Heterotropic effectors exert more significant strain on monoligated than on unligated hemoglobin

The effect of allosteric effecters, such as inositol hexakisphosphate and/or benafibrate, has been investigated on the unliganded human adult hemoglobin both spectroscopically (employing electronic absorption, circular dichroism, resonance Raman, and x-ray absorption near-edge spectroscopies) and functionally (following the kinetics of the first CO binding step up to a final 4% ligand saturation degree). All data indicate that the unliganded T-state is not perturbed by the interaction with either one or both effecters, suggesting that their functional influence is only exerted when a ligand molecule is bound to the heme. This is confirmed by the observation that CO dissociation from partially liganded hemoglobin ((Y) over bar less than or equal to 0.04) is strongly altered by the presence of either effector, and the effect is enhanced whenever the two effecters are simultaneously present. Altogether, these data are a direct demonstration of the occurrence of a strain induced by the presence of a ligand molecule bound to the heme, and for the first time there is a clear indication that the expression of the functional heterotropic effect by these non-heme ligands requires this strain, which is not present in the unliganded molecule.