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, fluorescein mono-beta-D-glucuronide (FMG), liberated the fluorescent product, fluorescein. FMG and fluorescein were detected by fluorescence, with excitation and emission at 480 and 520 nm, respectively. Microchip CE was used to separate FMG and fluorescein. Fluorescein production was monitored to assess beta-glucuronidase activity. Michaelis-Menten enzyme kinetics analysis yielded the Km value. The results were compared with those from experiments done by conventional CE. The Km value for beta-glucuronidase with FMG is being reported for the first time as 18 microM. The inhibition of beta-glucuronidase by the competitive inhibitor D-saccharic acid-1,4-lactone (SL) was also determined using microchip CE. Reactions were done with various concentrations of inhibitor and constant beta-glucuronidase and FMG concentrations. A dose-response plot was acquired and the IC50 value for SL was determined to be 3 microM.
Starkey
D E
DE
Department of Chemistry, University of Cincinnati, OH 45221-0172, USA.
Han
A
A
Bao
J J
JJ
Ahn
C H
CH
Wehmeyer
K R
KR
Prenger
M C
MC
Halsall
H B
HB
Heineman
W R
WR
D019075:Electrophoresis, Capillary / Q000295:instrumentation* / Q000379:methods; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005966:Glucuronidase / Q000032:analysis* / Q000037:antagonists & inhibitors; D007700:Kinetics; D008904:Miniaturization; D013050:Spectrometry, Fluorescence / Q000379:methods*
D004791:Enzyme Inhibitors; D005966:Glucuronidase