Hydrophobic interaction chromatography of proteins: thermodynamic analysis of conformational changes.
For BSA and beta-lactoglobulin adsorption to hydrophobic interaction chromatography (HIC) stationary phases leads to conformational changes. In order to study the enthalpy (DeltaH(ads)), entropy (DeltaS(ads)), free energy (DeltaG(ads)) and heat capacity (Deltac(p,ads)) changes associated with adsorption we evaluated chromatographic data by the non-linear van't Hoff model. Additionally, we performed isothermal titration calorimetry (ITC) experiments. van't Hoff analysis revealed that a temperature raise from 278 to 308K increasingly favoured adsorption seen by a decrease of DeltaG(ads) from -12.9 to -20.5kJ/mol for BSA and from -6.6 to -13.2kJ/mol for beta-lactoglobulin. Deltac(p,ads) values were positive at 1.2m (NH(4))(2)SO(4) and negative at 0.7m (NH(4))(2)SO(4). Positive Deltac(p,ads) values imply hydration of apolar groups and protein unfolding. These results further corroborate conformational changes upon adsorption and their dependence on mobile phase (NH(4))(2)SO(4) concentration. ITC measurements showed that DeltaH(ads) is dependent on surface coverage already at very low loadings. Discrepancies between DeltaH(ads) determined by van't Hoff analysis and ITC were observed. We explain this with protein conformational changes upon adsorption which are not accounted for by van't Hoff analysis.
Ueberbacher
Rene
R
Department of Biotechnology, University of Natural Resources and Applied Life Sciences Vienna, Muthgasse 18, Vienna, Austria.
Rodler
Agnes
A
Hahn
Rainer
R
Jungbauer
Alois
A
D000327:Adsorption; D000818:Animals; D002853:Chromatography, Liquid / Q000379:methods*; D057927:Hydrophobic and Hydrophilic Interactions; D007782:Lactoglobulins / Q000737:chemistry; D008962:Models, Theoretical; D011487:Protein Conformation; D017510:Protein Folding; D011506:Proteins / Q000737:chemistry*; D012685:Sepharose / Q000737:chemistry; D012710:Serum Albumin, Bovine / Q000737:chemistry; D013816:Thermodynamics
D007782:Lactoglobulins; D011506:Proteins; D012710:Serum Albumin, Bovine; D012685:Sepharose