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 pr esent study describes the preparation and properties of a noncovalent immob ilized artificial membrane (noncovalent IAM) stationary phase. The noncoval ent IAM phase was prepared by coating the C-18 chains of a reversed-phase H PLC column with the phospholipid ditetradecanoyl-sn-glycero-3-phosphocholin e. Lipid coating was achieved by pumping a lipid solution in water-2-propan ol through the column. The formation of a bilayer-like structure on the chr omatographic surface was confirmed by calculating the phospholipid surface density of the stationary phase. The surface density was determined to be a pproximately 1.95 mu mol m(-2), which is close to that of lipid vesicles. T he coating was found to be stable in chromatographic elution systems contai ning less than 35% of acetonitrile. Employing this new technique, we determ ined interaction parameters of a set of helical antibacterial magainin-2-am ide peptides with pairwise substitutions of adjacent amino acids by their D -enatiomers. The results demonstrate that the chromatographic retention beh avior of peptides on noncovalent IAM stationary phase shows an excellent co rrelation with lipid affinities to phospholipid vesicles. (C) 1999 Elsevier Science B.V. All rights reserved.