Oxygen-organophosphate linkage in hemoglobin A. The double hump effect

At low concentrations of chloride ions, and in the presence of nonsaturating concentrations of organophosphates, the oxygen equilibrium curves (OEC) for solutions of human adult hemoglobin exhibit a biphasic shape conveniently revealed by graphical analysis of the first derivative of the Hill equation with a characteristic form that we call "the double hump effect." This shape, observed for sub-saturating concentrations of organophosphates, stands in marked contrast to the simple lateral shifts of the OEC represented largely by scaling factors when pH or chloride are varied. In the case of protons or chloride, there is a self-buffering effect due to the presence of a large reservoir of proton or chloride binding sites not necessarily linked to oxygen, whereas such sites do not exist in the case of organophosphates. In addition, in the former case, we are dealing with curves measured at constant activity of the effector, while in the latter, at constant concentration. In the presence of saturating concentrations of inositol hexaphosphate (IHP), at low chloride concentration, the entire OEC is shifted to the right, including both its upper and lower asymptotes, indicating a decrease in the intrinsic oxygen affinities of both the T and R states. Theoretical considerations leading to a successful modeling of OEC obtained under nonsaturating and saturating concentrations of IHP required an expanded two-state allosteric model in which IHP-dependent variations in oxygen association constants for both the T and R conformations are taken into account