Distribution of molecular size Hb cross-links and polymers after H₂O₂ treatment. (A) Silver-stained SDS-PAGE analysis of nonoxidized and H₂O₂-treated Hb/Hb-Hp (5 μg Hb per lane). Hb was mixed with different concentrations of Hp (phenotype 1-1) to get Hp/Hb ratios between 2:1 and 0.25:1. The concentration of Hb was equal in all samples (250 μM in heme). Oxidation was performed with a 10-fold excess concentration of H₂O₂ over heme where indicated (H₂O₂+) for 60 minutes. (B) Western blot analysis of nonoxidized and H₂O₂-treated Hb and Hb-Hp with an anti–α-globin–specific monoclonal antibody (α-globin) and with a polyclonal anti-Hp antibody (Hp). Nonoxidized αα DBBF cross-linked Hb was used as a control to prove α-globin specificity of the anti-Hb antibody. With αα cross-linked Hb there appears a strong signal at approximately 30 kDa that represents αα dimers, but no signal can be observed at 15 kDa where fully dissociated α-globin chains (monomer) appear in HbA₀. (C) In contrast to HbA₀, α-α cross-linked Hb that does not bind to Hp is not protected from polymerization when exposed to H₂O₂ (left panel: [1] marker, [2] HbA0 + H₂O₂, [3] HbA0-Hp + H₂O₂; right panel: [1] α-α Hb + H₂O₂ [2/3] α-α Hb-Hp + H₂O₂ [2 independent reactions]). (D) MALDI-MS analysis of H₂O₂-treated Hb and Hb-Hp. As observed in the α-globin Western blot, α-chains at m/z approximately 15 290 disappear during H₂O₂ treatment of Hb but do not disappear if Hb is oxidized in the presence of equimolar concentrations of Hp.