Selective carboxymethylation of the α-amino groups of hemoglobin. Effect on functional properties

Alberto Di Donato, Wendy J. Fant, A. Seetharama Acharya, James M. Manning

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Abstract

Hemoglobin A hybrids with carboxymethyl groups at the α-NH2 termini of the α-chains or the β-chains or at the termini of both chains have been prepared by reductive alkylation of the protein with glyoxylate and NaCNBH3 under controlled conditions. A hemoglobin derivative, which was selectively carboxymethylated at the NH2-terminal residues, was separated into its α- and β-chains. These derivatized chains were recombined to yield α2(Cm)β2(Cm) or were combined with unmodified β- or α-chains, respectively, and purified to yield α2(Cm)β or α2β2(Cm). These hybrid tetramers retained their cooperativity and function (average n = 2.4). The hybrid α2(Cm)β2 had a lower oxygen affinity (p50 = 12 mm) than unmodified hemoglobin (p50 = 7 mm) and was reactive with 2,3-diphosphoglycerate (p50 = 48 mm). The oxygen affinity of the derivative α2β2(Cm) was lower (p50 = 17 mm) than unmodified hemoglobin and was affected only slightly by 2,3-diphosphoglycerate (p50 = 25 mm). The tetramer carboxymethylated at all 4 NH2-terminal residues, α2(Cm)β2(Cm), exhibited a very low intrinsic oxygen affinity (p50 = 37 mm) that was further lowered to a limiting value of 50 mm by saturating amounts of 2,3-diphosphoglycerate. Each carboxymethyl tetramer, except α2(Cm)β2(Cm), was reactive with chloride to lead to a lower oxygen affinity. These carboxymethylated hybrids (Hb-NH-CH2COO-) may provide a useful model system for studies on the binding of anions to hemoglobin or on the interaction of CO2 with hemoglobin to form the carbamate Hb-NH-COO-.

Original languageEnglish (US)
Pages (from-to)11890-11895
Number of pages6
JournalJournal of Biological Chemistry
Volume258
Issue number19
Publication statusPublished - Dec 27 1983

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ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Di Donato, A., Fant, W. J., Seetharama Acharya, A., & Manning, J. M. (1983). Selective carboxymethylation of the α-amino groups of hemoglobin. Effect on functional properties. Journal of Biological Chemistry, 258(19), 11890-11895.