Capillary electrophoresis of liposomes functionalized for protein binding.
CE enabled assessing the attachment of hexa-histidine-tagged proteins to functionalized phospholipid liposomes. The liposomes were made of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, phosphatidyl-ethanolamine, cholesterol and distearoyl-glycero-3-phosphoethanolamine-N-methoxy(polyethylene glycol) in a molar ratio of 29:26:40:5. The unilamellar vesicles, which had an average diameter of 170 nm, were labelled by inclusion of FITC-dextran for fluorescence detection. CE was carried out in poly(vinyl alcohol) (PVA)-coated capillaries at 25 degrees C with a BGE consisting of Tris-HCl (50 mM, pH 8.0). For conjugation of the liposomes with the proteins (soluble synthetic receptor fragments with molecular mass of 60 and 70 kDa, respectively), Ni(2+) was implanted into the vesicle surface by an anchor lipid containing a nitrilotriacetate acid (NTA) group as complexation agent for the metal ions. The difference in surface charge enabled the separation of the different species of interest by CE: plain vesicles, vesicles functionalised with Ni-NTA, vesicle-protein complexes and the species formed upon removal of the Ni-ions by complexation with EDTA. Loss of the Ni-ions resulted in the release of the proteins and the reappearance of the plain Ni-free NTA-liposome species in the electropherograms.
Bilek
Gerhard
G
Institute for Analytical Chemistry, University of Vienna, Vienna, Austria.
Kremser
Leopold
L
Blaas
Dieter
D
Kenndler
Ernst
E
D002784:Cholesterol / Q000737:chemistry; D019075:Electrophoresis, Capillary / Q000379:methods*; D006639:Histidine / Q000737:chemistry; D008081:Liposomes / Q000302:isolation & purification*; D008239:Lysine / Q000031:analogs & derivatives / Q000737:chemistry; D009532:Nickel / Q000737:chemistry; D009829:Oleic Acids / Q000737:chemistry; D010316:Particle Size; D011092:Polyethylene Glycols / Q000737:chemistry; D011485:Protein Binding; D011506:Proteins / Q000737:chemistry; D013386:Succinates / Q000737:chemistry
D008081:Liposomes; C476561:N(alpha),N(alpha)-bis(carboxymethyl)-N(epsilon)-(1,2-dioleyl-sn-glycero-3-succinyl)-L-lysine; D009829:Oleic Acids; D011506:Proteins; D013386:Succinates; D011092:Polyethylene Glycols; D006639:Histidine; D009532:Nickel; D002784:Cholesterol; D008239:Lysine