HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHIC ASSAY OF GLYCOSYLTRANSFERASESUSING FLAVONOIDS AS SUBSTRATE

An HPLC method for the determination of glycosyltransferase activity, alternative to the radioactive assay, is proposed. The method is suita ble for following the kinetics of consecutive enzymes that yield monog lucosides, diglucosides and triglucosides, as demonstrated with a pea seedling extract containing a mixture of three glucosyltransferases us ing flavonoids as substrate and UDP-glucose as carbohydrate donor. In this instance the HPLC determination of the three glucosides could be accomplished after separation of the aglycones by solid extraction on a Sep-Pak C-18 microcolumn. After isolation of the enzyme catalysing t he production of the monoglucoside of quercetin (isoquercitrin) or kae mpferol (astragalin), the kinetics of the reaction were determined by HPLC, following both the increase of the product and the disappearance of the substrate. The increasing amounts of isoquercitrin and astraga lin were consistent with the decrease in the amount of aglycone measur ed after direct injection of the reaction mixture into the HPLC system and its elution with a less polar solvent.