Representative reverse-phase HPLC chromatograms of oxidized (ox) and nonoxidized Hb-Hp products. Gray lines indicate spectra at 280 nm (protein) and black lines indicate spectra at 405 nm (heme). The chromatogram of nonoxidized Hb demonstrates a typical elution pattern of β- and α-globin chains at 22.0 and 23.0 minutes, respectively. H₂O₂ causes decreased absorbance of chains at 280 and coelution with heme at approximately 24.0 minutes. Hp(1-1) elutes at approximately 6.2 minutes (shown as dashed gray and dashed black lines). When complexed with Hb and exposed to H₂O₂, several newly formed Hp-related oxidation products elute between 7 and 8 minutes and at 22.5 minutes. In oxidized Hb-Hp (oxHb-Hp), the Hb β and α chains retain their elution times similar to nonoxidized Hb at 280 nm, indicating intact undamaged protein. Red indicates Hb's β- and α-globin chains, tan indicates heme, and blue indicates Hp.