Whiteman Matthew, Siau Jia Ling, Halliwell Barry
Department of Biochemistry, Faculty of Medicine, National University of Singapore, 8 Medical Dr., Singapore 117597, Republic of Singapore.
J Biol Chem. 2003 Mar 7;278(10):8380-4. doi: 10.1074/jbc.M211086200. Epub 2002 Dec 9.
Elevated levels of reactive nitrogen species (RNS) such as peroxynitrite have been implicated in over 50 diverse human diseases as measured by the formation of the RNS biomarker 3-nitrotyrosine. Recently, an additional RNS was postulated to contribute to 3-nitrotyrosine formation in vivo; nitryl chloride formed from the reaction of nitrite and neutrophil myeloperoxidase-derived hypochlorous acid (HOCl). Whether nitryl chloride nitrates intracellular protein is unknown. Therefore, we exposed intact human HepG2 and SW1353 cells or cell lysates to HOCl and nitrite and examined each for 3-nitrotyrosine formation by: 1) Western blotting, 2) using a commercial 3-nitrotyrosine enzyme-linked immunosorbent assay kit, 3) flow cytometric analysis, and 4) confocal microscopic analysis. With each approach, no significant 3-nitrotyrosine formation was observed in either whole cells or cell lysates. However, substantial 3-nitrotyrosine was observed when peroxynitrite (100 microm) was added to cells or cell lysates. These data suggest that nitryl chloride formed from the reaction of nitrite with HOCl does not contribute to the elevated levels of 3-nitrotyrosine observed in human diseases.
通过活性氮物种（RNS）生物标志物3-硝基酪氨酸的形成来衡量，过氧亚硝酸盐等RNS水平升高与50多种不同的人类疾病有关。最近，有人提出另一种RNS在体内对3-硝基酪氨酸的形成有作用；亚硝酸盐与中性粒细胞髓过氧化物酶衍生的次氯酸（HOCl）反应形成的硝酰氯。硝酰氯是否会使细胞内蛋白质硝化尚不清楚。因此，我们将完整的人HepG2和SW1353细胞或细胞裂解物暴露于HOCl和亚硝酸盐中，并通过以下方法检测每种物质中3-硝基酪氨酸的形成：1）蛋白质印迹法，2）使用商业3-硝基酪氨酸酶联免疫吸附测定试剂盒，3）流式细胞术分析，4）共聚焦显微镜分析。采用每种方法，在全细胞或细胞裂解物中均未观察到明显的3-硝基酪氨酸形成。然而，当向细胞或细胞裂解物中加入过氧亚硝酸盐（100微摩尔）时，观察到大量的3-硝基酪氨酸。这些数据表明，亚硝酸盐与HOCl反应形成的硝酰氯对人类疾病中观察到的3-硝基酪氨酸水平升高没有作用。
