Analysis of the Primary Reaction

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 off HPLC Assay for Enzymatic Reaction 

Select the mode of HPLC (size-exdusion, ion-exchange, reversed-phase) that will allow for separation of substrates from products. 

Make initial selection of mobile phase (pH, buffer, salt concentration) and method of delivery (isocratic or gradient elution). 

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Unfortunately, these requirements have not yet fully been met for any catalytic reaction, although for some simple catalytic reactions reasonable approaches are known. Such reactions are the oxidation of CO over a supported Rh catalyst [46,47], ammonia synthesis over iron [48, 49], and the HCN synthesis over a Pt gauze catalyst. More recently Wolf [50] carried out a micro-kinetic analysis of the primary reaction steps in the oxidative coupling of methane and also related the rate... 

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