Formation and Detection of Highly Oxidized Hemoglobin Forms in Biological Fluids during Hemolytic Conditions

Figure 4

Decay of ferrylHb leads to the regeneration of metHb and formation of gmoxHb multimers. (a–c) Purified human metHb (60 μmol/L heme) was oxidized with different concentrations of H₂O₂ (125, 250, and 500 μmol/L), and the concentrations of different redox states of Hb were determined every minute for 10 minutes. (a) Time- and dose-dependent formation and decomposition of ferrylHb in the reaction between metHb and H₂O₂ presented as of 3 independent experiments. (b) ferrylHb decomposition rate (μmol ferrylHb/min) was calculated at each H₂O₂ concentration from the kinetic measurements. Graph shows of 3 independent experiments. (c) Time-dependent inverse changes of ferrylHb and metHb levels in the course of metHb oxidation with H₂O₂ (500 μmol/L) presented as of 3 independent experiments. (d, e) Time- and dose-dependent formation of gmoxHb in the course of metHb oxidation with H₂O₂. Purified human metHb (60 μmol/L heme) was oxidized with different concentrations of H₂O₂ (125, 250, and 500 μmol/L) for 1 and 10 minutes at 37°C. (d) Representative western blot is shown. (e) Densitometric analysis of western blots was performed, and the percentages of gmoxHb monomers, dimers, and tetramers as a percent of total Hb were calculated. The bar graph shows from 3 independent experiments. values were calculated using one-way ANOVA followed by Tukey’s multiple comparison analysis. , , .

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