Avidin-biotin immobilization of unilamellar liposomes in gel beads for chromatographic analysis of drug-membrane partitioning.

To construct a homogeneous lipid membrane chromatographic phase, biotinylated unilamellar liposomes of small and large sizes (SUVs and LUVs, respectively) were immobilized in avidin- or streptavidin-derived gel beads in amounts up to 55 micromol phospholipid/ml gel bed at yields above 50%. The immobilized liposomes exhibited excellent stability due to avidin-biotin multiple-site binding. The trapped volume and size distribution of the immobilized liposomes (0.33-0.42 microl/micromol lipid and 20-30 nm diameter for SUVs, 1.7-1.9 microl/micromol lipid and 80-120 nm for LUVs) indicated the unilamellarity and integrity of the immobilized liposomes. Partitioning of 15 pharmaceutical drugs into the bilayers of LUVs immobilized in different gel matrices correlated very well, as shown by chromatographic drug retention analysis. The partitioning of several beta-blockers into the immobilized LUVs showed a close correlation with their partitioning, reported in the literature, into free liposomes. The avidin-biotin-immobilized unilamellar liposomes can thus be used for chromatographic analysis and screening of solute-membrane interactions.

Oriented immobilization of bacterial photosynthetic membrane.

We have examined a method for oriented immobilization of photosynthetic membrane fragments on a solid surface by specific avidin-biotin interaction. Photosynthetic membrane fragments from the purple non-sulphur photosynthetic bacterium Rhodopseudomonas viridis, of which the H-subunit of the photosynthetic reaction centre was biotinylated, was immobilized on an avidin-adsorbed plate. Orientation of the immobilized membrane on the plastic plate was checked by an antisera binding assay that could react to the respective sides of the membrane: the H-subunit side was selectively adsorbed on the plate. Light-induced potential and current responses could be measured when the membrane immobilized on the SnO2-coated glass plate was dried and sandwiched with a counter electrode of Hg. The electrical response in the immobilized membrane was much improved in comparison with the control (membranes were simply adsorbed on the plate), supporting the idea that the membranes have an orientation on solid surfaces.