Effects of heterotropic allosteric effectors on the equilibrium and kinetics of the reaction of a single ligand molecule with an α or β subunit of deoxygenated HbA

Symmetrical FeZn hybrids of human HbA have been used to measure K ₁(α) and K₁(β), the dissociation constants for the binding of a single molecule of oxygen to unliganded HbA at an α subunit and at a β subunit, respectively. The kinetic constants, l ₁′(α) and l₁′(β), for the combination of the first CO molecule to unliganded HbA at an α or a β subunit, respectively, were also measured. Measurements were carried out between pH 6 and pH 8 in the presence and absence of inositol hexaphosphate (IHP). Both equilibrium constants exhibit a significant Bohr effect in the absence of IHP. The addition of IHP to a concentration of 0.1 mM increases both dissociation constants in a pH-dependent manner with the result that both Bohr effects are greatly reduced. These results require a negative thermodynamic linkage between the binding of a single oxygen at either an α or β subunit and the binding of IHP to the T quaternary structure of HbA. Although the β hemes are relatively near the IHP binding site, a linkage between that site and the α hemes, such that the binding of a single oxygen molecule to the heme of one α subunit reduces the affinity of the T state for IHP, requires communication across the molecule. l₁′ (α) exhibits a very slight pH dependence, with a maximum variation of 20%, while l₁′(β) varies with pH three times as much. IHP has no effect on the pH dependence of either rate constant but reduces l ₁′(α) marginally, 20%, and l₁′(β) by 2-fold at all pH values.