Fig. 3.

Time- and concentration-dependent loss of OHEB carboxylation activity and determination of the partition ratio for CYP2J2 during inactivation by MS. (A) CYP2J2 in the reconstituted system was incubated with 0 (●), 1 (○), 3 (▴), 5 (Δ), 10 μM (▪), 20 (□), or 100 μM (▾) MS and aliquots were removed at the times indicated and assayed for residual OHEB metabolism as described under Materials and Methods. The catalytic activity at time zero was used as the 100% control to calculate the initial rate constants for the inactivation (k_obs) for each concentration of MS. (B) Shows the fitting of the initial rate constants as a function of the MS concentrations to the Michaelis-Menten equation. The K_I and k_inact were determined to be 6.1 μM and 0.22 min⁻¹, respectively. (C) Determination of the partition ratio for the inactivation of CYP2J2 by MS. The percentage of catalytic activity remaining was determined as a function of the molar ratio of MS to CYP2J2 as described under Materials and Methods. The partition ratio (∼10) was estimated from the intercept of the linear regression line from the lower ratios of MS to CYP2J2 and the straight line obtained from higher ratios of MS to CYP2J2. The data represent the average of two separate experiments done in duplicate.