The linkage between the four-step binding of oxygen and the binding of heterotropic anionic ligands in hemoglobin

The linkage between the four-step binding of oxygen and the binding of heterotropic anionic ligands in hemoglobin was investigated by accurately measuring and analyzing the oxygen equilibrium curves of human adult hemoglobin in the presence and absence of various concentrations of one or two of the following materials: chloride (Cl∼), 2, 3-diphosphoglycerate (DPG), and inositol hexaphosphate (IHP). Each equilibrium curve was analyzed according to the Adair equation to evaluate the four-step oxygen equilibrium constants (Adah constants) and the median oxygen pressure. The binding constants of the anions for the molecular species of hemoglobin carrying j oxygen molecules, Hb(O₂) j=0, 1,....,4), were evaluated from the dependences of the Adair constants and the median oxygen pressure on the anion concentration by introducing a model which takes the competitive binding of CI- and DPG or IHP into account. Assumptions made in the model are: (a) the hemoglobin molecule has two oxygen-linked binding sites for Cl- which are equivalent and independent and (b) no Cl- can be bound to hemoglobin to which DPG or IHP is already bound and vice versa. Thus, we could obtain values for the intrinsic binding constants of Cl- and DPG, i. e., the constants in the absence of other competitive anions. For IHP, only the binding constants and apparent binding constants for Hb and Hb(O₂)₄ were obtained. Values of the Cl- binding constants and apparent binding constants for DPG and IHP, i. e., the binding constants in the presence of Cl- for Hb and Hb(O₂)4, were in reasonable agreement with literature values. From the binding constants we calculated anion binding curves for Hb(O₂) (j=0, 1,....,4), the number of anions bound to Hb(O₂, and the relationship between fractional anion saturation of hemoglobin and fractional oxygen saturation. The numbers of released anions are not uniform with respect to oxygenation step. This non-uniformity is the reason for the changes in the shape of the oxygen equilibrium curve with anion concentration changes and for the non-uniform dependences of the Adair constants on anion concentration, and also results in non-linear relations between anion saturation and oxygen saturation. The anion binding constants and various binding properties of the anions derived from those constants are consistent with those observed by other investigators using different techniques, indicating that the present model describes the oxygen-linked competitive anion binding well.