As indicated in Table 4.1, the design of an HPLC assay system for an enzymatic activity begins with a complete analysis of the primary reaction—the reaction catalyzed by the enzyme under study. To begin this analysis, indicate all substrates, products, and cofactors of the reaction. If metals are required for catalysis, include them. In the case of the metals, however, it is useful to note whether they are an integral part of the substrate (e.g., when the complex MgATP is the substrate) or whether they are required for some other function (e.g., activation of the enzyme). It is also useful to indicate the pH of the reaction as well as the type and concentration of the buffer to be used. The goal of this analysis is to list all the components present in the reaction mixture before the start of the reaction.
To illustrate this approach, consider the assay of a pyrophosphohydrolase, an enzyme that catalyzes the reaction
TABLE 4.1 Steps in Design of HPLC Assay for Enzymatic Reaction
1. Analyze the primary reaction.
2. Analyze all secondary reactions.
3. Select the mode of HPLC (size-exclusion, ion-exchange, reversed-phase) that will allow for separation of substrates from products.
4. Make initial selection of mobile phase (pH, buffer, salt concentration) and method of delivery (isocratic or gradient elution).
5. Select appropriate detector. Determine whether it will be necessary to collect fractions.
MgATP is the substrate, and AMP and pyrophosphate (PPj) are the products. Since this activity is usually assayed at a pH of 7.5 using a Tris-HCl buffer system, the reaction tube will contain ATP, Mg, and Tris-HCl as illustrated in Figure 4.1.
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