Specific modification of the carboxyl groups of hemoglobin S

R. Seetharam, J. M. Manning, A. S. Acharya

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16 Scopus citations


The reactivity of the carboxyl groups of hemoglobin S to form amide bonds with glycine ethyl ester by carbodiimide-activated coupling, and the influence of this derivatization on the functional properties of the protein have been investigated. Incubation of carbonmonoxy or oxyhemoglobin S with 20 mM 1-ethyl-3-(3'-dimethylaminopropyl)carbodiimide in the presence of 100 mM [14C]glycine ethyl ester, at pH 6.0 and 23° C for 1 h resulted in the modification of, on an average three carboxyl groups of the protein. The Hill coefficient of the modified hemoglobin S was 2.7. indicating normal subunit interactions. The derivatization increased the oxygen affinity of the molecule (the p50 was lowered from 8.0 to 5.0). The derivatization also resulted in an increase in the minimum gelling concentration of hemoglobin S from 16 to 24 g/100 ml. The reaction conditions used for the derivatization of the carboxyl groups of hemoglobin S are very selective for the protein carboxyl groups; very little of the label is associated with the heme carboxyls. Tryptic peptide mapping of the modified hemoglobin S indicated that the peptide β T5, i.e. the segment representing amino acid residues 41 to 59 of β-chain, accounted for nearly 75% of the label associated with globin, demonstrating the high selectivity of the derivatization. Sequence analysis of the derivatized β T5 demonstrated that at least 65% of the label incorporated into hemoglobin S is targeted toward the carboxyl groups of Glu-43(β), identifying it as the most reactive carboxyl group of hemoglobins. The results suggest that modification of the carboxyl group of hemoglobin S, presumably the γ-carboxyl of Glu-43(β), reduces the propensity of deoxyhemoglobin S to polymerize.

Original languageEnglish (US)
Pages (from-to)14810-14815
Number of pages6
JournalJournal of Biological Chemistry
Issue number24
StatePublished - 1983
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology


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