Malinin V S, Haque M E, Lentz B R
Department of Biochemistry, University of North Carolina, Chapel Hill, NC 27599-7260, USA.
Biochemistry. 2001 Jul 27;40(28):8292-9. doi: 10.1021/bi010570r.
A number of fluorescent probes have been used to follow membrane fusion events, particularly intermixing of lipids. None of them is ideal. The most popular pair of probes is NBD-PE and Rh-PE, in which the fluorescent groups are attached to the lipid headgroups, making them sensitive to changes in the surrounding medium. Here we present a new assay for monitoring lipid transfer during membrane fusion using the acyl chain tagged fluorescent probes BODIPY500-PC and BODIPY530-PE. Like the NBD-PE/Rh-PE assay, this assay is based on fluorescence resonance energy transfer (FRET) between the donor, BODIPY500, and the acceptor, BODIPY530. The magnitude of FRET is sensitive to the probe surface concentration, allowing one to detect movement of probes from labeled to unlabeled vesicles during fusion. The high quantum yield of fluorescence, high efficiency of FRET (R(o) is estimated to be approximately 60 A), photostability, and localization in the central hydrophobic region of a bilayer all make this pair of probes quite promising for detecting fusion. We have compared this and two other lipid mixing assays for their abilities to detect the initial events of poly(ethylene glycol) (PEG)-mediated fusion of small unilamellar vesicles (SUVs). We found that the BODIPY500/530 assay showed lipid transfer rates consistent with those obtained using the DPHpPC self-quenching assay, while lipid mixing rates measured with the NBD-PE/Rh-PE RET assay were significantly slower. We speculate that the bulky labeled headgroups of NBD-PE and especially Rh-PE molecules hamper movement of probes through the stalk between fusing vesicles, and thus reduce the apparent rate of lipid mixing.
许多荧光探针已被用于追踪膜融合事件，尤其是脂质的混合。但它们都不理想。最常用的一对探针是NBD-PE和Rh-PE，其中荧光基团连接在脂质头部基团上，使其对周围介质的变化敏感。在此，我们展示了一种使用酰基链标记的荧光探针BODIPY500-PC和BODIPY530-PE监测膜融合过程中脂质转移的新方法。与NBD-PE/Rh-PE方法一样，该方法基于供体BODIPY500和受体BODIPY530之间的荧光共振能量转移（FRET）。FRET的大小对探针表面浓度敏感，使人们能够在融合过程中检测探针从标记囊泡向未标记囊泡的移动。荧光的高量子产率、FRET的高效率（估计R(o)约为60 Å）、光稳定性以及在双层膜中心疏水区域的定位，都使得这对探针在检测融合方面颇具前景。我们比较了该方法与其他两种脂质混合检测方法检测聚乙二醇（PEG）介导的小单层囊泡（SUV）融合初始事件的能力。我们发现，BODIPY500/530检测方法显示的脂质转移速率与使用DPHpPC自猝灭检测方法获得的结果一致，而用NBD-PE/Rh-PE RET检测方法测得的脂质混合速率明显较慢。我们推测，NBD-PE尤其是Rh-PE分子庞大的标记头部基团阻碍了探针通过融合囊泡之间的柄部移动，从而降低了脂质混合的表观速率。
