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

RADICALES LIBRES

OXIDANTES

CELULAS ENDOTIALES

PEROXINITRITO

BIBLIOGRAFIA NACIONAL QUIMICA

2016

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 1.7×106 M−1 s−1, a million times faster than the rate constant measured for H2O2 (k=1.7 M−1 s−1) and 2,700 faster than HOCl (6.2×102 M−1 s−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.

Free Radical Biology and Medicine v. 101, 2016. -- p. 284-295

Elsevier

2016

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DOI: 10.1016/j.freeradbiomed.2016.08.033

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