Oxidative reactions in normal and sickle hemoglobin-lipid mixtures under various conditions

Hemoglobin (Hb) can undergo autoxidation in erythrocytes releasing Superoxide anion (O{dollar}{bsol}sb2{bsol}sp-{dollar}) radicals. These radicals initiate lipid and protein oxidation causing membrane damage. Our understanding of Hb toxicity and oxidative stress in erythrocytes under physiological and pathophysiological conditions will depend on our knowledge on individual oxidative reactions and the exact role of reaction products generated from Hb oxidation and lipid peroxidation in erythrocytes. Therefore I studied the oxidative reactions in a Hb-small unilamellar vesicle (SUV) system under physiological (35 mM phosphate and 110 mM NaCl at pH 7.4 (P110N 7.4)) and non-physiological (10 mM tris and 5 mM KCl at pH 6.5 buffer (T5K 6.5)) conditions. Under P110N 7.4 buffer I observed adult Hb (HbA) oxidation, destruction of heme, release of free iron (total, in the aqueous phase and lipid phase) and formation of malondialdehyde (MDA). I also observed that Hb undergo cross-linking immediately ({dollar}{bsol}sim{dollar}5 min) after mixing with SUVs. The Hb cross-linking was directly related to Hb oxidation and occurred due to the oxygen radicals that was produced in the process of oxidation reacting with the amino acids at the site of their generation. The heme destruction also occurred due to radicals reacting with the heme and releasing free iron. The iron was present in both the aqueous phase and the lipid phase. Most of the free iron was present in the lipid phase with the increasing the incubation time. Following the t{dollar}{bsol}sb{lcub}1/2{rcub}{dollar} values of total free iron release and MDA formation it was clear that free iron initiated the lipid peroxidation. Sickle Hb (HbS) in SUVs showed similar results as HbA in high salt and high pH (P110N 7.4) conditions. When these experiments were performed in low salt and low pH conditions (T5K 6.5) we observed significant differences between HbA and HbS in oxyHb disappearance and Hb cross-linking and heme transfer into SUV. As the osmolarity was increased by adding KCl we observed a decrease in heme transferred. Similar effects were observed when low Cl{dollar}{bsol}sp-{dollar}, phosphate buffers were used. The percentage of heme transfer was less in phosphate buffers as compared in tris buffers. The differences between HbA and HbS was observed only under low osmolarity and pH in the presence of SUVs. Therefore if sickle RBCs undergo cellular changes decreasing the pH and ionic strength SUVs HbS will show higher rates of oxidation than HbA. It was also clear that the membrane abnormalities that observed in sickle membrane is most likely due to the hemachrome (denatured Hb) build up in the membrane and not due to lipid peroxidation.