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The activities of cytochrome c, a basic haem protein, to mediate fusion of membranes was investigated using transfer and fusion assays. Donor and acceptor vesicles were prepared and used for the assays. The experiments were monitored using radio labelled lipid {[14C]-lipid} and fluorescence lipid, N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dihexadecanoyl-snglycero-3-phosphoethanolamine (NBD-PE). A 100 µl of magnetic beads was adequate for separation of acceptor from donor vesicle. The loss of either radioactivity or fluorescence or both was used to assess the percentage fusion. The results show that mediation of membrane fusion by cytochrome c is enhanced at physiological temperature, acidic pH and in the presence of anionic phospholipids, bis(monoacylglycerol) phosphate (BMP). The fusion process was rapid, the fusion took place within 10 min. Cytochrome c could not mediate transfer of lipids in the membrane, but could assist in membrane fusion. Therefore, cytochrome c is a fusogenic and not a transfer protein.

Keywords: Cytochrome c; Membrane fusion; Fusion assay; Transfer assay; Radioactivity; Fluorescence; bis(monoacylglycero)phosphate (BMP).