Microassay of superoxide anion scavenging activity in vitro
Microassay of superoxide anion scavenging activity in vitro
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DOI：
10.1016/s1382-6689(96)00143-3
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发表时间：
1997-02-15
影响因子：
4.3
通讯作者：
Anggard, EE
中科院分区：
文献类型：
--
作者：
Laight, DW;Andrews, TJ;Anggard, EE
我们开发了一种光度法，基于平板阅读器的微量测定体外超氧阴离子清除活性。采用黄嘌呤氧化酶/次黄嘌呤体系生成超氧阴离子，并在550 nM下进行铁细胞色素c还原检测。对超氧化物歧化酶（SOD）、超氧化物阴离子清除剂铁和TEMPO及其衍生物的抑制活性进行了评估。黄嘌呤氧化酶(20 mU/ml)/次黄嘌呤（100 mU M）偶联铁色素c （100 mU M）在550 nm处产生的初始光密度（OD）变化率即初始反应速率被SOD （200 U/ml）、铁（10 mM）和TEMPO （0.3 mM）有效抵消，表明超氧阴离子的参与。TEMPO衍生物抑制初始反应速率的效价顺序为：TEMPO >4-羟基-TEMPO=4-羧基-TEMPO。相比之下，缺乏自由基氮氧化物功能的4-羟基- tempo在1mm内无活性。(C) 1997 Elsevier Science B.V.
We have developed a photometric, platereader-based microassay for superoxide anion scavenging activity in vitro. Superoxide anions were generated using a xanthine oxidase/hypoxanthine system and detected by following the reduction of ferricytochrome c at 550 nM. Inhibitory activity was assessed for superoxide dismutase (SOD) and the superoxide anion scavengers tiron and TEMPO together with a number of TEMPO derivatives. The initial rate of change in optical density (OD) at 550 nm, i.e., initial reaction rate, generated by xanthine oxidase (20 mU/ml)/hypoxanthine (100 mu M) coupled to ferricytochrome c (100 mu M) was effectively abolished by SOD (200 U/ml), tiron (10 mM) and TEMPO (0.3 mM), indicating the involvement of superoxide anions. TEMPO derivatives inhibited the initial reaction rate with the potency order: TEMPO >4-hydroxy-TEMPO=4-carboxy-TEMPO. In contrast, 4-hydroxy-TEMPO, which lacks the free radical nitroxide function, was inactive up to 1 mM. (C) 1997 Elsevier Science B.V.