Fluorogenic assay for beta-glucuronidase using microchip-based capillary electrophoresis

Microchip capillary electrophoresis (CE) was used with a model enzyme assay to demonstrate its potential application to combinatorial drug screening. Hydrolysis with beta -glucuronidase of the conjugated glucuronide, fluoresc ein mono-beta -D-glucuronide (FMG), liberated the fluorescent product, fluo rescein. FMG and fluorescein were detected by fluorescence, with excitation and emission at 480 and 520 nm, respectively. Microchip CE was used to sep arate FMG and fluorescein. Fluorescein production was monitored to assess b eta -glucuronidase activity. Michaelis-Menten enzyme kinetics analysis yiel ded the K-m value. The results were compared with those from experiments do ne by conventional CE. The K-m value for beta -glucuronidase with FMG is be ing reported for the first time as 18 muM. The inhibition of beta -glucuron idase by the competitive inhibitor D-saccharic acid-1,4-lactone (SL) was al so determined using microchip CE. Reactions were done with various concentr ations of inhibitor and constant beta -glucuronidase and FMG concentrations . A dose-response plot was acquired and the IC50 value for SL was determine d to be 3 muM. (C) 2001 Elsevier Science B.V. All rights reserved.