Silvius J R, Leventis R, Brown P M, Zuckermann M
Department of Biochemistry, McGill University, Montréal, Québec, Canada.
Biochemistry. 1987 Jul 14;26(14):4279-87. doi: 10.1021/bi00388a015.
A series of fluorescent phospholipids has been synthesized, by a general and versatile procedure, with various fluorescent groups attached to the methyl-terminal half of one acyl chain in an otherwise normal phospholipid structure. Phospholipids labeled with (dialkylamino)coumarin moieties, and to a slightly lesser extent those labeled with a bimane group, exhibit a strong and stable blue fluorescence in phospholipid dispersions that is relatively insensitive to the physical state of the lipid phase. The fluorescence of these labeled phospholipids is efficiently quenched by resonance energy transfer to lipids labeled with a [[(dimethylamino)phenyl]azo]phenyl or a methyl(nitrobenzoxadiazolyl)amino group when these acceptors are incorporated into the same bilayer as the donor species. Acyl chain labeled phospholipid probes, both of whose chains are at least sixteen carbons in length, exchange extremely slowly between lipid vesicles (less than 1% exchange/h). These properties allow various donor-acceptor combinations of probes to be employed in sensitive and reliable assays of lipid mixing accompanying membrane fusion. We demonstrate that, in two particularly demanding applications (assays of the calcium-mediated coalescence of phosphatidylserine vesicles and of the proton-triggered coalescence of phosphatidylethanolamine vesicles), some combinations of acyl chain labeled probes offer substantial advantages over the commonly used N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)phosphatidylethanolamine/N-(l issamine rhodamine B sulfonyl)phosphatidylethanolamine pair to monitor accurately the progress of lipid mixing between vesicles.
通过一种通用且灵活的方法，合成了一系列荧光磷脂，在正常磷脂结构的一条酰基链的甲基末端一半上连接了各种荧光基团。用（二烷基氨基）香豆素部分标记的磷脂，以及用双硫腙基团标记的磷脂在较小程度上，在磷脂分散体中呈现出强烈且稳定的蓝色荧光，这种荧光对脂质相的物理状态相对不敏感。当这些受体与供体物种掺入同一双层时，这些标记磷脂的荧光通过共振能量转移有效地猝灭到用[[（二甲基氨基）苯基]偶氮]苯基或甲基（硝基苯并恶二唑基）氨基标记的脂质上。两条链长度均至少为16个碳的酰基链标记的磷脂探针在脂质囊泡之间交换极其缓慢（小于1%交换/小时）。这些特性使得各种供体 - 受体组合的探针可用于伴随膜融合的脂质混合的灵敏且可靠的检测。我们证明，在两个特别苛刻的应用（磷脂酰丝氨酸囊泡的钙介导聚结和磷脂酰乙醇胺囊泡的质子触发聚结的检测）中，一些酰基链标记探针的组合相对于常用的N - （7 - 硝基苯并 - 2 - 恶唑 - 1,3 - 二氮杂 - 4 - 基）磷脂酰乙醇胺/N - （丽丝胺罗丹明B磺酰基）磷脂酰乙醇胺对在准确监测囊泡之间脂质混合的进展方面具有显著优势。
