Purification of chicken liver ferritin by two novel methods and structural comparison with horse spleen ferritin.
Ferritin was purified from chicken liver by two different methods: gel filtration on controlled-pore glass beads, and immunoaffinity chromatography employing a chicken ferritin-specific monoclonal antibody that did not cross-react with horse spleen ferritin. This antibody recognizes intact ferritin and an oligomeric 240 kDa form of the molecule after protein transfer to nitrocellulose, but not the 22 kDa chicken ferritin subunit. Chicken liver ferritin purified by these methods exhibited reduced migration on non-denaturing polyacrylamide gels compared with horse spleen ferritin. These results were consistent with the difference in calculated isoelectric points of chicken and horse ferritin subunits. By two-dimensional gel electrophoresis, chicken ferritin 22 kDa subunits exhibited isoelectric points from 6.1 to 6.6 whereas horse spleen ferritin subunits exhibited isoelectric points of 5.8-6.3. The 240 kDa form of the chicken ferritin molecule had an isoelectric point of 6.6 whereas the 210 kDa form of the horse ferritin molecule had isoelectric points of 5.1 and 4.9. Intact chicken liver ferritin particles were 13.4 +/- 0.8 nm (controlled-pore glass-purified) and 12.5 +/- 0.9 nm (affinity-purified) in diameter when viewed by electron microscopy. Horse spleen ferritin consisted of slightly smaller particles with an average diameter of 11.0 +/- 0.7 nm. However, ferritin from chicken liver and horse spleen co-migrated with an apparent molecular mass of 470 kDa when analysed by Sepharose 4B gel filtration chromatography. These results indicate that, consistent with results from other published purification methods, the chicken ferritin purified by the methods reported here exhibits both structural similarities to, and differences from, horse spleen ferritin.
Passaniti
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A
Department of Biological Sciences, University of Maryland, Baltimore County, Catonsville 21228.
Roth
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D000818:Animals; D000911:Antibodies, Monoclonal; D002645:Chickens; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D005293:Ferritins / Q000032:analysis* / Q000276:immunology; D006736:Horses; D007525:Isoelectric Focusing; D008099:Liver / Q000032:analysis*; D013154:Spleen
D000911:Antibodies, Monoclonal; D005293:Ferritins