Abstract
The aim of this study is to demonstrate the reliability of the use of FT-IR spectroscopy to monitor conformational changes when a protein (HSA) is adsorbed under chromatographic conditions on the silica material RP-C<sub>6</sub>. In the aqueous eluent (D<sub>2</sub>O), the amount of protein retained on the phase is minimal for 30% CH<sub>3</sub>CN, whereas no protein is retained for 40%. Structural results are deduced from quantitative analyses of the infrared Amide I' absorptions and from measurements of the peptide NH-ND exchange. Both are performed for HSA in solution and for HSA adsorbed on RP-C<sub>6</sub> in suspension in equivalent eluents. For the solutions, 30% CH<sub>3</sub>CN in the solvent weakly unfolds some structured loops of the protein. Hydration and aggregation are enhanced in 40% CH<sub>3</sub>CN, which significantly denatures α- and β-domains. When the protein is adsorbed with 0-30% CH<sub>3</sub>CN in the solvent, about one-tenth of HSA backbone unfolds. In the adsorbed state, the protein is more hydrated and self-associated than in the corresponding solutions. The larger contacts between HSA and RP-C<sub>6</sub> are observed when the retention amount is the weakest (30% CH<sub>3</sub>CN). Results show that retention should have a hydrophobic origin. The irreversibility of the retention is supposed to be dependent on the protein structural and solvation changes observed from the solutions to the adsorbed states. To explain the elution with organic solvent > ~38%, a competition between acetonitrile and the solid phase in solvating hydrophobic domains of the protein is suggested.
PDF Article
More Like This
Cited By
You do not have subscription access to this journal. Cited by links are available to subscribers only. You may subscribe either as an Optica member, or as an authorized user of your institution.
Contact your librarian or system administrator
or
Login to access Optica Member Subscription
Add to BibSonomy