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

Abstract

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 CO 2 and in the presence and absence of chloride (Cl −), 2,3-diphosphoglycerate (P 2G), 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 CO 2 on oxygen affinity and co-operativity are influenced by H +, Cl −, P 2G and IHP. The shape of the oxygen equilibrium curve varies with changes of CO 2 concentration; the four Adair constants are affected by CO 2 non-uniformly. Hence, the number of CO 2 molecules released upon oxygenation is not the same in the individual oxygenation steps. In the absence of added Cl −, CO 2 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 k 1, k 2 and k 3 without changing k 4. significantly. The effect of CO 2 on oxygen affinity becomes smaller with decrease in pH, disappearing below pH 6.5. The alkaline Bohr effect is reduced by CO 2. 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 [CO 2] 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 CO 2 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 CO 2 and Cl −. It was found to be useful to consider in this analysis that the bicarbonate ion introduced by added CO 2 exerts a heterotropic effect equivalent to that of Cl −. The combined effects of Cl −, CO 2 and IHP were not explained satisfactorily by the present analysis using linkage relations.