Skip to main content

FT-IR Analysis for Structural Characterization of Albumin Adsorbed on the Reversed-Phase Support RP-C6

Buy Article:

$29.00 plus tax (Refund Policy)

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-C6. In the aqueous eluent (D2O), the amount of protein retained on the phase is minimal for 30% CH3CN, 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-C6 in suspension in equivalent eluents. For the solutions, 30% CH3CN in the solvent weakly unfolds some structured loops of the protein. Hydration and aggregation are enhanced in 40% CH3CN, which significantly denatures α- and β-domains. When the protein is adsorbed with 0-30% CH3CN 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-C6 are observed when the retention amount is the weakest (30% CH3CN). 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.

Keywords: Conformation; Human serum albumin; Protein adsorption; Reversed-phase chromatography; Transmission FT-IR spectroscopy

Document Type: Research Article

DOI: http://dx.doi.org/10.1366/0003702953965885

Affiliations: 1: Laboratoire de Physico-Chimie des Biopolymères C.N.R.S., UMR 27, 8 rue Henri Dunant, 94320, Thiais, France 2: Laboratoire de Spectrochimie Infrarouge et Raman, C.N.R.S., UPR 2631 T, 8 rue Henri Dunant, 94320, Thiais, France

Publication date: December 1, 1995

More about this publication?
sas/sas/1995/00000049/00000012/art00004
dcterms_title,dcterms_description,pub_keyword
6
5
20
40
5

Access Key

Free Content
Free content
New Content
New content
Open Access Content
Open access content
Subscribed Content
Subscribed content
Free Trial Content
Free trial content
Cookie Policy
X
Cookie Policy
ingentaconnect website makes use of cookies so as to keep track of data that you have filled in. I am Happy with this Find out more