Modification of ferritin during iron loading

Recombinant human ferritin loaded with iron via its own ferroxidase activit y did not sediment through a sucrose-density gradient as a function of iron content. Analysis of the recombinant ferritin by native PAGE demonstrated an increase in altered migration pattern of the ferritins with increasing s edimentation, indicating an alteration of the overall charge of ferritin. A dditionally, analysis of the ferritin by SDS-PAGE under nonreducing conditi ons demonstrated that the ferritin had formed large aggregates, which sugge sts disulfide bonds are involved in the aggregation. The hydroxyl radical w as detected by electron spin resonance spectroscopy during iron loading int o recombinant ferritin by its own ferroxidase activity. However, recombinan t human ferritin loaded with iron in the presence of ceruloplasmin sediment ed through a sucrose-density gradient similar to native ferritin. This ferr itin was shown to sediment as a function of iron content. The addition of c eruloplasmin to the iron loading assay eliminated the detection of the DMPO -(OH)-O-. adduct observed during loading using the ferroxidase activity of ferritin. The elimination of the DMPO-(OH)-O-. adduct was determined to be due to the ability of ceruloplasmin to completely reduce oxygen to water du ring the oxidation of the ferrous iron. The implications of these data for the present models for iron uptake into ferritin are discussed. (C) 2001 El sevier Science Inc.