Journal of Pharmacopuncture (대한약침학회지)

KOREAN PHARMACOPUNCTURE INSTITUTE (대한약침학회)
권호 표지
DOI QR코드

DOI QR Code

Isolation and Characterization of a 32-kDa Fibrinolytic Enzyme (FE-32kDa) from Gloydius blomhoffii siniticus Venom -Fibrinolytic Enzyme from Gloydius blomhoffii siniticus Venom-

  • Kim, Joung-Yoon (Division of Animal Science and Biotechnology, Sangji University) ;
  • Lee, Seung-Bae (Division of Animal Science and Biotechnology, Sangji University) ;
  • Kwon, Ki Rok (Research Center, Korean Pharmacopuncture Institute) ;
  • Choi, Suk-Ho (Division of Animal Science and Biotechnology, Sangji University)
  • Received : 2013.12.31
  • Accepted : 2014.01.29
  • Published : 2014.03.31
Download PDF
⟨ Previous Next ⟩

Abstract

Objectives: This study was undertaken to isolate a fibrinolytic enzyme from the snake venom of Gloydius blomhoffii siniticus and to investigate its enzymatic characteristics and hemorrhagic activity as a potential pharmacopuncture agent. Methods: The fibrinolytic enzyme was isolated by using chromatography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and fibrin plate assay. The characteristics of the enzyme were investigated using fibrin plate assay, protein hydrolysis analysis, and hemorrhage assay. Its amino acid composition was determined. Results: The fibrinolytic enzyme with the molecular weight of 32kDa (FE-32kDa) from Gloydius blomhoffii siniticus showed a fibrin hydrolysis zone at the concentration of 0.2 mg/mL in the fibrin plate assay. The fibrin hydrolysis activity of the enzyme was inhibited completely by ethylenediaminetetraacetic acid (EDTA), ethyleneglycoltetraacetic acid (EGTA), and 1, 10-phenanthroline, thiothreitol and cysteine, and partially by phenylmethanesulfonylfluoride (PMSF). Metal ions such as $Fe^{2+}$ and $Hg^{2+}$ inhibited the fibrin hydrolysis completely, but $Zn^{2+}$ enhanced it. FE-32kDa hydrolyzed ${\alpha}$-chain but did not hydrolyze ${\beta}$-chain and ${\gamma}$-chain of fibrinogen. High-molecular-weight polypeptides of gelatin were hydrolyzed partially into low-molecular-weight polypeptides, but the extent of hydrolysis was limited. FE-32kDa induced hemorrhage beneath back skin of mice at the dose of $2{\mu}g$. Conclusions: FE-32kDa is a ${\alpha}$-fibrin(ogen)olytic metalloprotease that requires $Zn^{2+}$ for fibrinolytic activity and causes hemorrhage, suggesting that the enzyme is not appropriate for use as a clinical pharmacopuncture.

Keywords

References

  1. Swenson S, Markland FS Jr. Snake venom fibrin(ogen) olytic enzymes. Toxicon. 2005;45(8):1021-39. https://doi.org/10.1016/j.toxicon.2005.02.027
  2. Ramos OHP, Selistre-de-Araujo HS. Snake venom metalloproteases - structure and function of catalytic and disinterin domains. Comp Biochem Physiol C. 2006;142(3-4):328-46.
  3. Baramova EN, Shannon JD, Bjarnason JB, Fox JW. Degradation of extracellular matrix proteins by hemorrhagic metalloproteinases. Arch Biochem Biophys. 1989;275(1):63-71. https://doi.org/10.1016/0003-9861(89)90350-0
  4. Shannon JD, Baramova EN, Bjarnason JB, Fox JW. Amino acid sequence of a Crotalus atrox venom metalloproteinase which cleaves type IV collagen and gelatin. J Biol Chem. 1989;264(20):11575-83.
  5. Markland FS. Snake venom and the hemostatic system. Toxicon.1998;36(12):1749-800. https://doi.org/10.1016/S0041-0101(98)00126-3
  6. Markland FS, Swenson S. Fibrolase: trials and tribulations. Toxins (Basel). 2010;2(4):793-808. https://doi.org/10.3390/toxins2040793
  7. Anonymous. Mamusi. Wikipedia 2013. Retrieved Jan 2, 2014 from http://en.wikipedia.org/wiki/Gloydius_blomhoffii.
  8. Choi SH. [Characterization of fibrinolytic proteases from Gloydius blomhoffii siniticus venom]. Pharmacopuncture. 2011;14(3):71-9. Korean. https://doi.org/10.3831/KPI.2011.14.3.071
  9. Choi SH, Lee SB. Isolation from Gloydius blomhoffii siniticus venom of a fibrin(ogen)olytic enzyme consisting of two heterogenous polypeptides. Pharmacopuncture. 2013;16(2):46-54. https://doi.org/10.3831/KPI.2013.16.010
  10. Lee JW, Seu JH, Rhee IK, Jin I, Kawamura Y, Park W. Purification and characterization of brevinase, a heterogeneous two-chain fbrinolytic enzyme from the venom of Korean snake, Agkistrodon blomhoffii brevicaudus. Biochem Biophys Res Commun. 1999;260(3):665-70. https://doi.org/10.1006/bbrc.1999.0977
  11. Astrup T, Mullertz S. The fibrin plate method for estimating of fibrinolytic activity. Arch Biochem Biophys. 1952;40(2):346-51. https://doi.org/10.1016/0003-9861(52)90121-5
  12. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970; 227(5259):680-5. https://doi.org/10.1038/227680a0
  13. Terada S, Tsumura Y, Kawabata A. Characterization of brevilysin H1, an $\alpha$-fibrinogenolytic metalloproteinase from the venom of Chinese snake (Gloydius blomhoffii brevicaudus). Fukuoka Univ Sci Rep. 2011;41(2):195-202.
  14. Terada S, Kimoto E, Kawasaki H, Hao WX, Li HW. Purification of a metalloprotease from Chinese mamushi (Agkistrodon halys brevicaudus) venom. Fukuoka Univ Sci Rep.1981; 21(2):147-53.
  15. Fujimura S, Oshikaqa K, Terada S, Kimoto E. Primary structure and autoproteolysis of brevilysin H6 from the venom of Gloydius halys brevicaudus. J Biochem. 2000;128(2):167-73. https://doi.org/10.1093/oxfordjournals.jbchem.a022737
  16. Fujimura S, Rikimaru T, Baba S, Hori J, Hao XQ, Terada S, et al. Purification and characterization of non-hemorrhagic metalloprotease from Agkistrodon halys brevicaudus venom. Biochim Biophys Acta. 1995;1243(1):94-100. https://doi.org/10.1016/0304-4165(94)00115-E
  17. Deshimaru M, Ichihara M, Hattori T, Koba K, Terada S. Primary structure of brevilysin L4, an enzymatically active fragment of a disintegrin precursor from Gloydius halys brevicaudus venom. Toxicon. 2005;45(5):570-80.
  18. 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(1): 64-9. https://doi.org/10.1093/oxfordjournals.jbchem.a022269
  19. Anonymous. Gloydius ussuriensis. Wikipedia 2013. Retrieved Jan 2, 2014 from http://en.wikipedia.org/wiki/Ussuri_mamush.

Cited by

  1. Agkihpin, a novel SVTLE from Gloydius halys Pallas, promotes platelet aggregation in vitro and inhibits thrombus formation in vivo in murine models of thrombosis vol.122, 2016, https://doi.org/10.1016/j.toxicon.2016.09.017
  2. Structures and Functions of Snake Venom Metalloproteinases (SVMP) from Protobothrops venom Collected in Japan vol.22, pp.8, 2017, https://doi.org/10.3390/molecules22081305
  3. Severe Coagulopathy after Ingestion of “Snake Wine” vol.50, pp.6, 2016, https://doi.org/10.1016/j.jemermed.2015.11.037