title: Detection of peroxidase/H2O2-mediated oxidation with enhanced yellow fluorescent protein.
abstract: The ability to sense oxidative stress in live cells and organisms would have far-reaching implications for biotechnology, drug discovery, and potentially medical imaging. We hypothesized that tyrosine-containing fluorescent proteins could be used as switches for sensing oxidative stress, based on their sensitivity to environmental and structural variations. We therefore tested purified EGFP, EYFP, ECFP, and DsRed proteins against the heme-peroxidase/H(2)O(2) reaction. We found that peroxidase-mediated oxidation resulted in up to 99.5% quenching of EYFP fluorescence (but not that of other fluorescent proteins) in a dose-dependent manner. Western blotting revealed inter- and intramolecular cross-linking. The observed detection limit for hydrogen peroxide was approximately 100 nM, well below the extracellular levels previously reported to occur in mammalian tissue during signaling. Combined expression of EYFP (quenchable) and ECFP or EGFP (nonquenchable) is expected to allow sensitive monitoring of oxidative stress.
authors.affiliation: Center for Molecular Imaging Research, Massachusetts General Hospital, Charlestown, Massachusetts 02129, USA.
authors.forename: Andrew
authors.initials: A
authors.lastname: Tsourkas
authors.forename: Gail
authors.initials: G
authors.lastname: Newton
authors.forename: J Manuel
authors.initials: JM
authors.lastname: Perez
authors.forename: James P
authors.initials: JP
authors.lastname: Basilion
authors.forename: Ralph
authors.initials: R
authors.lastname: Weissleder
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry*; D006735:Horseradish Peroxidase / Q000737:chemistry / Q000378:metabolism*; D006861:Hydrogen Peroxide / Q000737:chemistry / Q000378:metabolism*; D008164:Luminescent Proteins / Q000737:chemistry*; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D013050:Spectrometry, Fluorescence
chemical_list: D001426:Bacterial Proteins; D008164:Luminescent Proteins; C054562:yellow fluorescent protein, Bacteria; D006861:Hydrogen Peroxide; D006735:Horseradish Peroxidase