 9

1.4 Chemiluminescence and Bioluminescence, 10

1.4.1 Chemiluminescence, 11

1.4.2 Bioluminescence, 13

1.5 Common Bioluminescence Systems, 14

1.5.1 Firefly Luciferase, 14

1.5.2 Renilla Luciferase, 16

1.5.3 Bacterial Luciferases, 18

1.5.4 Aequorin, 19

1.5.5 Other Luciferases and Photoproteins, 20

1.6 A Coupled Bioluminescent Reaction, 22

1.7 Summary, 23

2 Coupled Bioluminescent Reactions in Practice 24

2.1 Principles of Coupled Bioluminescent Reactions, 24

2.1.1 Requirements for Successful Coupled Bioluminescent Assays, 24

2.2 Instrumentation and Equipment for Coupled Bioluminescent Assays, 25

2.2.1 Instrument Testing, 27

2.2.2 Instrument Features, 28

2.2.3 Luminescent Microplates, 31

2.3 Coupled Bioluminescent Assay Procedures and Precautions, 32

2.3.1 Coupled Enzyme Assays, 32

2.3.2 Steady-State Kinetics, 36

2.3.3 Coupled Bioluminescent Reactions, 38

2.3.4 Recommended Precautions for Coupled Bioluminescent Reactions, 44

2.4 Data Handling for Coupled Bioluminescent Assays, 45

2.4.1 Analysis of Coupled Bioluminescent Assay Results by Time-Dependent Linear Regression, 46

2.4.2 Performing the Linear Regression, 49

2.4.3 Outliers, 50

2.4.4 Special Considerations Relating to High-Throughput Screening, 52

2.5 Comparison of Coupled Bioluminescent Assays with Other Methods, 53

2.5.1 Coupled Bioluminescent Assays Versus Spectrophotometric Assays, 53

2.5.2 Coupled Bioluminescent Assays Versus Fluorescent Assays, 54

2.5.3 Coupled Bioluminescent Assays Versus Chemiluminescent Assays, 55

Part II Biomedical Applications of Coupled Bioluminescence 57

3 Coupled Bioluminescent Cytotoxicity Assays 59

3.1 Introduction, 59

3.2 Membrane Integrity Assays, 60

3.2.1 Radioactive Isotope Methods, 60

3.2.2 Other Labeling Methods, 64

3.2.3 General Enzyme-Release Methods, 65

3.3 A Coupled Bioluminescent Assay for Enzyme Release: The G3PDH-Release Assay, 70

3.3.1 Development and Features, 70

3.3.2 Performance Characteristics, 75

3.4 Viability Assays, 82

3.4.1 ATP-Release Assay, 82

3.4.2 Assays of Vital Cellular Functions, 82

3.4.3 Simple Cell-Growth Assays, 83

3.4.4 Total Enzyme-Release Methods, 84

4 The Role of Coupled Bioluminescent Assays in Kinase Screening and Study 85

4.1 The Many Roles of Kinases in Biology, 85

4.2 Current Standard Kinase Assays, 86

4.2.1 Fluorometric Kinase Assays, 87

4.2.2 Radiolabeling Kinase Assays, 88

4.2.3 Fluorometric Assays Involving Detection of ADP, 89

4.3 Coupled Bioluminescent Kinase Assays, 90

4.3.1 A Historical Note: The Coupled Bioluminescent Creatine Kinase Assay, 90

4.3.2 ATP Depletion Kinase Assays, 90

4.3.3 Reverse CB Kinase Assays, 98

4.3.4 Kinases as Participants in Other CB Assays, 102

4.4 Conclusions, 102

5 Coupled Bioluminescent Phosphatase Assays 103

5.1 Introduction, 103

5.2 Phosphatases, 103

5.2.1 Catalytic Properties of Phosphatases, 104

5.2.2 Classification of Phosphatases, 104

5.3 Contemporary Phosphatase Assay Technologies, 105

5.3.1 Fluorometric Phosphatase Assays, 106

5.3.2 Colorimetric Phosphatase Assays, 117

5.3.3 Chemiluminescent Phosphatase Assays, 119

5.3.4 Radiolabeling Phosphatase Assays, 120

5.4 CB Phosphatase Assays, 122

5.4.1 Assays Using Phosphorylated Luciferin, 122

5.4.2 Assays Based on the GPL Reaction Series, 122

5.4.3 Challenges in the Development of the GPL-Coupled Bioluminescent System for Phosphate Detection, 127

5.4.4 Potential Drawbacks of GPL Phosphatase Assays, 129

5.5 Conclusions, 130

6 Acetylcholinesterase 131

6.1 Introduction, 131

6.2 Established AChE Assay Methods, 133

6.3 Recent Developments in AChE Assay Methods, 134

6.4 Evaluation of Current AChE High-Throughput Screening Methods, 135

6.5 Coupled Bioluminescent Assays of AChE Activity, 136

6.…