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), beta-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 beta-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.
Department of Biotechnology, University of Natural Resources and Applied Life Sciences Vienna, Vienna, Austria.
Rene
R
Ueberbacher
Emmerich
E
Haimer
Rainer
R
Hahn
Alois
A
Jungbauer
D000327:Adsorption; D000818:Animals; D002417:Cattle; D002845:Chromatography / Q000379:methods*; D057927:Hydrophobic and Hydrophilic Interactions; D007782:Lactoglobulins / Q000737:chemistry; D009113:Muramidase / Q000737:chemistry; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry*; D015203:Reproducibility of Results; D012710:Serum Albumin, Bovine / Q000737:chemistry; D017550:Spectroscopy, Fourier Transform Infrared; D013696:Temperature
D007782:Lactoglobulins; D011506:Proteins; D012710:Serum Albumin, Bovine; D009113:Muramidase