Properties of immobilized-liposome-chromatographic supports for interaction analysis

Abstract Liposomes, composed of egg yolk phospholipids or phosphatidylcholine, were sterically immobilized in Superdex 200 PG and TSK 6000 PW gel beads by freeze-thawing. The presence of Percoll improved and speeded up the separation of non-immobilized liposomes from gel beads during centrifugal washes. Depending on the concentration of the added liposome suspension and the type of gel beads and lipids used, the amount of lipids immobilized in the gel beads was 20–77 μmol/ml packed gel, with corresponding internal volumes of 20–150 μ1/ml. The specific internal volumes were 0.5–2.5 μl/μmol. The external surface area of the immobilized liposomes was dependent on the amount of lipids immobilized and increased during one week of storage, which indicates structural changes. Immobilization of liposomes in Superdex beads did not significantly affect the back-pressure over the columns (0.5 cm I.D.), which could be run at flow-rates up to 2 ml/min. For drug interaction studies the liposome columns were used for a period of two weeks, at flow-rates between 0.1 and 1 ml/min, for more than 100 runs with good reproducibility and less than 3% loss of lipids. The liposome-chromatographic supports are stable and can be used for the analysis of interactions with membranes and membrane proteins.