STUDY OF THE ADSORPTION OF SELF-ASSOCIATING PROTEINS ON AN ANION-EXCHANGER - APPLICATION TO THE CHROMATOGRAPHY OF BETA-LACTOGLOBULIN-B

The stoichiometric displacement model (SDM) for the adsorption of proteins on an ion exchanger was extended to describe the adsorption of self-associated molecules. The model based on the mass-balance equations assumes a monomer-dimer equilibrium with a rapid interconversion rate. The role of protein self-association in solution and in the adsorbed state is discussed in terms of adsorption isotherm shapes and of zonal elution chromatographic behaviours. The influence of the parameter Z, defined as the ratio of the protein valency to the displacing counter-ion valency, is demonstrated. The model was applied to analyse the zonal elution behaviour of beta-lactoglobulin B (beta-lact B) on a polymeric anion-exchange stationary phase. The influence of the counter-ion valency was studied by adding NaCl or Na2SO4 to the eluent. The adsorption isotherm of beta-lact B on the anion exchanger was determined at pH 7.5 from the non-linear zonal elution profiles observed with increasing sample size. Various aspects of the adsorption behaviour of beta-lact B, such as positive cooperativity, were deduced from the isotherm shape and the corresponding Scatchard plot. The column capacity, the association constant in solution and the parameter defining the association in the adsorbed phase were determined from the best it of the theoretical model to the experimental profiles. In agreement with the model, these parameters can be used to describe the experiments performed with a monovalent or a divalent counter ion. It is shown that protein-protein interactions exist in the adsorbed state and lead to an increase in the protein self-association.