Manipulation of the Affinity Between Protein and Metal Ions by Imidazole and PHfor Metal Affinity Purification of Protein c from Cohn Fraction IV-1

  • James J. Lee
  • Duane F. Bruley
  • Kyung A. Kang
Part of the Advances In Experimental Medicine And Biology book series (AEMB, volume 614)


Protein C (PC) is an important anticoagulant in blood plasma. Cohn Fraction IV-1 (CFIV-1) is an inexpensive PC source but contains a large amount of factor II (FII). Immobilized metal affinity chromatography (IMAC) utilizes metal ions to adsorb proteins primarily via their surface histidine. Two major operation parameters for IMAC are imidazole concentration and pH: imidazole is a histidine analog and pH controls the protein surface protonation level. The effects of these two parameters on the adsorption and elution of PC and FII were studied for each protein individually and also together as a mixture. For the individual proteins, low FII (16%) and high PC (98%) adsorption were achieved at 8 mM imidazole, pH 8.0. At 11 mM imidazole, 92% of the adsorbed FII was eluted, with only a 3% PC loss. At 40 mM, 97% of the adsorbed PC was recovered. For the protein mixture, very similar adsorption and elution results were obtained, but slightly greater PC loss (16%) during elution at 11 mM imidazole. This result shows that there is a high potential for the PC purification from CFIV-1 by appropriately adjusting the imidazole concentration and pH in the IMAC process.


Immobilize Metal Affinity Chromatography Iminodiacetic Acid Cupric Sulfate Sulfate Pentahydrate Imidazole Concentration 
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  1. 1.
    C. T. Esmon, The Anticoagulant and Anti-Inflammatory Roles of the Protein C Anticoagulant Pathway, J. Autoimmun. 15, 113–116 (2000).PubMedCrossRefGoogle Scholar
  2. 2.
    C. T. Esmon, Protein C anticoagulant pathway and its role in controlling microvascular thrombosis and inflammation, Crit. Care Med. 29(7), 48–51 (2001).CrossRefGoogle Scholar
  3. 3.
    D. E. Joyce, L. Gelbert, A. Ciaccia, B. DeHoff and B. W. Grinnell, Gene expression profile of antithrombotic protein C defines new mechanisms modulating inflammation and apoptosis. J. Biol. Chem. 276, 11199–11203 (2001).PubMedCrossRefGoogle Scholar
  4. 4.
    Rosen, K. Fukudome and B. V. Zlokovic, Activated protein C blocks p53-mediated apoptosis in ischemic human brain endothelium and its neuroprotective, Nat. Med. 9(3), 338–342 (2003).PubMedCrossRefGoogle Scholar
  5. 5.
    D. Liu, T. Cheng, H. Guo, J. A. Fernandez, J. H. Griffin, X. Song and B. V. Zlokovic, Tissue plasminogen activator neurovascular toxicity is controlled by activated protein C, Nat. Med. 10(12), 1379–1383 (2004).PubMedCrossRefGoogle Scholar
  6. 6.
    R. M. Bertina, Protein C and Related Proteins; Biochemical and Clinical Aspects, (Churchill Livingstone, New York, 1998), pp. 1–54.Google Scholar
  7. 7.
    D. F. Bruley and W. N. Drohan, Advances in Applied Biotechnology Series; Protein C and Related Anticoagulants, 11 (Gulf Publications, Houston, TX 1990).Google Scholar
  8. 8.
    S. Rezania, D. G. Ahn and K. A. Kang, Separation of Protein C from Cohn Fraction IV-1 by Mini-Antibody, Proceedings of the 33rd Annual ISOTT meeting 2005. Adv. Exp. Med. Biol.: Oxygen Transport to Tissue XXVIII, 599, (Maguire, D. J., Bruley, D. F., Harrison, D. K., eds.), p. 125–132, 2007–Q:AU: Please update.–.Google Scholar
  9. 9.
    L. H. Edmunds and E. W. Salzman, Hemostatic Problems, Transfusion Therapy, and Cardiopulmonary Bypass in Surgical Patients, Hemostasis and Thrombosis, 3rd ed., R. W. Colman, J. Hirsh, V. J. Marder and E. W. Salzman, (J. B. Lippincott Co, Philadelphia, 1994), p. 958.Google Scholar
  10. 10.
    G. J. Broze Jr. and J. P. Miletich, Biochemistry and Physiology of Protein C, Protein S, and Thrombomodulin, Hemostasis and Thrombosis, 3rd ed., R. W. Colman, J. Hirsh, V. J. Marder and E. W. Salzman, (J. B. Lippincott Co., Philadelphia, 1994), p. 262.Google Scholar
  11. 11.
    E. S. Hemdan, Y. J. Zhao, E. Sulkowski and J. Porath, Surface topography of hisitdine residues: A facile probe by immobilized metal ion affinity chromatography, Proceedings of the National Academy of Science USA, 86, 1811–1815 (1989).CrossRefGoogle Scholar
  12. 12.
    H. Wu and D. F. Bruley, Homologous human blood protein separation using immobilized metal affinity chromatography: protein C separation from prothrombin with application to the separation of factor IX and prothrombin, Biotechnol. Progr. 15, 928 (1999).Google Scholar
  13. 13.
    H. Wu, D. F. Bruley, K. A. Kang, Protein C Separation form human plasma Cohn fraction IV-1 using immobilized metal affinity chromatography, Adv. Exp. Med. & Bio.: Oxy. Trans. to Tis. XX, 454, (Plenum Press, New York, 1998), pp. 697–704.Google Scholar
  14. 14.
    J. J. Lee, D. F. Bruley, and K. A. Kang, Effect of pH and Imidazole on Protein C Purification from Cohn Fraction IV-1 by IMAC, Proceedings of the 33rd Annual ISOTT meeting 2005. Adv. Exp. Med. Biol.: Oxygen Transport to Tissue XXVIII, 599, (Maguire, D. J., Bruley, D. F., Harrison, D. K., eds), p. 53–60, 2007.Google Scholar

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© Springer Science+Business Media, LLC 2008

Authors and Affiliations

  • James J. Lee
    • 1
  • Duane F. Bruley
    • 2
    • 3
  • Kyung A. Kang
    • 1
  1. 1.Department of Chemical EngineeringUniversity of LouisvilleLouisville
  2. 2.Department of Chemical and Biochemical EngineeringUniversity of Maryland Baltimore CountyBaltimore
  3. 3.Synthesizer Inc.Ellicott City

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