Abstract

The cytochrome P450 (P450)-dependent conversion of phenytoin (PHT) to p-hydroxy phenytoin (pHPPH), and tolbutamide (TLB) to 4-hydroxy tolbutamide (hydroxy-TLB), in human liver microsomes was studied in the presence of increasing concentrations (0–4%) of bovine serum albumin (BSA). Therefore, the free fraction (f_u) of PHT and TLB varied. Whereas the f_u of PHT (5 μM) decreased, an increase (3-fold), rather than a decrease in the pHPPH formation rate was observed when BSA (<1%) was present. The stimulation was attributed to a significant decrease in apparentK_m. The change, however, was diminished as the BSA concentration reached 4% (PHTf_u = 0.2), in which the reaction velocity remained the same as that measured in the absence of BSA. Therefore, unchanged K_m (16.2 ± 0.7 μM) and V_max (9.4 ± 0.2 pmol/min/mg of protein) values were determined based on total PHT concentrations, whereas correction for f_u led to an unboundK_m (K_mu) of ∼3.2 μM. Similarly, the metabolism of TLB (50 μM) was enhanced (∼2-fold) in the presence of 0.25% BSA but remained only 35% of the control activity (no BSA) at 1% BSA. However, the remaining activity was higher (3-fold) than that determined with an equivalent free concentration of TLB (4 μM) calculated according to itsf_u (0.08). The difference became less significant when BSA concentration was 4% (f_u < 0.02). Collectively, the results suggest a 2-fold effect of BSA on PHT and TLB hydroxylation: first, facilitation of the reactions via a decrease inK_m; second, a decrease inf_u leading to a drop in reaction rate. For a given P450 reaction, therefore, the effect of BSA may depend upon enzyme affinity, catalytic capacity, and the extent of protein binding.