High-performance liquid chromatography of amino acids, peptides and proteins .170. Comparative studies on the isothermal characteristics of proteins adsorbed under batch equilibrium conditions to ion-exchange, immobilised metal ion affinity and dye affinity matrices with different ionic strength and temperature conditions

In these investigations, the influence of a range of experimental parameters on the isothermal characteristics of hen egg white lysozyme (HEWL) and human serum albumin (HSA) adsorbed to several different adsorbents has been examined. The adsorbents were selected to encompass the same basic types of silica support matrices, but with the ligand properties and surface characteristics adjusted so that the dominant mode of interaction between the protein and the ligand involved either electrostatic binding (i.e. as ion-exchange interactions with polyaspartic acid immobilised onto glycidoxypropyl-modified Fractosil 1000), mixed-mode binding with both hydrophobic and electrostatic effects contributing to the protein-ligand interaction (i.e. as dye-affinity interactions with Cibacron Blue F3G-A immobilised onto Lichroprep DIOL or onto glycidoxypropyl-modified Fractosil 1000), or lone pair coordination binding (i.e. as immobilised metal ion affinity (IMAC) interactions with Cu2+ ions complexed with iminodiacetic acid immobilised onto glycidoxypropyl-modified Fractosil 1000). In each case, the adsorbents exhibited similar ligand densities and had the same particle size ranges and silica surface pretreatment. The effect of the ionic strength of the adsorption buffer and temperature on the isothermal adsorption behaviour under batch equilibrium binding conditions of the two test proteins were determined. Consistent with previous observations with soft gel ion exchangers and triazine dye-based adsorbents that are used in packed bed chromatographic systems, the capacities of the silica-based ion-exchange adsorbents, as well as the Cibacron Blue F3G-A dye affinity adsorbents, for both HSA and HEWL were reduced as the salt concentration was increased under batch equilibrium binding conditions. Moreover, with both of these classes of adsorbents, as the ionic strength was increased under constant temperature conditions, the isothermal adsorption dependencies progressively approximated more closely a Langmuirean model of independent binding site interactions, typical of a mono-layer binding process. In contrast, with the silica-based immobilised metal ion affinity adsorbents as the ionic strength was increased the adsorption behaviour appeared to follow a Freundlich model, indicative of positive cooperativity in the binding process. In parallel experiments, the effect of changes in temperature under iso-ionic strength conditions was examined. With increasing temperature, different patterns of isothermal adsorption behaviour for both test proteins were observed, with the magnitude of these trends depending on the type of interaction involved between the immobilised ligand and the protein. Utilising first order Van't Hoff relationships to analyse the experimental data for these protein-ligand interactions, the apparent changes in enthalpy and entropy for these interactions have been derived from the dependency of the change in the apparent Gibbs free energy on 1/T.