Molecular and Cellular Biochemistry

, Volume 282, Issue 1–2, pp 147–155 | Cite as

Strong myotoxic activity of Trimeresurus malabaricus venom: Role of metalloproteases

  • C. D. Raghavendra Gowda
  • R. Rajesh
  • A. Nataraju
  • B. L. Dhananjaya
  • A. R. Raghupathi
  • T. V. Gowda
  • B. K. Sharath
  • B. S. Vishwanath


Trimeresurus malabaricus is an endemic snake found in the Southern region of Western Ghats section of India along with the more widely distributed species like Naja naja and Daboia russelii. T. malabaricus venom is not lethal when injected (i.p.) up to 20 mg/kg body weight in mice, but causes extensive local tissue degeneration. N. naja and D. russelii are highly toxic (i.p.) with minimum local tissue damage in experimental mice. In this study a comparative analysis of local tissue damage of T. malabaricus venom is made with N. naja and D. russelii snake venoms of the Southern regions of Western Ghats. T. malabaricus venom exhibits caseinolytic activity 16 and 24 times more than N. naja and D. russelii venom. Inhibition studies with specific protease inhibitors reveal that the major proteases belong to metalloproteases. T. malabaricus venom hydrolyses gelatin and induces strong hemorrhagic activity in mice. Both N. naja and D. russelii fail to hydrolyze gelatin even at very high concentration and did not induce any hemorrhagic activity. With D. russelii venom small hemorrhagic spot was observed at the site of injection. The hemorrhagic activity of T. malabaricus venom is completely neutralized by metalloprotease inhibitors and not by serine protease inhibitor. The i.m. injection of T. malabaricus venom causes extensive degradation of muscle tissue within 24 h. The light microscopic observation of muscle tissue showed congestion of blood vessels and hemorrhage at the early stage followed by extensive necrosis of muscle fibers. The elevated levels of serum CK and LDH activity further supported the muscle degeneration. Such pathological symptoms were not seen with N. naja and D. russelii snake venom. The hemorrhagic and the muscle necrosis was completely neutralized by metalloprotease inhibitors and not by serine protease inhibitor strongly suggests that the major toxin component in the T. malabaricus venom is metalloprotease and its activity can be easily neutralized using chelating agents and its use in the first aid as chelation therapy is beneficial.

Key words

gelatinase hemorrhage metalloprotease myotoxicity southern region of Western Ghats Trimeresurus malabaricus 



degree Celsius














lethal dose 50




















standard error mean


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  1. 1.
    Ishwar NM, Chellam R, Kumar A: Distribution of forest flora reptiles in the rain forest of Kalakad-mundanthuraia. Tiger reserve, south India. Curr Sci 80: 413–418, 2001Google Scholar
  2. 2.
    Sarvanakumar SV, Sukumar R, Prabhakar R: Survey and mapping of shoal forest and grasslands in the upper Niligiri plateau and assessment of human utilization of the vegetation report produced in collaboration with Center of Ecological Sciences IISc, WWR of nature, India, 1993Google Scholar
  3. 3.
    Sajeeth TO, D'souza CJM, Sharath BK: Snake bite incidence in Hassan District. A report submitted to University of Mysore, Mysore, India, 2003Google Scholar
  4. 4.
    Vishwanath BS, Kini RM, Gowda TV: Purification and partial biochemical characterization of an edema inducing phospholiase A2 from Vipera russelli (Russell's viper) snake venom. Toxicon 26: 713–720, 1988CrossRefPubMedGoogle Scholar
  5. 5.
    Rodrigues VM, Soares AM, Andriao-Escarso SH, Franceschi AM, Rucavado A, Gutierrez JM, Giglio JR: Pathological alterations induced by neuwiedase, a metalloproteinase isolated from Bothrops neuwiedi snake venom. Biochimie 83: 471–479, 2001CrossRefPubMedGoogle Scholar
  6. 6.
    Nijaguna B Prasad, Uma B, Subramanya KG Bhat, Gowda TV: Comparative characterization of Russell's viper (Dobaia/Vipera russelli) venoms from different regions of the Indian peninsula. Biochim Biophys Acta 1428: 121–136, 1999PubMedGoogle Scholar
  7. 7.
    Shashidhara murthy R, Jagadeesha DK, Girish KS, Kemparaju K: Variation in biochemical and pharmacological properties of Indian cobra (Naja naja) venom due to geographical distribution. Mol Cell Biochem 229: 93–101, 2002CrossRefGoogle Scholar
  8. 8.
    Lowry OH, Rosenbrough NJ, Farr AL, Randall RJ: Protein measurement with the Folin phenol reagent. J Bio Chem 193: 265–275, 1951Google Scholar
  9. 9.
    Meier J, Theakston RGD: Approximate LD50 determination of snake venoms using eight to ten experimental animals. Toxicon 19: 395–401, 1986CrossRefGoogle Scholar
  10. 10.
    Rothut B, Russo-Marie, Wood J, Di Rosa M, Flower RJ: Further characterization of the glucocorticoid-induced antiphospholipase protein “renocortin”. Biochem Biophys Res Commun 117: 878–884,1983CrossRefGoogle Scholar
  11. 11.
    Vishwanath BS, Frey FJ, Bradbury MJ: Dallman MF, Frey BM: Glucocorticoid deficiency increases phospholipase A2 activity in rats. J Clin Invest 92: 1974–1980, 1993PubMedCrossRefGoogle Scholar
  12. 12.
    Reissig JL, Stominger LF, Leloir A: A modified colorimetric method for the estimation of N-acetylamino sugars. J Bio Chem 217: 959–969, 1955Google Scholar
  13. 13.
    Murata J, Satake M, Suzuki T: Studies on snake venom XII. Distribution of proteinase activities among Japanese and Formosan snake venoms. J Biochem 53: 431–443, 1963Google Scholar
  14. 14.
    Heussen C, Dowdle EB: Electrophoretic analysis of plasminogen activators in polyacrylamide gels containing sodium dodecyl sulfate and co-polymerized substrates. Ana Biochem 102: 196–202, 1980CrossRefGoogle Scholar
  15. 15.
    Kondo H, Kondo S, Ikezawa H, Murata R, Ohasaka A: Studies on the quantitative method from determination of hemorrhagic activity of Habu snake venom. Jpn J Med Sci Biol 13: 43–51, 1969Google Scholar
  16. 16.
    Hughes BP: A method for the estimation of serum creatine kinase and its use in comparing creatine kinase and aldolase activity in normal and pathological sera. Clin Chim Acta. 7: 597–603, 1962CrossRefPubMedGoogle Scholar
  17. 17.
    King JC: Practical clinical enzymolgy. D. Von Nostrand, London, 1965Google Scholar
  18. 18.
    Dufton MJ: Kill and cure: the promising future for venom research. Ende 17: 138–140, 1993CrossRefGoogle Scholar
  19. 19.
    Girish KS, Shashidharamurthy R, Nagaraju S, Gowda TV, Kemparaju K: Isolation and characterization of hyaluronidase a “Spreading factor” from Indian cobra (Naja naja) venom. Biochimie 86: 193–202, 2004CrossRefPubMedGoogle Scholar
  20. 20.
    Iwasaki A, Sheih TC, Shimohigashi T, Waki M, Kihara H, Ohno M: Purification and characterization of coagulant enzyme, Okinoxobin I, from the venom Trimeresurus okinvenisis (Himehabu snake), which releases fibrinopeptide B. J Biochem 108: 822–823, 1990Google Scholar
  21. 21.
    Jagadeesha DK, Shashidhara murthy R, Girish KS, Kemparaju K: A non-toxic anticoagulant metallo protease: Purification and characterization from Indian cobra (Naja naja) venom. Toxicon 40: 667–675, 2002CrossRefPubMedGoogle Scholar
  22. 22.
    Gomis-Ruth FX, Kress LF, Bode W: First structure of a snake venom metalloproteinase: a prototype for matrix metalloproteinases/ collagenases. EMBO J 12: 4151–4157, 1993PubMedGoogle Scholar
  23. 23.
    Spinucci, Zucker S, Wieman JM, Lysik RM, Imhof B, Ramamurthy N, Liotta LA, Nagase H: Purification of a gelatin degrading type IV collagenase secreted by ras oncogene transformed fibroblasts. J Natl Cancer Inst 80: 1416–1420, 1998CrossRefGoogle Scholar
  24. 24.
    Matsui T, Fujimura Y, Titani K: Snake venom proteases affecting hemostasis and thrombosis. Biochim Biophys Acta. 1477: 146–156, 2000PubMedGoogle Scholar
  25. 25.
    Feitosa L, Gremski W, Veiga SS, Elias MC, Graner E, Mangili OC, Brentani RR: Detection and characterization of metalloproteinases with gelatinolytic, fibroncctinolytic and fibrinogenolytic activities in brown spider (Loxosceles intermedia) venom. Toxicon 36: 1039–1051, 1998CrossRefPubMedGoogle Scholar
  26. 26.
    Bee A, Theakston RD, Harrison RA, Carter SD: Novel in vitro assay for assessing the hemorrhagic activity of snake venoms and for demonstration of venom metalloproteinase inhibitors. Toxicon 39, 1429–1434, 2001CrossRefPubMedGoogle Scholar
  27. 27.
    de Roodt AR, Litwin S, Vidal JC: Hemorrhagic activity of venoms determined by two different methods and relationship with proteolytic activity on gelatin and lethality. Toxicon 41: 949–958, 2003PubMedCrossRefGoogle Scholar
  28. 28.
    Baramova EN, Shannon JD, Bjarnason JB, Fox JW: Degradation of extracellular matrix proteins by hemorrhagic metalloproteinases. Arch Biochem Biophys 275: 63–71, 1989CrossRefPubMedGoogle Scholar
  29. 29.
    Bjarnason JB, Fox JW: Hemorrhagic metalloproteinases from snake venoms. Pharmacol Ther 62: 325–372, 1994PubMedCrossRefGoogle Scholar
  30. 30.
    Rucavado A, Nunez J, Gutierrez JM: Blister formation and skin Damage induced by BaP1, a hemorrhagic metalloproteinase from the venom of the snake Bothrops asper. Int J Exp Pathol 79: 245–254, 1998PubMedGoogle Scholar
  31. 31.
    Garcia LT, Parreiras, Silva LT, Ramos OH, Carmona AK, Bersanetti PA, Selisterei-de-Araujo HS: The effect of post-translational modifications of the hemorrhagic activity of snake venom metalloproteinases. Comp Biochem Physiol 138: 23–32, 2004Google Scholar
  32. 32.
    Gutierrez JM, Arce V, Brenes F, Chaves F: Changes in myofibrillar components after skeletal muscle necrosis induced by myotoxin isolated from the venom of the Bothrops asper. Exp Mol Pathol 52: 25–37, 1990CrossRefPubMedGoogle Scholar
  33. 33.
    Moura-da-Silva AM, Laing GD, Paine MJI, Dennison JMTJ, Politi V, Crampton JM, Theakston RGD: Processing of pro-tumor necrosis factor-α by venom metalloproteinases: A hypothesis explaining local tissue damage following snakebite. Euro J Immunol 26: 2000–2005, 1996CrossRefGoogle Scholar
  34. 34.
    Rucavado A, Escalante T, Teixeira CFP, Tambourgi DV, Gutierrez JM: Increments in cytokines and matrix metalloproteinases in skeletal muscle after injection of tissue- damaging toxins from the venom of the snake Bothrops asper. Mediators Inflamm 11: 121–128, 2002CrossRefPubMedGoogle Scholar
  35. 35.
    Laing GD, Clissa PB, Theakston RGD: Inflammatory pathogenesis of snake venom metalloproteinase-induced skin necrosis. Euro J Immunol 33: 3458–3463, 2003CrossRefGoogle Scholar
  36. 36.
    Sathyanathan VP, Mathew MT: Rayanuds's phenomenon and gangrene following snake envenomation. J Assoc Physicians India, 41: 122–211, 1993PubMedGoogle Scholar
  37. 37.
    Noyez JF, Sinozobahamvya N, Kalangu K: Peripheral gangrene in African children: A clinical report of twelve cases. Acta Orthop Belg 62: 207–211, 1996PubMedGoogle Scholar
  38. 38.
    Milani Junior R, Jorge MT, de Campos FP, Martins FP, Bousso A, Cardoso JL, Ribeiro LA, Fan HW, France FO, Sano-Martins IS, Cardoso D, Ide Fernandez C, Fernandes JC, Aldred VL, Sandoval MP, Puorto G, Theakston RD, Warrell DA: Snake bites by the Jararacucu (Bothrosps Jararacussu): Clinical pathological studies of 29 proven cases in Sao Paulo State, Brazil. QJM 90: 323–334, 1997CrossRefPubMedGoogle Scholar
  39. 39.
    Kularatne SA, Ratnatunga N: Severe systemic effects of Merrem's hump nosed viper bite. Ceylon Med J 44: 169–170, 1999PubMedGoogle Scholar
  40. 40.
    Gutierrez JM, Racavado A: Snake venom metalloproteinases: Their role in the pathogenesis of local tissue damage. Biochimie 82: 841–850, 2000PubMedCrossRefGoogle Scholar
  41. 41.
    Rucavado A, Lomonte B, Ovadia M, Gutierrez JM: Local tissue damage induced by BaP1, a metalloproteinase isolated from Bothrops asper (terciopelo) snake venom. Exp Mol pathol 63: 186–199, 1995CrossRefPubMedGoogle Scholar
  42. 42.
    Chan JC, Kwok MM, Cockram CS, Prematileke MN, Tomlinson B, Critchley JA: Blood coagulation abnormalities associated with envenoming by T. albolabris in Hong Kong. Singapore Med J 34: 145–147, 1993PubMedGoogle Scholar
  43. 43.
    Bhat MK, Gowda TV: Purification and characterization of a myotoxic phospholipase A2 from Indian cobra (Naja naja naja) venom. Toxicon 27: 861–73, 1989CrossRefPubMedGoogle Scholar
  44. 44.
    Basavarajappa BS, Gowda TV: Comparative characterization of two toxic phospholipase A2 from Indian cobra (Naja naja naja) venom. Toxicon 30: 1227–1238, 1992CrossRefPubMedGoogle Scholar
  45. 45.
    Satish S, Tejaswini J, Krishnakantha TP, Veerabasappa Gowda T: Purification of a Class B1 platelet aggregation inhibitor phospholipase A2 from Indian cobra (Naja naja) venom. Biochimie 86: 203–210, 2004CrossRefPubMedGoogle Scholar
  46. 46.
    Uma B, Veerabasappa Gowda T: Molecular mechanism of lung hemorrhage induction by VRV-PL-VIIIa from Russell's viper (Vipera russelii) venom. Toxicon 38: 1129–1147, 2000CrossRefPubMedGoogle Scholar
  47. 47.
    Chakrabarty D, Datta K, Gomes A, Bhattacharya D: Hemorrhagic protein of Russell's viper venom with fibrinolytic and esterolytic activities. Toxicon 38: 1475–1490, 2000CrossRefPubMedGoogle Scholar
  48. 48.
    Cranton EM: A textbook on EDTA chelation therapy. J Adv Medici 2: 1–416, 1989Google Scholar
  49. 49.
    Borkow G, Gutierrez JM, Ovadia M: Inhibition of the hemorrhagic activity of Bothrops asper venom by a novel neutralizing mixture. Toxicon 35: 865–877, 1997CrossRefPubMedGoogle Scholar
  50. 50.
    Rucavado A, Escalante T, Franceschi A, Chayes F, Leon G, Cury Y, Ovadia M, Gutierrez JM: Inhibition of local hemorrhage and dermonecrosis induced by Bothrops asper snake venom: {Effectiveness} of early in situ administration of the peptidomimetic metalloproteinase inhibitor batimastat and the chelating agent CaNa2EDTA. Am J Trop Med 63: 313–319, 2000Google Scholar

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© Springer Science + Business Media, Inc. 2006

Authors and Affiliations

  • C. D. Raghavendra Gowda
    • 1
  • R. Rajesh
    • 1
  • A. Nataraju
    • 1
  • B. L. Dhananjaya
    • 1
  • A. R. Raghupathi
    • 2
  • T. V. Gowda
    • 1
  • B. K. Sharath
    • 3
  • B. S. Vishwanath
    • 1
  1. 1.Department of Studies in BiochemistryMysore UniversityManasagangotri, MysoreIndia
  2. 2.Department of PathologyGovernment Medical CollegeMysoreIndia
  3. 3.Department of Studies in BioscienceMysore UniversityHassanIndia

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