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.

"The chapters of the book are well balanced.... Recommended to scientist and students who want to improve their knowledge of bioluminescent coupled assays and bioluminescence in general." (Analytical and Bioanalytical Chemistry, August 2009)

• Preface xiiiPart I Background to Coupled Bioluminescent Assays 11 Introduction 31.1 Introduction to Coupled Bioluminescent Assays, 31.2 Luminescent Technologies of the Life Sciences, 41.3 Varieties of Fluorometric Assays, 71.3.1 Time-Resolved Fluorescence, 81.3.2 Fluorescence Resonance Energy Transfer, 81.3.3 Fluorescence Polarization, 91.4 Chemiluminescence and Bioluminescence, 101.4.1 Chemiluminescence, 111.4.2 Bioluminescence, 131.5 Common Bioluminescence Systems, 141.5.1 Firefly Luciferase, 141.5.2 Renilla Luciferase, 161.5.3 Bacterial Luciferases, 181.5.4 Aequorin, 191.5.5 Other Luciferases and Photoproteins, 201.6 A Coupled Bioluminescent Reaction, 221.7 Summary, 232 Coupled Bioluminescent Reactions in Practice 242.1 Principles of Coupled Bioluminescent Reactions, 242.1.1 Requirements for Successful Coupled Bioluminescent Assays, 242.2 Instrumentation and Equipment for Coupled Bioluminescent Assays, 252.2.1 Instrument Testing, 272.2.2 Instrument Features, 282.2.3 Luminescent Microplates, 312.3 Coupled Bioluminescent Assay Procedures and Precautions, 322.3.1 Coupled Enzyme Assays, 322.3.2 Steady-State Kinetics, 362.3.3 Coupled Bioluminescent Reactions, 382.3.4 Recommended Precautions for Coupled Bioluminescent Reactions, 442.4 Data Handling for Coupled Bioluminescent Assays, 452.4.1 Analysis of Coupled Bioluminescent Assay Results by Time-Dependent Linear Regression, 462.4.2 Performing the Linear Regression, 492.4.3 Outliers, 502.4.4 Special Considerations Relating to High-Throughput Screening, 522.5 Comparison of Coupled Bioluminescent Assays with Other Methods, 532.5.1 Coupled Bioluminescent Assays Versus Spectrophotometric Assays, 532.5.2 Coupled Bioluminescent Assays Versus Fluorescent Assays, 542.5.3 Coupled Bioluminescent Assays Versus Chemiluminescent Assays, 55Part II Biomedical Applications of Coupled Bioluminescence 573 Coupled Bioluminescent Cytotoxicity Assays 593.1 Introduction, 593.2 Membrane Integrity Assays, 603.2.1 Radioactive Isotope Methods, 603.2.2 Other Labeling Methods, 643.2.3 General Enzyme-Release Methods, 653.3 A Coupled Bioluminescent Assay for Enzyme Release: The G3PDH-Release Assay, 703.3.1 Development and Features, 703.3.2 Performance Characteristics, 753.4 Viability Assays, 823.4.1 ATP-Release Assay, 823.4.2 Assays of Vital Cellular Functions, 823.4.3 Simple Cell-Growth Assays, 833.4.4 Total Enzyme-Release Methods, 844 The Role of Coupled Bioluminescent Assays in Kinase Screening and Study 854.1 The Many Roles of Kinases in Biology, 854.2 Current Standard Kinase Assays, 864.2.1 Fluorometric Kinase Assays, 874.2.2 Radiolabeling Kinase Assays, 884.2.3 Fluorometric Assays Involving Detection of ADP, 894.3 Coupled Bioluminescent Kinase Assays, 904.3.1 A Historical Note: The Coupled Bioluminescent Creatine Kinase Assay, 904.3.2 ATP Depletion Kinase Assays, 904.3.3 Reverse CB Kinase Assays, 984.3.4 Kinases as Participants in Other CB Assays, 1024.4 Conclusions, 1025 Coupled Bioluminescent Phosphatase Assays 1035.1 Introduction, 1035.2 Phosphatases, 1035.2.1 Catalytic Properties of Phosphatases, 1045.2.2 Classification of Phosphatases, 1045.3 Contemporary Phosphatase Assay Technologies, 1055.3.1 Fluorometric Phosphatase Assays, 1065.3.2 Colorimetric Phosphatase Assays, 1175.3.3 Chemiluminescent Phosphatase Assays, 1195.3.4 Radiolabeling Phosphatase Assays, 1205.4 CB Phosphatase Assays, 1225.4.1 Assays Using Phosphorylated Luciferin, 1225.4.2 Assays Based on the GPL Reaction Series, 1225.4.3 Challenges in the Development of the GPL-Coupled Bioluminescent System for Phosphate Detection, 1275.4.4 Potential Drawbacks of GPL Phosphatase Assays, 1295.5 Conclusions, 1306 Acetylcholinesterase 1316.1 Introduction, 1316.2 Established AChE Assay Methods, 1336.3 Recent Developments in AChE Assay Methods, 1346.4 Evaluation of Current AChE High-Throughput Screening Methods, 1356.5 Coupled Bioluminescent Assays of AChE Activity, 1366.6 Comparison of Coupled Bioluminescent and Other Methods of Measuring AChE Activity, 1387 Measurement of Nitric Oxide Synthase Activity by Coupled Bioluminescence 1397.1 Introduction, 1397.1.1 Nitric Oxide Synthases, 1397.1.2 The Nitrate Ion in Medicine and NO Biology, 1417.1.3 NO Biology, 1417.1.4 Interrelationship Among Nitrate, Nitrite, and NO, 1427.1.5 Medical Aspects of Nitrate Biology, 1437.2 Current NOS Assays, 1447.2.1 Measuring NOS Activity by Assays for Nitrite and Nitrate, 1447.2.2 “Direct” Assays of NOS Activity, 1487.2.3 Coupled Enzymatic Assays of NOS Activity, 1487.3 Conclusions, 1498 The Coupled Bioluminescent Pyrophosphorolysis Assay 1508.1 Introduction, 1508.2 Genetic Variation in Modern Medicine, 1508.3 DNA Complementarity, 1528.3.1 DNA Hybridization, 1528.3.2 Primer Extension in SNP Analysis, 1538.3.3 Primer Degradation in SNP Analysis, 1548.4 The READIT ® Pyrophosphorolysis Assay, 1568.4.1 READIT ® : Description and Technical Aspects, 1568.4.2 Performing the READIT ® Assay, 1578.4.3 Competitive Position of READIT ® , 1589 Coupled Luminescent Assays of G-Protein-Coupled Receptors 1609.1 Introduction to G-Protein-Coupled Receptors, 1609.1.1 Structure and Function of GPCRs, 1619.1.2 GPCRs in Medicine, 1659.2 GPCR Assay Methods, 1669.2.1 Arrestins as Markers of GPCR Activity, 1669.2.2 Calcium Quantification in GPCR Assays, 1699.2.3 Quantitative cAMP Assays of GPCR Activity, 1759.2.4 GPCR Assays Involving Inositol Triphosphate Detection, 1829.2.5 GPCR Assays Involving GTP, 1839.2.6 GPCR Assays Involving Reporter Genes, 1869.2.7 Novel GPCR Assays and Other Strategies, 1899.3 Summary, 19010 Coupled Bioluminescent Protease Assays 19110.1 Introduction, 19110.1.1 Proteases, 19110.2 Protease Assays, 19310.2.1 Chromogenic Protease Assays, 19410.2.2 Fluorometric Protease Assays, 19410.2.3 Exotic Fluorometric Protease Assays, 19710.3 Coupled Bioluminescent Protease Assays, 19810.3.1 Protease Assays Using Luciferin Derivatives, 19810.3.2 Coupled Bioluminescent Protease Assays Employing Recombinant Luminescent Proteins, 19810.4 Summary, 19911 Coupled Luminescent Assays Involving Aequorin 20111.1 Introduction to Aequorin, 20111.1.1 Aequorin as the First Photoprotein, 20111.1.2 The Aequorin Reaction, 20211.2 Detection of Calcium in Practice, 20311.2.1 Fluorometric Quantification of Calcium, 20311.2.2 Coupled Bioluminescent Quantification of Calcium Using Aequorin, 20711.2.3 Real-Time Imaging of Calcium Flux with Aequorin, 20811.2.4 Other Uses of Aequorin in Coupled Bioluminescent Assays, 20812 Coupled Bioluminescent Reporter Assays 20912.1 Introduction to Reporter Assays, 20912.1.1 Brief History and Development of Reporter Assays, 21012.1.2 Other Non-Luminescent Reporter Proteins, 21112.2 Luciferases as Reporters of Promoter Activities, 21212.2.1 The Modern Luminescent Reporter Assay, 21312.2.2 Multicolor Light Emission by Luciferases, 21412.2.3 Dual-Mode Reporter Assays in Practice, 21412.3 Aequorin as a Reporter Enzyme, 21512.4 Vectors for Use in Reporter Assays, 21612.4.1 Systems for Study of Specific Transcriptional Modulators, 21612.4.2 Bacterial Reporter Systems, 21612.4.3 Viral Vectors, 21712.5 Summary, 21713 Coupled Bioluminescent Assays: Regulatory Concerns 21813.1 Introduction, 21813.2 Regulatory Aspects of Assay Development, 22013.2.1 Standard Operating Procedures, 22013.2.2 Philosophy of Assay Validation, 22113.2.3 Parameters for Assay Validation, 22313.2.4 Gaining Regulatory Acceptance of CB Methods, 24013.3 Summary, 240Part III other Applications of Coupled Bioluminescence 24114 Coupled Bioluminescent Determination of Bioburden and Sterility 24314.1 Introduction, 24314.2 Rapid Methods of Bioburden and Sterility Assessment, 24414.2.1 Bioburden Measurement by ATP-Release Assay, 24414.2.2 Bioburden Measurement by Protein Assay, 24514.2.3 Coupled Bioluminescent Methods of Measuring Bioburden, 24515 Environmental Applications of Coupled Bioluminescent Assays 24715.1 Introduction, 24715.2 Current Methods for Environmental Monitoring of Water Quality, 24815.3 Methods of Monitoring Stream Water and Lake Water, 24915.3.1 Monitoring of Phosphate in Freshwater, 24915.3.2 Monitoring of Nitrate in Groundwater, 25015.3.3 Monitoring of Pesticides in Freshwater, 25115.3.4 Monitoring of Bacterial Content of Freshwater, 25215.4 Methods of Monitoring Drinking Water, 25415.4.1 Current Drinking Water Reports, 25415.4.2 Biohazard Monitoring in Drinking Water, 25515.4.3 Monitoring of Chemical Hazards in Drinking Water, 257Appendix A: One-Letter Amino Acid Abbreviations 259Glossary 260Bibliography 265Index 291

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.

"The chapters of the book are well balanced.... Recommended to scientist and students who want to improve their knowledge of bioluminescent coupled assays and bioluminescence in general." (Analytical and Bioanalytical Chemistry, August 2009)

• Preface xiiiPart I Background to Coupled Bioluminescent Assays 11 Introduction 31.1 Introduction to Coupled Bioluminescent Assays, 31.2 Luminescent Technologies of the Life Sciences, 41.3 Varieties of Fluorometric Assays, 71.3.1 Time-Resolved Fluorescence, 81.3.2 Fluorescence Resonance Energy Transfer, 81.3.3 Fluorescence Polarization, 91.4 Chemiluminescence and Bioluminescence, 101.4.1 Chemiluminescence, 111.4.2 Bioluminescence, 131.5 Common Bioluminescence Systems, 141.5.1 Firefly Luciferase, 141.5.2 Renilla Luciferase, 161.5.3 Bacterial Luciferases, 181.5.4 Aequorin, 191.5.5 Other Luciferases and Photoproteins, 201.6 A Coupled Bioluminescent Reaction, 221.7 Summary, 232 Coupled Bioluminescent Reactions in Practice 242.1 Principles of Coupled Bioluminescent Reactions, 242.1.1 Requirements for Successful Coupled Bioluminescent Assays, 242.2 Instrumentation and Equipment for Coupled Bioluminescent Assays, 252.2.1 Instrument Testing, 272.2.2 Instrument Features, 282.2.3 Luminescent Microplates, 312.3 Coupled Bioluminescent Assay Procedures and Precautions, 322.3.1 Coupled Enzyme Assays, 322.3.2 Steady-State Kinetics, 362.3.3 Coupled Bioluminescent Reactions, 382.3.4 Recommended Precautions for Coupled Bioluminescent Reactions, 442.4 Data Handling for Coupled Bioluminescent Assays, 452.4.1 Analysis of Coupled Bioluminescent Assay Results by Time-Dependent Linear Regression, 462.4.2 Performing the Linear Regression, 492.4.3 Outliers, 502.4.4 Special Considerations Relating to High-Throughput Screening, 522.5 Comparison of Coupled Bioluminescent Assays with Other Methods, 532.5.1 Coupled Bioluminescent Assays Versus Spectrophotometric Assays, 532.5.2 Coupled Bioluminescent Assays Versus Fluorescent Assays, 542.5.3 Coupled Bioluminescent Assays Versus Chemiluminescent Assays, 55Part II Biomedical Applications of Coupled Bioluminescence 573 Coupled Bioluminescent Cytotoxicity Assays 593.1 Introduction, 593.2 Membrane Integrity Assays, 603.2.1 Radioactive Isotope Methods, 603.2.2 Other Labeling Methods, 643.2.3 General Enzyme-Release Methods, 653.3 A Coupled Bioluminescent Assay for Enzyme Release: The G3PDH-Release Assay, 703.3.1 Development and Features, 703.3.2 Performance Characteristics, 753.4 Viability Assays, 823.4.1 ATP-Release Assay, 823.4.2 Assays of Vital Cellular Functions, 823.4.3 Simple Cell-Growth Assays, 833.4.4 Total Enzyme-Release Methods, 844 The Role of Coupled Bioluminescent Assays in Kinase Screening and Study 854.1 The Many Roles of Kinases in Biology, 854.2 Current Standard Kinase Assays, 864.2.1 Fluorometric Kinase Assays, 874.2.2 Radiolabeling Kinase Assays, 884.2.3 Fluorometric Assays Involving Detection of ADP, 894.3 Coupled Bioluminescent Kinase Assays, 904.3.1 A Historical Note: The Coupled Bioluminescent Creatine Kinase Assay, 904.3.2 ATP Depletion Kinase Assays, 904.3.3 Reverse CB Kinase Assays, 984.3.4 Kinases as Participants in Other CB Assays, 1024.4 Conclusions, 1025 Coupled Bioluminescent Phosphatase Assays 1035.1 Introduction, 1035.2 Phosphatases, 1035.2.1 Catalytic Properties of Phosphatases, 1045.2.2 Classification of Phosphatases, 1045.3 Contemporary Phosphatase Assay Technologies, 1055.3.1 Fluorometric Phosphatase Assays, 1065.3.2 Colorimetric Phosphatase Assays, 1175.3.3 Chemiluminescent Phosphatase Assays, 1195.3.4 Radiolabeling Phosphatase Assays, 1205.4 CB Phosphatase Assays, 1225.4.1 Assays Using Phosphorylated Luciferin, 1225.4.2 Assays Based on the GPL Reaction Series, 1225.4.3 Challenges in the Development of the GPL-Coupled Bioluminescent System for Phosphate Detection, 1275.4.4 Potential Drawbacks of GPL Phosphatase Assays, 1295.5 Conclusions, 1306 Acetylcholinesterase 1316.1 Introduction, 1316.2 Established AChE Assay Methods, 1336.3 Recent Developments in AChE Assay Methods, 1346.4 Evaluation of Current AChE High-Throughput Screening Methods, 1356.5 Coupled Bioluminescent Assays of AChE Activity, 1366.6 Comparison of Coupled Bioluminescent and Other Methods of Measuring AChE Activity, 1387 Measurement of Nitric Oxide Synthase Activity by Coupled Bioluminescence 1397.1 Introduction, 1397.1.1 Nitric Oxide Synthases, 1397.1.2 The Nitrate Ion in Medicine and NO Biology, 1417.1.3 NO Biology, 1417.1.4 Interrelationship Among Nitrate, Nitrite, and NO, 1427.1.5 Medical Aspects of Nitrate Biology, 1437.2 Current NOS Assays, 1447.2.1 Measuring NOS Activity by Assays for Nitrite and Nitrate, 1447.2.2 “Direct” Assays of NOS Activity, 1487.2.3 Coupled Enzymatic Assays of NOS Activity, 1487.3 Conclusions, 1498 The Coupled Bioluminescent Pyrophosphorolysis Assay 1508.1 Introduction, 1508.2 Genetic Variation in Modern Medicine, 1508.3 DNA Complementarity, 1528.3.1 DNA Hybridization, 1528.3.2 Primer Extension in SNP Analysis, 1538.3.3 Primer Degradation in SNP Analysis, 1548.4 The READIT ® Pyrophosphorolysis Assay, 1568.4.1 READIT ® : Description and Technical Aspects, 1568.4.2 Performing the READIT ® Assay, 1578.4.3 Competitive Position of READIT ® , 1589 Coupled Luminescent Assays of G-Protein-Coupled Receptors 1609.1 Introduction to G-Protein-Coupled Receptors, 1609.1.1 Structure and Function of GPCRs, 1619.1.2 GPCRs in Medicine, 1659.2 GPCR Assay Methods, 1669.2.1 Arrestins as Markers of GPCR Activity, 1669.2.2 Calcium Quantification in GPCR Assays, 1699.2.3 Quantitative cAMP Assays of GPCR Activity, 1759.2.4 GPCR Assays Involving Inositol Triphosphate Detection, 1829.2.5 GPCR Assays Involving GTP, 1839.2.6 GPCR Assays Involving Reporter Genes, 1869.2.7 Novel GPCR Assays and Other Strategies, 1899.3 Summary, 19010 Coupled Bioluminescent Protease Assays 19110.1 Introduction, 19110.1.1 Proteases, 19110.2 Protease Assays, 19310.2.1 Chromogenic Protease Assays, 19410.2.2 Fluorometric Protease Assays, 19410.2.3 Exotic Fluorometric Protease Assays, 19710.3 Coupled Bioluminescent Protease Assays, 19810.3.1 Protease Assays Using Luciferin Derivatives, 19810.3.2 Coupled Bioluminescent Protease Assays Employing Recombinant Luminescent Proteins, 19810.4 Summary, 19911 Coupled Luminescent Assays Involving Aequorin 20111.1 Introduction to Aequorin, 20111.1.1 Aequorin as the First Photoprotein, 20111.1.2 The Aequorin Reaction, 20211.2 Detection of Calcium in Practice, 20311.2.1 Fluorometric Quantification of Calcium, 20311.2.2 Coupled Bioluminescent Quantification of Calcium Using Aequorin, 20711.2.3 Real-Time Imaging of Calcium Flux with Aequorin, 20811.2.4 Other Uses of Aequorin in Coupled Bioluminescent Assays, 20812 Coupled Bioluminescent Reporter Assays 20912.1 Introduction to Reporter Assays, 20912.1.1 Brief History and Development of Reporter Assays, 21012.1.2 Other Non-Luminescent Reporter Proteins, 21112.2 Luciferases as Reporters of Promoter Activities, 21212.2.1 The Modern Luminescent Reporter Assay, 21312.2.2 Multicolor Light Emission by Luciferases, 21412.2.3 Dual-Mode Reporter Assays in Practice, 21412.3 Aequorin as a Reporter Enzyme, 21512.4 Vectors for Use in Reporter Assays, 21612.4.1 Systems for Study of Specific Transcriptional Modulators, 21612.4.2 Bacterial Reporter Systems, 21612.4.3 Viral Vectors, 21712.5 Summary, 21713 Coupled Bioluminescent Assays: Regulatory Concerns 21813.1 Introduction, 21813.2 Regulatory Aspects of Assay Development, 22013.2.1 Standard Operating Procedures, 22013.2.2 Philosophy of Assay Validation, 22113.2.3 Parameters for Assay Validation, 22313.2.4 Gaining Regulatory Acceptance of CB Methods, 24013.3 Summary, 240Part III other Applications of Coupled Bioluminescence 24114 Coupled Bioluminescent Determination of Bioburden and Sterility 24314.1 Introduction, 24314.2 Rapid Methods of Bioburden and Sterility Assessment, 24414.2.1 Bioburden Measurement by ATP-Release Assay, 24414.2.2 Bioburden Measurement by Protein Assay, 24514.2.3 Coupled Bioluminescent Methods of Measuring Bioburden, 24515 Environmental Applications of Coupled Bioluminescent Assays 24715.1 Introduction, 24715.2 Current Methods for Environmental Monitoring of Water Quality, 24815.3 Methods of Monitoring Stream Water and Lake Water, 24915.3.1 Monitoring of Phosphate in Freshwater, 24915.3.2 Monitoring of Nitrate in Groundwater, 25015.3.3 Monitoring of Pesticides in Freshwater, 25115.3.4 Monitoring of Bacterial Content of Freshwater, 25215.4 Methods of Monitoring Drinking Water, 25415.4.1 Current Drinking Water Reports, 25415.4.2 Biohazard Monitoring in Drinking Water, 25515.4.3 Monitoring of Chemical Hazards in Drinking Water, 257Appendix A: One-Letter Amino Acid Abbreviations 259Glossary 260Bibliography 265Index 291