Interactions between a Classical Allosteric Protein and a Strong Effector Revisited

Abstract

According to the Two-State Model of Monod-Wyman-Changeux, the allosteric regulation in human tetrameric hemoglobin (Hb) is achieved upon binding of heterotrophic effectors, such as 2,3-bisphosphoglycerate (BPG), to the allosteric site located between the beta subunits of unliganded, low oxygen affinity T-structure Hb. As a result, the new complex exhibits an additional decrease in the affinity for oxygen when compared with Hb in the absence of BPG. On the other hand, liganded Hb in the high oxygen affinity R-structure does not bind BPG, and thus its oxygen affinity remains unaffected.We have studied the interactions between inositol hexakisphosphate (IHP), an allosteric effector stronger that DPG, and Hb, both in the liganded and unliganded forms, by isothermal titration calorimetry (ITC) and oxygen binding measurements in the equimolar range at pH 7.0 and 15°C. For the liganded “R”-structure, we chose the cyanmetHb derivative (Hb+CN-), and for the unliganded “T”-structure, the nickel-porphyrin Hb (NiHb). ITC experiments showed that IHP binds to both tetrameric derivatives in equimolar amount and with relatively high affinity.Tertiary/quaternary structural perturbations introduced systematically into Hb+CN- by removal of specific amino acid residues suggested that the IHP binding site is identical to that exhibited by NiHb, i.e., between the beta subunits. Under these conditions, we have not found any evidence that suggests IHP bind between the alpha subunits, or in the central cavity, as recent reports have suggested.At pH 8.2, we observed that while unliganded Hb with equimolar amounts of IHP remained practically invariable, the addition of above 100-fold molar excess indeed produced a pronounced decrease in the affinity for oxygen, suggesting the existence of an additional form of interaction. The function of liganded Hb, on the other hand, remained unchanged in the presence of IHP in excess.