Lee H C, Forte J G
Biochim Biophys Acta. 1979 Jul 5;554(2):375-87. doi: 10.1016/0005-2736(79)90378-x.
Fluorescamine and trinitrobenzenesulfonate were used as chemical probes to differentially label amino phospholipids in liposomes. At low concentrations, fluorescamine reacts primarily with amino lipids on the external half of the bilayer. Further increase in fluorescamine concentration resulted in a linear increase of labeling indicating penetration and reaction with the internal half of the bilayer. Because of the pH requirements of the fluorescamine reaction, internal labeling was eliminated with a H+ gradient: inside acidic/outside alkaline. Differential labeling was also achieved with trinitrobenzenesulfonate, which is normally not permeable but which can be transported by valinomycin-K+ complex and react with internal amines. Thus, either half of the bilayer can be labeled with the same or different reagents. When liposomes were double-labeled, the fluorescence of fluorescamine was quenched by the trinitrobenzenesulfonate label. This quenching was reversed by solubilizing the liposomes with acidic ethanol. No quenching occurred when fluorescamine-labeled liposomes were mixed with trinitrobenzenesulfonate-reacted liposomes (or trinitrophenylated methylamine) suggesting close proximity of two labels is required for quenching. Conditions which promoted vesicular fusion promptly produced quenching. These differential labeling procedures can be usefully applied to quantitate aminolipids on internal and external vesicular surface, monitor vesicular fusion, and assess liposomal structure.
荧光胺和三硝基苯磺酸被用作化学探针，以对脂质体中的氨基磷脂进行差异标记。在低浓度下，荧光胺主要与双层外部的氨基脂质发生反应。荧光胺浓度的进一步增加导致标记呈线性增加，表明其穿透并与双层内部发生反应。由于荧光胺反应对pH的要求，通过H⁺梯度（内部酸性/外部碱性）消除了内部标记。用三硝基苯磺酸也实现了差异标记，三硝基苯磺酸通常不可渗透，但可通过缬氨霉素-K⁺复合物转运并与内部胺反应。因此，双层的任何一半都可用相同或不同的试剂进行标记。当脂质体进行双重标记时，荧光胺的荧光会被三硝基苯磺酸标记淬灭。通过用酸性乙醇溶解脂质体，这种淬灭作用可以逆转。当荧光胺标记的脂质体与三硝基苯磺酸反应的脂质体（或三硝基苯基化甲胺）混合时，没有发生淬灭，这表明淬灭需要两个标记紧密相邻。促进囊泡融合的条件会迅速产生淬灭。这些差异标记程序可有效地用于定量囊泡内部和外部表面的氨基脂质、监测囊泡融合以及评估脂质体结构。
