Study of recombinant cytochrome P450 2C9 activity with diclofenac by MEKC.
Cytochrome P450 2C9 (CYP2C9) is one of the most important isoforms in human liver involved in the metabolism of a large number of therapeutic agents. The aim of this paper is to demonstrate the applicability of CE for the determination of the enzymatic activity of CYP2C9 with diclofenac as a probe substrate. MEKC with SDS as a pseudostationary phase was used for this purpose. Compared to other assays, the MEKC-based method is rapid, can be automated and requires only a small quantity of enzymes and substrate. Moreover, the enzymatic reaction can be monitored with high sensitivity and repeatability even when the reaction mixture is used for the analysis without any pretreatment. The kinetic study on the given enzymatic reaction was also performed since the basic characterization of drug biotransformation generally begins with the enzyme kinetic analysis of metabolite formation. As a result, the Michaelis constant and maximum reaction velocity were evaluated, the values 3.44 +/- 0.45 microM and 19.78 +/- 0.76 nmol min(-1) nmol(-1), respectively, were in agreement with the literature data. On the other hand, a slight deviation from typical Michaelis-Menten kinetics with a weak positive cooperativity was found at diclofenac concentrations below 2 microM. The same atypical kinetic behavior of CYP2C9 was also observed by other authors.
Konecný
Jiri
J
Department of Biochemistry, Faculty of Science, Masaryk University, Brno, Czech Republic.
Jurica
Jan
J
Tomandl
Josef
J
Glatz
Zdenek
Z
D001189:Aryl Hydrocarbon Hydroxylases / Q000378:metabolism*; D020374:Chromatography, Micellar Electrokinetic Capillary / Q000379:methods*; D065729:Cytochrome P-450 CYP2C9; D004008:Diclofenac / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D011994:Recombinant Proteins / Q000378:metabolism; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D011994:Recombinant Proteins; D004008:Diclofenac; C450260:CYP2C9 protein, human; D065729:Cytochrome P-450 CYP2C9; D001189:Aryl Hydrocarbon Hydroxylases