Rios Natalia, Piacenza Lucía, Trujillo Madia, Martínez Alejandra, Demicheli Verónica, Prolo Carolina, Álvarez María Noel, López Gloria V, Radi Rafael
Departamento de Bioquímica, Facultad de Medicina, Universidad de la República, Av. Gral. Flores 2125, Montevideo 11800, Uruguay; Center for Free Radical and Biomedical Research, Facultad de Medicina, Universidad de la República, Av. Gral. Flores 2125, Montevideo 11800, Uruguay; Departamento de Química Orgánica, Facultad de Ciencias-Facultad de Química, Universidad de la República, Iguá 4225, Montevideo 11400, Uruguay.
Departamento de Bioquímica, Facultad de Medicina, Universidad de la República, Av. Gral. Flores 2125, Montevideo 11800, Uruguay; Center for Free Radical and Biomedical Research, Facultad de Medicina, Universidad de la República, Av. Gral. Flores 2125, Montevideo 11800, Uruguay.
Free Radic Biol Med. 2016 Dec;101:284-295. doi: 10.1016/j.freeradbiomed.2016.08.033. Epub 2016 Sep 15.
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 (HO), hypochlorous acid (HOCl) and peroxynitrite were performed. The rate constant for the reaction of peroxynitrite with Fl-B was 1.7×10Ms, a million times faster than the rate constant measured for HO (k=1.7Ms) and 2,700 faster than HOCl (6.2×10Ms) 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 ∼ 0.1μMs, while immunostimulated macrophages do so in the order of ∼1μMs 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.
由于过氧亚硝酸盐（ONOO⁻/ONOOH）对多种生物分子具有高反应活性，因此在生物系统中对其进行特异性和灵敏的检测极具挑战性。在此，我们验证了在两种不同细胞模型中，使用荧光素硼酸酯（Fl-B）作为高灵敏荧光探针在生物相关条件下直接检测过氧亚硝酸盐的优势。Fl-B的合成通过非常简单的两步转化合成路线实现，纯度高（>99%）且总产率约为42%。对Fl-B与包括过氧化氢（H₂O₂）、次氯酸（HOCl）和过氧亚硝酸盐在内的相关生物氧化剂进行了反应活性分析。在37°C和pH 7.4条件下，过氧亚硝酸盐与Fl-B反应的速率常数为1.7×10⁶M⁻¹s⁻¹，比H₂O₂（k = 1.7M⁻¹s⁻¹）的速率常数快一百万倍，比HOCl（6.2×10³M⁻¹s⁻¹）快2700倍。即使在存在生理浓度的一氧化碳（一种已知的过氧亚硝酸盐反应物）的情况下，Fl-B与过氧亚硝酸盐的反应也很显著。实验和模拟动力学分析证实，Fl-B的主要氧化过程是通过直接双分子反应与过氧亚硝酸盐本身发生，而不是与过氧亚硝酸盐衍生的自由基发生。Fl-B已成功应用于检测内皮细胞内源性产生的过氧亚硝酸盐以及巨噬细胞吞噬的寄生虫中的过氧亚硝酸盐。此外，所生成的数据有助于估算过氧亚硝酸盐的实际细胞内通量。例如，离子霉素刺激的内皮细胞以约0.1μM s⁻¹的速率产生过氧亚硝酸盐，而免疫刺激的巨噬细胞在克氏锥虫感染的吞噬小体内以约1μM s⁻¹的速率产生过氧亚硝酸盐。Fl-B被证明在浓度高达1mM且作用24小时的情况下无毒。通过包括流式细胞术和落射荧光显微镜在内的传统基于实验室荧光的方法实现了细胞内过氧亚硝酸盐的检测。由于具有更高的摩尔吸收系数和量子产率，Fl-B比香豆素硼酸酯更灵敏。总体而言，我们的结果表明Fl-B是一种动力学选择性且高灵敏的探针，可用于直接检测细胞来源的过氧亚硝酸盐。
