COUPLED BIOLUMINESCENT ASSAYS
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1 COUPLED BIOLUMINESCENT ASSAYS Methods, Evaluations, and Applications MICHAEL J. COREY A John Wiley & Sons, Inc., Publication
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3 COUPLED BIOLUMINESCENT ASSAYS
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5 COUPLED BIOLUMINESCENT ASSAYS Methods, Evaluations, and Applications MICHAEL J. COREY A John Wiley & Sons, Inc., Publication
6 Copyright 2009 by John Wiley & Sons, Inc. All rights reserved Published by John Wiley & Sons, Inc., Hoboken, New Jersey Published simultaneously in Canada No part of this publication may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, recording, scanning, or otherwise, except as permitted under Section 107 or 108 of the 1976 United States Copyright Act, without either the prior written permission of the Publisher, or authorization through payment of the appropriate per-copy fee to the Copyright Clearance Center, Inc., 222 Rosewood Drive, Danvers, MA 01923, (978) , fax (978) , or on the web at Requests to the Publisher for permission should be addressed to the Permissions Department, John Wiley & Sons, Inc., 111 River Street, Hoboken, NJ 07030, (201) , fax (201) , or online at Limit of Liability/Disclaimer of Warranty: While the publisher and author have used their best efforts in preparing this book, they make no representations or warranties with respect to the accuracy or completeness of the contents of this book and specifically disclaim any implied warranties of merchantability or fitness for a particular purpose. No warranty may be created or extended by sales representatives or written sales materials. The advice and strategies contained herein may not be suitable for your situation. You should consult with a professional where appropriate. Neither the publisher nor author shall be liable for any loss of profit or any other commercial damages, including but not limited to special, incidental, consequential, or other damages. For general information on our other products and services or for technical support, please contact our Customer Care Department within the United States at (800) , outside the United States at (317) or fax (317) Wiley also publishes its books in a variety of electronic formats. Some content that appears in print may not be available in electronic formats. For more information about Wiley products, visit our web site at Library of Congress Cataloging-in-Publication Data: ISBN Printed in the United States of America
7 There are two ways of spreading light: to be the candle or the mirror that reflects it. Edith Wharton
8
9 CONTENTS PREFACE xiii PART I BACKGROUND TO COUPLED BIOLUMINESCENT ASSAYS 1 1 Introduction Introduction to Coupled Bioluminescent Assays, Luminescent Technologies of the Life Sciences, Varieties of Fluorometric Assays, Time-Resolved Fluorescence, Fluorescence Resonance Energy Transfer, Fluorescence Polarization, Chemiluminescence and Bioluminescence, Chemiluminescence, Bioluminescence, Common Bioluminescence Systems, Firefly Luciferase, Renilla Luciferase, Bacterial Luciferases, Aequorin, Other Luciferases and Photoproteins, A Coupled Bioluminescent Reaction, Summary, 23 vii
10 viii CONTENTS 2 Coupled Bioluminescent Reactions in Practice Principles of Coupled Bioluminescent Reactions, Requirements for Successful Coupled Bioluminescent Assays, Instrumentation and Equipment for Coupled Bioluminescent Assays, Instrument Testing, Instrument Features, Luminescent Microplates, Coupled Bioluminescent Assay Procedures and Precautions, Coupled Enzyme Assays, Steady-State Kinetics, Coupled Bioluminescent Reactions, Recommended Precautions for Coupled Bioluminescent Reactions, Data Handling for Coupled Bioluminescent Assays, Analysis of Coupled Bioluminescent Assay Results by Time-Dependent Linear Regression, Performing the Linear Regression, Outliers, Special Considerations Relating to High-Throughput Screening, Comparison of Coupled Bioluminescent Assays with Other Methods, Coupled Bioluminescent Assays Versus Spectrophotometric Assays, Coupled Bioluminescent Assays Versus Fluorescent Assays, Coupled Bioluminescent Assays Versus Chemiluminescent Assays, 55 PART II BIOMEDICAL APPLICATIONS OF COUPLED BIOLUMINESCENCE 57 3 Coupled Bioluminescent Cytotoxicity Assays Introduction, Membrane Integrity Assays, Radioactive Isotope Methods, Other Labeling Methods, General Enzyme-Release Methods, A Coupled Bioluminescent Assay for Enzyme Release: The G3PDH-Release Assay, Development and Features, Performance Characteristics, 75
11 CONTENTS ix 3.4 Viability Assays, ATP-Release Assay, Assays of Vital Cellular Functions, Simple Cell-Growth Assays, Total Enzyme-Release Methods, 84 4 The Role of Coupled Bioluminescent Assays in Kinase Screening and Study The Many Roles of Kinases in Biology, Current Standard Kinase Assays, Fluorometric Kinase Assays, Radiolabeling Kinase Assays, Fluorometric Assays Involving Detection of ADP, Coupled Bioluminescent Kinase Assays, A Historical Note: The Coupled Bioluminescent Creatine Kinase Assay, ATP Depletion Kinase Assays, Reverse CB Kinase Assays, Kinases as Participants in Other CB Assays, Conclusions, Coupled Bioluminescent Phosphatase Assays Introduction, Phosphatases, Catalytic Properties of Phosphatases, Classification of Phosphatases, Contemporary Phosphatase Assay Technologies, Fluorometric Phosphatase Assays, Colorimetric Phosphatase Assays, Chemiluminescent Phosphatase Assays, Radiolabeling Phosphatase Assays, CB Phosphatase Assays, Assays Using Phosphorylated Luciferin, Assays Based on the GPL Reaction Series, Challenges in the Development of the GPL-Coupled Bioluminescent System for Phosphate Detection, Potential Drawbacks of GPL Phosphatase Assays, Conclusions, Acetylcholinesterase Introduction, Established AChE Assay Methods, Recent Developments in AChE Assay Methods, 134
12 x CONTENTS 6.4 Evaluation of Current AChE High-Throughput Screening Methods, Coupled Bioluminescent Assays of AChE Activity, Comparison of Coupled Bioluminescent and Other Methods of Measuring AChE Activity, Measurement of Nitric Oxide Synthase Activity by Coupled Bioluminescence Introduction, Nitric Oxide Synthases, The Nitrate Ion in Medicine and NO Biology, NO Biology, Interrelationship Among Nitrate, Nitrite, and NO, Medical Aspects of Nitrate Biology, Current NOS Assays, Measuring NOS Activity by Assays for Nitrite and Nitrate, Direct Assays of NOS Activity, Coupled Enzymatic Assays of NOS Activity, Conclusions, The Coupled Bioluminescent Pyrophosphorolysis Assay Introduction, Genetic Variation in Modern Medicine, DNA Complementarity, DNA Hybridization, Primer Extension in SNP Analysis, Primer Degradation in SNP Analysis, The READIT Pyrophosphorolysis Assay, READIT : Description and Technical Aspects, Performing the READIT Assay, Competitive Position of READIT, Coupled Luminescent Assays of G-Protein-Coupled Receptors Introduction to G-Protein-Coupled Receptors, Structure and Function of GPCRs, GPCRs in Medicine, GPCR Assay Methods, Arrestins as Markers of GPCR Activity, Calcium Quantification in GPCR Assays, Quantitative camp Assays of GPCR Activity, GPCR Assays Involving Inositol Triphosphate Detection, GPCR Assays Involving GTP, GPCR Assays Involving Reporter Genes, Novel GPCR Assays and Other Strategies, Summary, 190
13 CONTENTS xi 10 Coupled Bioluminescent Protease Assays Introduction, Proteases, Protease Assays, Chromogenic Protease Assays, Fluorometric Protease Assays, Exotic Fluorometric Protease Assays, Coupled Bioluminescent Protease Assays, Protease Assays Using Luciferin Derivatives, Coupled Bioluminescent Protease Assays Employing Recombinant Luminescent Proteins, Summary, Coupled Luminescent Assays Involving Aequorin Introduction to Aequorin, Aequorin as the First Photoprotein, The Aequorin Reaction, Detection of Calcium in Practice, Fluorometric Quantification of Calcium, Coupled Bioluminescent Quantification of Calcium Using Aequorin, Real-Time Imaging of Calcium Flux with Aequorin, Other Uses of Aequorin in Coupled Bioluminescent Assays, Coupled Bioluminescent Reporter Assays Introduction to Reporter Assays, Brief History and Development of Reporter Assays, Other Non-Luminescent Reporter Proteins, Luciferases as Reporters of Promoter Activities, The Modern Luminescent Reporter Assay, Multicolor Light Emission by Luciferases, Dual-Mode Reporter Assays in Practice, Aequorin as a Reporter Enzyme, Vectors for Use in Reporter Assays, Systems for Study of Specific Transcriptional Modulators, Bacterial Reporter Systems, Viral Vectors, Summary, 217
14 xii CONTENTS 13 Coupled Bioluminescent Assays: Regulatory Concerns Introduction, Regulatory Aspects of Assay Development, Standard Operating Procedures, Philosophy of Assay Validation, Parameters for Assay Validation, Gaining Regulatory Acceptance of CB Methods, Summary, 240 PART III OTHER APPLICATIONS OF COUPLED BIOLUMINESCENCE Coupled Bioluminescent Determination of Bioburden and Sterility Introduction, Rapid Methods of Bioburden and Sterility Assessment, Bioburden Measurement by ATP-Release Assay, Bioburden Measurement by Protein Assay, Coupled Bioluminescent Methods of Measuring Bioburden, Environmental Applications of Coupled Bioluminescent Assays Introduction, Current Methods for Environmental Monitoring of Water Quality, Methods of Monitoring Stream Water and Lake Water, Monitoring of Phosphate in Freshwater, Monitoring of Nitrate in Groundwater, Monitoring of Pesticides in Freshwater, Monitoring of Bacterial Content of Freshwater, Methods of Monitoring Drinking Water, Current Drinking Water Reports, Biohazard Monitoring in Drinking Water, Monitoring of Chemical Hazards in Drinking Water, 257 Appendix A: One-Letter Amino Acid Abbreviations 259 Glossary 260 Bibliography 265 Index 291
15 PREFACE Every scientist with a career of sufficient length has a favorite story of serendipity. In the 1990s, while studying acceleration of the complement system by antibodies directed against its regulators, I became dissatisfied with the available means of measuring cell death. Either I had to load up the cells with a radioactive label, hope it would not perturb the results, and then clean it up; or I had to wait a long time for a weak signal from lactate dehydrogenase released by the dying cells. It was then that my mentor Bob Kinders said, Why don t you come up with a luminescent assay? Five minutes after Bob posed his question, I was racing upstairs to try the method that eventually became acella-tox TM,acoupled bioluminescent (CB) general cytotoxicity assay. After much help from many people, I obtained my own patent (an experience every life scientist should have at least once), published, found partners, got it on the market, and finally wrote this book. Today, it is not difficult to come up with a new CB assay, but in a sense, all of the developments derive to some degree either from Arne Lundin s ideas as expressed in the late 1970s and 1980s regarding the many possible ways of expanding the use of luciferase in coupled assays or from the important work of Shimomura in isolating and characterizing the first photoproteins. Other academic workers and, notably, vendors such as Promega have driven the field far beyond the initial applications, to the point where there is probably a CB alternative for more than half of all biochemical measurements proposed. The activities of enzymes that are critical in today s medicine, including kinases, phosphatases, proteases, acetylcholinesterase, nitric oxide synthase (NOS), and many others, may be measured rapidly and with extraordinary sensitivity by using this approach. In other cases, such as reporter assays, luciferase activity simply serves as a robust quantitative marker. The power of CB methods to provide information with extraordinary speed and sensitivity is growing xiii
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