Figure 3

Biochemical characterization of the H₂O₂ reactions with human Hb, bvHb, α-α-cross-linked Hb, and the Hb/Hp complex. (a) Time-resolved analysis of the reactions of the different types of Hb in complete endothelial cell culture medium at 37 °C. Spectra were recorded at 2-min intervals and deconvoluted to calculate the concentrations of the individual Hb oxidation states. (b) Heme release and transfer to Hpx from ferric Hb. Serial spectra were recorded from a mixture of ferric Hb (12.5 μM) with a 10-fold molar excess of Hpx. The first spectrum is red (ferric Hb) and the last recorded spectrum is blue (heme-Hpx). The insert shows the reference spectra of ferric Hb (red) and heme-Hpx (blue), respectively. (c) Decay of ferric Hb over time. (d) Radical generation during Hb-H₂O₂ reactions was explored with spin-trapping EPR under the same conditions used for endothelial cell culture (cell culture medium, Hb (2 mg/ml), and GOX (2.5 mU/ml)) in the presence of 50 mM DEPMPO. EPR spectra (left panel) of the different experimental samples were recorded sequentially over time, and the integrated spectral values are plotted in the right panel. The insert shows an anti-DMPO Western blot of Hbs reacted with H2O2 in the presence of DMPO. The signals indicate generation of protein based radicals