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

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 β-carotene and sodium azide minimized GFP photobleaching. 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 COS7 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 chlorine e₆.