Bagatolli L, Gratton E, Khan T K, Chong P L
Laboratory for Fluorescence Dynamics, Department of Physics, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA.
Biophys J. 2000 Jul;79(1):416-25. doi: 10.1016/S0006-3495(00)76303-X.
The effects of temperature and pH on Laurdan (6-lauroyl-2-(dimethylamino)naphthalene) fluorescence intensity images of giant unilamellar vesicles (GUVs) ( approximately 20-150 microm in diameter) composed of the polar lipid fraction E (PLFE) from the thermoacidophilic archaebacteria Sulfolobus acidocaldarius have been studied using two-photon excitation. PLFE GUVs made by the electroformation method were stable and well suited for microscopy studies. The generalized polarization (GP) of Laurdan fluorescence in the center cross section of the vesicles has been determined as a function of temperature at pH 7.23 and pH 2.68. At all of the temperatures and pHs examined, the GP values are low (below or close to 0), and the GP histograms show a broad distribution width (> 0.3). When excited with light polarized in the y direction, Laurdan fluorescence in the center cross section of the PLFE GUVs exhibits a photoselection effect showing much higher intensities in the x direction of the vesicles, a result opposite that previously obtained on monopolar diester phospholipids. This result indicates that the chromophore of Laurdan in PLFE GUVs is aligned parallel to the membrane surface. The x direction photoselection effect and the low GP values lead us to further propose that the Laurdan chromophore resides in the polar headgroup region of the PLFE liposomes, while the lauroyl tail inserts into the hydrocarbon core of the membrane. This unusual L-shaped disposition is presumably caused by the unique lipid structures and by the rigid and tight membrane packing in PLFE liposomes. The GP exhibited, at both pH values, a small but abrupt decrease near 50 degrees C, suggesting a conformational change in the polar headgroups of PLFE. This transition temperature fully agrees with the d-spacing data recently measured by small-angle x-ray diffraction and with the pyrene-labeled phosphatidylcholine and perylene fluorescence data previously obtained from PLFE multilamellar vesicles. Interestingly, the two-photon Laurdan fluorescence images showed snowflake-like lipid domains in PLFE GUVs at pH 7.23 and low temperatures (<20 degrees C in the cooling scan and <24 degrees C in the heating scan). These domains, attributable to lipid lateral separation, were stable and laterally immobile at low temperatures (<23 degrees C), again suggesting tight membrane packing in the PLFE GUVs.
利用双光子激发研究了温度和pH对由嗜热嗜酸古细菌酸热硫化叶菌的极性脂质组分E（PLFE）构成的巨型单层囊泡（GUV，直径约20 - 150微米）的劳丹（6 - 月桂酰 - 2 - （二甲基氨基）萘）荧光强度图像的影响。通过电形成法制备的PLFE GUV稳定且非常适合显微镜研究。已确定囊泡中心横截面中劳丹荧光的广义极化（GP）随pH 7.23和pH 2.68时温度的变化情况。在所研究的所有温度和pH值下，GP值都很低（低于或接近0），并且GP直方图显示出较宽的分布宽度（> 0.3）。当用沿y方向偏振的光激发时，PLFE GUV中心横截面中的劳丹荧光表现出光选择效应，在囊泡的x方向上强度高得多，这一结果与先前在单极二酯磷脂上得到的结果相反。该结果表明PLFE GUV中劳丹的发色团与膜表面平行排列。x方向的光选择效应和低GP值使我们进一步提出，劳丹发色团位于PLFE脂质体的极性头部区域，而月桂酰尾部插入膜的烃核中。这种不寻常的L形排列大概是由独特的脂质结构以及PLFE脂质体中刚性且紧密的膜堆积引起的。在两个pH值下，GP在接近50℃时都出现了小而突然的下降，这表明PLFE的极性头部基团发生了构象变化。这个转变温度与最近通过小角x射线衍射测量的d间距数据以及先前从PLFE多层囊泡获得的芘标记磷脂酰胆碱和苝荧光数据完全一致。有趣的是，双光子劳丹荧光图像显示在pH 7.23和低温（冷却扫描中<20℃，加热扫描中<24℃）下PLFE GUV中存在雪花状脂质域。这些归因于脂质侧向分离的域在低温（<23℃）下是稳定且横向不可移动的，这再次表明PLFE GUV中膜堆积紧密。
