Dissociation of hemoglobin into subunits: Ligand-linked dissociation at neutral pH

Abstract

The dimer-tetramer association-dissociation equilibrium of hemoglobin is strongly ligand-linked at neutral pH; the ratio of the association constant of unliganded to that of oxyhemoglobin is estimated from ultracentrifuge data to be not less than 10 3 in sodium chloride solutions and probably not less than 10 5 in sodium iodide solutions. This finding affords an explanation of the fact that the ligand binding characteristics of hemoglobin are to a first approximation independent of the degree of dissociation of oxyhemoglobin—the “salt paradox”. Sedimentation equilibrium and velocity studies were routinely performed at concentrations as low as 10 μg/ml. (6.2 × 10 −7 m-heme) using the photoelectric scanning absorption optical system. This system permitted strict spectral control of the integrity of the protein, especially important in the case of unliganded hemoglobin. In oxyhemoglobin, dissociation into monomers was not sufficient to be detectable even at high salt concentrations. In both sodium chloride and sodium iodide solutions, the association-dissociation equilibrium conformed well to a simple dimer-tetramer equilibrium. For each set of conditions, the equilibrium constant and dimer molecular weight, expressed as the buoyancy term M 2 ( ∂ϱ ∂c 2 ) μ , were computed simultaneously by direct fitting of c versus r 2 data using a non-linear least squares procedure. When the values of the density increment, ( ∂ϱ ∂c 2 ) μ , determined independently bypycnometry in 2 m-sodium chloride and 1 m-sodium iodide, were combined in accordance with three-component theory with the corresponding experimentally determined buoyancy terms, dimer molecular weights of 33,100 and 31,000 daltons were calculated respectively. In unliganded hemoglobin, the dissociation into dimer was not sufficient to be detectable. In 0.09 and 2.0 m-sodium chloride and in 1 m-sodium iodide solutions, plots of In c versus r 2 in the range 10 to 100 μg/ml. (6.2 × 10 −7 to 6.2 × 10 −6 m-heme) were linear, corresponding to molecular weights of 63,800, 61,300 and 62,000 daltons respectively. Sedimentation velocity experiments performed at 20 μg/ml. (l.24 × 10 −6 m-heme) yielded S 20,W values of 4.81 s, 4.68 s and 4.82 s respectively.