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Hydrophobic interaction chromatography selectivity changes among three stable proteins: conformation does not play a
Tara Tibbs Jones1, Erik J Fernandez
1Department of Chemical Engineering, University of Virginia, 102 Engineers' Way, PO Box 400741, Charlottesville, Virginia 22904-4741, USA.
Biotechnology and Bioengineering
|July 29, 2004
Summary
Hydrophobic interaction chromatography (HIC) selectivity changes in proteins are not due to conformational changes. Stable proteins studied showed minimal structural alterations upon adsorption to HIC media, suggesting other factors influence protein retention.
Area of Science:
- Biochemistry
- Analytical Chemistry
- Protein Science
Background:
- Hydrophobic interaction chromatography (HIC) is widely used for protein separation.
- Observed variations in protein retention and selectivity in HIC suggest potential structural changes during adsorption.
- Understanding these changes is crucial for optimizing chromatographic methods.
Purpose of the Study:
- To investigate the role of protein conformational changes in selectivity variations during HIC.
- To assess solvent accessibility of stable proteins adsorbed onto different HIC media using hydrogen-deuterium isotope exchange mass spectrometry.
- To determine if structural alterations are responsible for observed changes in protein retention.
Main Methods:
- Utilized hydrogen-deuterium isotope exchange detected by mass spectrometry (HDX-MS) to measure protein solvent accessibility.
- Studied lysozyme, chymotrypsinogen A, and ovalbumin adsorbed onto various HIC media (Butyl Sepharose, Phenyl Sepharose, Toyopearl Butyl).
- Analyzed hydrogen exchange kinetics (EX2) to infer conformational flexibility and solvent exposure.
Main Results:
- Lysozyme exhibited EX2 kinetics in solution and on HIC media, with a slight increase in solvent accessibility upon adsorption.
- Three stable proteins showed enhanced solvent accessibility when adsorbed, but no differences were observed between different HIC adsorbents.
- No significant structural changes were detected in lysozyme before or during elution from HIC media.
Conclusions:
- The HIC media examined do not significantly alter the structure of these stable proteins.
- Conformational differences are unlikely to be the primary cause of selectivity changes observed in HIC.
- Factors such as binding orientation, pore structure, or surface chemistry variations likely account for altered protein retention and selectivity in HIC.