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

Andras Zsigmond Komaromy, Chadin Kulsing, Reinhard I Boysen, Milton Thomas William Hearn

Research output: Contribution to journalArticleResearchpeer-review

3 Citations (Scopus)


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.
Original languageEnglish
Pages (from-to)417-426
Number of pages10
JournalBiotechnology Journal
Issue number3
Publication statusPublished - 2015


  • Circular dichroism
  • Hydrophobic interaction chromatography
  • Protein-protein interaction
  • Protein shape
  • Small angle x-ray scattering

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