Strategies for Enzyme Activity Measurements

2 Detecting Enzyme Activity A Case Study of Polygalacturonase Cl.2.1 

Basic Protocol 2 Viscosity-Based Assay for Polygalacturonase Activity C 1.2.5 

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Figure 4.1 Overview of strategy for design of an HPLC method to determine enzymatic activity. The reaction tube contains a mix preparation to measure the activity of an ATP pyrophosphohydrolase, which catalyzes the formation of AMP and PPj from ATP. The mix contains the substrate, ATP the buffer, Tris-HCl and magnesium, a metal cofactor. The addition of a sample from the enzyme fraction initiates the primary reaction and also several secondary reactions. Samples of the incubation mixture are withdrawn at intervals (r( and r2), and the reaction is terminated by injection of the samples onto the HPLC column. A representative analysis of each sample is shown. The amount of each component can be calculated from the area of its peak and is graphed as a function of reaction time.

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