A quantitative model for using acridine orange as a transmembrane pH gradient probe
A quantitative model for using acridine orange as a transmembrane pH gradient probe
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DOI：
10.1006/abio.1998.2639
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发表时间：
1998-05-15
影响因子：
2.9
通讯作者：
Barenholz, Y
中科院分区：
文献类型：
--
作者：
Clerc, S;Barenholz, Y
利用光学探针可以很容易地实现质子泵监测膜囊泡内部空间的酸化。跨膜pH梯度导致阳离子染料吖啶橙的吸收光谱蓝移和荧光猝灭。据推测，这些变化是由小泡内染料的积累和聚集引起的。我们使用具有跨膜浓度梯度的硫酸铵脂质体作为模型体系来验证这一假说。与脂质体孵育的吖啶橙溶液的荧光强度受梯度大小、囊泡捕获体积和温度的影响。将这些实验数据与一个理论模型进行比较，该理论模型描述了根据质子浓度的内外比在囊泡中积累的吖啶橙单体，以及泡内形成三明治状的吖啶橙阳离子堆积的情况。该理论模型定量地预测了吖啶橙跨膜pH梯度与光谱变化之间的关系。因此，对染料在生物囊泡管腔内聚集的充分表征，为使用吖啶橙作为光学探针来量化跨膜pH梯度提供了理论基础。(C)1998年学术出版社。
Monitoring the acidification of the internal space of membrane vesicles by proton pumps can be achieved easily with optical probes. Transmembrane pH gradients cause a blue-shift in the absorbance spectrum and the quenching of the fluorescence of the cationic dye acridine orange. It has been postulated that these changes are caused by accumulation and aggregation of the dye inside the vesicles. We tested this hypothesis using liposomes with transmembrane concentration gradients of ammonium sulfate as model system. Fluorescence intensity of acridine orange solutions incubated with liposomes was affected by magnitude of the gradient, volume trapped by vesicles, and temperature. These experimental data were compared 60 a theoretical model describing the accumulation of acridine orange monomers in the vesicles according to the inside-to-outside ratio of proton concentrations, and the intravesicular formation of sandwich-like piles of acridine orange cations. This theoretical model predicted quantitatively the relationship between the transmembrane pH gradients and spectral changes of acridine orange. Therefore, adequate characterization of aggregation of dye in the lumen of biological vesicles provides the theoretical basis for using acridine orange as an optical probe to quantify transmembrane PH gradients. (C) 1998 Academic Press.