Figure 5.

Oxidation decreases Cp ferroxidase activity. A, Ferroxidase activity was analyzed by Btp assay. Purified Cp (1.25 μg) was incubated with 60 μm FeCl₃ (ferrous form) and analyzed at five different times (0, 15, 30, 45, 60 min) with a solution of 1 mm Btp. The decrease in absorbance at 535 nm of Btp-Fe²⁺ complex is due to ferrous iron oxidation into ferric form (Fe³⁺). Ferroxidase activity was also analyzed after oxidation by H₂O₂ treatments (1, 5, 10 mm). By way of control, the assay was performed in the presence of buffer alone (No Cp). Means with SE are indicated (n = 5). The inset shows linear regression of the Btp-Fe²⁺ complex optical density at 535 nm with different Fe²⁺ micromolar concentrations. B, Btp assay performed with Cp after 50 mm H₂O₂ treatments, with heat-denatured Cp (Den Cp), and with buffer alone containing 20 nm Cu²⁺, the concentration reached by the Cu released from 1.25 μg of denatured Cp.