Conformational aspects of the interaction of polyanions with liganded β chains of human hemoglobin

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Abstract
The interaction of CO .beta. chains with 2 allosteric effectors, namely inositol hexaphosphate and benzenehexacarboxylate, was studied. The sedimentation coefficient (s20,w) of the liganded .beta. chains was the same in the presence and absence of the 2 effectors, suggesting that the protein existed as a tetramer under the conditions of these titration and optical studies. The binding of benzenehexacarboxylate to the liganded .beta. chains was investigated by potentiometric titration in the pH range 6.7-8.0. The results at pH 7.4 showed a binding of 2 mol of benzene-hexacarboxylate per tetramer, with an association constant of 1.26 .times. 104 l mol-1 at 20.degree. C. The Hill coefficient for the binding was 0.73. Similar experiments on the interaction of inositol hexaphosphate with the .beta. chains showed a binding of 2 mol of the effector/tetramer with identical Hill coefficient (0.737) and comparable association constants (0.88 .times. 104 l mol-1). The value below unity of the Hill coefficient, found for the binding of the 2 effectors to the protein, probably reflected an anticooperativity produced by the different net electric charges of the free protein and the protein-effector complex. The difference in protons bound per mole of heme by the .beta. subunits in the presence and absence of benzenehexacarboxylate appeared consistent with the proposal that 2 groups per chain changed their pK from 6.6 to 7.4 upon the interaction. In the presence of benzenehexacarboxylate, the protonation of these groups appeared to be cooperative, suggesting a conformational change of the protein upon the binding. The absorption spectra of CO .beta. chains in the Soret region was markedly altered by benzenehexacarboxylate and inositol hexaphosphate. The features in the difference spectra of the protein obtained with the 2 effectors were qualitatively identical and indicated changes in the heme environment produced by the interaction of the effectors with the .beta. chains. Concomitant changes in circular dichroism and optical rotatory dispersion of the liganded .beta. chains caused by the addition of the 2 effectors provided supporting evidence for the conformational change in the protein produced by the binding of the effectors.

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