Oxidative damage and tyrosine nitration from peroxynitrite
Oxidative damage and tyrosine nitration from peroxynitrite
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DOI：
10.1021/tx9501445
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发表时间：
1996-07-01
影响因子：
4.1
通讯作者：
Beckman, JS
中科院分区：
文献类型：
--
作者：
Beckman, JS
羟基自由基是高度反应性的，当然能够破坏试管中分离的DNA，蛋白质或脂质。然而，高活性物质不一定是高毒性的。在羟基自由基的情况下，与每个有机分子的反应速率接近扩散极限，范围为109至1010 M-1·s-1。因此，在简单的磷酸盐缓冲溶液中，羟基自由基会随机损伤分离的DNA。DNA结合蛋白的结合保护序列免受羟基自由基的侵害，使分子生物学家能够识别与蛋白质相互作用的DNA的特定区域。随着越来越多的细胞成分被添加到混合物中，DNA或任何关键的细胞靶点被高反应性物质击中的机会变得越来越小。辐射化学家计算出，在一个细胞中，羟基自由基的扩散距离仅为3 nm（2）。羟基自由基会随机攻击非关键的细胞成分，因为它在扩散限制速率下的广泛反应性使羟基自由基成为一种不加区分的物质。生物化学在清洁、孤立的系统中工作的还原论性质夸大了羟基自由基的明显毒性，因为替代的非关键目标被从系统中移除。另一个困难是，天然抗氧化剂和清除酶，如超氧化物歧化酶和过氧化氢酶，是缺乏体外系统，但存在于体内。尽管如此，向动物和人类注射外源性超氧化物歧化酶或过氧化氢酶可以减少缺血和炎症造成的损伤，这表明氧化过程参与了许多内源性抗氧化防御无法预防的疾病过程（3，4）。然而，超氧化物驱动的芬顿反应是一个相对缓慢的反应，在体外很容易通过加入少量的过氧化氢酶或超氧化物歧化酶来停止。在体内，高达1%的可溶性蛋白质是超氧化物歧化酶（5，6），而过氧化氢酶和谷胱甘肽过氧化物酶也很丰富，并有效地去除过氧化氢。即使是亚微摩尔浓度的过氧化氢也不会在体内长期存在。然而，在大多数体外实验中，毫摩尔浓度被称为低剂量。为了了解体内氧化剂的毒性，人们需要寻找足够快的自由基反应，以胜过内源性抗氧化剂防御。反应必须足够快，超氧化物歧化酶和过氧化氢酶都不会停止反应。其他氧化机制必须在体内运作。在本综述中，我们将集中在一个反应的超氧化物与一氧化氮的扩散限制反应。更多的肯定还有待调查。目前，唯一已知的生物分子，是在足够高的浓度产生，并能与超氧化物反应足够快，以超过内源性超氧化物歧化酶是一氧化氮（6）。一氧化氮是一种自由基，通过自由基-自由基偶联与超氧化物结合形成过氧亚硝酸根阴离子（7）：反应速率为6.7× 109 M-1·s-1，比超氧化物歧化酶与超氧化物反应至少快3倍（8，9）。超氧化物歧化酶清除超氧化物的能力部分地被生理水平的氯离子所降低，氯离子屏蔽了吸引超氧化物到活性位点的静电场。氯化物使超氧化物的清除率降低2-3倍（9，10），但对一氧化氮和超氧化物的自由基-自由基偶联没有影响。因此，与在磷酸盐缓冲液中进行的反应相比，在生理条件下过氧亚硝酸盐的形成稍微更有利。过氧亚硝酸盐本身不是一种自由基，因为...
Hydroxyl radical is highly reactive and certainly capable of destroying isolated DNA, protein, or lipid in a test tube. However, a highly reactive species is not necessarily highly toxic. In the case of hydroxyl radical, the rate of reaction with every organic molecule is near the diffusion limit, ranging from 109 to 1010 M-1 ‚s-1. Thus, hydroxyl radical will randomly damage isolated DNA in a simple phosphate-buffered solution. Binding of DNA binding proteins protects sequences from hydroxyl radical, enabling molecular biologists to identify specific regions of the DNA that interact with the protein. As more and more components of a cell are added to the mixture, the chances that DNA or any critical cellular target will be hit by a highly reactive species become smaller and smaller. Radiation chemists calculate that the diffusion distance of hydroxyl radical is only 3 nm in a cell (2). Hydroxyl radical will randomly attack noncritical cellular components because its broad reactivity at diffusionlimited rates makes hydroxyl radical an indiscriminant species. The reductionist nature of biochemistry to work on clean, isolated systems exaggerates the apparent toxicity of hydroxyl radical because alternative noncritical targets are removed from the system. An additional difficulty is that natural antioxidants and scavenging enzymes like superoxide dismutase and catalase are absent from in vitro systems but are present in vivo. Still, the injection of exogenous superoxide dismutase or catalase into animals and humans can reduce injury from ischemia and inflammation, which establishes that oxidative processes are involved in many disease processes that cannot be prevented by endogenous antioxidant defenses (3, 4). However, the superoxide-driven Fenton reaction is a relatively slow reaction that is easily stopped in vitro by adding small amounts of catalase or superoxide dismutase. In vivo, up to 1% of soluble protein is superoxide dismutase (5, 6), while catalase and glutathione peroxidase are also abundant and effectively remove hydrogen peroxide. Even submicromolar concentrations of hydrogen peroxide will not exist for long in vivo. Yet, millimolar concentrations are called low doses in most in vitro experiments. To understand oxidant toxicity in vivo, one needs to look for free radical reactions that are fast enough to outcompete endogenous antioxidant defenses. The reactions must be fast enough that neither superoxide dismutase nor catalase will stop the reaction. Other oxidative mechanisms must be operating in vivo. In the present review, we will focus upon only one reactionsthe diffusion-limited reaction of superoxide with nitric oxide. More certainly remain to be investigated. At present, the only known biological molecule that is produced in high enough concentrations and can react fast enough with superoxide to outcompete endogenous superoxide dismutase is nitric oxide (6). Nitric oxide is a free radical that combines by radical-radical coupling with superoxide to form peroxynitrite anion (7):The reaction rate is 6.7× 109 M-1 ‚s-1, which is at least 3 times faster than superoxide dismutase reacts with superoxide (8, 9). The scavenging of superoxide by superoxide dismutase is partially reduced by physiological levels of chloride ions, which screen the electrostatic field that attracts superoxide to the active site. Chloride decreases the scavenging of superoxide by a factor of 2-3 (9, 10), but should have no effect upon the radical-radical coupling of nitric oxide and superoxide. Therefore, the formation of peroxynitrite is slightly more favorable under physiological conditions compared to reactions conducted in phosphate buffer. Peroxynitrite itself is not a free radical because the …