NOVEL CAPILLARY ELECTROPHORESIS TECHNIQUE FOR THE STUDY OF PLANT PHENOLIC ENZYMATIC OXIDATION MECHANISMS

A new enzyme assay for the study of phenolic oxidation in reaction mix tures containing more than one substrate is described. The assay uses capillary electrophoresis to directly measure the disappearance of the substrate(s) over time. The appearance of product can be studied at t he same time. The method is rapid, able to be automated, and requires only small amounts of substrate and enzyme. This study found that the rate of phenolic oxidation of a given substrate is influenced by the p resence of other substrates in the reaction mixture. When chlorogenic acid was assayed in a binary mixture containing either apigenin, isoor ientin, or quercetin, both the rate of disappearance and the overall a mount of chlorogenic acid to disappear were reduced, compared to when it was assayed alone. The presence of chlorogenic acid on the other su bstrates tested caused an increase in their rate of disappearance and in the overall amount consumed compared to when they were assayed alon e. This assay system, utilizing rapid separation by capillary electrop horesis, has potential for use with other enzymes, especially when bot h the substrate and product have similar spectral characteristics, pre venting the use of standard spectrophotometric assay systems.