QUANTITATION OF ENZYMES AND THEIR SUBSTRATES in Visual Studio .NET

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QUANTITATION OF ENZYMES AND THEIR SUBSTRATES
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of the reaction. Manometric methods have been largely supplanted by more selective instrumental techniques. 3.5.3.3. Calorimetry. Isothermal titration calorimetry (ITC) has recently been proposed as a general method for the determination of enzymatic reaction rates.20 This method is based on the relationship between the power needed to maintain constant temperature and the number of moles of substrate converted. Power, the measured quantity, is related to heat (Q) by differentiation: Power dQ=dt 3:34
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Heat is then directly related to the number of moles of substrate converted, with a proportionality constant of Happ , an experimentally determined apparent molar enthalpy for the reaction: Q n Happ 3:35
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Thus, the measured thermal power for an enzyme reaction may be directly related to the enzymatic reaction rate: Rate d P =dt 1= V Happ dQ=dt 3:36
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where V is the solution volume and [P] is the concentration of reaction product. The sensitivity of this method is directly related to the apparent molar enthalpy of reaction, so that very endo- or exothermic reactions will be most readily followed. Examples of the application of this method to the determination of enzyme kinetic parameters include dihydrofolate reductase, creatine phosphokinase, hexokinase, urease, trypsin, HIV-1 protease, heparinase, and pyruvate carboxylase. 3.6. ULTRA-HIGH-THROUGHPUT ASSAYS (HTA)21 23 Recent developments in the design of miniaturized well plates have demonstrated that many reactions that can be followed using conventional 96-well microtiter plates can be adapted to newer 384- and 1536-well microplates. Motivation for this increased assay capacity has come largely from the pharmaceutical industry, where screening of a large number of drug candidates produced by combinatorial synthesis allows higher productivity and more rapid identi cation of lead compounds. Challenges in high-throughput assay development involve the reduction in assay volume from 200 (96-well) to <1 mL (1536-well) with the concomitant problems of solvent evaporation and the development of instrumentation to reliably determine assay signals from such small sample volumes. Absorbance-based assays are currently the most readily adapted, since path length decreases by only a factor of 2 for the 1536-well plates as compared to the conventional 96-well plates. Such assays have been demonstrated for phosphatase, ATPase, and beta-lactamase, as well as their inhibitors, under conditions where the 1536-well plates had completely lled wells (10-mL total volume).21,22 Further developments are underway to
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produce 9600-well plates for ultra-HTS methods. An excellent summary of HTS and ultra-HTS robotics and instrumentation has recently been published.23 3.7. PRACTICAL CONSIDERATIONS FOR ENZYMATIC ASSAYS With any assay involving enzymes, care must be taken to avoid denaturation and loss of activity due to improper handling and storage conditions. In particular, the following conditions should be avoided for most enzymes: temperatures in excess of 40  C, pH values >9 or <5, and the presence of organic solvents, surfactants, and trace metals. When adjusting the pH of an enzyme solution, acids or bases should be added dropwise along the sides of the vessel, with stirring, to avoid local pH extremes. Enzyme activity is preserved by cold storage. Lyophilized (freeze-dried) proteins are stored in a freezer or refrigerator, while dilute solutions are stored at 2 5  C. Concentrated suspensions of enzymes in ammonium sulfate are usually stable for long periods at 2 5  C. Some enzymes strongly adsorb to glassware. If this is the case, solutions are commonly prepared in the presence of a large (100-fold) excess of an inert protein, such as albumin, to avoid activity losses that would lead to miscalibrations. The reduced forms of the pyridine coenzymes, NADH and NADPH, are sensitive to light (20% loss over 7 weeks), moisture (50% loss in 24 h), and high temperatures (10% loss over 3 weeks at 33  C). The oxidized forms of these coenzymes are more stable, but should also be stored at low temperature. Flavin cofactors are light sensitive, and should be stored in the dark. Buffered solutions should be prepared under sterile conditions, and stored after ultra ltration. Many biochemical buffer systems are excellent growth media for bacteria. SUGGESTED REFERENCES
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H. Bergmeyer, Ed. Methods of Enzymatic Analysis, 3rd ed., Vols. I XII, VCH Publishers, Weinheim, Germany, 1986. R. Eisenthal and M. J. Danson, Eds., Enzyme Assays: A Practical Approach, Oxford University Press, New York, 1992. D. C. Harris, Quantitative Chemical Analysis (6th ed.), W. H. Freeman, New York, 2003. G. G. Guilbault, Enzymatic Methods of Analysis, Pergamon Press, New York, 1970.
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