Contribution of the γ-carboxyl group of glu-43(β) to the alkaline bohr effect of Hemoglobin A

Glu-43(β) of hemoglobin A exhibits a high degree of chemical reactivity around neutral pH for amidation with nucleophiles in the presence of carbodiimide. Such a reactivity is unusual for the side-chain carboxyl groups of proteins. In addition, the reactivity of Glu-43(β) is also sensitive to the ligation state of the protein [Rao, M. J., & Acharya, A. S. (1991) J. Protein Chem. 10, 129-138]. The influence of deoxygenation of hemoglobin A on the chemical reactivity of the γ-carboxyl group of Glu-43(β) has now been investigated as a function of pH (from 5.5 to 7.5). The chemical reactivity of Glu-43(β) for amidation increases upon deoxygenation only when the modification reaction is carried out above pH 6.0. The pH-chemical reactivity profile of the amidation of hemoglobin A in the deoxy conformation reflects an apparent pK_a of 7.0 for the γ-carboxyl group of Glu-43(β). This pK_a is considerably higher than the pK_a of 6.35 for the oxy conformation. The deoxy conformational transition mediated increase in the pK_a of the γ-carboxyl group of Glu-43(β) implicates this carboxyl group as an alkaline Bohr group. The amidated derivative of hemoglobin A with 2 mol of glycine ethyl ester covalently bound to the protein was isolated by CM-cellulose chromatography. The chemical characterization of this derivative of hemoglobin A, involving the separation of the modified β-globin by RPHPLC, isolation of the modified tryptic peptide by RPHPLC, and amino acid sequencing of the modified tryptic peptide, has established that this is a homogeneous derivative of hemoglobin in which both γ-carboxyl groups of Glu-43(β) have been amidated. The amidation of Glu-43(β) increases the O₂ affinity slightly without any influence on the Hill coefficient. A comparison of the Bohr proton titration of hemoglobin A with that of the disubstituted derivative as a function of pH established the contribution of the γ-carboxyl group of Glu-43(β) to the alkaline Bohr effect of the protein. Glu-43(β) is located at the α₁\β₁ interface of hemoglobin A, and the increase in the pK_a of the γ-carboxyl group of Glu-43(β) upon deoxygenation of the protein apparently reflects an increase in the hydrophobicity of the α₁\β₁ interface of the molecule.