In vitro loading of apoferritin.
This study compared the effect of loading apoferritin either with ferrous ammonium sulfate in various buffers or with ceruloplasmin and chelated ferrous iron. It was shown that loading of apoferritin with ferrous ammonium sulfate was dependent on buffer and pH, and was directly related to the rate of iron autoxidation. The ceruloplasmin-dependent loading of apoferritin, however, was unaffected by these factors. Isoelectric focusing and amino acid analysis of the differently loaded ferritins showed that ferrous ammonium sulfate loading of apoferritin resulted in the depletion of the basic amino acids, lysine and histidine, probably as a result of protein oxidation. No significant differences in amino acid composition was noted for ceruloplasmin-loaded ferritin. Furthermore, ferritin loaded with ferrous ammonium sulfate released more iron than either native or ceruloplasmin-loaded ferritin when either paraquat or EDTA was used as an iron mobilizing agent. We suggest that the loading of apoferritin with ferrous ammonium sulfate occurred as a result of iron autoxidation and may result in oxidation of amino acids and loss of integrity of the protein, and that ceruloplasmin may act as a catalyst for the incorporation of iron into apoferritin in a manner more closely related to that occurring in vivo.
de Silva
D
D
Biotechnology Center, Utah State University, Logan 84322-4705.
Miller
D M
DM
Reif
D W
DW
Aust
S D
SD
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001052:Apoferritins / Q000737:chemistry*; D002021:Buffers; D002570:Ceruloplasmin / Q000737:chemistry; D005296:Ferrous Compounds / Q000494:pharmacology; D006736:Horses; D006863:Hydrogen-Ion Concentration; D010100:Oxygen / Q000737:chemistry; D000644:Quaternary Ammonium Compounds / Q000494:pharmacology
D000596:Amino Acids; D002021:Buffers; D005296:Ferrous Compounds; D000644:Quaternary Ammonium Compounds; D001052:Apoferritins; C038178:ammonium ferrous sulfate; D002570:Ceruloplasmin; D010100:Oxygen