Laboratoire de phathologie moléculaire, Département de pathologie, Université de Montréal, Succ. Centre-ville, C.P. 6128, H3C 3J7, Montréal, Québec, Canada.
J Fluoresc. 1996 Dec;6(4):209-19. doi: 10.1007/BF00732824.
Steady-state and time-resolved spectroscopic properties of rhodamine-123 (rh123) and 4,5-dibromorhodamine methyl ester (dbr123) bound to different cell lines are evaluated. Studies are also performed on the dye bound to extracted mitochondria. Results are compared with those obtained in homogeneous and microheterogeneous media. Results suggest that these dyes can specifically bind only with cell mitochondria. As a result of binding, excitation and emission spectra are red shifted by 10 to 12 nm. The fluorescence decay of these dyes bound to mitochondria shows two lifetimes. Values are about 4.0 and 2.0 ns forrh123 and about 1.9 and 0.5 ns fordbr123. Detailed global analysis of emission wavelength and dye concentration dependences of the fluorescence decay is performed. Results indicate that these dyes are bound to two different binding sites at mitochondria. The decay-associated fluorescence spectrum for the species corresponding to each binding site is recovered. Species1, corresponding to the longer lifetime, is found to be more red shifted compared to species2. The fluorescence of species2 is heavily quenched. The origin of this quenching is explained in terms of resonance energy transfer between donor species2 and acceptor species1. The possible nature of the two binding sites is also discussed.
评估了罗丹明-123（rh123）和 4,5-二溴罗丹明甲酯（dbr123）与不同细胞系结合的稳态和时间分辨光谱性质。还对与提取的线粒体结合的染料进行了研究。将结果与均相和微相异介质中获得的结果进行了比较。结果表明，这些染料只能特异性地与细胞线粒体结合。结合后，激发和发射光谱分别红移 10 到 12nm。这些与线粒体结合的染料的荧光衰减显示出两个寿命。对于 rh123，值约为 4.0 和 2.0ns，对于 dbr123，值约为 1.9 和 0.5ns。对发射波长和染料浓度对荧光衰减的依赖性进行了详细的全局分析。结果表明，这些染料结合在线粒体的两个不同结合位点上。恢复了与每个结合位点相对应的物种的衰减相关荧光光谱。与较长寿命相对应的物种 1 被发现比物种 2 红移更大。物种 2 的荧光被严重猝灭。这种猝灭的起源可以根据供体物种 2 和受体物种 1 之间的共振能量转移来解释。还讨论了两个结合位点的可能性质。
