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Biological Trace Element Research

, Volume 61, Issue 3, pp 263–275 | Cite as

Separation of cellular iron containing compounds by electrophoresis

  • Daniel Vyoral
  • Jirí Petrák
  • Antonín Hradilek
Original Articles

Abstract

High resolution separation of metalloproteins and other iron compounds based on native gel electrophoresis followed by59Fe autoradiography is described. Lysates of mouse spleen erythroid cells metabolically labeled with59Fe-transferrin were separated on 3–20% polyacrylamide gradient gels in the presence of Triton X100 and detected by autoradiography. In addition to ferritin and hemoglobin, several compounds characterized by their binding of iron under different conditions were described. Iron chelatable by desferrioxamine migrated in the region where several high-molecular weight compounds were detected by silver staining. The technique is nondissociative, allowing identification of iron compounds with the use of specific antibodies. Cellular iron transport and the action of iron chelators on specific cellular targets can be investigated in many small biological samples in parallel.

Index Entries

59Fe iron chelatable iron ferritin heme desferrioxamine native electrophoresis 

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References

  1. 1.
    C. Hershko, Control of disease by selective iron depletion: a novel therapeutic strategy utilizing iron chelators,Baillieeres Clin. Haematol. 7, 965–1000 (1994).CrossRefGoogle Scholar
  2. 2.
    G. J. Kontoghiorghes and E. D. Weinberg, Iron: mammalian defence systems, mechanisms of disease, and chelation therapy approaches,Blood Rev. 9, 33–45 (1995).PubMedCrossRefGoogle Scholar
  3. 3.
    T. A. Rouault, D. J. Haile, W. E. Downey, C. C. Philpott, C. Tang, F. Samaniego, J. Chin, I. Paul, J. B. Harford, and R. D. Klausner, An iron-sulfur cluster plays a novel regulatory role in the iron-responsive element binding protein,Biometals 5, 131–140 (1992).PubMedCrossRefGoogle Scholar
  4. 4.
    J. V. Primosigh and E. D. Thomas, Studies on the partition of iron in bone marrow cells,J. Clin. Invest. 47, 1473–1482 (1968).PubMedCrossRefGoogle Scholar
  5. 5.
    M. T. Nunez, I. Pinto, and J. Glass, Assay and characteristics of the iron binding moiety of reticulocyte endocytic vesicles,J. Membrane Biol. 107, 129–135 (1989).CrossRefGoogle Scholar
  6. 6.
    J. Weaver and S. Pollack, Low-Mr iron isolated from guinea pig reticulocytes as AMP-Fe and ATP-Fe complexes,Biochem. J. 261, 787–792 (1989).PubMedGoogle Scholar
  7. 7.
    E. R. Giblett, C. G. Hickman, and O. Smithies, Serum transferrins,Nature 183, 1589–1590 (1959).PubMedCrossRefGoogle Scholar
  8. 8.
    Y. Chen and J. Drysdale, Detection of iron binding proteins by a blotting technique,Anal. Biochem. 212, 47–49 (1993).PubMedCrossRefGoogle Scholar
  9. 9.
    P. Owen, G. J. Kaczorowski, and H. R. Kaback, Resolution and identification of ironcontaining antigens in membrane vesicles from Escherichia coli,Biochemistry 19, 596–600 (1980).PubMedCrossRefGoogle Scholar
  10. 10.
    B. Crowe and P. Owen, Immunochemical analysis of respiratory-chain components of Micrococcus luteus (lysodeikticus),J. Baderiol. 153, 498–505 (1983).Google Scholar
  11. 11.
    J. Mengaud and M. A. Horwitz, The major iron-containing protein of Legionella pneumophila is an aconitase homologous with the human iron-responsive elementbinding protein,J. Bacteriol. 175, 5666–5676 (1993).PubMedGoogle Scholar
  12. 12.
    W. Scher, J. G. Holland, and C. Friend, Hemoglobin synthesis in murine virusinduced leukemic cells in vitro. I. Partial purification and identification of hemoglobins,Blood 37, 428–437 (1971).PubMedGoogle Scholar
  13. 13.
    J. Margolis and K. G. Kenrick, Polyacrylamide gel electrophoresis in a continuous molecular sieve gradient,Anal. Biochem. 25, 347–362 (1968).PubMedCrossRefGoogle Scholar
  14. 14.
    C. J. Eaves, G. Krystal, and A. C. Eaves, Erythropoietic cells,Biblthca Haemat. 48, 81–111 (1984).Google Scholar
  15. 15.
    D. Vyoral, A. Hradilek, and J. Neuwirt, Transferrin and iron distribution in subcellular fractions of K562 cells in the early stages of transferrin endocytosis,Biochim. Biophys. Acta 137, 148–154 (1992).Google Scholar
  16. 16.
    P. Ponka, A. Wilczynska, and H. M. Schulman, Iron utilization in rabbit reticulocytes. A study using succinylacetone as an inhibitor of heme synthesis,Biochim. Biophys. Acta 720, 96–105 (1982).PubMedCrossRefGoogle Scholar
  17. 17.
    J. Sambrook, E. F. Fritsch, and T. Maniatis,Molecular Cloning-A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor (1989).Google Scholar
  18. 18.
    P. J. Wirth and A. Romano, Staining methods in gel electrophoresis, including the use of multiple detection methods,J. Chromatography A 698, 123–143 (1995).CrossRefGoogle Scholar
  19. 19.
    D. R. Richardson, P. Ponka, and D. Vyoral, Distribution of iron in reticulocytes after inhibition of heme synthesis with succinylacetone. Examination of cytoplasmic and mitochondrial intermediates involved in iron metabolism, Blood84, Suppl. 1 (1994).Google Scholar
  20. 20.
    A. Jacobs, Low molecular weight intracellular iron transport compounds,Blood 50, 433–439 (1977).PubMedGoogle Scholar
  21. 21.
    S. Pollack, T. Campana, and J. Weaver, Low molecular weight iron in guinea pig reticulocytes,Am. J. Hematol. 19, 75–84 (1985).PubMedCrossRefGoogle Scholar
  22. 22.
    D. Hemmaplardh and E. Morgan, The mechanism of iron exchange between synthetic iron chelators and rabbit reticulocytes,Biochim. Biophys. Acta 373, 84–99 (1974).PubMedCrossRefGoogle Scholar
  23. 23.
    M. J. Pippard, D. K. Johnson, and C. A. Finch, Hepatocyte iron kinetics in the rat explored with an iron chelator,Brit. J. Haematol. 52, 211–224 (1982).Google Scholar
  24. 24.
    O. Gabriel and D. M. Gersten, Staining for enzymatic activity after gel electrophoresis,Anal. Biochem. 203, 1–21 (1992).PubMedCrossRefGoogle Scholar
  25. 25.
    U. Novak and L. Paradiso, Identification of proteins in DNA-protein complexes after blotting of EMSA gels,BioTechniques 19, 54–55 (1995).PubMedGoogle Scholar
  26. 26.
    H. Schagger, W. A. Cramer, and G. von Jagow, Analysis of molecular masses and oligomeric states of protein complexes by blue native electrophoresis and isolation of membrane protein complexes by two-dimensional native electrophoresis,Anal. Biochem. 217, 220–230 (1994).PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc 1998

Authors and Affiliations

  • Daniel Vyoral
    • 1
  • Jirí Petrák
    • 1
  • Antonín Hradilek
    • 1
  1. 1.Institute of Hematology and Blood TransfusionPrague 2Czech Republic

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