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.