Abstract
Although very high performance separation of biomolecules such as protein isoforms is possible with ion-exchange and hydrophobic interaction chromatography, the fundamental mechanisms involved in ligand-protein interaction is still not clear. In this study, ligand-protein interaction was analyzed by three different methods, isothermal titration calorimetry (ITC), batch adsorption isotherm measurements and column gradient elution experiments. It was found that proteins having a rigid structure such as lysozyme and those having a soft (flexible) structure such as myoglobin have different interaction properties to the ligands of the base matrix. The latter shows a stronger hydrophobic interaction and greater binding affinity. The gradient elution data also showed that quite a large number of binding sites are involved, and in some cases the adsorption is irreversible (proteins are not eluted).
