title: Protein instability during HIC: evidence of unfolding reversibility, and apparent adsorption strength of disulfide bond-reduced alpha-lactalbumin variants.
abstract: A two-conformation, four-state model has been proposed to describe protein adsorption and unfolding behavior on hydrophobic interaction chromatography (HIC) resins. In this work, we build upon previous study and application of a four-state model to the effect of salt concentration on the adsorption and unfolding of the model protein alpha-lactalbumin in HIC. Contributions to the apparent adsorption strength of the wild-type protein from native and unfolded conformations, obtained using a deuterium labeling technique, reveal the free energy change and kinetics of unfolding on the resin, and demonstrate that surface unfolding is reversible. Additionally, variants of alpha-lactalbumin in which one of the disulfide bonds is reduced were synthesized to examine the effects of conformational stability on apparent retention. Below the melting temperatures of the wild-type protein and variants, reduction of a single disulfide bond significantly increases the apparent adsorption strength (approximately 6-8 kJ/mol) due to increased instability of the protein. Finally, the four-state model is used to accurately predict the apparent adsorption strength of a disulfide bond-reduced variant.
authors.affiliation: Department of Chemical Engineering, University of Virginia, 102 Engineers' Way, Charlottesville, Virginia 22904-4741, USA.
authors.forename: R W
authors.initials: RW
authors.lastname: Deitcher
authors.forename: Y
authors.initials: Y
authors.lastname: Xiao
authors.forename: J P
authors.initials: JP
authors.lastname: O'Connell
authors.forename: E J
authors.initials: EJ
authors.lastname: Fernandez
mesh_terms: D000327:Adsorption; D002845:Chromatography / Q000379:methods*; D003903:Deuterium; D004220:Disulfides / Q000737:chemistry*; D057927:Hydrophobic and Hydrophilic Interactions; D007700:Kinetics; D007768:Lactalbumin / Q000737:chemistry* / Q000302:isolation & purification*; D008958:Models, Molecular; D010084:Oxidation-Reduction; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D013194:Staining and Labeling
chemical_list: D004220:Disulfides; D007768:Lactalbumin; D003903:Deuterium