Hydrophobic interaction chromatography of proteins: V. Quantitative assessment of conformational changes

Protein adsorption during hydrophobic interaction chromatography (HIC) may induce conformational changes. We analyzed conformational changes in three model proteins, bovine serum albumin (BSA), β-lactoglobulin, and lysozyme by attenuated total reflectance Fourier transform infrared (ATR FT-IR) spectroscopy and pulse response experiments. Conformational changes occurred in the secondary structure of BSA, the tertiary structure of β-lactoglobulin, and no changes occurred in lysozyme under the adsorption conditions investigated. Protein unfolding varied substantially among proteins, caused incomplete isocratic elution in HIC, and was confirmed by in situ assessments. Lower temperatures and binding capacities significantly reduced protein unfolding; the activation energy for unfolding ranged from 47 to 125 kJ/mol.