High resolution nuclear magnetic resonance spectra of hemoglobin: III. The half-ligated state and allosteric interactions

Abstract

The high resolution proton nuclear magnetic resonance spectra of the halfligated valency hybrids ( α III CN β II ) 2 and ( α II β III CN) 2 of human hemoglobin have been studied at 220 MHz. It has been possible to resolve and identify resonances from the α III CN or the β III CN subunits and from the deoxygenated subunits. Depending upon solution conditions, the cyanoferric subunits of both hybrids gave spectra which were either very similar to or definitely changed from those observed in fully ligated hemoglobin. For example, in the ( α III CN β II ) 2 hybrid at pH > 7.3 in the absence of phosphates, the α III CN subunit spectrum looked very much like that observed in ( α III CN β II O 2) 2. Upon addition of one mole of 2, 3-diphosphoglycerate per tetramer of hemoglobin the nuclear magnetic resonance spectrum changed. A similar change was observed at pH 7.1 in 0.1 m-phosphate. The other hybrid ( α II β III CN) 2 did not change its spectrum significantly under these conditions but did change with the addition of a slight molar excess of inositol hexaphosphate. Under certain conditions, the changed and unchanged spectra of the ( α III CN β II ) 2 hybrid were only slowly exchanging with each other, since in the presence of less than stoichiometric amounts of inositol hexaphosphate the two types of spectra were superimposed. These two distinct nuclear magnetic resonance spectra of the hybrids were interpreted as reflecting the two quaternary structures. The forms with the changed spectra, reflecting the deoxy quaternary, were stabilized by the organic phosphates which preferentially bind to deoxyhemoglobin. The stabilization of the deoxy quaternary in the ( α II β III CN) 2 hybrid was more difficult than in the ( α III CN β II ) 2 hybrid, reflecting differences between the a and β subunits, and therefore, a stronger allosteric effector, inositol hexaphosphate, was necessary to switch the structure. Comparison of the two types of nuclear magnetic resonance spectra with the rate of CO combination in these hybrids ( Cassoly, Gibson, 1971, Ogawa & Shulman, 1971) showed that the changed spectra arose from a form with slow CO combination rate, characteristic of the first ligand binding to hemoglobin and the unchanged spectra corresponded to a fast rate characteristic of the fourth ligand. From these experiments, we concluded that the two quaternary forms of hemoglobin can be observed for the halfligated states of hemoglobin and that the switch between the two forms is responsible for the co-operativity. It is also shown that the change of ligand binding energy during co-operativity does not show up in the iron-ligand bond of the ligated heme.