J-AGGREGATE FORMATION OF A CARBOCYANINE AS A QUANTITATIVE FLUORESCENT INDICATOR OF MEMBRANE-POTENTIAL
J-AGGREGATE FORMATION OF A CARBOCYANINE AS A QUANTITATIVE FLUORESCENT INDICATOR OF MEMBRANE-POTENTIAL
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DOI：
10.1021/bi00232a015
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发表时间：
1991-05-07
期刊：
影响因子：
2.9
通讯作者：
CHEN, LB
中科院分区：
文献类型：
--
作者：
REERS, M;SMITH, TW;CHEN, LB
本文报道了一种新型膜电位敏感探针JC-1在缓冲液和离体心肌线粒体中的光谱特性。JC-1是一种带有离域正电荷的碳菁。在适宜的条件下，它形成了由J-聚集体组成的浓度依赖性荧光相。当在490 nm处激发时，单体在527 nm处表现出最大发射，J-聚集体在590 nm处表现出最大发射。增加JC-1的浓度超过一定浓度引起J-聚集体荧光的线性上升，而单体荧光保持恒定。通电线粒体的膜电位（内部为负）促进JC-1定向摄取到基质中，随后也形成J-聚集体。J-聚集体荧光对ADP诱导的瞬时膜电位变化和氧化磷酸化代谢抑制剂敏感。发现J-聚集体荧光在7.15-8.0的生理pH范围内与pH无关，并且可以用缬氨霉素诱导的K+扩散电位线性校准。JC-1与罗丹明和其他碳菁相比的优点是，随着膜电位的增加，其颜色可逆地从绿色变为红色。这可以用于在显微镜的载物台上对活线粒体进行成像。
The spectral properties of a novel membrane potential sensitive probe (JC-1) were characterized in aqueous buffers and in isolated cardiac mitochondria. JC-1 is a carbocyanine with a delocalized positive charge. It formed under favorable conditions a concentration-dependent fluorescent nematic phase consisting of J-aggregates. When excited at 490 nm, the monomers exhibited an emission maximum at 527 nm and J-aggregates at 590 nm. Increasing concentrations of JC-1 above a certain concentration caused a linear rise in the J-aggregate fluorescence, while the monomer fluorescence remained constant. The membrane potential of energized mitochondria (negative inside) promoted a directional uptake of JC-1 into the matrix, also with subsequent formation of J-aggregates. The J-aggregate fluorescence was sensitive to transient membrane potential changes induced by ADP and to metabolic inhibitors of oxidative phosphorylation. The J-aggregate fluorescence was found to be pH independent within the physiological pH range of 7.15-8.0 and could be linearly calibrated with valinomycin-induced K+ diffusion potentials. The advantage of JC-1 over rhodamines and other carbocyanines is that its color altered reversibly from green to red with increasing membrane potentials. This can be exploited for imaging live mitochondria on the stage of a microscope.