Identification of oxidatively modified hemoglobin in vivo. Panels A throuch C demonstrate oxidative modification to Hb following hemoglobinuria (Hb-U). (A) The reverse-phase HPLC of hemoglobin within dog urine. During the initial elution time (0-10 minutes) altered heme is shown. At approximately 23 minutes, a heme-altered protein product elutes, consistent with an identical HPLC analysis of CSF.³¹  Neither altered heme nor heme protein adducts are observed in native highly purified stroma-free hemoglobin from plasma (right panel). (B) α-Globin chain cross-links ([M + H] = 30.9 m/z) and stabilized tetramer ([M + H] = 62.6 m/z) from a representative Hb-U sample. This spectra is identical to a representative oxidized Hb sample (1:10, HbA₀/H₂O₂) (OxHb) and demonstrates a similar αα cross-link ion ([M + H] = 30.2 m/z) found in αXLHb. The high-mass ions are absent in nonmodified Hb, which demonstrates only single α- and β-globin chain ions. (C) The full scan spectra of the reduced and alkylated Cys112 peptide (L₁₀₅LGNVLVC₁₁₂ [CH2CONH2]VLAHHFGK₁₂₀ [M + 3H] = 593.14, M = 1719.95 + 56 = 1775.95) shown to be nonoxidized in circulating plasma Hb. Within urine, the same Hb peptide is almost exclusively found as a trioxidation product on Cys112 (cysteic acid) (L₁₀₅LGNVLVC₁₁₂[O3]VLAHHFGK₁₂₀ [M + 3H] = 590.2, M = 1719.95 + 48 [3 oxygens], = 1767.95).