Gel Permeation Analysis of Macromolecular Association by an Equilibrium Method

Abstract

Abstract An equilibrium method for determining macromolecular association by gel permeation is described. A thermodynamic description of the simplest case, dimerization, is presented in terms of the equilibrium constant for association, and equilibrium partition coefficients for species present. The theoretical analysis yields an equation relating the equivalents of monomer in the external phase to total equivalents, from which the equilibrium constant for association may be obtained. Those parameters critical in experimental design, gel swelling time, and centrifugal collapse point are determined for Sephadex gels of porosity G-25 to G-200. Application of the method to human hemoglobin gives a value of K′ α = 5 × 10−6 for dissociation of oxyhemoglobin at pH 7.0 in 0.1 M buffer.