Premium
New capillary electrophoretic method for on‐line screenings of drug metabolism mediated by cytochrome P 450 enzymes
Author(s) -
Řemínek Roman,
Zeisbergerová Marta,
Langmajerová Monika,
Glatz Zdeněk
Publication year - 2013
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.201300124
Subject(s) - chemistry , cytochrome , enzyme , laminar flow , cytochrome p450 , chromatography , substrate (aquarium) , analytical chemistry (journal) , biochemistry , thermodynamics , biology , physics , ecology
A new method for the determination of kinetic and inhibition parameters of cytochromes P 450 reactions by means of on‐line CE was developed. It is based on transverse diffusion of laminar flow profiles methodology introduced by K rylov et al. that injection procedure was modified. The solutions of an enzyme and its substrates are injected by hydrodynamic pressure as a series of repeated consecutive plugs. Proposed injection of three plugs of enzyme surrounded with plugs of substrates represents a certain trade‐off to obtain the reaction mixture with the satisfying homogeneity by the short‐injection procedure as possible. Mathematical modeling confirmed the assumption of a consistent distribution of reactants in the final reaction mixture. Kinetic and inhibition studies of cytochrome P 450 isoform 2 C 9's reaction with diclofenac as a probe substrate and sulfaphenazole as a probe inhibitor were conducted in order to prove the practical applicability of the proposed method for on‐line screenings of drug metabolism mediated by cytochrome P 450 enzymes. As a result, an apparent M ichaelis constant of 2.66 ± 0.18 μM, apparent maximum reaction velocity of 7.91 ± 0.22 nmol min −1 nmol −1 , H ill coefficient of 1.59 ± 0.16, half maximal inhibitory concentration of 0.94 ± 0.04 μM and apparent inhibition constant of 0.39 ± 0.07 μM were determined. All these values are in agreement with literature data obtained using different techniques. In addition, less than 30 nL of cytochrome P 450 2 C 9 solution was consumed per analysis in the kinetic and inhibition studies using this method.