Acute and long-term stability studies of deoxy hemoglobin and characterization of ascorbate-induced modifications

The reaction of ascorbate with recombinant hemoglobin (rHb1.1) in the prese nce of differing partial pressures of oxygen was studied. In the presence o f 15 000 ppm (1.5%) residual oxygen, ascorbate/oxygen-mediated reactions re sulted in an increased rate of autoxidation, modification of the beta-globi n, increased oxygen affinity and decreased maximum Hill coefficient. One of the observed modifications to the beta-globin was a`72 Da addition to its N-terminus. Detailed characterization indicates the modification was an imi dazolidinone type structure. Thorough deoxygenation of the hemoglobin solut ion to <150 ppm of oxygen prior to addition of ascorbate was required to pr event these modifications. Addition of ascorbate to the deoxy hemoglobin (d eoxyHb) at pH 8 induced aggregation, eventually leading to precipitation. N o such precipitation was observed at pH 7. Long-term storage of the hemoglo bin was carried out by addition of ascorbate to deoxyHb at pH 7. The level of methemoglobin remained at <2% for up to 1 year at 4 degrees C, with no d etectable precipitation of the protein. Modifications similar to those obse rved by the acute studies were observed over the 1-year period and correlat ed with disappearance of the added ascorbate.