FT-IR ANALYSIS FOR STRUCTURAL CHARACTERIZATION OF ALBUMIN ADSORBED ONTHE REVERSED-PHASE SUPPORT RP-C-6

The aim of this study is to demonstrate the reliability of the use of FT-IR spectroscopy to monitor conformational changes when a protein (H SA) is adsorbed under chromatographic conditions on the silica materia l RP-C-6. 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 ad sorbed on RP-C-6 in suspension in equivalent eluents. For the solution s, 30% CH3CN in the solvent weakly unfolds some structured loops of th e protein. Hydration and aggregation are enhanced in 40% CH3CN, which significantly denatures alpha- and beta-domains. When the protein is a dsorbed with 0-30% CH3CN in the solvent, about one-tenth of HSA backbo ne unfolds. In the adsorbed state, the protein is more hydrated and se lf-associated than in the corresponding solutions. The larger contacts between HSA and RP-C-6 are observed when the retention amount is the weakest (30% CH3CN). Results show that retention should have a hydroph obic origin. The irreversibility of the retention is supposed to be de pendent on the protein structural and solvation changes observed from the solutions to the adsorbed states. To explain the elution with orga nic solvent > similar to 38%, a competition between acetonitrile and t he solid phase in solvating hydrophobic domains of the protein is sugg ested.