Cyanide Insensitive Iron Superoxide Dismutase in Euglena gracilis Comparison of the Reliabilities of Different Test Systems for Superoxide Dismutases

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Abstract
Two proteins (P1 and P2, with MW of 57,500 and 27,500, respectively) were isolated from E. gracilis. Both proteins show cyanide-insensitive superoxide dismutase activity in the classical superoxide dismutase assay, using xanthine-xanthine oxidase O2.- generator. If O2.- is generated chemically (autoxidation of reduced anthraquinone), photochemically (illuminated riboflavine) or pulse radiolytically, only protein P1 but not P2 shows SOD activity. Protein P1 contains 1 g atom (determined: 0.82) Fe (no MN or Cu)/mol protein and may thus be defined as iron-superoxide dismutase. Protein P2, showing the spectral properties of a flavoprotein, exhibits the activities of ferredoxin-NADP-oxidoreductase and diaphorase. The cyanide-insensitive SOD-activity of this diaphorase in the xanthine oxidase-assay for superoxide dismutase makes this classical and commonly used test unreliable for assaying cyanide insensitive SOD activities. The existence of the prokaryote-type of superoxide dismutase (Fe-SOD) in E. gracilis is exceptional for an eukaryotic, autotrophically grown organisms.