A recombinant human hemoglobin with asparagine‐102(β) substituted by alanine has a limiting low oxygen affinity, reduced marginally by chloride

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Abstract
A recombinant (r) mutant hemoglobin (Hb) with Asn‐102(β) replaced by an Ala (N102A(β)) has been prepared by PCR amplification of a mutagenic DNA fragment and expression of the recombinant protein in yeast. The side chain of Asn‐102(β) is part of an important region of the α1β2 interface that undergoes large structural changes in the transition between the deoxy and oxy conformations. Three natural mutant Hbs with neutral substitutions of Thr, Ser, or Tyr at this site have low oxygen affinities because a hydrogen bond between Asn‐102(β) and Asp‐94(α) in normal HbA was considered to be absent in these mutants, thereby destabilizing the oxy conformation in favor of the deoxy conformation. This proposal has been tested by expression of an rHb containing alanine at position 102(0); alanine was chosen because its methyl side chain cannot participate in hydrogen bond formation, yet it is small enough not to disrupt the subunit interface. The nature of the desired replacement was established by sequencing the entire mutated β‐globin gene as well as the tryptic peptide containing the substitution. Further characterization by SDS‐PAGE, isoelectric focusing, HPLC analysis, mass spectrometry, amino acid analysis, and sequencing of the mutant tryptic peptide confirmed the purity of the rHb. Its oxygen binding curve (2.4 mM in heme) in the absence of chloride showed that it had a very low oxygen affinity with a P50 of 42 mm Hg. In the presence of added chloride (0.5 M), its oxygen affinity was further reduced only slightly to a P50 of 49 mm Hg. In contrast, the oxygen affinity of HbA was lowered two‐ to threefold by the same concentration of chloride. Comparison of the properties of the rHb N102A(β) with those of the rHb with Asp‐99(β), which is at this same subunit interface, substituted by a Lys (Yanase H, et al., 1994, Protein Sci 3:1213–1223), demonstrates how the judicious choice of the amino acid substitution based upon the properties of natural mutations at a particular site can further enhance our understanding of the role of certain amino acid side chains in Hb function.