Figure 7.

Dependence of the activity of H₂O₂ production by HLMs and CYP2C9 baculosomes upon human recombinant cyt b5 addition. (A) Soluble human recombinant cyt b5 strongly inhibited the NADPH-dependent production of H₂O₂ by HLMs. Representative kinetic traces (linear traces) of the increase of the fluorescence (excitation and emission wavelengths of 530 and 590 nm, respectively) monitoring the production of H₂O₂ using the Amplex Red assay in the absence of NADPH and after the addition of only 50 μM NADPH or 50 μM NADPH plus 5 μM cyt b5. The difference between the slope of the linear traces after the addition of 50 μM NADPH minus the slope before the addition of NADPH was used for calculations of the rate of NADPH-dependent H₂O₂ production. The assays were done with 5 μg/ml of HLM protein, see section "Materials and Methods" for further experimental details. (B) Titration with soluble human recombinant cyt b5 of the NADPH-dependent production of H₂O₂ by HLMs. Human recombinant cyt b5 was added at the concentrations indicated in the abscissae to the assay medium for H₂O₂ measurements containing 5 μg/ml of HLM protein and 50 μM NADPH. The results obtained are presented as the percentage of the rate of NADPH-dependent H₂O₂ production measured before the addition of soluble human recombinant cyt b5 (solid squares). Non-linear regression fit of the inhibition curve (solid line) yielded a maximum inhibition of 100% and an inhibition constant of 1.21 ± 0.25 μM cyt b5 (R-square = 0.9613). (C) Soluble human recombinant cyt b5 strongly inhibited the NADPH-dependent production of H₂O₂ by CYP2C9 baculosomes. Representative kinetic traces (linear traces) of the increase of fluorescence (excitation and emission wavelengths of 530 and 590 nm, respectively) monitoring the production of H₂O₂ using the Amplex Red assay in the absence of NADPH and after the addition of only 50 μM NADPH or 50 μM NADPH plus 5 μM cyt b5. The difference between the slope of the linear trace after the addition of 50 μM NADPH minus the slope before the addition of NADPH was used for calculations of the rate of NADPH-dependent H₂O₂ production. The assays were done with 4.25 μg/ml of protein of CYP2C9 baculosomes, see section "Materials and Methods" for further experimental details. (D) Titration with soluble human recombinant cyt b5 of the NADPH-dependent production of H₂O₂ by CYP2C9 baculosomes. Human recombinant cyt b5 was added at the concentrations indicated in the abscissae to the assay medium for H₂O₂ measurements containing 4.25 μg/ml (solid squares) or 17 μg/ml (open squares) of protein of CYP2C9 baculosomes and 50 μM NADPH. The results obtained are presented as a percentage of the rate of NADPH-dependent H₂O₂ production measured before the addition of soluble human recombinant cyt b5. Non-linear regression fit of the inhibition curves (dashed lines) was good (R-square = 0.937 for solid squares and 0.976 for open squares), and yielded a maximum inhibition of 100% and the same inhibition constant, namely 0.53 ± 0.08 and 0.54 ± 0.03 μM cyt b5 with 4.25 and 17 μg/ml of protein of CYP2C9 baculosomes, respectively.