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

Abstract

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.

Optimal Design of Microarray Immunoassays to Compensate for Kinetic Limitations

“…For example, the energy for adsorption of a 13-residue peptide on colloidal silica was measured to be about 36 kJ/mol (41); binding of relatively small proteins such as lysozyme (14.3 kDa) or RNase A (13.7 kDa) to different chromatographic solid supports was indicated in the range of 13-34 kJ/mol (42)(43)(44), and the binding energy of a series of proteins to the aluminum salt ranges from about 30 to 40 kJ/mol (45). Assuming E b ϭ 20 kJ/mol and d corresponds to the molecular size of the IFNG molecules (about 4-nm diameter), the two-stage diffusion mechanism can only be significant in our incubation chamber with R c Ϸ 0.35 cm for R of only a few m or even less (the dominating mechanism if R Ͻ (DЈ/D)10 Ϫ4 cm).…”

Evaluation of selectivity changes in HIC systems using a preferential interaction based analysis

“…In their study, the solute affinities on HIC stationary phases were also found to have a linear relationship with the lyotropic series. Lin et al (2000Lin et al ( , 2001Lin et al ( , 2002 studied the effects of salt, ligand hydrophobicity, and protein structure on the interaction between protein and ligand using microcalorimetric measurement. Byun et al (2000) explained the salt effects on peptide binding on a SynChropak column and found that a salting-in parameter and a surface tension parameter could be very useful for peptide binding mechanism study.…”

Studies of lysozyme binding to histamine as a ligand for hydrophobic charge induction chromatography

“…11 The similar conclusion was affirmed in adsorption isotherm and chromatographic retention of BSA by Gao et al 12 In a combination with equilibrium binding analysis and isothermal titration microcalorimetry (ITC) measurements, Lin et al found that aromatic stacking contributed to protein adsorption on aromatic-based HIC adsorbents (e.g., Phenyl Sepharose). 13 Besides that, a typical HCIC chromatographic process is also influenced by other factors, such as the composition of the liquid phase, pH, and temperature. 2,11,12 Therefore, a better understanding of the mechanism of protein adsorption on HCIC beads is indispensable for the development and application of HCIC technology.…”

“…ITC has been successfully used to acquire thermodynamic data in protein adsorption on various ion exchange and hydrophobic interaction adsorbents. [13][14][15] For this purpose, we synthesized a histamine-Sepharose (HA-S) gel with higher ligand density than the histamine-ligand chromatography media reported previously. [16][17][18] Lysozyme was chosen for adsorption onto HA-S because it has been used extensively as a model protein for fundamental researches on chromatographic techniques.…”