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). Th e GFP-chromophore damage caused by singlet oxygen was studied in COS 7 kidn ey cells and E. coli bacteria following light irradiation. Electron spin re sonance (ESR) revealed the generation of endogenous singlet oxygen (O-1(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 O-1(2) quenchers p-carotene and sodium azide minimized GFP photobleaching. Gel electrophoresis of photosensitized GFP followed by flu orescence multi-pixel spectral imaging revealed the binding of chlorin e(6) to GFP, affecting the photobleaching efficacy. Fluorescence multi-pixel sp ectral imaging of GFP-transfected COS7 cells demonstrated the presence of G FP in the cytoplasm and nucleus, while chlorin e(6) was found to be concent rated in the perinuclear vesicles. Exposure of the cells to light induced G FP photobleaching in the close vicinity of chlorin e(6) vesicles. We conclu de that photoactivated GFP generates endogenous O-1(2), inducing chromophor e damage,, which can be enhanced by the cooperation of exogenous chlorin e( 6).