Sensitive detection and estimation of cell-derived peroxynitrite fluxes using fluorescein-boronate

Publication date: Available online 15 September 2016
Source:Free Radical Biology and Medicine
Author(s): Natalia Rios, Lucía Piacenza, Madia Trujillo, Alejandra Martínez, Verónica Demicheli, Carolina Prolo, María Noel Álvarez, Gloria V. López, Rafael Radi
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 x 10⁶ M^-1s^-1, a million times faster than the rate constant measured for H2O2 (k = 1.7 M^-1s^-1) and 2,700 faster than HOCl (6.2 x 10² M^-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 ∼ 0.1 μM s^-1, while immunostimulated macrophages do so in the order of ∼ 1 μM s^-1 inside T. cruzi-infected phagosomes. Fl-B revealed not to be toxic in concentrations up to 1 mM for 24 h. 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.



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