FIG. 2.

H₂O₂ generation by CPRs and by SUPERMIX. H₂O₂ was quantified using AR/HRP in the presence of NADPH and an NADPH-regenerating system. At the indicated time points, acetonitrile (25%, final concentration) was added to terminate the reactions. To detect H₂O₂, AR/HRP was then added to the reactions and resorufin fluorescence quantified (530 nm exitation/585 nm emission). The amount of H₂O₂ formed in the reactions was calculated using a standard curve. The values are the means ± SD of at least two independent experiments assayed in triplicate. (Panel A) H₂O₂ generation by CPRs. Equal amounts (20 units/well) of recombinant human (dotted line) and purified rabbit enzymes (solid line) were assayed. Note that the human recombinant enzyme is coexpressed with human cytochrome b5. (Panel B) H₂O₂ production by P450-free microsomal preparations from genetically engineered insect cells. Open bars, no protein additions, NADPH only; hatched bars, microsomal proteins (5.0 μg protein) from negative control insect cells (note that these preparations have very low cytochrome c reductase activity, 42 and 29 nmol cytochrome c reduced/min/mg of protein, this is at least 20-fold less than the cytochrome c reductase activity in all other preparations used in these studies), gray bars, recombinant human CPR (40 units, 4.4 μg protein). H₂O₂ production was measured at zero time and after 5 and 10 min. The panel also shows the effects of 10μM DPI on the H₂O₂ generating activity of human CPR after 10 min. (Panel C) Lack of effect of cytochrome b5 on H₂O₂ generation by CPRs. Open bars, “negative control” preparations (5.0 μg protein) enriched with 10 pmol of purified human cytochrome b5; gray bars, 30 units of human CPR coexpressed with cytochrome b5; hatched bars, 20 units of purified rabbit CPR, black bars, 20 units of purified rabbit CPR enriched with 10 pmol of purified human cytochrome b5. (Panel D) Comparison of the H₂O₂ generating activity of CPR (Hm OR + b5) and SUPERMIX, a microsomal preparation containing CPR coexpressed with a numbers of cytochrome P450 enzymes. Microsomal proteins in both experiments contained 20 units CPR activity.