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

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^-.