Quenching and dequenching of octadecyl Rhodamine B chloride fluorescence in Ca(2+)-induced fusion of phosphatidylserine vesicles: effects of poly(ethylene glycol).

Ca(2+)-induced fusion of SUV and LUV composed of ox brain phosphatidylserine (PS) was studied as a function of temperature and concentration of Ca2+ using octadecyl Rhodamine B chloride (R-18). Ca2+ was added to a 1:1 mixture of labelled (8 mol%) and unlabelled vesicles (assay conditions) or to samples containing only labelled liposomes (control conditions). Both, in SUV and LUV the dependence of differences in fluorescence between assay and control samples on temperature can be divided into three regions. At temperatures lower than 20 degrees C the differences in fluorescence increase only slightly in SUV or remain unchanged in LUV after the addition of Ca2+. At 28 degrees C and higher temperatures the differences of fluorescence intensities increase much more drastically, whereby SUV exhibit higher fusion rates than LUV. Between 20 degrees C and 28 degrees C exists an intermediate region for both SUV and LUV. Here the fluorescence changes continuously from one behaviour to the other independent of the concentration of Ca2+. A drastic quenching of R-18 fluorescence occurs in LUV composed of PS below 10 degrees C, where the lipids are in the gel state. In SUV the fluorescence is only weakly changed in this temperature region. It is assumed that a demixing between dye and phospholipid molecules occurs below phase transition. During fusion the phase transition of PS is shifted from 8-10 degrees C to about 24-28 degrees C as revealed by polarization measurements using diphenylhexatriene. Because the differences in R-18 fluorescence between assay and control samples depend strongly on temperature we assume that the shift in phase transition temperature of PS occurs immediately after the addition of Ca2+ to SUV or LUV. Poly(ethylene glycol) 6000 accelerates fusion in both SUV and LUV under all conditions where a fusion takes place. Further, the threshold concentration of Ca2+ to induce fusion is diminished from about 1 mmol/l without polymers to about 0.5 mmol/l in the presence of 10% (w/v) PEG 6000. The intermediate region of changes in fluorescence properties of R-18 in the Ca(2+)-induced fusion of PS is not changed by PEG.