Protein instability during HIC: Evidence of unfolding reversibility, and apparent adsorption strength of disulfide bond-reduced α-lactalbumin variants

Authors

  • R.W. Deitcher,

    1. Department of Chemical Engineering, University of Virginia, 102 Engineers' Way, Charlottesville, Virginia 22904-4741; telephone: 434-924-7778; fax: 434-982-2658
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  • Y. Xiao,

    1. Department of Chemical Engineering, University of Virginia, 102 Engineers' Way, Charlottesville, Virginia 22904-4741; telephone: 434-924-7778; fax: 434-982-2658
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  • J.P. O'Connell,

    1. Department of Chemical Engineering, University of Virginia, 102 Engineers' Way, Charlottesville, Virginia 22904-4741; telephone: 434-924-7778; fax: 434-982-2658
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  • E.J. Fernandez

    Corresponding author
    1. Department of Chemical Engineering, University of Virginia, 102 Engineers' Way, Charlottesville, Virginia 22904-4741; telephone: 434-924-7778; fax: 434-982-2658
    • Department of Chemical Engineering, University of Virginia, 102 Engineers' Way, Charlottesville, Virginia 22904-4741; telephone: 434-924-7778; fax: 434-982-2658.
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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 α-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 α-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 (∼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. Biotechnol. Bioeng. 2009;102: 1416–1427. © 2008 Wiley Periodicals, Inc.

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