Lipid-vesicle-surface chromatography.
Egg-yolk phospholipid vesicles (liposomes) containing stearylamine cations or phosphatidylserine anions, were formed and entrapped in agarose gel beads (Sepharose 6B) by a dialysis procedure. On a column of entrapped phospholipid-stearylamine (4:1) (cationic) vesicles, 0.36 mg of ferritin was bound per mumol lipids at 0.05 M ionic strength and pH 7. About 30% of the vesicle surface thus became covered with ferritin. Only 0.04 mg of citraconylated myoglobin was bound per mumol lipids, as myoglobin is much smaller than ferritin. Haeme groups were readily inserted into the lipid bilayers. An excess amount of bovine serum albumin (BSA) or ribonuclease A was applied to entrapped ionic vesicles and the bound proteins were eluted by increasing the ionic strength from 0.01 to 0.2 or 0.5 M. After three to five runs, 82-88% of the vesicles (the phospholipids) remained entrapped. The capacity of the cationic vesicle-column for BSA decreased more than did the amount of entrapped vesicles, which indicates a preferential loss of stearylamine. Ion-exchange experiments were done with human plasma and with BSA monomers and dimers on entrapped cationic vesicles. Plasma proteins could be separated. BSA dimers were eluted later than BSA monomers in a sodium chloride gradient and the separation was better than on DEAE-Sepharose. The contact area between the protein and the vesicle surface is important for the binding strength. Protein-vesicle surface interactions can be studied by chromatography on entrapped vesicles.
Department of Biochemistry, University of Uppsala, Sweden.
Q
Q
Yang
M
M
Wallstén
P
P
Lundahl
D001798:Blood Proteins / Q000032:analysis; D002852:Chromatography, Ion Exchange; D007201:Indicator Dilution Techniques; D008081:Liposomes; D010743:Phospholipids; D011485:Protein Binding; D011506:Proteins / Q000032:analysis*; D012710:Serum Albumin, Bovine / Q000032:analysis; D013499:Surface Properties
D001798:Blood Proteins; D008081:Liposomes; D010743:Phospholipids; D011506:Proteins; D012710:Serum Albumin, Bovine