FIGURE 1.
Effects of ligands on the visible absorption spectra of P450 2D6. A, spectra of P450 2D6 isolated in the presence of 50 μm thioridazine (blue), quinidine (green), or prinomastat (red) or in the absence of an exogenous ligand (black) are shown. For comparison, spectral absorbance is expressed as millimolar absorptivity for each complex, and the four spectra were normalized to each other at 477 nm to reduce the effects of experimental variation. The scale for the apparent millimolar absorptivity is shown on the left for wavelengths below 477 nm and on the right for longer wavelengths. The thioridazine and quinidine complexes show an increase in the high spin state of the enzyme as judged by the increased absorbance at 390 and 650 nm as well as decreases in the absorbance at 418 and 568 nm. In contrast, prinomast produces a low spin enzyme as judged by the loss of the 650 nm band and shift of the Soret band to 421 nm. B, concentration-dependent effects of thioridazine on the spectrum of the 10 μm P450 2D6 prepared in the absence of an exogenous ligand are displayed. Thioridazine in water was added to the sample and reference cuvettes in small increments to final concentrations of 10, 21, 31, 40, 50, 60, 70, 80, 99, 149, 198, and 247 μm. The spectrum (blue) of P450 2D6 isolated in the presence of 50 μm thioridazine exhibits a larger proportion of high spin enzyme than evident for P450 2D6 prepared in the absence of an exogenous ligand at saturating concentrations. C, concentration-dependent effects of prinomastat on 10 μm P450 2D6 prepared without exogenous ligands are depicted. The final concentrations of prinomast were 2, 4, 8, 16, 32, 64, and 127 μm. The maximum concentration of DMSO was 0.66% v/v. The arrows indicate the direction of change in response to addition of each ligand in B and C. Prinomastat converts P450 2D6 to a low spin form similar to that isolated in the presence of prinomastat. Estimates of the Kd and ΔAmax for multiple determinations are reported in the text.