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Characterization of Fibrinolytic Proteases from Gloydius blomhoffii siniticus Venom

  • Choi, Suk-Ho (Division of Animal Resources and Life Science, Sangji University)
  • Received : 2011.05.16
  • Accepted : 2011.07.18
  • Published : 2011.09.30

Abstract

Objectives : This study was undertaken to identify fibrinolytic proteases from Gloydius blomhoffii siniticus venom and to characterize a major fibrinolytic protease purified from the venom. Methods : The venom was subjected to chromatography using columns of Q-Sepharose and Sephadex G-75. The molecular weights of fibrinolytic proteases showing fibrinolytic zone in fibrin plate assay were determined in SDS-PAGE (Sodium dodecyl sulfate-polyacrylamide gel electrophoresis) The effects of inhibitors and metal ions on fibrinolytic protease and the proteolysis patterns of fibrinogen, gelatin, and bovine serum albumin were investigated. Results : 1) The fibrinolytic fractions of the three peaks isolated from Gloydius blomhoffii siniticus venom contained two polypeptides of 46 and 59 kDa and three polypeptides of 32, 18, and 15 kDa and a major polypeptide of 54 kDa, respectively. 2) The fibrinolytic activity of the purified protease of 54 kDA was inhibited by metal chelators, such as EDTA, EGTA, and 1,10-phenanthroline, and disulfhydryl-reducing compounds, such as dithiothreitol and cysteine. 3) Calcium chloride promoted the fibrinolytic activity of the protease, but mercuric chloride and cobalt(II) chloride inhibited it. 4) The fibrinolytic protease cleaved preferentially A${\alpha}$-chain and slowly B${\beta}$-chain of fibrinogen. It also hydrolyzed gelatin but not bovine serum albumin. Conclusions : The Gloydius blomhoffii siniticus venom contained more than three fibrinolytic proteases. The major fibrinolytic protease was a metalloprotease which hydrolyzed both fibrinogen and gelatin, but not bovine serum albumin.

References

  1. Markland FS. Snake venoms and the hemostatic system. Toxicon. 1998;36:1749-800. https://doi.org/10.1016/S0041-0101(98)00126-3
  2. Bjarnason JB and Fox JW. Hemorrhagic metalloproteinases from snake venoms. Pharmacol. Ther. 1994;62:325-72. https://doi.org/10.1016/0163-7258(94)90049-3
  3. Marsh NA. Snake venom affecting the haemostatic mechanism - a consideration of their mechanisms, practical applications and biological significance, Blood Coagul. Fibrinolysis. 1994;5:399-410.
  4. Kamiguti AS, Hay CRM, Theakston RDG, and Zuzel M. Insights into the mechanism of haemorrhage caused by snake venom metalloproteinases. Toxicon. 1996;34:627-42. https://doi.org/10.1016/0041-0101(96)00017-7
  5. Suhr SM, Kim DS. Identification of the snake venom substance that induces apoptosis. Biochem. Biophys. Res. Commun. 1996;224:134-9. https://doi.org/10.1006/bbrc.1996.0996
  6. Matsui T, Fujimura Y, Titani K. Snake venom proteases affecting hemostasis and thrombosis, Biochim. Biophys. Acta. 2000;1477:146-56. https://doi.org/10.1016/S0167-4838(99)00268-X
  7. Swenson S. Markland Jr FS. Snake venom fibrin(ogen)olytic enzymes. Toxicon. 2005;45:1021-39. https://doi.org/10.1016/j.toxicon.2005.02.027
  8. Ramos OHP, Selistre-de-Araujo HS. Snake venom metalloproteases - structure and function of catalytic and disintegrin domains. Comp. Biochem. Physiol. Part C. 2006;142:328-46.
  9. Marsh N, Willams V. Practical applications of snake venom toxins in haemostasis. Toxicon. 2005;45:1171-81. https://doi.org/10.1016/j.toxicon.2005.02.016
  10. McDiarmid RW, Campbell JA, Toure T. Snake species of the world: A taxonomic and geographic reference, vol. 1. Washington : The Herpetologists' league. 1999.
  11. Mehrtens JM. Living snakes of the world in color. New York : Sterling Publishers. 1987.
  12. Terada S, Kimoto E, Kawasaki H, Hao WX, and Li HW. Purification of a metalloprotease from Chinese Mamushi (Agkistrodon halys brevicaudus) venom. Fukuoka Univ. Sci. Rep. 1981;21:147-53.
  13. Fujimura S, Rikimaru T, Baba S, Hori J, Hao X-Q, Terada S, Kimoto E. Purification and characterization of non-hemorrhagic metalloprotease from Agkistrodon halys brevicaudus ve-nom. Biochim. Biophys. Acta. 1995;1243:94-100. https://doi.org/10.1016/0304-4165(94)00115-E
  14. Terada S, Hori J, Fujimura S, Kimoto E. Purification and amino acid sequence of Brevilysin L6, a non-hemorrhagic metalloprotease from Agkistrodon halys brevicaudus venom. J. Biochem. 1999;125:64-9. https://doi.org/10.1093/oxfordjournals.jbchem.a022269
  15. Matsui T, Sakurai Y, Fujimura Y, Hayashi I, Oh-Ishi S, Suzuki M, Hamako J, Yamamoto Y, Yamazaki J, Kinoshita M, and Titani K. Purification and amino sequence of halystase from snake venom of Agkistrodon halys brevicaudus, a serine protease that cleaves specifically fibrinogen and kininogen. Eur. J. Biochem. 1998;252:69-575.
  16. Lee JW, Seu JH, Rhee IK, Jin I, Kawamura Y, Park W. Purificiation and characterization of brevinase, a heterogenous two-chain fibrinolytic enzyme from the venom of Korean snake, Agkistroden blomhoffii brevicaudus. Biochem. Biophys. Res. Commun. 1999;260:665-70. https://doi.org/10.1006/bbrc.1999.0977
  17. Astrup T, Mullertz S. The fibrin plate method for estimating of fibrinolytic activity. Archs. Biochem. Biophys. 1952;40:346-51. https://doi.org/10.1016/0003-9861(52)90121-5
  18. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T-4. Nature (London). 1970;227: 680-5. https://doi.org/10.1038/227680a0
  19. Baramova EN, Shannon JD, Bjarnason JB, Fox JW. Degradation of extracellular matrix proteins by hemorrhagic metalloproteinases. Arch. Biochem. Biophys. 1989;275:63-71. https://doi.org/10.1016/0003-9861(89)90350-0
  20. Shannon JD, Baramova EN, Bjarnason JB, Fox JW. Amino acid sequence of a Croatalus atrox venom metalloproteinase which cleaves type IV collagen and gelatin. J. Biol. Chem. 1989;264:11575-83.
  21. Johnson EK, Ownby CL. Isolation of hemorrhagic toxin from the venom of Agkistrodon contortrix laticinetus (broad-banded copperhead) and pathogenesis of the hemorrhage induced by the toxin in mice. Int. J. Biochem. 1993;25:267-78. https://doi.org/10.1016/0020-711X(93)90016-8

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