Conformational aspects of the interaction of polyanions with liganded beta chains of human hemoglobin.

The interaction of carbon monoxide beta chains with two allosteric effectors, namely inositol hexaphosphate and benzenehexacarboxylate, was studied. The sedimentation coefficient (s20,w) of the liganded beta chains was measured to be the same both in the presence and absence of the two effectors suggesting that the protein exists as a tetramer under the conditions of our 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 benzenehexacarboxylate per tetramer, with an association constant of 1.26 X 10(4) 1. mol-1 at 20 degrees C. The Hill coefficient for the binding was determined to be 0.73. Similar experiments on the interaction of inositol hexaphosphate with the beta chains showed a binding of 2 mol of the effector per tetramer with identical Hill coefficient (0.737) and comparable association constants (0.88 X 10(4)1. mol-1). The value below unity of the Hill coefficient, found for the binding of the two 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 two 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 carbon monoxide 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 two 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 two effectors provided supporting evidence for the conformational change in the protein produced by the binding of the effectors.