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

6.6 Comparison of Coupled Bioluminescent and Other Methods of Measuring AChE Activity, 138

7 Measurement of Nitric Oxide Synthase Activity by Coupled Bioluminescence 139

7.1 Introduction, 139

7.1.1 Nitric Oxide Synthases, 139

7.1.2 The Nitrate Ion in Medicine and NO Biology, 141

7.1.3 NO Biology, 141

7.1.4 Interrelationship Among Nitrate, Nitrite, and NO, 142

7.1.5 Medical Aspects of Nitrate Biology, 143

7.2 Current NOS Assays, 144

7.2.1 Measuring NOS Activity by Assays for Nitrite and Nitrate, 144

7.2.2 “Direct” Assays of NOS Activity, 148

7.2.3 Coupled Enzymatic Assays of NOS Activity, 148

7.3 Conclusions, 149

8 The Coupled Bioluminescent Pyrophosphorolysis Assay 150

8.1 Introduction, 150

8.2 Genetic Variation in Modern Medicine, 150

8.3 DNA Complementarity, 152

8.3.1 DNA Hybridization, 152

8.3.2 Primer Extension in SNP Analysis, 153

8.3.3 Primer Degradation in SNP Analysis, 154

8.4 The READIT ® Pyrophosphorolysis Assay, 156

8.4.1 READIT ® : Description and Technical Aspects, 156

8.4.2 Performing the READIT ® Assay, 157

8.4.3 Competitive Position of READIT ® , 158

9 Coupled Luminescent Assays of G-Protein-Coupled Receptors 160

9.1 Introduction to G-Protein-Coupled Receptors, 160

9.1.1 Structure and Function of GPCRs, 161

9.1.2 GPCRs in Medicine, 165

9.2 GPCR Assay Methods, 166

9.2.1 Arrestins as Markers of GPCR Activity, 166

9.2.2 Calcium Quantification in GPCR Assays, 169

9.2.3 Quantitative cAMP Assays of GPCR Activity, 175

9.2.4 GPCR Assays Involving Inositol Triphosphate Detection, 182

9.2.5 GPCR Assays Involving GTP, 183

9.2.6 GPCR Assays Involving Reporter Genes, 186

9.2.7 Novel GPCR Assays and Other Strategies, 189

9.3 Summary, 190

10 Coupled Bioluminescent Protease Assays 191

10.1 Introduction, 191

10.1.1 Proteases, 191

10.2 Protease Assays, 193

10.2.1 Chromogenic Protease Assays, 194

10.2.2 Fluorometric Protease Assays, 194

10.2.3 Exotic Fluorometric Protease Assays, 197

10.3 Coupled Bioluminescent Protease Assays, 198

10.3.1 Protease Assays Using Luciferin Derivatives, 198

10.3.2 Coupled Bioluminescent Protease Assays Employing Recombinant Luminescent Proteins, 198

10.4 Summary, 199

11 Coupled Luminescent Assays Involving Aequorin 201

11.1 Introduction to Aequorin, 201

11.1.1 Aequorin as the First Photoprotein, 201

11.1.2 The Aequorin Reaction, 202

11.2 Detection of Calcium in Practice, 203

11.2.1 Fluorometric Quantification of Calcium, 203

11.2.2 Coupled Bioluminescent Quantification of Calcium Using Aequorin, 207

11.2.3 Real-Time Imaging of Calcium Flux with Aequorin, 208

11.2.4 Other Uses of Aequorin in Coupled Bioluminescent Assays, 208

12 Coupled Bioluminescent Reporter Assays 209

12.1 Introduction to Reporter Assays, 209

12.1.1 Brief History and Development of Reporter Assays, 210

12.1.2 Other Non-Luminescent Reporter Proteins, 211

12.2 Luciferases as Reporters of Promoter Activities, 212

12.2.1 The Modern Luminescent Reporter Assay, 213

12.2.2 Multicolor Light Emission by Luciferases, 214

12.2.3 Dual-Mode Reporter Assays in Practice, 214

12.3 Aequorin as a Reporter Enzyme, 215

12.4 Vectors for Use in Reporter Assays, 216

12.4.1 Systems for Study of Specific Transcriptional Modulators, 216

12.4.2 Bacterial Reporter Systems, 216

12.4.3 Viral Vectors, 217

12.5 Summary, 217

13 Coupled Bioluminescent Assays: Regulatory Concerns 218

13.1 Introduction, 218

13.2 Regulatory Aspects of Assay Development, 220

13.2.1 Standard Operating Procedures, 220

13.2.2 Philosophy of Assay Validation, 221

13.2.3 Parameters for Assay Validation, 223

13.2.4 Gaining Regulatory Acceptance of CB Methods, 240

13.3 Summary, 240

Part III other Applications of Coupled Bioluminescence 241

14 Coupled Bioluminescent Determination of Bioburden and Sterility 243

14.1 Introduction, 243

14.2 Rapid Methods of Bioburden and Sterility Assessment, 244

14.2.1 Bioburden Measurement by ATP-Release Assay, 244

14.2.2 Bioburden Measurement by Protein Assay, 245

14.2.3 Coupled Bioluminescent Methods of Measuring Bioburden, 245

15 Environmental Applications of Coupled Bioluminescent Assays 247

15.1 Introduction, 247

15.2 Current Methods for Environmental Monitoring of Water Quality, 248

15.3 Methods of Monitoring Stream Water and Lake Water, 249

15.3.1 Monitoring of Phosphate in Freshwater, 249

15.3.2 Monitoring of Nitrate in Groundwater, 250

15.3.3 Monitoring of Pesticides in Freshwater, 251

15.3.4 Monitoring of Bacterial Content of Freshwater, 252

15.4 Methods of Monitoring Drinking Water, 254

15.4.1 Current Drinking Water Reports, 254

15.4.2 Biohazard Monitoring in Drinking Water, 255

15.4.3 Monitoring of Chemical Hazards in Drinking Water, 257

Appendix A: One-Letter Amino Acid Abbreviations 259

Glossary 260

Bibliography 265

Index 291