X-ray diffraction analysis of deoxyhemoglobin S crystals has implicated that a number of carboxyl groups of the protein are present at or near the intermolecular contact regions. The reactivity of these or other carboxyl groups of hemoglobin S for the amidation with an amino sugar, i.e., glucosamine, and the influence of amidation on the oxygen affinity and polymerization have been investigated. Reaction of oxyhemoglobin S at pH 6.0 and 23 °C with 20 mM l-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC) and 100 mM [3H]glucosamine for 1 h resulted in an incorporation of nearly two residues of glucosamine per tetramer. The amidation was very specific for the carboxyl groups of globin; the glucosamine was not incorporated into the heme carboxyls. Derivatization of hemoglobin S by glucosamine increased the O2 affinity of the protein but had no influence on either the Hill coefficient or the Bohr effect. Amidation by glucosamine also increased the solubility of deoxyhemoglobin S by about 55%. Tryptic peptide mapping of the modified hemoglobin S indicated that the peptides β-T3 and β-T5 contained the glucosamine incorporated into the protein. Sequence analysis of glucosamine-modified β-T3 and β-T5 demonstrated that the γ-carboxyl groups of Glu-22 and Glu-43, respectively, had been derivatized with glucosamine. The residue Glu-43(β) shows a high selectivity toward glycine ethyl ester also, whereas Glu-22(β) is not reactive toward this amine. The results demonstrate that the selectivity of amidation by glucosamine is distinct from that of glycine ethyl ester and is apparently related to the differences in propensity of the carbodiimide-activated γ-carboxyl groups of Glu-22(β) and Glu-43 (β) to undergo aminolysis with these two amines.
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