Study of atypical kinetic behaviour of cytochrome P450 2C9 isoform with diclofenac at low substrate concentrations by sweeping-MEKC combination.

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  μm 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 BGE. 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 with 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±0.10  μM, maximum reaction velocity of 9.16±0.16  nmol/min/nmol and Hill coefficient of 1.66±0.08 were determined. This value of Hill coefficient confirms the presence of a positive cooperativity at low diclofenac concentrations and supports the hypothesis of two substrates binding at or near the active site.