Development of electrophoretically mediated microanalysis method for the kinetics study of flavin-containing monooxygenase in a partially filled capillary

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Authors

HAI Xin KONEČNÝ Jiří ZEISBERGEROVÁ Marta ADAMS Erwin HOOGMARTENS Jos VAN SCHEPDAEL Ann

Year of publication 2008
Type Article in Periodical
Magazine / Source Electrophoresis
MU Faculty or unit

Faculty of Science

Citation
Field Biochemistry
Keywords Clozapine EMMA flavin containing monooxygenase kinetics
Description An electrophoretically mediated microanalysis method with a partial filling technique was developed for flavin-containing monooxygenase, form 3 (FMO3). The in-line enzymatic reaction was performed in 100 mM phosphate reaction buffer (pH 7.4) whereas 150 mM phosphate buffer (pH 3.3) was used as a background electrolyte. A long plug of cofactor NADPH dissolved in reaction buffer was hydrodynamically injected into a fused-silica capillary, followed by enzyme and substrate solution. The reaction was initiated at 37C in the thermostated part of the cartridge by the application of 9 kV for 0.9 min. The voltage was turned off to increase the product amount (zero-potential amplification) and again turned on at a constant voltage of 10 kV to elute all the components. Direct detection was performed at 191 nm. The developed electrophoretically mediated microanalysis method was applied for the kinetics study of FMO3 using clozapine as a substrate probe. A Michaelis-Menten constant (Km) of 410.3 uM was estimated from the corrected peak area of the product, clozapine N-oxide. The calculated value of the maximum reaction velocity (Vmax) was found to be 1.86 nmol/nmol enzyme/min. The acquired FMO3 kinetic parameters are in accordance with the published literature data.
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