Arnhold J, Wiegel D, Hussler O, Arnold K
Institute of Medical Physics and Biophysics, School of Medicine, University of Leipzig, Germany.
Biochim Biophys Acta. 1994 May 11;1191(2):375-83. doi: 10.1016/0005-2736(94)90189-9.
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.
利用十八烷基罗丹明B氯化物（R - 18），研究了由牛脑磷脂酰丝氨酸（PS）组成的小单室囊泡（SUV）和大单室囊泡（LUV）在Ca(2+)诱导下的融合情况，该融合情况是温度和Ca2+浓度的函数。将Ca2+添加到标记的（8 mol%）和未标记的囊泡的1:1混合物中（测定条件），或添加到仅含有标记脂质体的样品中（对照条件）。在SUV和LUV中，测定样品和对照样品之间荧光差异对温度的依赖性均可分为三个区域。在低于20℃的温度下，添加Ca2+后，SUV中的荧光差异仅略有增加，而LUV中的荧光差异保持不变。在28℃及更高温度下，荧光强度差异急剧增加，其中SUV的融合速率高于LUV。在20℃至28℃之间，SUV和LUV都存在一个中间区域。在此区域，荧光从一种行为连续变化到另一种行为，与Ca2+浓度无关。在低于10℃时，由PS组成的LUV中会发生R - 18荧光的剧烈猝灭，此时脂质处于凝胶态。在该温度区域，SUV中的荧光仅发生微弱变化。据推测，在相变温度以下，染料和磷脂分子之间会发生相分离。使用二苯基己三烯进行偏振测量表明，在融合过程中，PS的相变温度从8 - 10℃转变为约24 - 28℃。由于测定样品和对照样品之间R - 18荧光差异强烈依赖于温度，我们推测在向SUV或LUV中添加Ca2+后，PS的相变温度立即发生了转变。在所有发生融合的条件下，聚乙二醇6000都会加速SUV和LUV中的融合。此外，诱导融合所需的Ca2+阈值浓度从无聚合物时的约1 mmol/l降低到存在10%（w/v）PEG 6000时的约0.5 mmol/l。PEG不会改变PS在Ca(2+)诱导融合过程中R - 18荧光特性变化的中间区域。
