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 p rocesses of proteins in hydrophobic interaction chromatography (HIC). Compa rative data acquired by the equilibrium binding analysis and by isothermal titration microcalorimetry (ITC) are presented. In particular, thermodynami c parameters, including the enthalpy changes, related to the interactions b etween several globular proteins and various Toyopearl 650 M sorbents under solvent conditions containing either 2.0 M ammonium sulfate or 2.0 M sodiu m sulfate at pH 7.0 and 298.15 K have been evaluated in terms of the molecu lar properties of these systems. The results reveal that the dependence of the free energy change, DeltaG(ads), for protein adsorption to HIC sorbents on the salt composition can be mainly attributed to the enthalpy changes a ssociated with protein and sorbent dehydration and hydrophobic interactions . Differences in binding mechanisms between the n-butyl- and phenyl-HIC sor bents were evident. In the latter case, the participation of,T-x hydrophobi c interactions leads to significant differences in the associated enthalpy and entropy changes. Furthermore, an increase in the hydrophobicity of eith er the sorbent or the protein resulted in more negative values for the free energy change, which arose mostly from dehydration processes. Entropic eff ects favoring HIC adsorption increased with an increase in the exposed nonp olar surface area of the protein. Consequently, an increased contribution f rom 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 mechanis m from a binding event dominated by adsorption to a partitioning-like proce ss. Data extracted from the ITC measurements also provided insight into the interaction mechanisms that occur between proteins and hydrophobic solid s urfaces, yielding information that can be applied to the HIC purification o f proteins according to the concept of critical hydrophobicity of the syste m and its thermodynamic consequences.