Question

When running enzyme assays, why do you need to normalize enzyme activity to total protein concentration? What does the specific activity mean in the research lab where enzymes are used? (Hint: are enzymes always pure?)

Answer 1

The enzyme assays include checking or validating the enzyme identity(if the protein preparation contains the desired enzyme), mass purity( percentage of protein in the mixture that can be the targeted enzyme) and activity purity( fraction of activity observed which is performed by a single enzyme). The former( enzyme identity) and the latter( enzyme activity) both are the most critical factors because it is possible that for an assay which has no mass purity the observed activity is 100% which is coming from the target enzyme. This means that there might be enzyme contamination. Therefore it is important that during enzyme assay, the enzyme activity is first normalised to total protein concentration by SDS-PAGE, Reversed phase HPLC, Mass spectrometry.

Specific activity = enzymatic activity (given in defined units)/mass of protein (mg)

It is defined as the measurement of purity of an enzyme.

Specific activity is relative.

The enzyme activity means the amount of substrate which is being converted by an enzyme per unit time of reaction.