Spectral changes during the oxidation of Hb by H₂O₂. Spectral changes during the oxidation of Hb by H₂O₂. (A,B) Hb (50 μM in heme) was incubated with 250 μM H₂O₂ at 37°C in 50 mM phosphate buffer, pH 7.4. Spectra were collected in a fast scanning diode-array spectrophotometer. The representative spectra of oxy Hb in the beginning, intermediate ferryl Hb, and ferric Hb at the end of the reaction were shown. (A) Hb without Hp and (B) Hb preincubated with 50 μM Hp. (C-F) The rate of reaction of ferric Hb with H₂O₂ to form ferryl Hb. The reaction rate was evaluated using a stopped-flow equipped with a photodiode array detector (Applied Photophysics). Spectral changes in the Soret and visible regions were recorded as a function of time and H₂O₂ concentration. (C) Representative spectral changes recorded after mixing Hb (4 μM) with excess H₂O₂. (D) Reconstructed spectra of the main reaction species after the global fitting computation of the data set. (E) Concentration changes of the main reaction species over time obtained by the global analysis. The reactions by ferric Hb only or by ferric Hb preincubated with slight molar excess of haptoglobin were carried out as a dependent of [H₂O₂], and plotted in panel F. (F) Second-order rate constants obtained under our experimental conditions for met HbA₀ (●) and met HbA₀ with haptoglobin (○). The derived rate constants are 42 M⁻¹s⁻¹ and 43.6 M⁻¹s⁻¹, respectively.