Green fluorescent protein photobleaching: a model for protein damage by endogenous and exogenous singlet oxygen

Characterization of protein damage during photosensitization of chlorin e(6)-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 ((1)O(2)) by photoactivated GFP, an effect similar to that produced by the exogenous photosensitizer chlorin e(6). A light dose-dependent photobleaching effect of GFP was pronounced at low pH or upon photosensitization with chlorin e(6). However, the (1)O(2) quenchers p-carotene and sodium azide minimized GFP photobleaching. Gel electrophoresis of photosensitized GFP followed by fluorescence multi-pixel spectral imaging revealed the binding of chlorin e(6) to GFP, affecting the photobleaching efficacy. Fluorescence multi-pixel spectral imaging of GFP-transfected COS7 cells demonstrated the presence of GFP in the cytoplasm and nucleus, while chlorin e(6) was found to be concentrated in the perinuclear vesicles. Exposure of the cells to light induced GFP photobleaching in the close vicinity of chlorin e(6) vesicles. We conclude that photoactivated GFP generates endogenous (1)O(2), inducing chromophore damage,, which can be enhanced by the cooperation of exogenous chlorin e(6).