Noncovalent immobilized artificial membrane chromatography, an improved method for describing peptide-lipid bilayer interactions

A promising approach in assessing hydrophobic peptide-membrane interactions is the use of reversed-phase highperformance liquid chromatography. The present study describes the preparation and properties of a noncovalent immobilized artificial membrane (noncovalent IAM) stationary phase. The noncovalent IAM phase was prepared by coating the C-18 chains of a reversed-phase HPLC column with the phospholipid ditetradecanoyl-sn-glycero-3-phosphocholine. Lipid coating was achieved by pumping a lipid solution in water-2-propanol through the column. The formation of a bilayer-like structure on the chromatographic surface was confirmed by calculating the phospholipid surface density of the stationary phase. The surface density was determined to be approximately 1.95 mu mol m(-2), which is close to that of lipid vesicles. The coating was found to be stable in chromatographic elution systems containing less than 35% of acetonitrile. Employing this new technique, we determined interaction parameters of a set of helical antibacterial magainin-2-amide peptides with pairwise substitutions of adjacent amino acids by their D-enatiomers. The results demonstrate that the chromatographic retention behavior of peptides on noncovalent IAM stationary phase shows an excellent correlation with lipid affinities to phospholipid vesicles. (C) 1999 Elsevier Science B.V. All rights reserved.