Protein Interaction with Immobilized Metal Ion Affinity Ligands under High Ionic Strength Conditions

Abstract

An investigation into the adsorption behavior under batch equilibrium binding conditions of hen egg white lysozyme (HEWL) with the immobilized metal ion affinity chromatographic adsorbent, Cu2+–IDA Sepharose CL-4B, in the presence of high concentrations of NaCl or KCl has been undertaken. When the concentration of NaCl or KCl in the adsorption buffer was ≤0.2M, the adsorption data correlated with predictions based on the Langmuir model. However, when the salt concentration was ≥0.5M, the adsorption data appeared to follow the Freundlich–Langmuir model under these higher ionic strength conditions. Under elevated ionic strength conditions, the protein–ligand interactions were shown to involve positive cooperativity by Scatchard plot analysis of the experimental data. The number,n, of interacting protein molecules increased with the salt concentration, while the apparent dissociation constantKdunder these high ionic strength conditions was less dependent on the type of salt present in the adsorption buffer. These studies reveal that the binding of Cu2+–IDA Sepharose CL-4B to HEWL acceptor molecules under conditions of high ionic strength is characteristic of general heterogeneous interactions, whereby the immobilized metal ion affinity chromatography (IMAC) ligands bind to a protein acceptor undergoing isodesmic indefinite self-association and multisite attachment. As a consequence, these results on the adsorption behavior of HEWL have important general implications for the preparative use of immobilized metal ion affinity adsorbents in packed and expanded bed systems, particularly with regard to the resolution and productivity of the separation for the target protein. In addition, the results from these studies are relevant to investigations involving analytical affinity chromatographic or biosensor assessment of the interaction of proteins with immobilized IMAC ligands when high ionic strength equilibration feedstocks or conditions are employed.