title: Systematic in vitro assessment of responses of roGFP2-based probes to physiologically relevant oxidant species.
abstract: The genetically encoded probes roGFP2-Orp1 and Grx1-roGFP2 have been designed to be selectively oxidized by hydrogen peroxide (H2O2) and glutathione disulfide (GSSG), respectively. Both probes have demonstrated such selectivity in a broad variety of systems and conditions. In this study, we systematically compared the in vitro response of roGFP2, roGFP2-Orp1 and Grx1-roGFP2 to increasing amounts of various oxidant species that may also occur in biological settings. We conclude that the previously established oxidant selectivity is highly robust and likely to be maintained under most physiological conditions. Yet, we also find that hypochlorous acid, known to be produced in the phagocyte respiratory burst, can lead to non-selective oxidation of roGFP2-based probes at concentrations ≥2µM, in vitro. Further, we confirm that polysulfides trigger direct roGFP2 responses. A side-by-side comparison of all three probes can be used to reveal micromolar amounts of hypochlorous acid or polysulfides.
authors.affiliation: Institute of Biochemistry and Pathobiochemistry - Microbial Biochemistry, Ruhr-University Bochum, 44780 Bochum, Germany. Electronic address: alexandra.mueller-2@ruhr-uni-bochum.de.
authors.forename: Alexandra
authors.initials: A
authors.lastname: Müller
authors.affiliation: Institute of Biochemistry and Pathobiochemistry - Microbial Biochemistry, Ruhr-University Bochum, 44780 Bochum, Germany.
authors.forename: Jannis F
authors.initials: JF
authors.lastname: Schneider
authors.affiliation: Institute of Biochemistry and Pathobiochemistry - Microbial Biochemistry, Ruhr-University Bochum, 44780 Bochum, Germany.
authors.forename: Adriana
authors.initials: A
authors.lastname: Degrossoli
authors.affiliation: Institute of Biochemistry and Pathobiochemistry - Microbial Biochemistry, Ruhr-University Bochum, 44780 Bochum, Germany.
authors.forename: Nataliya
authors.initials: N
authors.lastname: Lupilova
authors.affiliation: Division of Redox Regulation, DKFZ-ZMBH Alliance, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, 69120 Heidelberg, Germany.
authors.forename: Tobias P
authors.initials: TP
authors.lastname: Dick
authors.affiliation: Institute of Biochemistry and Pathobiochemistry - Microbial Biochemistry, Ruhr-University Bochum, 44780 Bochum, Germany.
authors.forename: Lars I
authors.initials: LI
authors.lastname: Leichert
mesh_terms: D054477:Glutaredoxins / Q000737:chemistry; D005978:Glutathione / Q000737:chemistry / Q000378:metabolism; D019803:Glutathione Disulfide / Q000737:chemistry* / Q000302:isolation & purification; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000235:genetics*; D006861:Hydrogen Peroxide / Q000737:chemistry / Q000302:isolation & purification*; D009569:Nitric Oxide / Q000737:chemistry / Q000378:metabolism; D016877:Oxidants / Q000737:chemistry* / Q000378:metabolism; D010084:Oxidation-Reduction; D030421:Peroxynitrous Acid / Q000737:chemistry / Q000378:metabolism*; D010586:Phagocytes / Q000378:metabolism; D026361:Reactive Nitrogen Species / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism
chemical_list: C516008:GLRX2 protein, human; D054477:Glutaredoxins; D016877:Oxidants; D026361:Reactive Nitrogen Species; D017382:Reactive Oxygen Species; D030421:Peroxynitrous Acid; D049452:Green Fluorescent Proteins; D009569:Nitric Oxide; D006861:Hydrogen Peroxide; D005978:Glutathione; D019803:Glutathione Disulfide
keywords: Genetically encoded redox probes; Glutathione; H(2)O(2); HOCl; Nitric oxide; Peroxynitrite; Polysulfides