Study of Atypical Kinetic Behaviour of Cytochrome P450 2C9 Isoform with Diclofenac at Low Substrate Concentrations by Sweeping-MEKC Combination
| Authors | |
|---|---|
| Year of publication | 2010 |
| Type | Article in Periodical |
| Magazine / Source | Journal of Separation Science |
| MU Faculty or unit | |
| Citation | |
| Field | Biochemistry |
| Keywords | Sweeping; Cytochrome P450; Diclofenac; Drug metabolism. |
| Description | In view of the fact that several studies have shown that diclofenac hydroxylation by cytochrome P450 2C9 deviated from Michaelis-Menten kinetics at low substrate concentrations, sweeping combined with MEKC was applied for the kinetic study of this pharmacologically important reaction. A 50 um fused silica capillary (56 cm effective length) was used to carry out all separations. 70 mM SDS in 20 mM phosphate 20 mM tetraborate buffer pH 8.6 was used as the background electrolyte. Injection was accomplished by the application of 50 mbar (5 kPa) pressure to the sample vial for 52 s. Separation was performed at 22 kV (positive polarity), with a capillary temperature of 25 C and detection at 200 nm. The higher sensitivity of the sweeping-MEKC combination compared to the simple MEKC method enabled this reaction to be fitted to a Hill kinetic model and confirmed the findings of other authors. A Michaelis constant of 2.91 uM, maximum reaction velocity of 9.16 nmol/min/nmol and Hill coefficient of 1.66 were found. |
| Related projects: |