Green Fluorescent Protein Photobleaching: a Model for Protein Damage by Endogenous and Exogenous Singlet Oxygen
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Lior Greenbaum
Abstract
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 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.
Copyright © 2000 by Walter de Gruyter GmbH & Co. KG
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- Content Index
- Author Index
- Subject Index
Articles in the same Issue
- Circadian Rhythms of Sterol 12α-Hydroxylase, Cholesterol 7α-Hydroxylase and DBP Involved in Rat Cholesterol Catabolism
- Mannosidase Action, Independent of Glucose Trimming, Is Essential for Proteasome-Mediated Degradation of Unassembled Glycosylated Ig Light Chains
- Characterization of a Receptor for Heat Shock Protein 70 on Macrophages and Monocytes
- Yeast Translational Activator Cbs2p: Mitochondrial Targeting and Effect of Overexpression
- Mutational Scanning of a Hairpin Loop in the Tryptophan Synthase β-Subunit Implicated in Allostery and Substrate Channeling
- The Difference in the Carboxy-Terminal Sequence Is Responsible for the Difference in the Activity of Chicken and Rat Liver Fructose-2,6-Bisphosphatase
- Crystal Structure of the Caspase Activator Human Granzyme B, a Proteinase Highly Specific for an Asp-P1 Residue
- Primary Structure of Potato Kunitz-Type Serine Proteinase Inhibitor
- Activation of proPHBSP, the Zymogen of a Plasma Hyaluronan Binding Serine Protease, by an Intermolecular Autocatalytic Mechanism
- Human and Rat Dipeptidyl Peptidase III: Biochemical and Mass Spectrometric Arguments for Similarities and Differences
- Recombinant Anti-Stefin A Fab Fragment: Sequence Analysis of the Variable Region and Expression in Escherichia coli
- Green Fluorescent Protein Photobleaching: a Model for Protein Damage by Endogenous and Exogenous Singlet Oxygen
- 193 nm Photolysis of Aromatic and Aliphatic Dipeptides in Aqueous Solution: Dependence of Decomposition Quantum Yield on the Amino Acid Sequence
- NADPH:Protochlorophyllide Oxidoreductase Uses the General Import Route into Chloroplasts
- Superoxide Reactivates Nitric Oxide-Inhibited Catalase
- Erratum: The following abstract was unfortunately omitted from the GBM Fall Meeting section of the Biological Chemistry Special Supplement, Vol. 381, September 2000
- Content Index
- Author Index
- Subject Index
