Quantitative resolution of Cu,Zn- and Mn-superoxide dismutase activities

Abstract

Assays of superoxide dismutase on total soluble extracts of eucaryotic cells generally result in the sum of activities of the Cu,Zn- and Mn-enzymes. Accurate quantitative resolution of the total activity on the basis of inhibition of the Cu,Zn-enzyme with cyanide may require that a correction is introduced for incomplete saturation with inhibitor at the finite concentration of cyanide used. This correction is calculated using the apparent dissociation constant of one to one complex formation with cyanide, K′, under the conditions of the assay. A linear relationship describing the activity in the presence of graded concentrations of cyanide enables the determination of K′ on soluble extracts without isolation of the Cu,Zn-enzyme. An evaluation of the precision, based on a propagation of error analysis, of different methods for the quantitative resolution of the two dismutases is presented. This reveals most notably that the random error in the resolution in terms of McCord and Fridovich activity units is reduced by carrying out the resolution at high pH (where a lower K′ for cyanide applies) followed by the conversion of the result to pH 7.8.