Fragmentation of human Cu,Zn-superoxide dismutase by peroxidative reaction

We investigated the fragmentation of human Cu,Zn-superoxide dismutase (SOD) by H2O2. When Cu,Zn-SOD was incubated with H2O2, the fragmentation of protein proceeded rapidly within 1 min. The amounts of .OH radical formed in the Cu,Zn-SOD/H2O2 system reached a maximum in about 3 min. This result suggested that .OH was implicated in the fragmentation step. Copper ions released from Cu,Zn-SOD were gradually increased up to 30 min after starting the incubation with H2O2 in a time-dependent manner. However, the fragmentation was not inhibited by 5 mM DTPA. The results suggested that the fragmentation of Cu,Zn-SOD by H2O2 was due to the peroxidative reaction of SOD rather than the Fenton-like reaction by free copper released from oxidatively damaged SOD. A radical scavenger, azide anion, inhibited the fragmentation of Cu,Zn-SOD and the formation of .OH whereas ethanol did not. These results indicated that azide anions had easy access inside the active channel of Cu,Zn-SOD and thus protected the damage of the enzyme by .OH radicals whereas neutral alcohols stayed outside the active channel and could hardly intercept the newly-formed .OH radicals. Thus we conclude that the fragmentation of Cu,Zn-SOD by H2O2, is due to the oxidative damage resulting from free .OH radicals generated by the peroxidative reaction of SOD.