Bovine erythrocyte superoxide dismutase (BESOD) has been extensively succinylated with succinic anhydride. Succinylated BESOD has an identical electron paramagnetic resonance (EPR) spectrum but only 10% as much activity as the native enzyme, showing that an increase of the negative charge of the protein surface lowers the activity without alteration of the active site structure. On the other hand, sodium dodecyl sulfate (NaDodSO4)-polyacrylamide gel electrophoresis indicates that interaction between subunits is strongly weakened by succinylation. NaDodSO4 has no effect on either the activity or EPR spectrum of the protein. BESOD was immobilized by coupling to a Sepharose matrix with no alteration of the EPR spectrum. Succinylation of the immobilized protein led to detachment from the gel of approximately 50% of the molecules, as estimated by parallel EPR measurements of the gel and activity determinations on the eluate. It is concluded the succinylation leads to dissociation of BESOD into nondenatured subunits, having lower activity than the native protein possibly because of charge effects on the enzyme-O2-interaction.
