Selective carboxymethylation of the alpha-amino groups of hemoglobin. Effect on functional properties.
Hemoglobin A hybrids with carboxymethyl groups at the alpha-NH2 termini of the alpha-chains or the beta-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 alpha- and beta-chains. These derivatized chains were recombined to yield alpha 2 Cm beta 2 Cm or were combined with unmodified beta- or alpha-chains, respectively, and purified to yield alpha 2 Cm beta 2 or alpha 2 beta 2 Cm. These hybrid tetramers retained their cooperativity and function (average n = 2.4). The hybrid alpha 2 Cm beta 2 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 alpha 2 beta 2 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, alpha 2 Cm beta 2 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 alpha 2 Cm beta 2 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-.
D019794:2,3-Diphosphoglycerate; D000328:Adult; D000596:Amino Acids / Q000032:analysis; D002250:Carbon Radioisotopes / Q000032:analysis; D004163:Diphosphoglyceric Acids / Q000494:pharmacology; D006038:Glyoxylates / Q000378:metabolism; D006441:Hemoglobin A / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D046911:Macromolecular Substances; D010084:Oxidation-Reduction; D010100:Oxygen / Q000097:blood; D010446:Peptide Fragments; D014357:Trypsin
D000596:Amino Acids; D002250:Carbon Radioisotopes; D004163:Diphosphoglyceric Acids; D006038:Glyoxylates; D046911:Macromolecular Substances; D010446:Peptide Fragments; D019794:2,3-Diphosphoglycerate; D006441:Hemoglobin A; D014357:Trypsin; C031150:glyoxylic acid; D010100:Oxygen