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

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.

Development of a CD‐MEKC method for investigating the metabolism of tamoxifen by flavin‐containing monooxygenases and the inhibitory effects of methimazole, nicotine and DMXAA

“…In this approach, the main advantages are extremely minute sample consumption (nL) and complete automation by the integration of all the assay steps. Recently, several in‐line CE methods have been reported for FMO studies . Unfortunately, it was not possible to work in‐line because of the excessive hydrophobic character of TAM.…”

A rapid and sensitive CE method with field‐enhanced sample injection and in‐capillary derivatization for selenomethionine metabolism catalyzed by flavin‐containing monooxygenases

“…The key issue of in-capillary derivatization is the rate of product formation. It is also important to note that high temperature is very difficult to obtain at the capillary inlet where the derivatization takes place, due to the uneven temperature control of a capillary cartridge [35,36]. Thus, fast reaction under mild conditions without using highly toxic chemicals is the specific request for selection of the derivatization reagent.…”

Electrophoretically mediated microanalysis assay for sirtuin enzymes

“…Another crucial consideration in in-capillary enzyme assays is the efficient mixing of substrate and enzyme solutions, which is achieved by the application of a mixing voltage for a short period of time [23][24][25][26][27]. If the electrophoretic mobility of all components is available the voltage and period of time of voltage application can be calculated for the known plug lengths of enzyme and substrate in order to ensure maximum plug overlapping and, thus, a high efficiency of the enzymatic reaction [29,30]. However, the electrophoretic mobility of SIRT1 is not known.…”