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, ki (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 (pm) and increases the co-operativity expressed by Hill's coefficient (nmax) 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 pm 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 pp 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.
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