Figure 7.

HPLC profiles of the products of the reaction of HPr⁺ with various oxidants. Incubations contained HPr⁺ (10 μM) and 0.1 mM DTPA in phosphate buffer (50 mM, pH 7.4) and various oxidants as indicated: chloranil (5 μM), ferricyanide anion (20 μM), peroxynitrite (20 μM), ferricytochrome c (5 μM), horseradish peroxidase (HRP, 5 mU/ml) /H₂O₂ (5μM), Fenton’s reagent [Fe²⁺ (20 μM) and H₂O₂ (1 mM)], or H₂O₂ alone (1 mM). With ferricyanide anion, peroxynitrite and HRP/H₂O₂ system, incubation mixtures were analyzed immediately after mixing the components. With respect to chloranil, ferricytochrome, the Fenton’s reagent or H₂O₂-dependent oxidation of HPr⁺, HPLC analysis was performed after incubation at room temperature for 15–60 min. In the case of ferricytochrome, the reaction was stopped by mixing (1:1) with an ice-cold solution of 0.2 M HClO₄ in MeOH and the supernatant was diluted with phosphate buffer 1 M (pH = 2.6). HPLC traces were recorded at 290 nm. Because of differences in the intensities of absorption peaks, the scale was set individually for each HPLC trace, as shown on the figure.