Modification of ferritin during iron loading.
Recombinant human ferritin loaded with iron via its own ferroxidase activity 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 sedimentation, indicating an alteration of the overall charge of ferritin. Additionally, analysis of the ferritin by SDS-PAGE under nonreducing conditions demonstrated that the ferritin had formed large aggregates, which suggests disulfide bonds are involved in the aggregation. The hydroxyl radical was detected by electron spin resonance spectroscopy during iron loading into recombinant ferritin by its own ferroxidase activity. However, recombinant human ferritin loaded with iron in the presence of ceruloplasmin sedimented through a sucrose-density gradient similar to native ferritin. This ferritin was shown to sediment as a function of iron content. The addition of ceruloplasmin to the iron loading assay eliminated the detection of the DMPO-*OH adduct observed during loading using the ferroxidase activity of ferritin. The elimination of the DMPO-*OH adduct was determined to be due to the ability of ceruloplasmin to completely reduce oxygen to water during the oxidation of the ferrous iron. The implications of these data for the present models for iron uptake into ferritin are discussed.
Welch
K D
KD
Biotechnology Center, Utah State University, Logan, UT 84322-4705, USA.
Van Eden
M E
ME
Aust
S D
SD
D001052:Apoferritins / Q000378:metabolism*; D002499:Centrifugation, Density Gradient / Q000379:methods; D002570:Ceruloplasmin / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D004789:Enzyme Activation / Q000502:physiology; D005293:Ferritins / Q000378:metabolism*; D006801:Humans; D017665:Hydroxyl Radical / Q000378:metabolism*; D007501:Iron / Q000378:metabolism*; D046911:Macromolecular Substances; D011485:Protein Binding / Q000502:physiology; D011994:Recombinant Proteins / Q000378:metabolism; D013050:Spectrometry, Fluorescence / Q000379:methods; D013154:Spleen / Q000737:chemistry
D046911:Macromolecular Substances; D011994:Recombinant Proteins; D017665:Hydroxyl Radical; D005293:Ferritins; D001052:Apoferritins; D007501:Iron; D002570:Ceruloplasmin