| Author |
Rios, Natalia Piacenza, Lucía Trujillo, Madia Martínez, Alejandra Demicheli, Verónica Prolo, Carolina Álvarez, María Noel López, Gloria V. Radi, Rafael |
| Description |
Author Affiliation: Rios N ( Departamento de Bioquímica, Facultad de Medicina, Universidad de la República, Av. Gral. Flores 2125, Montevideo 11800, Uruguay); Piacenza L ( Departamento de Bioquímica, Facultad de Medicina, Universidad de la República, Av. Gral. Flores 2125, Montevideo 11800, Uruguay); Trujillo M ( Departamento de Bioquímica, Facultad de Medicina, Universidad de la República, Av. Gral. Flores 2125, Montevideo 11800, Uruguay); Martínez A ( Departamento de Bioquímica, Facultad de Medicina, Universidad de la República, Av. Gral. Flores 2125, Montevideo 11800, Uruguay); Demicheli V ( Departamento de Bioquímica, Facultad de Medicina, Universidad de la República, Av. Gral. Flores 2125, Montevideo 11800, Uruguay); Prolo C ( Departamento de Bioquímica, Facultad de Medicina, Universidad de la República, Av. Gral. Flores 2125, Montevideo 11800, Uruguay); Álvarez MN ( Departamento de Bioquímica, Facultad de Medicina, Universidad de la República, Av. Gral. Flores 2125, Montevideo 11800, Uruguay); López GV ( Center for Free Radical and Biomedical Research, Facultad de Medicina, Universidad de la República, Av. Gral. Flores 2125, Montevideo 11800, Uruguay); Radi R ( Departamento de Bioquímica, Facultad de Medicina, Universidad de la República, Av. Gral. Flores 2125, Montevideo 11800, Uruguay) |
| 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 (â¼42%). Reactivity analysis of Fl-B with relevant biological oxidants including hydrogen peroxide (H O ), hypochlorous acid (HOCl) and peroxynitrite were performed. The rate constant for the reaction of peroxynitrite with Fl-B was 1.7×10 M s , a million times faster than the rate constant measured for H O (k=1.7M s ) and 2,700 faster than HOCl (6.2×10 M s ) at 37°C and pH 7.4. The reaction of Fl-B with peroxynitrite was significant even in the presence of physiological concentrations of CO , 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 â¼ 0.1µMs , while immunostimulated macrophages do so in the order of â¼1µMs inside 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. |
