Greenbaum L, Rothmann C, Lavie R, Malik Z
Microscopy Unit, Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan, Israel.
Biol Chem. 2000 Dec;381(12):1251-8. doi: 10.1515/BC.2000.153.
Characterization of protein damage during photosensitization of chlorin e6-treated cells was performed using the green fluorescent protein (GFP). The GFP-chromophore damage caused by singlet oxygen was studied in COS 7 kidney cells and E. coli bacteria following light irradiation. Electron spin resonance (ESR) revealed the generation of endogenous singlet oxygen (1O2) by photoactivated GFP, an effect similar to that produced by the exogenous photosensitizer chlorin e6. A light dose-dependent photobleaching effect of GFP was pronounced at low pH or upon photosensitization with chlorin e6. However, the 1O2 quenchers beta-carotene and sodium azide minimized GFP photo-bleaching. Gel electrophoresis of photosensitized GFP followed by fluorescence multi-pixel spectral imaging revealed the binding of chlorin e6 to GFP, affecting the photobleaching efficacy. Fluorescence multi-pixel spectral imaging of GFP-transfected COS 7 cells demonstrated the presence of GFP in the cytoplasm and nucleus, while chlorin e6 was found to be concentrated in the perinuclear vesicles. Exposure of the cells to light induced GFP photobleaching in the close vicinity of chlorin e6 vesicles. We conclude that photoactivated GFP generates endogenous 1O2, inducing chromophore damage, which can be enhanced by the cooperation of exogenous chlorin e6.
利用绿色荧光蛋白（GFP）对二氢卟吩e6处理的细胞在光致敏过程中的蛋白质损伤进行了表征。在光照后，对COS 7肾细胞和大肠杆菌中由单线态氧引起的GFP发色团损伤进行了研究。电子自旋共振（ESR）显示光活化的GFP会产生内源性单线态氧（1O2），这一效应与外源性光敏剂二氢卟吩e6产生的效应相似。在低pH值或用二氢卟吩e6进行光致敏时，GFP的光剂量依赖性光漂白效应很明显。然而，1O2淬灭剂β-胡萝卜素和叠氮化钠可使GFP的光漂白作用降至最低。对光致敏的GFP进行凝胶电泳，随后进行荧光多像素光谱成像，结果显示二氢卟吩e6与GFP结合，影响了光漂白效果。对转染了GFP的COS 7细胞进行荧光多像素光谱成像，结果表明GFP存在于细胞质和细胞核中，而二氢卟吩e6则集中在核周小泡中。细胞暴露于光下会在二氢卟吩e6小泡附近诱导GFP光漂白。我们得出结论，光活化的GFP会产生内源性1O2，诱导发色团损伤，外源性二氢卟吩e6的协同作用可增强这种损伤。
