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Enzyme Assays | Open Access Journals
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Journal of Bioanalysis & Biomedicine

ISSN: 1948-593X

Open Access

Enzyme Assays

Enzyme assays are used are used to study the rates of enzyme catalyzed reactions. The essential requirements for enzyme assays are described and frequently occurring errors and pitfalls as well as their avoidance are discussed. The main factors, which must be considered for assaying enzymes, are temperature, pH, ionic strength and the proper concentrations of the essential components like substrates and enzymes. Standardization of these parameters would be desirable, but the diversity of the features of different enzymes prevents unification of assay conditions. Nevertheless, many enzymes, especially those from mammalian sources, possess a pH optimum near the physiological pH of 7.5, and the body temperature of about 37 °C can serve as assay temperature, although because of experimental reasons frequently 25 °C is preferred. But in many cases the particular features of the individual enzyme dictate special assay conditions, which can deviate considerably from recommended conditions. In addition, exact values for the concentrations of assay components such as substrates and enzymes cannot be given, unless general rules depending on the relative degree of saturation can be stated. Rules for performing the enzyme assay, appropriate handling, methodical aspects, preparation of assay mixtures and blanks, choice of the assay time, are discussed and suggestions to avoid frequent and trivial errors are given. Particularities of more complex enzyme assays, including reversible reactions and coupled tests are considered. Finally the treatment of experimental data to estimate the enzyme activity is described. The procedure for determining the initial enzyme velocity and its transformation into defined enzyme units as well as suggestions for documentation of the results are presented.

 

 

 

Enzyme assays are used to study the rates of enzyme catalyzed reactions. The essential requirements for enzyme testing are described and the frequently encountered errors and pitfalls and their avoidance are discussed. The main factors, which must be taken into account for the determination of enzymes, are temperature, pH, ionic strength and appropriate concentrations of essential components such as substrates and enzymes. The normalization of these parameters would be desirable, but the diversity of the characteristics of the different enzymes prevents the unification of the test conditions. However, many enzymes, particularly those from mammalian sources, have an optimal pH close to the physiological pH of 7.5, and the body temperature of around 37 ° C can be used as a test temperature, although for experimental reasons, often 25 ° C is preferred. But in many cases, the particular characteristics of the individual enzyme dictate special test conditions, which can deviate considerably from the recommended conditions. In addition, the exact values ​​of the concentrations of test components such as substrates and enzymes cannot be provided, unless general rules depending on the relative degree of saturation can be stated. The rules for carrying out the enzymatic assay, the appropriate handling, the methodical aspects, the preparation of the mixtures and the assay blanks, the choice of the assay time, are discussed and suggestions for avoiding frequent and trivial errors are given. The particularities of more complex enzyme assays, including reversible reactions and coupled tests, are taken into account. Finally, the processing of experimental data to estimate the enzymatic activity is described. The procedure for determining the initial rate of the enzyme and its transformation into defined enzyme units as well as suggestions for documenting the results are presented.

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Citations: 2821

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