The William Harvey Research Institute, Charterhouse Square, London EC1M 6BQ, UK.
Environ Toxicol Pharmacol. 1997 Feb 15;3(1):65-8. doi: 10.1016/s1382-6689(96)00143-3.
We have developed a photometric, platereader-based microassay for superoxide anion scavening 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 μM) coupled to ferricytochrome c (100 μM) 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.
我们开发了一种基于比色法和微孔板读数的体外超氧阴离子清除活性检测方法。超氧阴离子由黄嘌呤氧化酶/次黄嘌呤系统产生，并通过检测 550nm 处细胞色素 c 的还原来检测。评估了超氧化物歧化酶（SOD）、超氧阴离子清除剂 Tiron 和 TEMPO 以及一些 TEMPO 衍生物的抑制活性。黄嘌呤氧化酶（20mU/ml）/次黄嘌呤（100μM）与细胞色素 c（100μM）偶联产生的超氧阴离子（200U/ml）、Tiron（10mM）和 TEMPO（0.3mM）可有效消除在 550nm 处的光密度（OD）的初始变化率（即初始反应速率），表明超氧阴离子的参与。TEMPO 衍生物对初始反应速率的抑制作用的效价顺序为：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 直到 1mM 时才失活。
