Lior Greenbaum, Chana Rothmann, Ronit Lavie, Zvi Malik
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
?-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 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 chlorine6.
Biological Chemistry, Walter de Gruyter
Print ISSN: 1431-6730
Volume: 381, 12/2000
Pages: 1251 - 1258
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