Structural basis of peroxide-mediated changes in human hemoglobin - A novel oxidative pathway

Hydrogen peroxide (H2O2) triggers a redox cycle between ferric and ferryl hemoglobin (Hb) leading to the formation of a transient protein radical and a covalent hemeprotein cross-link. Addition of H2O2 to highly purified human hemoglobin (HbA(0)) induced structural changes that primarily resided within beta subunits followed by the internalization of the heme moiety within a subunits. These modifications were observed when an equal molar concentration of H2O2 was added to HbA(0) yet became more abundant with greater concentrations of H2O2. Mass spectrometric and amino acid analysis revealed for the first time that beta Cys-93 and beta Cys-112 were oxidized extensively and irreversibly to cysteic acid when HbA(0) was treated with H2O2. Oxidation of further amino acids in HbA(0) exclusive to the beta-globin chain included modification of beta Trp-15 to oxyindolyl and kynureninyl products as well as beta Met-55 to methionine sulfoxide. These findings may therefore explain the premature collapse of the beta subunits as a result of the H2O2 attack. Analysis of a tryptic digest of the main reversed phase-high pressure liquid chromatography fraction revealed two a-peptide fragments (alpha 128 - alpha 139) and a heme moiety with the loss of iron, cross-linked between alpha Ser-138 and the porphyrin ring. The novel oxidative pathway of HbA(0) modification detailed here may explain the diverse oxidative, toxic, and potentially immunogenic effects associated with the release of hemoglobin from red blood cells during hemolytic diseases and/or when cell-free Hb is used as a blood substitute.