Back to Search
Start Over
Sensitive detection and estimation of cell-derived peroxynitrite fluxes using fluorescein-boronate.
- Source :
-
Free Radical Biology & Medicine . Dec2016, Vol. 101, p284-295. 12p. - Publication Year :
- 2016
-
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 (H 2 O 2 ), hypochlorous acid (HOCl) and peroxynitrite were performed. The rate constant for the reaction of peroxynitrite with Fl-B was 1.7×10 6 M −1 s −1 , a million times faster than the rate constant measured for H 2 O 2 (k=1.7 M −1 s −1 ) and 2,700 faster than HOCl (6.2×10 2 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 CO 2 , 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. [ABSTRACT FROM AUTHOR]
Details
- Language :
- English
- ISSN :
- 08915849
- Volume :
- 101
- Database :
- Academic Search Index
- Journal :
- Free Radical Biology & Medicine
- Publication Type :
- Academic Journal
- Accession number :
- 120146143
- Full Text :
- https://doi.org/10.1016/j.freeradbiomed.2016.08.033