title: Sensitive detection and estimation of cell-derived peroxynitrite fluxes using fluorescein-boronate.
abstract: The specific and sensitive detection of peroxynitrite (ONOO-/ONOOH) in biological systems is a great challenge due to its high reactivity towards several biomolecules. Herein, we validated the advantages of using fluorescein-boronate (Fl-B) as a highly sensitive fluorescent probe for the direct detection of peroxynitrite under biologically-relevant conditions in two different cell models. The synthesis of Fl-B was achieved by a very simply two-step conversion synthetic route with high purity (>99%) and overall yield (∼42%). Reactivity analysis of Fl-B with relevant biological oxidants including hydrogen peroxide (H2O2), hypochlorous acid (HOCl) and peroxynitrite were performed. The rate constant for the reaction of peroxynitrite with Fl-B was 1.7×106M-1s-1, a million times faster than the rate constant measured for H2O2(k=1.7M-1s-1) and 2,700 faster than HOCl (6.2×102M-1s-1) at 37°C and pH 7.4. The reaction of Fl-B with peroxynitrite was significant even in the presence of physiological concentrations of CO2, a well-known peroxynitrite reactant. Experimental and simulated kinetic analyses confirm that the main oxidation process of Fl-B takes place with peroxynitrite itself via a direct bimolecular reaction and not with peroxynitrite-derived radicals. Fl-B was successfully applied for the detection of endogenously-generated peroxynitrite by endothelial cells and in macrophage-phagocyted parasites. Moreover, the generated data allowed estimating the actual intracellular flux of peroxynitrite. For instance, ionomycin-stimulated endothelial cells generated peroxynitrite at a rate of ∼ 0.1μMs-1, while immunostimulated macrophages do so in the order of ∼1μMs-1inside T. cruzi-infected phagosomes. Fl-B revealed not to be toxic in concentrations up to 1mM for 24h. Cellular peroxynitrite detection was achieved by conventional laboratory fluorescence-based methods including flow cytometry and epi-fluorescence microscopy. Fl-B was shown to be more sensitive than the coumarin boronate due to a higher molar absorption coefficient and quantum yield. Overall, our results show that Fl-B is a kinetically selective and highly sensitive probe for the direct detection of cell-derived peroxynitrite.
authors.affiliation: Departamento de Bioquímica, Facultad de Medicina, Universidad de la República, Av. Gral. Flores 2125, Montevideo 11800, Uruguay; Center for Free Radical and Biomedical Research, Facultad de Medicina, Universidad de la República, Av. Gral. Flores 2125, Montevideo 11800, Uruguay; Departamento de Química Orgánica, Facultad de Ciencias-Facultad de Química, Universidad de la República, Iguá 4225, Montevideo 11400, Uruguay.
authors.forename: Natalia
authors.initials: N
authors.lastname: Rios
authors.affiliation: Departamento de Bioquímica, Facultad de Medicina, Universidad de la República, Av. Gral. Flores 2125, Montevideo 11800, Uruguay; Center for Free Radical and Biomedical Research, Facultad de Medicina, Universidad de la República, Av. Gral. Flores 2125, Montevideo 11800, Uruguay.
authors.forename: Lucía
authors.initials: L
authors.lastname: Piacenza
authors.affiliation: Departamento de Bioquímica, Facultad de Medicina, Universidad de la República, Av. Gral. Flores 2125, Montevideo 11800, Uruguay; Center for Free Radical and Biomedical Research, Facultad de Medicina, Universidad de la República, Av. Gral. Flores 2125, Montevideo 11800, Uruguay.
authors.forename: Madia
authors.initials: M
authors.lastname: Trujillo
authors.affiliation: Departamento de Bioquímica, Facultad de Medicina, Universidad de la República, Av. Gral. Flores 2125, Montevideo 11800, Uruguay; Center for Free Radical and Biomedical Research, Facultad de Medicina, Universidad de la República, Av. Gral. Flores 2125, Montevideo 11800, Uruguay.
authors.forename: Alejandra
authors.initials: A
authors.lastname: Martínez
authors.affiliation: Departamento de Bioquímica, Facultad de Medicina, Universidad de la República, Av. Gral. Flores 2125, Montevideo 11800, Uruguay; Center for Free Radical and Biomedical Research, Facultad de Medicina, Universidad de la República, Av. Gral. Flores 2125, Montevideo 11800, Uruguay.
authors.forename: Verónica
authors.initials: V
authors.lastname: Demicheli
authors.affiliation: Departamento de Bioquímica, Facultad de Medicina, Universidad de la República, Av. Gral. Flores 2125, Montevideo 11800, Uruguay; Center for Free Radical and Biomedical Research, Facultad de Medicina, Universidad de la República, Av. Gral. Flores 2125, Montevideo 11800, Uruguay.
authors.forename: Carolina
authors.initials: C
authors.lastname: Prolo
authors.affiliation: Departamento de Bioquímica, Facultad de Medicina, Universidad de la República, Av. Gral. Flores 2125, Montevideo 11800, Uruguay; Center for Free Radical and Biomedical Research, Facultad de Medicina, Universidad de la República, Av. Gral. Flores 2125, Montevideo 11800, Uruguay.
authors.forename: María Noel
authors.initials: MN
authors.lastname: Álvarez
authors.affiliation: Center for Free Radical and Biomedical Research, Facultad de Medicina, Universidad de la República, Av. Gral. Flores 2125, Montevideo 11800, Uruguay; Departamento de Química Orgánica, Facultad de Ciencias-Facultad de Química, Universidad de la República, Iguá 4225, Montevideo 11400, Uruguay.
authors.forename: Gloria V
authors.initials: GV
authors.lastname: López
authors.affiliation: Departamento de Bioquímica, Facultad de Medicina, Universidad de la República, Av. Gral. Flores 2125, Montevideo 11800, Uruguay; Center for Free Radical and Biomedical Research, Facultad de Medicina, Universidad de la República, Av. Gral. Flores 2125, Montevideo 11800, Uruguay. Electronic address: rradi@fmed.edu.uy.
authors.forename: Rafael
authors.initials: R
authors.lastname: Radi
mesh_terms: D000818:Animals; D001011:Aorta / Q000166:cytology / Q000378:metabolism; D001897:Boronic Acids / Q000737:chemistry*; D002417:Cattle; D002460:Cell Line; D042783:Endothelial Cells / Q000166:cytology / Q000378:metabolism; D005452:Fluoresceins / Q000737:chemistry*; D005456:Fluorescent Dyes / Q000138:chemical synthesis*; D006861:Hydrogen Peroxide / Q000378:metabolism; D006997:Hypochlorous Acid / Q000378:metabolism; D007700:Kinetics; D008264:Macrophages / Q000166:cytology / Q000378:metabolism* / Q000469:parasitology; D051379:Mice; D010084:Oxidation-Reduction; D030421:Peroxynitrous Acid / Q000032:analysis* / Q000096:biosynthesis; D010587:Phagocytosis / Q000502:physiology; D061251:Primary Cell Culture; D012680:Sensitivity and Specificity; D014349:Trypanosoma cruzi
chemical_list: D001897:Boronic Acids; D005452:Fluoresceins; D005456:Fluorescent Dyes; D030421:Peroxynitrous Acid; D006997:Hypochlorous Acid; D006861:Hydrogen Peroxide
keywords: Endothelial cells; Fluorescein-boronate; Fluorescent probes; Free radicals; Macrophage infection; Oxidants; Peroxynitrite