Development of high-performance liquid chromatographic systems for the separation of radiolabelled carotenes and precursors formed in specific enzymatic reactions

High-performance liquid chromatographic (HPLC)systems were developed to separate radiolabelled carotenes and precursors formed in the following carotenogenic enzyme reactions: geranylgeranyl pyrophosphate synthase, phytoene synthase, phytoene desaturase(s), lycopene cyclase and p-carotene hydroxylase. Separations were carried out on reversed- (C,,)and normal-phase columns, using mobile phase mixtures of acetonitrile, methanol and hexane with appropriate modifiers. An isocratic mode of elution was employed throughout, although in several instances isocratic steps were necessary to achieve the desired resolution. The methods developed are specific for each enzyme reaction, resolving substrate from its reaction products and any interfering radiolabelled compounds, thus permitting reliable and accurate determination of enzyme activities. The new separation systems will facilitate further studies on the characterization of these proteins.