Archives of Pharmacal Research

The Pharmaceutical Society of Korea (대한약학회)
권호 표지

Purification and Characterization of Anticoagulant Protein from the Tabanus, Tabanus bivittatus

  • Ahn Mi-Young (Department of Agricultural Biology, National Institute of Agricultural Science and Technology) ;
  • Hahn Bum-Soo (Department of Molecular Physiology and Biochemistry, National Institute of Agricultural Biotechnology) ;
  • Lee Pyeong-Jae (Department of Agricultural Biology, National Institute of Agricultural Science and Technology) ;
  • Wu Song-Ji (Natural Products Research Institute, College of Pharmacy, Seoul National University) ;
  • Kim Yeong-Shik (Natural Products Research Institute, College of Pharmacy, Seoul National University)
  • Published : 2006.05.01
Download PDF
⟨ Previous Next ⟩

Abstract

Tabanus anticoagulant protein (TAP) was isolated from the whole body of the tabanus, Tabanus bivittatus, using three purification steps (ammonium sulfate fractionation, gel filtration on Bio-Gel P-60, and ion exchange chromatography on DEAE Sephadex gel). The purified TAP, with a molecular weight of 65 kDa, was assessed to be homogeneous by SDS-polyacrylamide gel electrophoresis, and an isoelectric point of 7.9 was determined by isoelectric focusing. The internal amino acid sequence of the purified protein was composed of Ser-Leu-Asn-Asn-Gln-Phe-Ala-Ser-Phe-lle-Asp-Lys-Val-Arg. The protein was activated by $Cu^{2+}\;and\;Zn^{2+}$, and the optimal conditions were found to be at pH $3\sim6\;and\;40\sim70^{\circ}C$. Standard coagulation screen assays were used to determine thrombin time and activated partial thromboplastin time. Chromogenic substrate assays were performed for thrombin and factor Xa activity. TAP considerably prolonged human plasma clotting time, especially activated partial thromboplastin time in a dose-dependent manner; it showed potent and specific antithrombin activity in the chromogenic substrate assay. Specific anti-factor Xa activity in TAP was not detected. Overall, this result suggested that TAP has significant anticoagulant activity on blood coagulation system.

Keywords

References

  1. Ahn, M. Y., Ryu, K. S., Lee, Y. W., and Kim, Y.S., Cytotoxicity and L-amino acid oxidase activity crude insect drugs. Arch. Pharm. Res., 5, 477-481 (2000)
  2. Ahn, M. Y., Hahn, B. S., Ryu, K. S., and Cho, S. I., Effects of insect crude drugs on blood coagulation and fibrinolysis system. Natural Product Sciences, 8, 66-70 (2002)
  3. Ahn, M. Y., Hahn, B. S., Ryu, K. S., Kim, J. W., Kim, I., and Kim, Y. S., Purification and characterization of a serine protease with fibrinolytic activity from dung beetles, Catharsius molossus, Thrombosis Research, 112, 339-347 (2003) https://doi.org/10.1016/j.thromres.2004.01.005
  4. Astrup, T. and Mullertz, S., The fibrin plate method for estimating of fibrinolytic activity. Arch Biochem Biophys, 40, 346-351 (1952) https://doi.org/10.1016/0003-9861(52)90121-5
  5. Bradford, M. M., A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem, 72, 248-254 (1976) https://doi.org/10.1016/0003-2697(76)90527-3
  6. Grevelink, S. A., Youssef, D. E., Loscalzo, J., and Lerner, E. A., Salivary gland extracts from the deerfly contain a potent inhibitor of platelet aggregation. PNAS, 90, 9155-9158 (1993)
  7. Kazimirova, M., Sulanova, M., Trimnell, A. R., Kozanek, M., VidliCka, L., Labuda M., and Nuttall, P. A., Anticoagulant activities in salivary glands of tabanid flies. Kazimirova, M., Sulanova, M., Trimnell, A. R., Kozanek, M., VidliCka, L., Labuda M., and Nuttall, P. A., Anticoagulant activities in salivary glands of tabanid flies. Med. Vet. Entomol., 16, 301-309 (2002) https://doi.org/10.1046/j.1365-2915.2002.00379.x
  8. Kazimirova, M., S¡ulanova, M., Kozanek, M., Takac, P., Labuda, M., and Nuttall, P. A., Identification of Anticoagulant Activities in Salivary Gland Extracts of Four Horsefly Species (Diptera, Tabanidae). Haemostasis, 31, 294-305 (2001)
  9. Laemmli, U. K., Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227, 680- 685 (1970) https://doi.org/10.1038/227680a0
  10. Markwardt, F. and Leberecht, E., Untersuchungen über den blutgerinnung-shemmenden Wirkstoff der Tabaniden. Naturwissenschaften, 46, 17-18 (1959)
  11. Markwardt, F. and Schulz, E., Uber den Mechanismus der blutgerinnungshemmenden Wirkung des Tabanins. Naunyn- Schmiedeberg's Archiv fur Experimentelle Pathologie Undpharmakologie, 238, 320-328 (1960)
  12. Markwardt, F., Coagulation inhibitors from animals feeding on blood. Rev Iberoamer Tromb Hemost, 7, 225-231 (1994)
  13. Ouriel, K., Current status of thrombolysis for peripheral arterial occlusive disease. Ann Vas Surg, 16, 797-804 (2002) https://doi.org/10.1007/s10016-001-0318-y
  14. Reddy, V. B., Kounga, K., Mariano, F., and Lerner, E. A., Chrysoptin is a potent glycoprotein Iib/IIIa fibrinogen receptor antagonist present in salivary gland extracts of the deerfly. J. Biol. Chem., 275, 15861-15867(2000) https://doi.org/10.1074/jbc.275.21.15861
  15. Ribeiro, J. M. C., Vachereau, A., Modi, G. B., and Tesh, R. B., A novel vasodilatory peptide from the salivary glands of the sand fly Lutzomyia longipalpis. Science, 243, 212-214 (1989) https://doi.org/10.1126/science.2783496