Figure 9.

k_offmeasurements of P450 51A1–sterol complexes. In each case, an equimolar concentration of P450 51A1 and each sterol, in one syringe, were mixed with a 20 μM concentration of ketoconazole in the other syringe, in an OLIS RSM1000 stopped-flow spectrophotometer. Full spectra were collected, and the A₃₉₀ and A₄₃₀ data were used in the calculations. At least five individual traces were averaged. Fits were to single exponentials using the OLIS GlobalWorks program. The error estimates were made (for each curve) in the program. A, dihydrolanosterol, 0.054 ± 0.010 s⁻¹; B, 14α-CH₂OH dihydrolanosterol, 0.022 ± 0.001 s⁻¹; C, 14α-CHO dihydrolanosterol, 0.059 ± 0.001 s⁻¹; D, dihydro FF-MAS, 2.0 ± 0.1 s⁻¹. In (D), the change observed after 60 s was not much greater than shown at 4 s (≥90% complete), and accordingly, the 4 s trace was used in the calculation of the k_off rate. FF-MAS, follicular fluid meiosis-activating sterol ((4β,5α)-4,4-dimethyl-cholesta-8,14,24-trien-3-ol).