Effects of polyvalent anion binding to hemoglobin on oxygen and oxidation-reduction equilibria and their relevance to allosteric transition

Abstract

The reaction of human hemoglobin and some of its derivatives with aliphatic and aromatic compounds carrying from two to six carboxylate groups has been studied. The effect of the polycarboxylates as well as of three co-ordinate anions (respectively tri-, tetra- and pentavalent) on the oxygenation and oxidation-reduction equilibria and optical spectra have been compared to those of 2,3-diphosphoglycerate and inositol hexaphosphate. All the polyvalent anions raise the P 0.5 ‡ ‡ Abbreviation used: P 0.5, oxygen pressure at 50% oxygenation; E m, half-reduction potential; Hb BME bis( N-maleimidomethyl) ether derivative of human hemoglobin; Hb NES desArg, N-ethylsuccinimide derivative of human hemoglobin subjected to the action of carboxypeptidase B, which splits the C-terminal arginine residues of α chains; DPG, 2,3-diphosphoglycerate; IHP, inositol hexaphosphate; BHC, benzene hexacarboxylate; BPC, benzene pentacarboxylate. and the E m values of human hemoglobin and are thus bound more strongly to deoxyhemoglobin than to oxy- or methemoglobin. Binding of benzene hexacarboxylate and benzene pentacarboxylate to oxyhemoglobin is demonstrated through a study of oxygenation curves, that of these reagents, and ferrocyanide, to methemoglobin through their effect on redox potential as well as on optical spectra. Methemoglobin and oxyhemoglobin are shown to bind more than one molecule of the carboxylates at high anion concentrations. Results bearing on the anion binding site for deoxy- as well as for methemoglobin are reported. Two appropriate human hemoglobin derivatives, namely Hb BME and Hb NES desArg have been examined in search of relations between the effect of anions on oxygen equilibria and that on quaternary structure: in both of these derivatives the chemical modifications inhibit quaternary conformational change that would result from oxygen binding, the deoxy structure being strongly destabilized. Several of the polyanions significantly raise the P 0.5 values of these derivatives but do not modify the quaternary structure, as judged from the absence of characteristic spectral changes. The results imply that anion binding by these proteins somehow inhibits the change in tertiary structure produced by oxygen binding; similar considerations may also apply in the case of the normal hemoglobin-diphosphoglycerate complex.