Enzyme Assays : A Practical Approach, [vol. 257] / edited by Robert Eisenthal and Michael J. Danson.

Enhanced descriptions from Syndetics:

Enzyme assays are among the most frequently performed procedures in biochemistry and are routinely used to estimate the amount of enzyme present in a cell or tissue, to follow the purification of an enzyme, or to determine the kinetic parameters of a system. The range of techniques used to measure the rate of an enzyme-catalysed reaction is limited only by the nature of the chemical change and the ingenuity of the investigator. This book describes the design and execution of enzyme assays, covering both general principles and specific chapters.Building upon the highly popular first edition, this book combines revised or rewritten chapters with entirely new contributions. Topics include experimental protocols covering photometric, radiometric, HPLC, and electrochemical assays, along with methods for determining enzyme assays after gel electrophoresis. The theory underlying each method is outlined, together with a description of the instrumentation, sensitivity and sources of error. Also included are chapters on the principles of enzyme assay and kinetic studies; techniques for enzyme extraction; high- throughout screening; statistical analysis of enzyme kinetic data; and the determination of active site concentration.This second edition of Enzyme Assays will be valuable not only to biochemists, but to researchers in all areas of the life sciences.

Table of contents provided by Syndetics

• List of protocols (p. xiii)
• Abbreviations (p. xvii)
• 1 Principles of enzyme assay and kinetic studies (p. 1)
• 1 Introduction (p. 1)
• 2 Behaviour of assays (p. 1)
• Reaction progress curves (p. 1)
• Initial rate measurements (p. 5)
• Integrated rate equations (p. 6)
• Bursts and lags in progress curves (p. 7)
• Blank rates (p. 11)
• 3 The effects of enzyme concentration (p. 15)
• Direct proportionality (p. 15)
• Upward curvature (p. 17)
• Downward curvature (p. 18)
• 4 Expression of enzyme activity (p. 18)
• Units and specific activity (p. 19)
• The katal (p. 19)
• Stoichiometry (p. 19)
• Conditions for activity measurements (p. 20)
• 5 The effects of substrate concentration (p. 20)
• The Michaelis-Menten relationship (p. 20)
• Failure to obey the Michaelis-Menten equation (p. 21)
• 6 Experimental approaches (p. 29)
• Type of assay (p. 29)
• Choice of assay method (p. 38)
• The effects of pH (p. 39)
• Practical considerations (p. 40)
• Conclusions (p. 44)
• References (p. 44)
• 2 Photometric assays (p. 49)
• 1 Introduction (p. 49)
• 2 Absorption (p. 49)
• Terminology (p. 49)
• Absorbance (p. 50)
• Limitations and sources of error (p. 52)
• Absorbance range (p. 55)
• Measurement of low rates of absorbance change (p. 55)
• Use of absorbance coefficient (p. 56)
• Continuous assays (p. 58)
• Discontinuous assays (p. 63)
• Examples of enzymes assayed by absorbance change (p. 64)
• 3 Turbidimetry (p. 70)
• 4 Fluorescence (p. 71)
• The fluorescence spectrometer (p. 72)
• Quantitation of fluorescence (p. 72)
• Causes of non-linearity - the inner filter effect (p. 73)
• Examples of fluorimetric enzyme assays (p. 73)
• References (p. 77)
• 3 Radiometric assays (p. 79)
• 1 Introduction (p. 79)
• 2 Techniques (p. 80)
• Ion-exchange methods (p. 80)
• Precipitation of macromolecules (p. 81)
• Solvent extraction methods (p. 81)
• Paper and thin-layer chromatographic (TLC) methods (p. 81)
• Electrophoretic methods (p. 81)
• Scintillation Proximity Assay (SPA) (p. 82)
• 3 Experimental design (p. 98)
• 4 Microplate technology (p. 99)
• 5 Measurement of radioactivity (p. 100)
• 6 Automation of assays (p. 100)
• Acknowledgements (p. 100)
• References (p. 100)
• 4 High performance liquid chromatographic assays (p. 103)
• 1 Introduction (p. 103)
• 2 Theory of HPLC (p. 103)
• Introduction (p. 103)
• Chromatographic parameters (p. 104)
• 3 Retention mechanism (p. 106)
• Characteristics of silica (p. 106)
• Polymeric packings (p. 107)
• Reverse phase chromatography (p. 108)
• Influence of composition of mobile phase (p. 111)
• Effect of pH and salts (p. 112)
• Influence of temperature (p. 112)
• Ion-pair chromatography (p. 113)
• Ion-exchange resins (p. 114)
• Size-exclusion chromatography (p. 116)
• 4 Instrumentation (p. 117)
• Essential components of an HPLC system (p. 117)
• Pumps (p. 117)
• Biocompatibility (p. 118)
• Sample injection (p. 118)
• 5 Detectors (p. 119)
• UV/visible detectors (p. 119)
• Fluorescent detectors (p. 120)
• Refractive-index (RI) detectors (p. 121)
• Electrochemical detectors (p. 122)
• Radioactivity monitors (p. 122)
• 6 Practical considerations (p. 123)
• Selection of a chromatographic mode (p. 123)
• Solvent selection (p. 123)
• De-gassing and filtration of solvents (p. 124)
• Sample preparation (p. 124)
• Column packing (p. 124)
• Column protection (p. 125)
• Tubing (p. 125)
• 7 Application of HPLC to enzymatic analysis (p. 125)
• Hydrolases (p. 125)
• Isomerases (p. 127)
• Lyases (p. 130)
• Ligases (p. 133)
• Oxidoreductases (p. 136)
• Transferases (p. 136)
• References (p. 137)
• 5 Electrochemical assays: the oxygen electrode (p. 141)
• 1 Introduction (p. 141)
• 2 Theory and principles (p. 141)
• 3 Current/voltage relationships (p. 142)
• 4 Sensitivity (p. 142)
• 5 Calibration (p. 142)
• 6 Electrode systems (p. 144)
• 7 Polarographic assays (p. 145)
• Tissue/organelle respiration studies (p. 145)
• Specific enzyme studies (p. 146)
• References (p. 148)
• 6 Electrochemical assays: the nitric oxide electrode (p. 149)
• 1 Introduction (p. 149)
• 2 Principles of detection (p. 149)
• 3 Principles of selectivity and sensitivity (p. 149)
• 4 Environmental influences (p. 150)
• Temperature (p. 150)
• Electrical interference (p. 150)
• 5 Membrane integrity and maintenance (p. 151)
• 6 Calibration (p. 151)
• Calibration for liquid measurements (p. 151)
• Calibration for gas-phase measurements (p. 153)
• 7 NO and cellular respiration studies (p. 153)
• References (p. 155)
• 7 Electrochemical assays: the pH-stat (p. 157)
• 1 Introduction (p. 157)
• 2 The basis of pH-stat methodology (p. 157)
• Principle and general approach (p. 157)
• pH-stat components and their functions (p. 158)
• Some limitations and sources of error (p. 159)
• 3 Commercial and custom-made pH-stat assemblies (p. 159)
• The range of equipment (p. 159)
• Some pH-stat systems described in the literature (p. 159)
• 4 General pH-stat procedure and specific protocols for some individual enzymes (p. 162)
• Procedures (p. 162)
• 5 A systematic error in pH-stat assays of enzymes in haemolysates (p. 168)
• 6 Concluding comment (p. 168)
• References (p. 168)
• 8 Enzyme assays after gel electrophoresis (p. 171)
• 1 Introduction (p. 171)
• 2 Preparation of enzyme extracts (p. 171)
• Extraction of microorganisms (p. 171)
• Animal soft tissues (p. 172)
• Mammalian blood (p. 173)
• Insects (p. 173)
• Plant tissues (p. 173)
• 3 Principles of enzyme visualization (p. 175)
• Methods to visualize oxidative enzymes (p. 175)
• Methods to visualize transferases (p. 177)
• Methods to visualize hydrolases (p. 178)
• Methods to visualize lyases, isomerases and ligases (p. 180)
• 4 A compliation of protocols to visualize enzymes following electrophoretic separation (p. 180)
• Staining protocols (p. 184)
• Buffer systems for electrophoresis (p. 201)
• References (p. 206)
• 9 Techniques for enzyme extraction (p. 209)
• 1 Introduction: scope of the chapter (p. 209)
• 2 Disruption of tissues and cells (p. 210)
• Choice of tissue (p. 210)
• Disruption of tissue and separation of cells (p. 211)
• Disruption of cells (p. 212)
• 3 Protection of enzyme activity (p. 215)
• Control of pH (p. 215)
• Control of temperature (p. 215)
• Control of proteolysis (p. 216)
• Protection of thiol groups (p. 217)
• Protection against heavy metals (p. 217)
• Control of mechanical stress (p. 218)
• Effects of dilution (p. 218)
• 4 Assays of enzymes in unfractionated cell-extracts (p. 219)
• The presence of endogenous inhibitors (p. 219)
• Interference from other reactions (p. 219)
• Removal of substrate (p. 219)
• Turbidity of extract (p. 220)
• 5 Concluding remarks (p. 220)
• References (p. 220)
• Appendix 1 Buffers and control of pH (p. 221)
• Appendix 2 The determination of protein (p. 223)
• References for Appendices (p. 224)
• 10 Determination of active site concentration (p. 225)
• 1 Introduction (p. 225)
• 2 Areas of application (p. 225)
• 3 Categories of titration methods (p. 226)
• Activity bursts (p. 226)
• Inhibitor titration (p. 228)
• Special techniques (p. 230)
• References (p. 233)
• 11 High throughput screening--considerations for enzyme assays (p. 235)
• 1 Introduction (p. 235)
• 2 The drug discovery process (p. 235)
• A historical perspective (p. 235)
• A model of drug discovery (p. 236)
• 3 High throughput screening (p. 237)
• Compounds for screening (p. 239)
• Considerations for high throughput assays (p. 240)
• 4 Enzymatic considerations (p. 245)
• 5 Assay formats for enzymatic HTS (p. 246)
• 6 Automation (p. 246)
• 7 Developments (p. 247)
• Higher density plates (p. 247)
• References (p. 247)
• 12 Statistical analysis of enzyme kinetic data (p. 249)
• 1 Introduction (p. 249)
• 2 Derivation of relationships (p. 250)
• 3 Defining objectives (p. 250)
• 4 Basic assumptions of least squares (p. 251)
• 5 Fitting the Michaelis-Menten equation (p. 252)
• 6 Equations with more than two parameters (p. 258)
• 7 Detecting lack of fit (p. 258)
• 8 Estimating pure error (p. 260)
• 9 Distribution-free methods (p. 262)
• 10 Residual plots (p. 264)
• 11 A note about rounding (p. 267)
• References (p. 268)
• List of suppliers (p. 269)
• Enzyme index (p. 273)
• General index (p. 277)

Author notes provided by Syndetics