title: Microassay of superoxide anion scavenging activity in vitro.
abstract: 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.
authors.affiliation: The William Harvey Research Institute, Charterhouse Square, London EC1M 6BQ, UK.
authors.forename: D W
authors.initials: DW
authors.lastname: Laight
authors.forename: T J
authors.initials: TJ
authors.lastname: Andrews
authors.forename: A I
authors.initials: AI
authors.lastname: Haj-Yehia
authors.forename: M J
authors.initials: MJ
authors.lastname: Carrier
authors.forename: E E
authors.initials: EE
authors.lastname: Anggård