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Development of electrophoretically mediated microanalysis method for the kinetics study of flavin‐containing monooxygenase in a partially filled capillary
Author(s) -
Hai Xin,
Konečnỳ Jiří,
Zeisbergerová Marta,
Adams Erwin,
Hoogmartens Jos,
Schepdael Ann Van
Publication year - 2008
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.200800138
Subject(s) - chemistry , microanalysis , substrate (aquarium) , capillary electrophoresis , chromatography , flavin group , analytical chemistry (journal) , kinetics , reaction rate constant , michaelis–menten kinetics , monooxygenase , capillary action , elution , enzyme , enzyme assay , materials science , organic chemistry , oceanography , physics , quantum mechanics , geology , cytochrome p450 , composite material
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°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 µM 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.