Thermodynamic nonideality and the dependence of partition coefficient upon solute concentration in exclusion chromatography. II. An improved theory of equilibrium partitioning of concentrated protein solutions. Application to hemoglobin.

An improved theory for the partitioning of protein between concentrated bulk solution and solution sequestered in a porous medium is presented. The theory is based upon the assumption that the sequestered solution may be formally represented as a sum of three compartments: (1) a compartment which Is inaccessible to protein, but accessible to solvent and small molecule solutes; (2) a surface layer immediately adjacent to the pore boundary, within which protein molecules are constrained to quasi-two-dimensional motion; and (3) the remaining volume accessible to protein, within which the protein molecules behave as if in bulk solution. The dependence of the partition coefficient of hemoglobin upon protein concentration over the range 10-225 g/l, calculated using the theory presented, is found to agree quantitatively with experimental data presented previously (R.J. Siezen, L.W. Nichol and D.J. Winzor, Biophys. Chem. 14 (1981) 221) without invoking self-association of hemoglobin molecules.