Analysis of Oxygen Equilibrium of Hemoglobin and Control Mechanism of Organic Phosphates

Abstract

Oxygen equilibrium curves of hemoglobins precisely determined by the automatic recording apparatus have been analyzed according to Adair's (Adair, G. S. (1925), J. Biol. Chem. 63, 529) stepwise oxygenation theory, and four successive association constants (k values) for the binding of oxygen have been estimated by the least-squares method. 2,3-Diphosphoglycerate markedly reduces k1, k2, and k3 for human adult hemoglobin without affecting k4. In contrast, inositol hexaphosphate enormously reduces all four k values. The glycerate also shows a similar but weaker effect on human fetal hemoglobin. The effect of 0.1 m NaCl on the oxygen equilibrium parameters is qualitatively similar to that of the glycerate, except that the neutral salt increases k4 slightly. Calculation of the hemoglobin fraction of various intermediate oxygenation stages reveals that the fraction of Hb(O2)3 is negligibly small in the presence of the organic phosphates and/or NaCl except at very high oxygen saturation. Analysis of k values in the presence of 0.1 M NaCl and various concentrations of 2,3-diphosphoglycerate indicates that the phosphates combine not only with the fully deoxygenated hemoglobin but also with the intermediates, Hb(O2)1 and Hb(O2)2, with considerable affinity. The binding constant of the phosphate for the fully deoxygenated human adult hemoglobin at pH 7.4 and 25° has been estimated as 3.1 X 104 M-1, which agrees well with that obtained by the direct binding experiment. The overall free energy of interaction among oxygen combining sites is increased by 2 mM 2,3-diphosphoglycerate in the absence of added NaCl by about 5100 and 2700 cal/mol in the adult and fetal hemoglobins, respectively, which corresponds to the formation of four and two, respectively, additional salt bridges between the glycerate and the deoxyhemoglobins. These results are consistent with the model proposed by Perutz [Perutz, M. F. (1970), Nature (London) 228, 726] for the cooperative oxygen binding of hemoglobin. © 1973, American Chemical Society. All rights reserved.