Indicator reaction, enzymatic analysis

The advantages of CE for analysis of enzymes are the use of small volumes, versatility, and ability to avoid the extra steps of indicator reactions. We found in some instances, for example, analysis of glutathione transferase it is easier to assign enzymatic activity units (lU) based on the CE because both the substrate and the products can be monitored at the same wavelength. Unfortunately, researchers did not take full advantage of the CE for enzyme analysis. In practice, kinetic spectrophotometric methods remain to be most widely used for routine work while the CE is reserved for those difficult and specialized tests. 

The pH of the buffer solution in which the analysis takes place must also be controlled. Most enzymes have a pH dependence of their activity of the type shown in Figure 4 B, and pH should be controlled to within 0.02. The optimum pH, and the range over which the enzyme is active, vary widely from one enzyme to another. These phenomena arise from the effects of pH on the structure of the enzyme itself, on the affinity constant between the enzyme and the substrate, on V ax. and also on any coupled indicator reaction that is used. The choice of buffer recipe for any given pH may be important. The ionic strength of the buffer and the salts contained in it can influence the rate and mechanism of the main and coupled reactions, sometimes with unpredictable results. It may be necessary to choose a buffer whose properties represent a compromise between the ideal conditions for the main reaction, and the ideal conditions for the indicator leaction(s). Immobilized enzymes (Section 9.1.7) often have pH (and temperature) dependence significantly different from their soluble counterparts. This all points to the value of establishing and maintaining a well-defined buffer system for enzymatic analyses. 

The oxidized and reduced forms of the pyridine cosubstrate are readily distinguished by absorbance readings at 340 nm (Fig. 2.5). Therefore, whenever possible, enzymatic reactions which are difficult to measure directly are coupled with an NAD(P)-dependent indicator reaction (cf. 2.6.1.1) for food analysis. 

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. 

The enzymatic assay is then described, including buffers and pH, the method for initiating the reaction, and the process used for termination. Next, the methods used in the preparation of the sample for HPLC analysis are described, including centrifugation, filtration, or any type of purification preceding injection into the HPLC system. For many of the assays, time span and range of protein concentration for which the reaction is linear are also indicated. 

As already indicated, a special problem with esters is their preparation from two natural precursor molecules by a chemical ester synthesis. Such products have to be labelled nature-identical. For an interesting positional H-NMR study on ethyl butyrate from enzymatic esterification of beet ethanol with butyric acid from milk see [317]. Another chance to detect a corresponding adulteration would be a positional carbon and oxygen isotope analysis of the ester components. Isotope effects on the esterification reaction in question seem to influence characteristically the 8-values of the atoms involved, and hence form a basis for the origin assignment of these compounds (for further details see 6.2.2.4.4). 

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