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 presence of differing partial pressures of oxygen was studied. In the presence of 15 000 ppm (1.5%) residual oxygen, ascorbate/oxygen‐mediated reactions resulted in an increased rate of autoxidation, modification of the β‐globin, increased oxygen affinity and decreased maximum Hill coefficient. One of the observed modifications to the β‐globin was a 72 Da addition to its N‐terminus. Detailed characterization indicates the modification was an imidazo‐lidinone type structure. Thorough deoxygenation of the hemoglobin solution to <150 ppm of oxygen prior to addition of ascorbate was required to prevent these modifications. Addition of ascorbate to the deoxy hemoglobin (deoxyHb) at pH 8 induced aggregation, eventually leading to precipitation. No such precipitation was observed at pH 7. Long‐term storage of the hemoglobin 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 °C, with no detectable precipitation of the protein. Modifications similar to those observed by the acute studies were observed over the 1‐year period and correlated with disappearance of the added ascorbate.