Studies on the Chemistry of Human Hemoglobin

This dissertation is concerned with the confirmation of adult human hemoglobin in solution. It is now well established that one form of the hemoglobin molecule is composed of four polypeptide chains, two α chains, and two β chains, held together by non-covalent bonds. The linear sequence of amino acids in both the α chains and the β chains of human hemoglobin and the three-dimensional arrangement of the polypeptide chains in the crystal of horse hemoglobin have been recently elucidated. Nevertheless, the relationships between the structure and the function of the molecule, although subjected to numerous investigations, are yet to be well understood especially on the basis of the Adair hypothesis. The function of hemoglobin is closely related to the state of the SH groups in the molecule. It was shown that of the six.SH groups in the hemoglobin tetramer, the two reactive ones in the native molecule at neutral pH are those of cysteine residue 93 on each β chain. When these SH groups have reacted with either iodoacetamide or N-ethylmaleimide, the chromatographic properties (and by inference the conformation) of the hemoglobin changes dramatically. The lack of reactivity of the other four SH groups in the tetramer, one on each α chain and one on each β chain, was attributed to the interactions between the polypeptide chains. The mechanism of dissociation of hemoglobin into subunits was then studied both by analyzing the reactivity, and chromatographic behavior of the products of the reaction, of hemoglobin with iodoacetamide and Nethylmaleimide under various conditions of pH and ionic strength and by the technique of thin film dialysis. The dissociation was found to be symmetrical according to the equations: where K1 and K2 are the dissociation constants. By the method of thin film dialysis, the values of the constants K1 and K2 were found for a variety of solvent conditions. The pH dependence of the dissociation in solutions of moderate ionic strength was found to resemble closely the data in the literature. However, in strong salt solutions at neutral and acid pH\u27s, no evidence for a shift of the equilibrium in favor of dimers was found. It was concluded that under all conditions hemoglobin is to be regarded as a solute in rapid association-dissociation equilibrium. The implications of the dynamic feature of the molecule with regard to its function were discussed as a possible alternative to the Adair hypothesis