Schneider K, Zimmermann H W
Institut für Physikalische Chemie, Universität Freiburg, Germany.
Histochemistry. 1994 Feb;101(2):135-42. doi: 10.1007/BF00269360.
A micromethod was developed for investigating the interactions between fluorescent dyes and cellular proteins. The lipophilic cationic dye APMC (azopentylmethylcarbocyanine) contains a photosensitive diazirine ring and is suitable for photoaffinity labelling. By combining photoaffinity labelling of cultured cells, micro-gel electrophoresis and detection of the fluorescence with a microfluorimeter, we established a highly sensitive and rapid procedure to identify APMC labelled proteins. Cells which had been incubated for 10 min with 10(-8) M APMC could be analysed for APMC binding without difficulty. Under our experimental conditions this corresponds to about 0.2 nmol APMC per mg protein. The lipophilic APMC specifically stains the mitochondria in living HeLa and LM cells. The fluorescing mitochondria can be easily detected under a fluorescence microscope. By photoaffinity labelling we were able to show that at low dye concentrations APMC preferentially marks four proteins with apparent molecular masses of 31, 40, 66, and 74 kDa. In order to establish that these are mitochondrial proteins, we isolated and analysed the mitochondria from incubated HeLa and LM cells; again, the same four proteins were detected. They are most probably proteins of the inner mitochondrial membranes, which accumulate the lipophilic APMC cations.
开发了一种微量方法用于研究荧光染料与细胞蛋白质之间的相互作用。亲脂性阳离子染料APMC（氮杂戊基甲基碳菁）含有一个光敏重氮环，适用于光亲和标记。通过将培养细胞的光亲和标记、微量凝胶电泳以及用微量荧光计检测荧光相结合，我们建立了一种高灵敏度且快速的程序来鉴定APMC标记的蛋白质。用10⁻⁸ M APMC孵育10分钟的细胞能够毫无困难地进行APMC结合分析。在我们的实验条件下，这相当于每毫克蛋白质约0.2 nmol APMC。亲脂性的APMC能特异性地对活的HeLa细胞和LM细胞中的线粒体进行染色。在荧光显微镜下可以很容易地检测到发荧光的线粒体。通过光亲和标记我们能够表明，在低染料浓度下，APMC优先标记四种表观分子量分别为31、40、66和74 kDa的蛋白质。为了确定这些是线粒体蛋白质，我们从孵育后的HeLa细胞和LM细胞中分离并分析了线粒体；同样，检测到了相同的四种蛋白质。它们很可能是线粒体内膜的蛋白质，能积累亲脂性的APMC阳离子。
