Properties of nonfused liposomes immobilized on an L1 Biacore chip and their permeabilization by a eukaryotic pore-forming toxin

Abstract

The L1 chip is used intensively for protein–membrane interaction studies in Biacore surface plasmon resonance systems. The exact form of captured lipid membranes on the chip is, however, not precisely known. Evidence exists that the vesicles both remain intact after the binding to the chip and fuse to form a large single-bilayer membrane. In this study, we were able to bind up to approximately 11,500 resonance units of zwitterionic liposomes (100 nm in diameter) at a low flow rate. We show by fluorescence microscopy that the entire surface of the flow cell is covered homogeneously by liposomes. Negatively charged vesicles (i.e., those composed of phosphatidylcholine/phosphatidylglycerol [1:1]) always deposited less densely, but we were able to increase the density slightly with the use of calcium chloride that promotes fusion of the vesicles. Finally, we used zwitterionic liposomes loaded with fluorescent probe calcein to show that they remain intact after the capture on the L1 chip. The fluorescence was lost only after we used equinatoxin, a well-studied pore-forming toxin, to perform on-chip permeabilization of vesicles. The characteristics of permeabilization process for chip-immobilized liposomes are similar to those of liposomes free in solution. All results collectively suggest that liposomes do not fuse to form a single bilayer on the surface of the chip.