Publication details
Kinetic study of cytochrome P450 2C9 reaction with diclofenac by MEKC combined with a sweeping technique
| Authors | |
|---|---|
| Year of publication | 2009 |
| Type | Article in Proceedings |
| Conference | Proceedeing of XVI. Euroanalysis symposium |
| MU Faculty or unit | |
| Citation | |
| Field | Biochemistry |
| Keywords | cytochrome P450 2C9; diclofenac; sweeping; MEKC; |
| Description | The early knowledge about the metabolism of a new drug and its affinity to certain drug-metabolising enzymes are especially important in the drug development process. The drug discovery thus continually creates a demand for a development of a new generation of high throughput assays. The majority of these studies are oriented on cytochromes P450 (CYP) because these enzymes play a key role in drug metabolism at human. The aim of this work was to demonstrate the possibility of using MEKC for kinetic study of CYP2C9, one of the most important isoforms in human liver, with diclofenac as a probe substrate. In view of the fact that several studies showed that diclofenac hydroxylation deviated slightly from typical Michaelis-Menten kinetics at low substrate concentration, the combination of sweeping with MEKC was applied in this study. The enzymatic reaction thus could be simply monitored by the analysis of the reaction mixture without any pre-treatment; moreover high sensitivity and repeatability of the separations were preserved. A 50 um fused silica capillary (56 cm effective length) was used to carry out all separations. 60 mM SDS in 20 mM phosphate 20 mM tetraborate buffer pH 8.6 was used as BGE. Injection was accomplished by an application of 50 mbar pressure to the sample vial for 48 s. Separation was performed at 24 kV (positive polarity), with the temperature of capillary 25 oC and detection at 200 nm. As the result Michaelis constant 4.62 uM, maximum reaction velocity 18.35 nmol.min-1.nmol-1 and Hill coefficient 1.22 were determined, which were in agreement with the literature data. Value of Hill coefficient confirms a presence of weak positive cooperativity in low substrate concentration region. The explanation could be found in binding of two substrates in or near the active site of CYP2C9. |
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