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. 2018 Dec 28;294(8):2700–2713. doi: 10.1074/jbc.RA118.007095

Figure 3.

Figure 3.

A sensitive assay for SOD inhibitors reveals an enhanced sensitivity of copper-only SOD5 to TTM and DETC copper chelators. A, schematic showing the basis for the SOD assay whereby superoxide produced by xanthine oxidase reduces WST-1 to formazan, a reaction blocked by SOD. Inhibitors of SOD allow WST-1 formazan formation in a dose-dependent manner. B, application of the WST-1 reduction assay for SOD activity in a 96-well format shows equal reactivity of bovine Cu,Zn-SOD1 and fungal copper-only SOD5. Controls are no xanthine oxidase (No XO) and denatured enzyme, heated for 1 h at 90 °C (DN). C and D, ammonium tetrathiomolybdate (TTM, C) and diethyldithiocarbamate (DETC, D) were tested for dose-dependent inhibition against Cu,Zn-SOD1 and copper-only SOD5 in the WST-1 reduction assay. The chelators were added at t = 0 to the SOD activity assay containing 1.5 μg/ml SOD (90 nm SOD monomers), and the reaction proceeded for 45 min at 37 °C as described under “Experimental procedures.” Results are the averages of duplicate samples from three independent experiments. Error bars, S.E.