Linkage between carbon dioxide binding and four-step oxygen binding to hemoglobin

In order to elucidate the molecular mechanism of the effect of carbon dioxide on the four-step oxygenation equilibria of hemoglobin, accurate oxygen equilibrium curves of human adult hemoglobin were determined at different concentrations of CO₂ and in the presence and absence of chloride (Cl^-), 2,3-diphosphoglycerate (P₂G), and/or inositol hexaphosphate (IHP) and were analyzed according to Adair's stepwise oxygenation scheme to evaluate the four Adair constants, k_i (i = 1 to 4). The effects of CO₂ on oxygen affinity and co-operativity are influenced by H⁺, Cl^-, P₂G and IHP. The shape of the oxygen equilibrium curve varies with changes of CO₂ concentration; the four Adair constants are affected by CO₂ non-uniformly. Hence, the number of CO₂ molecules released upon oxygenation is not the same in the individual oxygenation steps. In the absence of added Cl^-, CO₂ lowers the overall oxygen affinity expressed by median oxygen pressure (p_m) and increases the co-operativity expressed by Hill's coefficient (n_max) by reducing k₁, k₂ and k₃ without changing k₄. significantly. The effect of CO₂ on oxygen affinity becomes smaller with decrease in pH, disappearing below pH 6.5. The alkaline Bohr effect is reduced by CO₂. The first oxygenation step contributes to the reduction of the Bohr effect more than the fourth step. When log p_m is plotted against log [CO₂] at several constant Cl^- concentrations, the plots converge to a common point that is named "iso-effective point". When log p_p is plotted against log [Cl^-] at several constant CO₂ concentrations, the plots also converge to an iso-effective point. This phenomenon can be explained in terms of linkage relations in oxygen-linked competitive binding of CO₂ and Cl^-. It was found to be useful to consider in this analysis that the bicarbonate ion introduced by added CO₂ exerts a heterotropic effect equivalent to that of Cl^-. The combined effects of Cl^-, CO₂ and IHP were not explained satisfactorily by the present analysis using linkage relations.