Microcalorimetric studies on the interaction mechanism between proteins and hydrophobic solid surfaces in hydrophobic interaction chromatography: effects of salts, hydrophobicity of the sorbent, and structure of the protein.

This study examines the effects of different salts as well as the influence of the relative hydrophobicities of different sorbents on the adsorption processes of proteins in hydrophobic interaction chromatography (HIC). Comparative data acquired by the equilibrium binding analysis and by isothermal titration microcalorimetry (ITC) are presented. In particular, thermodynamic parameters, including the enthalpy changes, related to the interactions between several globular proteins and various Toyopearl 650 M sorbents under solvent conditions containing either 2.0 M ammonium sulfate or 2.0 M sodium sulfate at pH 7.0 and 298.15 K have been evaluated in terms of the molecular properties of these systems. The results reveal that the dependence of the free energy change, deltaGads, for protein adsorption to HIC sorbents on the salt composition can be mainly attributed to the enthalpy changes associated with protein and sorbent dehydration and hydrophobic interactions. Differences in binding mechanisms between the n-butyl- and phenyl-HIC sorbents were evident. In the latter case, the participation of pi-pi hydrophobic interactions leads to significant differences in the associated enthalpy and entropy changes. Furthermore, an increase in the hydrophobicity of either the sorbent or the protein resulted in more negative values for the free energy change, which arose mostly from dehydration processes. Entropic effects favoring HIC adsorption increased with an increase in the exposed nonpolar surface area of the protein. Consequently, an increased contribution from the entropy change to the respective change in free energy occurs when HIC sorbents or proteins of higher hydrophobicity are employed, with these larger entropy changes consistent with a change in the interaction mechanism from a binding event dominated by adsorption to a partitioning-like process. Data extracted from the ITC measurements also provided insight into the interaction mechanisms that occur between proteins and hydrophobic solid surfaces, yielding information that can be applied to the HIC purification of proteins according to the concept of critical hydrophobicity of the system and its thermodynamic consequences.