Determination of Oxygen Binding Properties of the Individual Subunits of Intact Human Adult Hemoglobin

Abstract

Abstract We determined the oxygen (O2) binding properties of the individual subunits of intact human adult hemoglobin (Hb A) through the combined use of 1H NMR and conventional spectrophotometric methods. We found that the O2 affinities of the α and β subunits of Hb A are almost indistinguishable in the absence of an allosteric effector, inositol hexaphosphate (IHP), and lower by factors of ≈8.4 and ≈10, respectively, in the presence of IHP at pH 7.40, as judged from the estimated P50 values, which represent the partial O2 pressure required to achieve 50% oxygenation of the individual subunits. These results demonstrated that the binding of IHP to the protein resulted in remarkable enhancement of the degree of nonequivalence in functional properties between the constituent subunits, which could be relevant to its cooperative ligand binding. This finding provides novel insights for elucidation of the molecular mechanisms responsible for the cooperativity in this allosteric protein.