Anion-linked polymerization of the tetrameric hemoglobin from Scapharca inaequivalvis. Characterization and...

The anion-linked polymerization of the tetrameric hemoglobin from Scapharca inaequivalvis has been characterized by means of sedimentation velocity experiments in terms of its dependence on the binding of other protein ligands, oxygen, and protons. The linkage with oxygen manifests itself at any given anion concentration in the markedly different sedimentation behavior of the oxygenated and deoxygenated protein; whereas the former sediments always as a single peak (congruent to 4.3 S), the latter exhibits bimodal Schlieren patterns with a fast peak that attains congruent to 10 S. A comparison of experimental and computer-simulated (Cox, D. J. (1971) Arch. Biochem. Biophys. 146, 181-195) boundaries shows that the behavior of oxyhemoglobin can be represented by a rapidly reversible dimerization of the native 4.3 S molecule, whereas the behavior of the deoxygenated protein can be described adequately in terms of the polymerization of the native structure into tetramers and octamers. The interplay between the binding of protons and anions is brought out by the different dependence of polymer formation on anion concentration at different pH values. In the case of chloride, polymerization goes through a maximum at around 20-50 mM Cl- at pH 6.3 but decreases monotonically above 5 mM at pH 5.5. On the basis of these data and of the effect of other anions such as phosphate and perchlorate, a tentative picture of the high affinity anion-binding sites has been proposed. From a functional point of view, the hemoglobin polymers are characterized by a lower oxygen affinity and a higher cooperativity than the tetrameric structure; hence, polymerization results in a shift of the lower asymptote of the Hill plots while the upper asymptote is unaltered. The effect of polymerization on oxygen binding has been analyzed in terms of the polysteric linkage scheme (Colosimo, A., Brunori, M., and Wyman, J. (1974) Biophys. Chem. 2, 338-343). The data obtained at pH 6.3 as a function of chloride concentration could be fitted satisfactorily by taking the self-association behavior of the protein into account.