A simple assay of the superoxide generation rate with Tiron as an EPR-visible radical scavenger

Tiron (1,2-dihydroxybenzene-3,5-disulfonate) is oxidized to an EPR-visible semiquinone by superoxide radicals produced by xanthine oxidase. The steady-state level of the Tiron radicals increases with an increased xanthine oxidase concentration. A calibration plot has been obtained relating the steady-state concentration of the Tiron semiquinone determined by EPR-spectroscopy to the rate of 0.2 production as measured by the superoxide dismutase-sensitive cytochrome c reduction. This approach allows for a simple and sensitive assay of 0.2 generation rate in biological systems in the range of ca.0.1-4.0 microM/min using Tiron as a spin trap. The rate of 0.2 generation by antimycin-inhibited ischemic rat heart mitochondria has been measured by this method.