MICROASSAY OF SUPEROXIDE ANION SCAVENGING ACTIVITY IN-VITRO

We have developed a photometric, platereader-based microassay for supe roxide anion scavenging activity in vitro. Superoxide anions were gene rated using a xanthine oxidase/hypoxanthine system and detected by fol lowing the reduction of ferricytochrome c at 550 nM. Inhibitory activi ty was assessed for superoxide dismutase (SOD) and the superoxide anio n scavengers tiron and TEMPO together with a number of TEMPO derivativ es. The initial rate of change in optical density (OD) at 550 nm, i.e. , initial reaction rate, generated by xanthine oxidase (20 mU/ml)/hypo xanthine (100 mu M) coupled to ferricytochrome c (100 mu M) was effect ively abolished by SOD (200 U/ml), tiron (10 mM) and TEMPO (0.3 mM), i ndicating the involvement of superoxide anions. TEMPO derivatives inhi bited the initial reaction rate with the potency order: TEMPO >4-hydro xy-TEMPO=4-carboxy-TEMPO. In contrast, 4-hydroxy-TEMPO, which lacks th e free radical nitroxide function, was inactive up to 1 mM. (C) 1997 E lsevier Science B.V.