KINETICS OF CALCIUM-INDUCED MIXING OF LIPIDS AND AQUEOUS CONTENTS OF LARGE UNILAMELLAR PHOSPHATIDYLSERINE VESICLES

Ca2+-induced fusion events in suspensions of large unilamellar phosphatidylserine (PS) liposomes were monitored by fluorescence methods. Mixing of vesicle contents was studied by measuring the increase in Tb emission intensity due to formation of a complex between Tb3+ ions and dipicolinic acid trapped in the liposomes. Lipid redistribution was determined with the aid of the resonance transfer of excitation energy using dipalmitoylphosphatidylethanolamine labeled with the donor N-(7-nitro-2,1,3-benzoxadiazol-4-yl) or the acceptor tetramethylrhodamine at the free amino group. The 2 methods yielded significantly different results. While recombination of contents could not be detected at Ca2+ concentrations below 2.5 mM the threshold concentrations for lipid mixing was 1 mM. For saturating Ca2+ concentrations (> 5 mM Ca2+) initial rates were higher by almost an order of magnitude for lipid mixing than for recombination of liposome contents. The observation of rapid lipid mixing phenomena does not allow conclusions to be drawn as to the fate of the enclosed volumes.