Improvement of thin-layer chromatography for enzyme assay of geranylgeraniol 18-hydroxylase from Croton stellatopilosus Ohba

Abstract

Plaunotol, an acyclic diterpenoid present in Croton stellatopilosus Ohba leaves, is a product of hydroxylation, catalyzed by geranylgeraniol-18- hydroxylase (GGOH-18-hydroxylase), in the last step of plaunotol biosynthesis. The activity of GGOH-18-hydroxylase in cell-free extracts of C. stellatopilosus Ohba leaves was previously determined by quantification of plaunotol separated by TLC with chloroform-n-propanol as mobile phase. Interference from endogenous constituents caused variation of GGOH-18-hydroxylase activity, however, which affected the accuracy of this enzyme assay. In this study, we investigated new appropriate conditions for sample preparation and TLC to improve the separation of plaunotol in the reaction mixture of the crude enzyme extracts. By using ethyl acetate as mobile phase for TLC analysis we could determine GGOH-18-hydroxylase activity in both the 20,000g and 100,000g precipitates of the crude extracts of C. stellatopilosus. Ethyl acetate could, moreover, also be used to separate plaunotol from a variety of cytochrome P-450 inhibitors (ancymidol, metyrapone, and miconazole) frequently used for biochemical characterization of the hydroxylase enzymes by TLC. © 2009 Akadémiai Kiadó.