We report here a novel finding that norvaline can be incorporated in place of leucine in recombinant human hemoglobin expressed in Escherichia coli. The presence of the norvaline was confirmed by several analytical methods such as amino acid analysis, peptide mapping, electrospray mass spectrometry, and Edman protein sequencing. It appears that substitution is distributed across both the ␤-and di-␣-globins in purified recombinant hemoglobin. The level of misincorporation correlated with the ratio of the free norvaline/leucine pool available in the cell culture. This suggests that the incorporation of norvaline for leucine occurs through misaminoacylation of tRNA Leu , similar to the misincorporation of norleucine for methionine found in many recombinant proteins expressed in E. coli.

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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 beta-globin, 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 imidazolidinone 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 degreesC, 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.