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The Biochemical journal1989; 258(2); 413-419; doi: 10.1042/bj2580413

Purification of chicken liver ferritin by two novel methods and structural comparison with horse spleen ferritin.

Abstract: 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.
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Publication Date: 1989-03-01 PubMed ID: 2705992PubMed Central: PMC1138378DOI: 10.1042/bj2580413Google Scholar: Lookup
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  • Comparative Study
  • Journal Article

Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

This study focuses on the purification of ferritin (a protein in animal tissues responsible for storing iron) from chicken liver through novel methods and then comparing the structure of the purified ferritin to horse spleen ferritin. The researchers used techniques such as gel filtration on controlled-pore glass beads, and immunoaffinity chromatography with a chicken-specific antibody.

Methods of Purification

  • The researchers utilized two distinct methods for the purification of ferritin from chicken liver. Gel filtration on controlled-pore glass beads and immunoaffinity chromatography were used.
  • For the immunoaffinity chromatography process, they used a specific monoclonal antibody that attaches only to the chicken liver ferritin and doesn’t interact with the horse spleen ferritin.
  • This specific antibody can recognize the whole ferritin molecule as well as an oligomeric 240 kDa form of the protein after it’s transferred to nitrocellulose; however, it couldn’t recognize the 22 kDa chicken ferritin subunit.

Comparison of Chicken and Horse Spleen Ferritin

  • Chicken liver ferritin that underwent purification using these methods showed reduced migration on non-denaturing polyacrylamide gels compared to horse spleen ferritin.
  • These observations were in alignment with the differences in the calculated isoelectric points of ferritin subunits found in chicken and horse spleen. Researchers determined the chicken ferritin 22 kDa subunits exhibited isoelectric points ranging from 6.1 to 6.6, while the horse spleen ferritin subunits showed isoelectric points of 5.8-6.3.
  • The 240 kDa form of the chicken ferritin molecule had an isoelectric point of 6.6, signifying its overall net electric charge, whereas the 210 kDa form of the horse ferritin molecule had isoelectric points of 5.1 and 4.9.

Structural Observations

  • Researchers observed that intact particles of chicken liver ferritin measured 13.4 +/- 0.8 nm (controlled-pore glass-purified) and 12.5 +/- 0.9 nm (affinity-purified) in terms of diameter through electron microscopy.
  • On the contrary, horse spleen ferritin particles were a bit smaller, having an average diameter of 11.0 +/- 0.7 nm.
  • Regardless of their size differences, when tested by Sepharose 4B gel filtration chromatography, ferritin from both chicken liver and horse spleen ran together as a single band with an apparent molecular weight of 470 kDa.
  • The comparison results indicate similarities and differences in the structural composition of the chicken and horse spleen ferritin.

Cite This Article

APA
Passaniti A, Roth TF. (1989). Purification of chicken liver ferritin by two novel methods and structural comparison with horse spleen ferritin. Biochem J, 258(2), 413-419. https://doi.org/10.1042/bj2580413

Publication

The Biochemical journal
ISSN: 0264-6021
NlmUniqueID: 2984726R
Country: England
Language: English
Volume: 258
Issue: 2
Pages: 413-419

Researcher Affiliations

Passaniti, A
  • Department of Biological Sciences, University of Maryland, Baltimore County, Catonsville 21228.
Roth, T F

    MeSH Terms

    • Animals
    • Antibodies, Monoclonal
    • Chickens
    • Chromatography, Affinity
    • Electrophoresis, Polyacrylamide Gel
    • Ferritins / analysis
    • Ferritins / immunology
    • Horses
    • Isoelectric Focusing
    • Liver / analysis
    • Spleen

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