Isolated Hb Providence β82Asn and β82Asp fractions are more stable than native HbA(0) under oxidative stress conditions.

We have previously shown that hydrogen peroxide (H(2)O(2)) triggers irreversible oxidation of amino acids exclusive to the β-chains of purified human hemoglobin (HbAo). However, it is not clear, whether α- or β-subunit Hb variants exhibit different oxidative resistance to H(2)O(2) when compared to their native HbAo. Hb Providence contains two β-subunit variants with single amino acid mutations at βLys82→Asp (βK82D) and at βLys82→Asn (βK82N) 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 β-mutant fractions with HbAo. Relative to HbAo, both βK82N and βK82D fractions showed similar autoxidation kinetics and similar initial oxidation reaction rates with H(2)O(2). 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 β-subunits, and α-α dimer formation in the presence of excess H(2)O(2). Mass spectrometric and amino acid analysis revealed that βCys93 and βCys112 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 βK82D 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 H(2)O(2) and safely internalizing radicals through the ferric/ferryl pseudoperoxidase cycle.