Figure 3.

Oxidation of HE with cyt c³⁺ and ferricyanide. A) HPLC-EC chromatogram of a mixture of HE (50 μM) and cyt c³⁺ (5 μM) incubated for 40 min in phosphate buffer (50 mM, pH 7.4) containing DTPA (0.1 mM). The reaction was stopped and protein precipitated by mixing (1:1) with an ice-cold solution of 0.2 M HClO₄ in MeOH. For HPLC analysis the supernatant was diluted (1:1) with phosphate buffer 1 M, pH 2.6. B) Effect of cyt c³⁺ concentration on HPLC-EC peak areas of selected analytes measured from reaction mixtures containing HE (10 μM) and cyt c³⁺ (0−0.2 mM) incubated for 15 min. Samples were processed as above. C) The dependence of the HPLC peak areas of HE and its oxidation products on the time of incubation with 5 μM cyt c³⁺ and H₂O₂ (50 μM). Other conditions and sample processing are as in A. D) HPLC-EC chromatogram of a reaction mixture consisting of HE (40 μM) and 20 μM K₃Fe(CN)₆ incubated for 10 min in phosphate buffer (50 mM, pH 7.4) containing DTPA (0.1 mM). Prior to analysis, the sample was diluted (1:9) with ice-cold phosphate buffer (1 M, pH 2.6).