QUENCHING AND DEQUENCHING OF OCTADECYL RHODAMINE-B CHLORIDE FLUORESCENCE IN CA2+-INDUCED FUSION OF PHOSPHATIDYLSERINE VESICLES - EFFECTS OF POLY(ETHYLENE GLYCOL)
QUENCHING AND DEQUENCHING OF OCTADECYL RHODAMINE-B CHLORIDE FLUORESCENCE IN CA2+-INDUCED FUSION OF PHOSPHATIDYLSERINE VESICLES - EFFECTS OF POLY(ETHYLENE GLYCOL)
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DOI：
10.1016/0005-2736(94)90189-9
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发表时间：
1994-05-11
影响因子：
3.4
通讯作者：
ARNOLD, K
中科院分区：
文献类型：
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作者：
ARNHOLD, J;WIEGEL, D;ARNOLD, K
用十八烷基罗丹明B氯化物(R-18)研究了钙离子诱导牛脑磷脂酰丝氨酸(PS)组成的SUV和LUV的融合随温度和钙离子浓度的变化。将Ca~(2+)加入标记的囊泡和未标记的囊泡的1：1混合物中(分析条件)或仅含有标记的脂质体的样品(对照条件)。在SUV和LUV中，测定样品和对照样品之间的荧光差异对温度的依赖可以分为三个区域。在低于20摄氏度的温度下，加入钙离子后，SUV的荧光差异仅略有增加，而LUV的荧光差异保持不变。在28摄氏度和更高的温度下，荧光强度的差异增加得更快，因此SUV表现出比LUV更高的融合率。在20摄氏度到28摄氏度之间，SUV和LUV都存在一个中间区域。在这里，荧光从一种行为持续变化到另一种行为，与钙离子的浓度无关。在低于10摄氏度的PS组成的LUV中，R-18荧光急剧猝灭，此时脂质处于凝胶状态。在SUV中，荧光只在这个温度区有微弱的变化。假设染料和磷脂分子之间的分离发生在相变以下。在聚变过程中，PS的相变从8-10℃移动到约24-28℃，这是由二苯基己三烯的偏振测量所揭示的。由于测定样品和对照样品之间R-18荧光的差异强烈地依赖于温度，我们假设PS的相变温度在向SUV或LUV中添加钙离子后立即发生。聚乙二醇6000在发生熔化的所有条件下都能在SUV和LUV中加速熔化。此外，在10%(w/v)PEG6000存在下，诱导融合的Ca~(2+)阈值浓度从没有聚合物的约1 mmol/l降低到约0.5 mmol/l。在Ca~(2+)诱导的PS融合过程中，R-18荧光性质变化的中间区域不受聚乙二醇组分的影响。
Ca2+-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/1 without polymers to about 0.5 mmol/1 in the presence of 10% (w/v) PEG 6000. The intermediate region of changes in fluorescence properties of R-18 in the Ca2+-induced fusion of PS is not changed by PEG.