Sieracki Nathan A, Gantner Benjamin N, Mao Mao, Horner John H, Ye Richard D, Malik Asrar B, Newcomb Martin E, Bonini Marcelo G
Department of Pharmacology, University of Illinois at Chicago, Chicago, IL 60612, USA.
Department of Pharmacology, University of Illinois at Chicago, Chicago, IL 60612, USA; Section of Cardiology, Department of Medicine, University of Illinois at Chicago, Chicago, IL 60612, USA.
Free Radic Biol Med. 2013 Aug;61:40-50. doi: 10.1016/j.freeradbiomed.2013.02.020. Epub 2013 Mar 7.
Peroxynitrite, a highly reactive biological oxidant, is formed under pathophysiologic conditions from the diffusion-limited reaction of nitric oxide and superoxide radical anion. Peroxynitrite has been implicated as the mediator of nitric oxide toxicity in many diseases and as an important signaling disrupting molecule (L. Liaudet et al., Front. Biosci.14, 4809-4814, 2009) [1]. Biosensors effective at capturing peroxynitrite in a specific and fast enough manner for detection, along with readouts compatible with in vivo studies, are lacking. Here we report that the boronic acid-based bioluminescent system PCL-1 (peroxy-caged luciferin-1), previously reported as a chemoselective sensor for hydrogen peroxide (G.C. Van de Bittner et al., Proc. Natl. Acad. Sci. USA107, 21316-21321, 2010) [2], reacts with peroxynitrite stoichiometrically with a rate constant of 9.8±0.3×10(5)M(-1)s(-1) and a bioluminescence detection limit of 16nM, compared to values of 1.2±0.3M(-1)s(-1) and 231nM for hydrogen peroxide. Further, we demonstrate bioluminescent detection of peroxynitrite in the presence of physiological competitors: carbon dioxide, glutathione, albumin, and catalase. We also demonstrate the utility of this method to assess peroxynitrite formation in mammalian cells by measuring peroxynitrite generated under normal culture conditions after stimulation of macrophages with bacterial endotoxin lipopolysaccharide. Thus, the PCL-1 method for measuring peroxynitrite generation shows superior selectivity over other oxidants under in vivo conditions.
过氧亚硝酸盐是一种高反应性的生物氧化剂，在病理生理条件下由一氧化氮和超氧阴离子自由基的扩散受限反应形成。在许多疾病中，过氧亚硝酸盐被认为是一氧化氮毒性的介质，也是一种重要的信号干扰分子（L. Liaudet等人，《生物科学前沿》14，4809 - 4814，2009年）[1]。目前缺乏能够以特定且足够快速的方式捕获过氧亚硝酸盐用于检测的生物传感器，以及与体内研究兼容的读数。在此我们报告，基于硼酸的生物发光系统PCL - 1（过氧笼形荧光素 - 1），先前被报道为过氧化氢的化学选择性传感器（G.C. Van de Bittner等人，《美国国家科学院院刊》107，21316 - 21321，2010年）[2]，与过氧亚硝酸盐发生化学计量反应，反应速率常数为9.8±0.3×10⁵M⁻¹s⁻¹，生物发光检测限为16 nM，而过氧化氢的值分别为1.2±0.3 M⁻¹s⁻¹和231 nM。此外，我们展示了在存在生理竞争物（二氧化碳、谷胱甘肽、白蛋白和过氧化氢酶）的情况下对过氧亚硝酸盐的生物发光检测。我们还通过测量用细菌内毒素脂多糖刺激巨噬细胞后在正常培养条件下产生的过氧亚硝酸盐，证明了该方法在评估哺乳动物细胞中过氧亚硝酸盐形成方面的实用性。因此，用于测量过氧亚硝酸盐生成的PCL - 1方法在体内条件下对其他氧化剂显示出卓越的选择性。
