Redox-active metal ions such as Fe(II)\(III) and Cu(I)\(II) have been proposed to activate reactive oxygen and nitrogen species (RONS) and thus, perpetuate oxidative damage. Here, we show that concentrations of metal ions and EDTA complexes with superoxide-destroying activities equivalent to 1 U SOD are Fe(III) 5.1 muM, Mn(II) 0.77 muM, Cu(II)-EDTA 3.55 muM, Fe(III)-EDTA 2.34 muM, and Mn(II)-EDTA 1.38 muM. The most active being the aquated Cu(II) species which exhibited superoxide-destroying activity equivalent to 2 U of SOD at 0.29 muM. Hydrogen peroxide-destroying activities were as follows Fe(Ill)-EDTA ca. 70 U/mg and aquated Fe(III) 141 U/mg. In contrast, DTPA prevented superoxide-destroying activity and significantly depleted hydrogen peroxide-destroying activity. In conclusion, non-protein bound transition metal ions may have significant anti-oxidant effects in biological systems. Caution should be employed in bioassays when chelating metal ions. Our results demonstrate that DTPA is preferential to EDTA for inactivating redox-active metal ions in bioassays. (C) 2004 Elsevier Inc. All rights reserved.