STERIC IMMOBILIZATION OF LIPOSOMES IN CHROMATOGRAPHIC GEL BEADS AND INCORPORATION OF INTEGRAL MEMBRANE-PROTEINS INTO THEIR LIPID BILAYERS

Interactions between water-soluble substances and immobilized liposomes or proteoliposomes can be analyzed by chromatographic procedures. Methods were therefore developed to sterically immobilize large amounts of liposomes in gel beads. First, freeze-thawing of moist Sephacryl S-1000 beads mixed with egg yolk phospholipid liposomes of 200-300 mM lipid concentration immobilized liposomes representing 60-90 mumol lipid with an internal volume of 60-150 mul per milliliter gel bed. The same procedure with freeze-dried beads immobilized liposomes representing more lipid but with lower internal volumes. Other lipid mixtures could also be used. Second, mixtures of Sephacryl S-1000 beads and liposomes were freeze-dried and rehydrated. Large amounts of liposomes became immobilized even with dilute liposome suspensions. Third, a mixture of diethylether-dried Sephacryl S-1000 beads and an emulsion of an aqueous solution in diethylether with lipids was subjected to reverse-phase evaporation. Liposomes of high specific internal volume became immobilized. Integral human red cell membrane proteins could be incorporated into the liposomal lipid bilayers. Upon mixing octyl glucoside-solubilized proteins with moist beads containing immobilized liposomes, up to 0.4 mg protein became incorporated per milliliter gel bed (yield 34%). Proteins could also be incorporated during immobilization by reverse-phase evaporation. Detergents could be removed efficiently from immobilized (proteo)liposomes in a gel bed. (C) 1994 Academic Press, Inc.