Enzyme assay
Enzyme assays are laboratory methods for measuring enzymatic activity. They are vital for the study of enzyme kinetics and enzyme inhibition.
The quantity or concentration of an enzyme can be expressed in molar amounts, as with any other chemical, or in terms of activity in enzyme units.
Enzyme activity = moles of substrate converted per unit time = rate × reaction volume. Enzyme activity is a measure of the quantity of active enzyme present and is thus dependent on conditions, which should be specified. The SI unit is the katal, 1 katal = 1 mol s−1, but this is an excessively large unit. A more practical and commonly used value is enzyme unit (U) = 1 μmol min−1. 1 U corresponds to 16.67 nanokatals.
Enzyme activity as given in katal generally refers to that of the assumed natural target substrate of the enzyme. Enzyme activity can also be given as that of certain standardized substrates, such as gelatin, then measured in gelatin digesting units (GDU), or milk proteins, then measured in milk clotting units (MCU). The units GDU and MCU are based on how fast one gram of the enzyme will digest gelatin or milk proteins, respectively. 1 GDU equals approximately 1.5 MCU.
An increased amount of substrate will increase the rate of reaction with enzymes, however once past a certain point, the rate of reaction will level out because the amount of active sites available has stayed constant.
The specific activity of an enzyme is another common unit. This is the activity of an enzyme per milligram of total protein (expressed in μmol min−1mg−1). Specific activity gives a measurement of enzyme purity in the mixture. It is the moles of product formed by an enzyme in a given amount of time (minutes) under given conditions per milligram of total proteins. Specific activity is equal to the rate of reaction multiplied by the volume of reaction divided by the mass of total protein. The SI unit is katal kg−1, but a more practical unit is μmol mg−1 min−1. Specific activity is a measure of enzyme processivity, at a specific (usually saturating) substrate concentration, and is usually constant for a pure enzyme. For elimination of errors arising from differences in cultivation batches and/or misfolded enzyme etc. an active site titration needs to be done. This is a measure of the amount of active enzyme, calculated by e.g. titrating the amount of active sites present by employing an irreversible inhibitor. The specific activity should then be expressed as μmol min−1 mg−1 active enzyme. If the molecular weight of the enzyme is known, the turnover number, or μmol product per second per μmol of active enzyme, can be calculated from the specific activity. The turnover number can be visualized as the number of times each enzyme molecule carries out its catalytic cycle per second.
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