Reactivity of Glu-22(beta) of hemoglobin S for amidation with glucosamine.
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 degrees C with 20 mM 1-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 beta-T3 and beta-T5 contained the glucosamine incorporated into the protein. Sequence analysis of glucosamine-modified beta-T3 and beta-T5 demonstrated that the gamma-carboxyl groups of Glu-22 and Glu-43, respectively, had been derivatized with glucosamine. The residue Glu-43(beta) shows a high selectivity toward glycine ethyl ester also, whereas Glu-22(beta) is not reactive toward this amine.(ABSTRACT TRUNCATED AT 250 WORDS)
Acharya
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Seetharam
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D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D005944:Glucosamine / Q000097:blood*; D005971:Glutamates / Q000378:metabolism; D018698:Glutamic Acid; D006451:Hemoglobin, Sickle / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D010100:Oxygen / Q000097:blood; D010446:Peptide Fragments / Q000032:analysis; D011487:Protein Conformation; D014357:Trypsin; D014961:X-Ray Diffraction / Q000379:methods
D000596:Amino Acids; D005971:Glutamates; D006451:Hemoglobin, Sickle; D010446:Peptide Fragments; D018698:Glutamic Acid; D014357:Trypsin; D005944:Glucosamine; D010100:Oxygen