A MICROTITER ASSAY FOR CU ZN-SUPEROXIDE AND MN-SUPEROXIDE DISMUTASE/

We present a microtiter assay system for monitoring the Cu/Zn-Superoxi de dismutase (SOD) and MnSOD activity as a micro version of ferri-cyto chrome c reduction assay of SOD using SOFT max software for 96 well MA X line microplate reader. The assay presented is in a 100 mu l reactio n volume, ten fold smaller volume than that of a standard cytochrome c reduction assay. The reaction kinetics of SOD inhibitable cytochsome c reduction was standardized using Cu/Zn-SOD (type I, 3000 units/mg pr otein). For measuring SOD activity a cytochrome c reduction of 7.5 mOD /minute was well suited in xanthine-xanthine oxidase reaction system. The inhibition of, O-2(-) mediated,cytochrome c reduction by Cu/Zn-SOD was inhibitable by 0.2 mM KCN. The assay was used to measure Cu/Zn-SO D and Mn-SOD (total SOD activity-KCN inhibitable activity) levels in n ormal (CL.7), and leukemic (3 R.1, 6R.1) clones. The method presented here is rapid, reproducible, and senstive and yields SOD activity usin g 1/10th of reagents as compared to a standard 1 ml spectrophotometric SOD assay system.