Study of recombinant cytochrome P450 2C9 activity with diclofenac by micellar electrokinetic capillary chromatography
| Title in English | tudy of recombinant cytochrome P450 2C9 activity with diclofenac by micellar electrokinetic capillary chromatography |
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| Authors | |
| Year of publication | 2007 |
| Type | Article in Periodical |
| Magazine / Source | Electrophoresis |
| MU Faculty or unit | |
| Citation | |
| Field | Biochemistry |
| Keywords | drug metabolism; cytochrome P450 2C9; diklofenac; MEKC |
| Description | Cytochrome P450 2C9 (CYP2C9) is one of the most important isoform in human liver involved in the metabolism of a large number of therapeutic agents. The aim of this communication is to demonstrate the applicability of capillary electrophoresis for the determination of the enzymatic activity of CYP2C9 with diclofenac as a probe substrate. Micellar electrokinetic capillary chromatography (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 small quantity of enzymes and substrate. Moreover, the enzymatic reaction can be monitored with high sensitivity and reproducibility even when the reaction mixture is used for the analysis without any pre-treatment. The kinetic study on the given enzymatic reaction was also performed since basic characterization of drug biotranformation generally begins with the enzyme kinetic analysis of metabolite formation. As a result Michaelis constant and maximum reaction velocity were evaluated, the values Km 3.44 uM, respectively 19.78 nmol.min-1.nmol-1 were in agreement with the literature data. On the other hand the slight deviation from typical Michaelis-Menten kinetics with a weak positive cooperativity was found at diclofenac concentration below 2 uM. The same atypical kinetic behaviour of CYP2C9 was also observed by other authors. |
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