Title: Development of electrophoretically mediated microanalysis method for the kinetics study of flavin-containing monooxygenase in a partially filled capillary
Authors: Hai, Xin ×
Konecny, Jirí
Zeisbergerová, Marta
Adams, Erwin
Hoogmartens, Jos
Van Schepdael, Ann #
Issue Date: Sep-2008
Series Title: Electrophoresis vol:29 issue:18 pages:3817-3824
Abstract: 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 37 degrees C 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 (K(m)) of 410.3 microM was estimated from the corrected peak area of the product, clozapine N-oxide. The calculated value of the maximum reaction velocity (V(max)) was found to be 1.86 nmol/nmol enzyme/min. The acquired FMO3 kinetic parameters are in accordance with the published literature data.
ISSN: 0173-0835
Publication status: published
KU Leuven publication type: IT
Appears in Collections:Pharmaceutical Analysis
× corresponding author
# (joint) last author

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