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

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^-1 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.