Fragmentation of human ceruloplasmin induced by hydrogen peroxide

We investigated the fragmentation of human ceruloplasmin induced by H 2O 2 to study its oxidative damage. When ceruloplasmin was incubated with H 2O 2, the frequency of the protein fragmentation increased in a proportion to the concentration of H 2O 2. It also increased in a time-dependent manner and was accompanied by gradual loss of the oxidase activity. Hydroxyl radical scavengers such as azide and mannitol inhibited the fragmentation of ceruloplasmin. The deoxyribose assay showed that hydroxyl radicals were generated in the reaction of ceruloplasmin with H 2O 2. Incubation of ceruloplasmin with H 2O 2 resulted in a time-dependent release of copper ions. The released copper ion may participate in a Fenton-like reaction to produce hydroxyl radical, which enhanced the fragmentation. The protection of the fragmentation by copper chelators such as diethylenetriaminepentaacetic acid and bathocuproine indicates a role for copper ion in the reaction. These results suggest that the fragmentation of ceruloplasmin induced by H 2O 2 is due to hydroxyl radicals formed by a copper-dependent Fenton-like reaction.