Rapid microplate assay for superoxide scavenging efficiency

Here we report a method to determine superoxide scavenging efficiency, using kinetic analysis of cytochrome c reduction and an automated UV/vis microtiter plate reader. Superoxide (O(2)(-&z. rad;)) was generated by xanthine oxidase metabolism of hypoxanthine, and quantified by following reduction of cytochrome c by O(2)(-&z. rad;) as increasing absorbance at 550 nm. Reaction conditions were established that provided a linear increase in O(2)(-&z.rad;) generation for more than 20 min, and good reproducibility over time. The majority of cytochrome c reduction was blocked by superoxide dismutase, indicating cytochrome c reduction derived predominantly from O(2)(-&z.rad;). Although EDTA is commonly included in this assay to eliminate undesirable Fenton side-reactions with H(2)O(2) (a co-product of reactions that use xanthine oxidase to produce O(2)(-&z.rad;)), we found that catalase, but not EDTA, blocked suicide elimination of cytochrome c from the reaction. Finally, we demonstrate the feasibility of evaluating superoxide scavenging abilities on small samples extracted from two types of neuronal cultures, a hypothalamic neuronal cell line (GT1 trk cells) and primary mouse cortical cell cultures. This assay allows rapid, high throughput assessments of superoxide scavenging efficacy for small molecules of interest, as well as for cell or tissue extracts.