INCORPORATION OF CYTOCHROME B5 INTO SUPPORTED PHOSPHOLIPID-BILAYERS BY VESICLE FUSION TO SUPPORTED MONOLAYERS

Supported phospholipid bilayers were formed by first depositing a single monolayer of 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) on a quartz slide by the Langmuir-Blodgett technique, followed by the adsorption and fusion of small unilamellar vesicles with and without the integral membrane protein cytochrome b5. Adsorption, fusion, and desorption reactions were monitored in real time by total internal reflection fluorescence microscopy and the resulting single planar bilayers were characterized by lateral diffusion measurements using the technique of fluorescence recovery after photobleaching. When vesicles which contained 0.2 mol% of cytochrome b5 were used for bilayer formation, the bilayers were completed more rapidly than without the protein and no additional vesicles adsorbed to the bilayer. The measured lateral diffusion coefficient of the lipids was 4 x 10(-8) cm2/s in both leaflets of the bilayer. Photobleaching experiments revealed two populations of cytochrome b5 in these films: one fraction (about 35%) was laterally mobile with a diffusion coefficient of (2.6 +/- 0.7) x 10(-10) cm2/s and one fraction (about 65%) which was immobilized. From its reduced lateral diffusion coefficient, we conclude that the mobile fraction of cytochrome b5 engages in weak interactions with the quartz support.