Determination of the dissociation constant of oligomeric proteins by size-exclusion high-performance liquid chromatography: application to human haemoglobin.

The measurement of protein retention volumes on a size-exclusion chromatographic column offers the possibility of determining dissociation constants for oligomeric proteins, as changes in the retention volume, depending on the concentration of the protein, are due to a dissociation equilibrium. The retention volume may be calibrated in terms of dissociation constant by using either extreme concentration conditions or chemical modifications that shift the equilibrium towards a single species. When zonal chromatography is used, the dilution during elution modifies the equilibrium state. In contrast, the saturation method permits the concentrations of the different species to be kept constant. These two methods were compared and the elution factor that must be used in zonal chromatography on high-performance size-exclusion columns (LiChrospher Diol) was obtained. The tetramer-dimer dissociation constants of normal and modified haemoglobins were measured by this method, and the results are in accordance with flash photolysis measurements.