Selective Carboxymethylation of the alfa-Amino Groups of Hemoglobin. Effect on Functional Properties.

Hemoglobin A hybrids with carboxymethyl groups at the a-NH2 termini of the alfa-chain or the beta-chain or at the termini of both chains have been prepared by reductive alkylation of the protein with glyoxylated with NaCNBH3 under controlled conditions. A hemoglobin derivative, which was selectively carboxymethylated at the NH2-terminal residues, was separated into its alfa and beta-chains. These derivatized chains were recombined to yield homogeneous carboxymethylated tetramers or were combined with unmodified beta or alfa-chains, respectively, and purified to yield tetramers carboxymethylated exclusively at alfa or beta-chains. These hybrid tetramers retained their cooperativity and function (average n = 2.4). The hybrid carboxymethylated only at the alfa-chain 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 carboxymethylated only at the beta-chain 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 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 the one carboxymethylated at all 4 NH2-terminal residues was reactive with chloride to lead to a lower oxygen affinity. These carboxymethylated hybrids (Hb-NH-CH,COO-) may provide a useful model system for studies on the binding of anions to hemoglobin or on the interaction of COz with hemoglobin to form the carbamate Hb-NHCOO-.