Observation of the Dissociation of Unliganded Hemoglobin

Abstract

Abstract A method is described for determining the equilibrium constant of the tetramer-dimer dissociation reaction of unliganded hemoglobin. The method is based on the large difference between the ligand binding affinities of the dimer and the unliganded tetramer, and utilizes the ligand binding affinity of dilute hemoglobin solutions as a sensitive assay for the fraction of heme in the dimeric state. The affinities for CO of hemoglobin solutions containing 0.05 to 8.0 µm heme have been determined, and show the dependence on heme concentration expected for a tetramer-dimer dissociation equilibrium. In 0.1 m phosphate, pH 7.0, this dissociation equilibrium is described by a dissociation constant of 3 x 10-12 m. Experiments with hemoglobin solutions in 2.0 m NaCl, 0.1 m phosphate, pH 7.0, indicate an increase in the dissociation constant to 7 x 10-11 m. The large amount of tetramer present in these dilute solutions combined with the capacity of the tetramer-dimer equilibrium to generate cooperativity in the ligand binding reaction accounts quantitatively for the highly cooperative binding of ligand by these dilute solutions (Hill's constant, n = 2.3). In addition, the ratio of the dissociation constants of liganded and unliganded hemoglobin yields a value for the allosteric constant L. This ratio is 6.7 x 105, a value which is in good agreement with the earlier estimates of L.