Interactions of hemoglobin with hydrogen peroxide alters thiol levels and course of endothelial cell death

We investigated cellular injury and death induced by ultrapure human Hb (HbA(0)) and its diaspirin cross-linked derivative DBBF-Hb in normal and glutathione (GSH)-depleted bovine aortic endothelial cells subjected to hydrogen peroxide (H2O2). HbA(0) underwent extensive degradation and heme loss, whereas DBBF-Hb persisted longer in its ferryl (Fe4+) form. The formation of ferryl HbA(0) or ferryl DBBF-Hb was associated with a significant decrease in endothelial cell GSH compared with the addition of H2O2 or Hbs alone. This effect was inhibited by catalase, but not by superoxide dismutase or deferoxamine mesylate. The presence of HbA(0) and DBBF-Hb reduced H2O2-induced apoptosis, as measured by cell morphology, annexin V binding assay, and caspase inhibition, consistent with the ability to consume H2O2 in an enzyme-like fashion. However, the pattern of cell death and injury produced by HbA(0) and DBBF-Hb appeared to be distinctly different among proteins as well as among cells with and without GSH. These findings may have important implications for the use of cell-free Hb as oxygen therapeutics in patients with coexisting pathologies who may lack antioxidant protective mechanisms.