The Use of HPLC to Establish Optimal Conditions for

2 THE USE OF HPLC TO ESTABLISH OPTIMAL CONDITIONS FOR THE ENZYMATIC REACTION 

The HPLC assay method is particularly useful when it is necessary to obtain initial rate data for a study of an enzymatic activity. Optimal assay conditions for the HPLC must be established first. Usually, the optimization process involves the determination of several variables, such as the optimal substrate concentration, pH, temperature, and enzyme concentration. It is assumed that the reader is familiar with the problems associated with assay conditions such as pH, buffer, and temperature. This chapter discusses only factors that might present problems for the HPLC assay method. For additional information, see the works cited in the General References. 

When studying an enzymatic activity for which an apparent Km value can be obtained from the literature, the determination of the optimum substrate concentration becomes somewhat easier. Thus, a concentration two to three times the Km value (assuming the absence of substrate inhibition ) is adequate for use in early experiments. The literature can provide descriptions of other parameters, such as the pH range, the requirement for activators, and the optimal temperature for the incubation. Armed with this information, 

Remaining to be determined prior to the initiation of the reaction by addition of the enzyme are the amount of enzyme to be added, the time course of the reaction, the time between samplings of the reaction mixture, and the volume of these samples. All four questions can be answered by trial and error using the following scheme. First, an arbitrary enzyme concentration is selected. While any concentration can be used, it should be remembered that in work involving a crude extract, excess protein can clog the column. Therefore, choose the lowest concentration possible. 

Having chosen an enzyme concentration, add enzyme meeting this specification to the reaction mixture to start the reaction. Sampling of the reaction mixture, by the withdrawal of a predetermined quantity for analysis, can begin at any time. 

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This chapter presents a strategy for the design of an HPLC assay system. Section 4.1, Setting up the Assay, focuses on the enzymatic reaction and the steps leading to the development of the assay. These steps are previewed in Table 4.1. Section 4.2, The Use of HPLC to Establish Optimal Conditions for the Enzymatic Reaction, discusses the procedure for monitoring the activity of an enzyme with the HPLC method and the use of HPLC assays to determine the parameters required for obtaining optimal activity. 

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