Characterization of free radical generation by xanthine oxidase. Evidence for hydroxyl radical generation.
Characterization of free radical generation by xanthine oxidase. Evidence for hydroxyl radical generation.
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DOI：
10.1016/s0021-9258(18)81740-9
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发表时间：
1989-06
期刊：
影响因子：
--
通讯作者：
P. Kuppusamy;J. Zweier
中科院分区：
文献类型：
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作者：
P. Kuppusamy;J. Zweier
黄嘌呤氧化酶被认为是生物自由基产生的重要来源。该酶产生超氧自由基，·O2−，并已被广泛用作·O2−产生系统;然而，该酶也可以产生其他形式的还原氧。利用电子顺磁共振（EPR）技术，以5，5 ′-二甲基-1-吡咯啉-N-氧化物（DMPO）为自旋捕获剂，研究了黄嘌呤氧化酶产生的自由基种类沿着产生机制.在黄嘌呤与黄嘌呤氧化酶反应时，观察到DMPO-OOH和DMPO-OH自由基。在乙醇或二甲基亚砜存在下，分别产生α-羟乙基或甲基自由基，表明直接从OH而不是简单地从DMPO-OOH的分解产生显著的DMPO-OH。超氧化物歧化酶完全清除DMPO-OOH信号，但不清除DMPO-OH信号，表明·O2−不是·OH产生所必需的。过氧化氢酶显著降低DMPO-OH信号，而超氧化物歧化酶+过氧化氢酶完全清除所有自由基的产生。因此，黄嘌呤氧化酶通过O2还原为H2 O2产生·OH，H2 O2又被还原为·OH。在厌氧制剂中，该酶将H2 O2还原为·OH，如纯DMPO-OH信号的出现所证明的。酶中的黄素是产生·O2−和·OH所必需的，这证实了黄素是O2还原的位点。·O2−和·OH生成的比例受溶解O2和H2 O2相对浓度的影响。因此，黄嘌呤氧化酶可以产生高活性的·OH自由基以及低活性的·O2−自由基。黄嘌呤氧化酶在含有这种酶的细胞和组织中直接产生·OH，可以解释超氧化物歧化酶不能阻止的氧化性细胞损伤的存在。
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, α-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 O2to H2O2, which in turn is reduced to•OH. In anaerobic preparations, the enzyme reduces H2O2to•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 O2reduction. The ratio of•O2−and•OH generation was affected by the relative concentrations of dissolved O2and 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.