Isolated Hb Providence β82Asn and β82Asp Fractions Are More Stable than Native HbA0 under Oxidative Stress Conditions

We have previously shown that hydrogen peroxide (H2O2) triggers irreversible oxidation of amino acids exclusive to the beta-chains of purified human hemoglobin (HbAo). However, it is not clear, whether alpha- or beta-subunit Hb variants exhibit different oxidative resistance to H2O2 when compared to their native HbAo. Hb Providence contains two beta-subunit variants with single amino acid mutations at beta Lys82 -> Asp (beta K82D) and at beta Lys82 -> Asn (beta K82N) positions and binds oxygen at lower affinity than wild type HbA. We have separated Hb Providence into its 3 component fractions, and contrasted oxidative reactions of its beta-mutant fractions with HbAo. Relative to HbAo, both beta K82N and beta K82D fractions showed similar autoxidation kinetics and similar initial oxidation reaction rates with H2O2. However, a more profound pattern of changes was seen in HbAo than in the two Providence fractions. The structural changes in HbAo include a collapse of beta-subunits, and alpha-alpha dimer formation in the presence of excess H2O2. Mass spectrometric and amino acid analysis revealed that beta Cys93 and beta Cys112 were oxidized in the HbAo fraction, consistent with oxidative pathways driven by a ferrylHb and its protein radical. These amino acids were oxidized at a lesser extent in beta K82D fraction. While the 3 isolated components of Hb Providence exhibited similar ligand binding and oxidation reaction kinetics, the variant fractions were more effective in consuming H2O2 and safely internalizing radicals through the ferric/ferryl pseudoperoxidase cycle.