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

The effect of allosteric effecters, such as inositol hexakisphosphate and/o r benafibrate, has been investigated on the unliganded human adult hemoglob in both spectroscopically (employing electronic absorption, circular dichro ism, resonance Raman, and x-ray absorption near-edge spectroscopies) and fu nctionally (following the kinetics of the first CO binding step up to a fin al 4% ligand saturation degree). All data indicate that the unliganded T-st ate is not perturbed by the interaction with either one or both effecters, suggesting that their functional influence is only exerted when a ligand mo lecule is bound to the heme. This is confirmed by the observation that CO d issociation from partially liganded hemoglobin ((Y) over bar less than or e qual to 0.04) is strongly altered by the presence of either effector, and t he 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 f unctional heterotropic effect by these non-heme ligands requires this strai n, which is not present in the unliganded molecule.