Kuppusamy P, Zweier J L
Department of Medicine, Johns Hopkins Medical Institutions, Baltimore, Maryland 21224.
J Biol Chem. 1989 Jun 15;264(17):9880-4.
Xanthine oxidase has been hypothesized to be an important source of biological free radical generation. The enzyme generates the superoxide radical, .O2- and has been widely applied as a .O2- generating system; however, the enzyme may also generate other forms of reduced oxygen. We have applied electron paramagnetic resonance (EPR) spectroscopy using the spin trap 5,5'-dimethyl-1-pyrroline-N-oxide (DMPO) to characterize the different radical species generated by xanthine oxidase along with the mechanisms of their generation. Upon reaction of xanthine with xanthine oxidase equilibrated with air, both DMPO-OOH and DMPO-OH radicals are observed. In the presence of ethanol or dimethyl sulfoxide, alpha-hydroxyethyl or methyl radicals are generated, respectively, indicating that significant DMPO-OH generation occurred directly from OH rather than simply from the breakdown of DMPO-OOH. Superoxide dismutase totally scavenged the DMPO-OOH signal but not the DMPO-OH signal suggesting that .O2- was not required for .OH generation. Catalase markedly decreased the DMPO-OH signal, while superoxide dismutase + catalase totally scavenged all radical generation. Thus, xanthine oxidase generates .OH via the reduction of O2 to H2O2, which in turn is reduced to .OH. In anaerobic preparations, the enzyme reduces H2O2 to .OH as evidenced by the appearance of a pure DMPO-OH signal. The presence of the flavin in the enzyme is required for both .O2- and .OH generation confirming that the flavin is the site of O2 reduction. The ratio of .O2- and .OH generation was affected by the relative concentrations of dissolved O2 and H2O2. Thus, xanthine oxidase can generate the highly reactive .OH radical as well as the less reactive .O2- radical. The direct production of .OH by xanthine oxidase in cells and tissues containing this enzyme could explain the presence of oxidative cellular damage which is not prevented by superoxide dismutase.
黄嘌呤氧化酶被认为是生物自由基产生的一个重要来源。该酶能产生超氧阴离子自由基·O₂⁻，并已被广泛用作·O₂⁻产生系统；然而，该酶也可能产生其他形式的还原态氧。我们应用电子顺磁共振（EPR）光谱，使用自旋捕获剂5,5'-二甲基-1-吡咯啉-N-氧化物（DMPO）来表征黄嘌呤氧化酶产生的不同自由基种类及其产生机制。当黄嘌呤与在空气中平衡的黄嘌呤氧化酶反应时，可观察到DMPO-OOH和DMPO-OH自由基。在乙醇或二甲基亚砜存在的情况下，分别产生了α-羟乙基或甲基自由基，这表明大量DMPO-OH的产生是直接由OH产生的，而不是简单地由DMPO-OOH分解产生。超氧化物歧化酶完全清除了DMPO-OOH信号，但没有清除DMPO-OH信号，这表明·OH的产生不需要·O₂⁻。过氧化氢酶显著降低了DMPO-OH信号，而超氧化物歧化酶+过氧化氢酶则完全清除了所有自由基的产生。因此，黄嘌呤氧化酶通过将O₂还原为H₂O₂，进而将H₂O₂还原为·OH来产生·OH。在厌氧制剂中，该酶将H₂O₂还原为·OH，这由纯DMPO-OH信号的出现所证明。该酶中黄素的存在对于·O₂⁻和·OH的产生都是必需的，这证实了黄素是O₂还原的位点。·O₂⁻和·OH产生的比例受溶解的O₂和H₂O₂相对浓度的影响。因此，黄嘌呤氧化酶可以产生高反应性的·OH自由基以及反应性较低的·O₂⁻自由基。黄嘌呤氧化酶在含有该酶的细胞和组织中直接产生·OH，可以解释超氧化物歧化酶无法预防的氧化细胞损伤的存在。
