Dissociation of hemoglobin into subunits. Monomer formation and the influence of ligands

The formation of monomer from several hemoglobins has been investigated by sedimentation equilibrium. The use of the split-beam photoelectric scanning absorption optical system has enabled observations to be made routinely down to 1 μg/ml. (6.2 × 10⁻⁸m-heme) with strict spectral control of the integrity of the hemoglobin molecule. The results show that the dissociation constant of dimer to monomer at neutral pH and moderate ionic strength is so small that monomer is present in reversible equilibrium with dimer only in fractions too small to be detectable. Any appreciable monomer formation is irreversible and accompanied by usually pronounced spectral changes. This irreversible monomer formation is probably a consequence of the presence of heavy-metal ions in solution and may be inhibited by 10⁻³m-EDTA. Hemoglobin ligands possessing chelating ability also inhibit monomer formation.