title: Salts employed in hydrophobic interaction chromatography can change protein structure - insights from protein-ligand interaction thermodynamics, circular dichroism spectroscopy and small angle X-ray scattering.
abstract: Key requirements of protein purification by hydrophobic interaction chromatography (HIC) are preservation of the tertiary/quaternary structure, maintenance of biological function, and separation of the correctly folded protein from its unfolded forms or aggregates. This study examines the relationship between the HIC retention behavior of hen egg white lysozyme (HEWL) in high concentrations of several kosmotropic salts and its conformation, assessed by circular dichroism (CD) spectroscopy. Further, the physicochemical properties of HEWL in the presence of high concentrations of ammonium sulfate, sodium chloride and magnesium chloride were investigated by small angle X-ray scattering (SAXS) at different temperatures. Radii of gyration were extrapolated from Guinier approximations and the indirect transform program GNOM with protein-protein interaction and contrast variation taken into account. A bead model simulation provided information on protein structural changes using ab initio reconstruction with GASBOR. These results correlated to the secondary structure content obtained from CD spectroscopy of HEWL. These changes in SAXS and CD data were consistent with heat capacity ΔCp -values obtained from van't Hoff plot analyses of the retention data. Collectively, these insights enable informed decisions to be made on the choice of chromatographic conditions, leading to improved separation selectivity and opportunities for innovative column-assisted protein refolding methods.
authors.affiliation: School of Chemistry, Monash University, Melbourne, Victoria, Australia.
authors.forename: Andras Z
authors.initials: AZ
authors.lastname: Komaromy
authors.forename: Chadin
authors.initials: C
authors.lastname: Kulsing
authors.forename: Reinhard I
authors.initials: RI
authors.lastname: Boysen
authors.forename: Milton T W
authors.initials: MT
authors.lastname: Hearn
mesh_terms: D000645:Ammonium Sulfate / Q000494:pharmacology; D000818:Animals; D002645:Chickens; D002942:Circular Dichroism / Q000379:methods; D057927:Hydrophobic and Hydrophilic Interactions; D015636:Magnesium Chloride / Q000494:pharmacology; D008958:Models, Molecular; D009113:Muramidase / Q000737:chemistry* / Q000187:drug effects*; D017433:Protein Structure, Secondary; D012492:Salts / Q000494:pharmacology*; D053838:Scattering, Small Angle; D012965:Sodium Chloride / Q000494:pharmacology; D013816:Thermodynamics; D014961:X-Ray Diffraction / Q000379:methods
chemical_list: D012492:Salts; D015636:Magnesium Chloride; D012965:Sodium Chloride; C421864:hen egg lysozyme; D009113:Muramidase; D000645:Ammonium Sulfate
keywords: Circular dichroism; Hydrophobic interaction chromatography; Protein shape; Protein-protein interaction; Small angle x-ray scattering