BINDING OF LYSOZYME ON THE SURFACE OF ENTRAPPED PHOSPHATIDYLSERINE-PHOSPHATIDYLCHOLINE VESICLES AND AN EXAMPLE OF HIGH-PERFORMANCE LIPID VESICLE SURFACE CHROMATOGRAPHY

Phospholipid vesicles (liposomes) composed of phosphatidylserine (PS) and phosphatidylcholine (PC) in five different molar ratios were formed and entrapped in agarose gel beads (Sepharose 6B and Superose 6). For this purpose a previous dialysis-entrapment procedure was improved by use of an apparatus with continuous buffer flow and rotating dialysis cells. By rotary dialysis combined with a method of vesicles fusion with the aid of Ca2+ ions, the capacity of entrapped vesicles composed of 80-85% phosphatidylserine from bovine brain [denoted "PS (brain extract)"] was increased. The entrapment of PSPC vesicles increased as the content of PS was increased. Lysozyme was applied to a PSPC vesicle-Sepharose 6B column. The amount of bound lysozyme was about 9 nmol per μmole of lipids for entrapped PSPC vesicles with 20 and 45 mol-% PS contents. The amounts of bound lysozyme increased to 29, 30 and 40 nmol per μmole of lipids as the fraction of PS in the vesicles was increased to 65, 80-85 and 100 mol-% PS, respectively. The lysozyme molecules thus closely packed on the surface of the vesicles with the higher PS contents. The losses of the entrapped vesicles in the first two chromatographic lysozyme-binding experiments were 5% for 80-85% PS (brain extract) vesicles, 10-12% for vesicles with 20 to 65 mol-% PS content and 23% for pure PS vesicles. PS (brain extract) vesicles were also formed and entrapped in Superose 6 gel beads (diameter 13 μm). Ribonuclease A, lysozyme and cytochrome c were well separated on this PS-vesicle-Superose column at an ionic strength of 0.05-0.15 M. © 1990.