A study of ribonuclease activity in venom of vietnam cobra

  • Nguyen, Thiet Van (Institute of Biotechnology, Vietnam Academy of Science and Technology (VAST)) ;
  • Osipov, A.V. (Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences (RAS))
  • Received : 2017.03.08
  • Accepted : 2017.06.22
  • Published : 2017.09.30


Background: Ribonuclease (RNase) is one of the few toxic proteins that are present constantly in snake venoms of all types. However, to date this RNase is still poorly studied in comparison not only with other toxic proteins of snake venom, but also with the enzymes of RNase group. The objective of this paper was to investigate some properties of RNase from venom of Vietnam cobra Naja atra. Methods: Kinetic methods and gel filtration chromatography were used to investigate RNase from venom of Vietnam cobra. Results: RNase from venom of Vietnam cobra Naja atra has some characteristic properties. This RNase is a thermostable enzyme and has high conformational stability. This is the only acidic enzyme of the RNase A superfamily exhibiting a high catalytic activity in the pH range of 1-4, with $pH_{opt}=2.58{\pm}0.35$. Its activity is considerably reduced with increasing ionic strength of reaction mixture. Venom proteins are separated by gel filtration into four peaks with ribonucleolytic activity, which is abnormally distributed among the isoforms: only a small part of the RNase activity is present in fractions of proteins with molecular weights of 12-15 kDa and more than 30 kDa, but most of the enzyme activity is detected in fractions of polypeptides, having molecular weights of less than 9 kDa, that is unexpected. Conclusions: RNase from the venom of Vietnam cobra is a unique member of RNase A superfamily according to its acidic optimum pH ($pH_{opt}=2.58{\pm}0.35$) and extremely low molecular weights of its major isoforms (approximately 8.95 kDa for RNase III and 5.93 kDa for RNase IV).


Acidic optimum pH;Conformational stability;Gel filtration;Ionic strength;Isoforms;Thermostability;Venom RNase;Vietnam cobra


Supported by : Vietnam Academy of Science and Technology, Russian Foundation for Basic Research


  1. Rosamond W, Flegal K, Friday G, Furie K, Go A, Greenlund K, et al. Heart disease and stroke statistics-2007 update. A report from the American Heart Association statistics committee and stroke statistics subcommittee. Circulation. 2007;115:e69-e171. Accessed 5 July 2017
  2. Patlak M. From viper's venom to drug design: treating hypertension. FASEB J 2004;18(3):421. PMID: 15003987. Accessed 5 July 2017.
  3. Gomes A, Ghosh S, Ghosh S. et al. Anti-osteoarthritic activity of Bungarus fasciatus venom fraction BF-F47 involving molecular markers in the rats. Toxicon 2016;118:43-46. Accessed 5 July 2017.
  4. Jain D, Kumar S. Snake venom: a potent anticancer agent. Asian Pac J Cancer Prev. 2012;13(10):4855-60. 233937869_Snake_Venom_A_Potent_Anticancer_Agent. Accessed 5 July 2017
  5. Vyas VK, Brahmbhatt K, Bhatt H, Parmar U, Patidar R. Therapeutic potential of snake venom in cancer therapy: current perspectives. Asian Pac J Trop Biomed. 2013;3(2):156-62.
  6. Zouari-Kessentini R, Srairi-Abid N, Bazaa A, El Ayeb M, Luis J, Marrakchi N. Antitumoral potential of Tunisian snake venoms secreted phospholipases A2. Biomed Res Int. 2013;2013:9. Accessed 12 June 2017
  7. Making Medicines from Snake Venom. Accessed 5 July 2017.
  8. Van Thiet N. Study on ribonucleolytic activity of cobra. J Med Mater. 2002; 7(6):181-5.
  9. Dhananjaya BL, D'souza CJM. An overview on nucleases (DNase, RNase, and phosphodiesterase) in snake venoms. Biochem Mosc. 2010;75(1):1-6. Accessed 5 July 2017
  10. Snake Venom. Types of Snake Venom. webprojects2003/stoneley/types.htm. Accessed 5 July 2017.
  11. Beintema JJ, Kleineidam RG. The ribonuclease a superfamily: general discussion. Cell Mol Life Sci. 1998;54:825-32.
  12. Sorrentino S, Libonati M. Structure-function relationships in human ribonucleases: main distinctive features of the major RNase types. FEBS Lett. 1997;404(1):1-5. Accessed 5 July 2017
  13. Cho S, Zhang J. Zebrafish Ribonucleases are bactericidal: implications for the origin of the vertebrate RNase a Superfamily. Mol Biol Evol. 2007;24(5):1259-68.
  14. Hooper LV, Stappenbeck TS, Hong CV, Gordon JI. Angiogenins: a new class of microbicidal proteins involved in innate immunity. Nat Immunol. 2003;4(3):69-73.
  15. Lehrer RI, Szklarek D, Barton A, Ganz T, Hamann KJ, Gleich GJ. Antibacterial properties of eosinophil major basic protein and eosinophil cationic protein. J Immunol. 1989;142(12):4428-34. Accessed 5 July 2017
  16. Pulido D, Arranz-Trullen J, Prats-Ejarque G, Velazquez D, Torrent M, Moussaoui M, et al. Insights into the antimicrobial mechanism of action of human RNase6: structural determinants for bacterial cell agglutination and membrane permeation. Int J Mol Sci. 2016;17:552.
  17. Pulido D, Torrent M, Andreu D, Nogues MV, Boix E. Two human host defense ribonucleases against mycobacteria, the eosinophil cationic protein (RNase 3) and RNase 7. Antimicrob Agents Chemother. 2013;57:3797-805.
  18. Rudolph B, Podschun R, Sahly H, Schubert S, Schroder JM, Harder J. Identification of RNase 8 as a novel human antimicrobial protein. Antimicrob Agents Chemother. 2006;50(9):3194-6. Accessed 5 July 2017
  19. Tao F, Fan M, Zhao W, Lin Q, Ma R. A novel cationic ribonuclease with antimicrobial activity from Rana dybowskii. Biochem Genet. 2011;49:369-84.
  20. Bedoya VI, Boasso A, Hardy AW, Rybak S, Shearer GM, Rugeles MT. Ribonucleases in HIV type 1 inhibition: effect of recombinant RNases on infection of primary T cells and immune activation-induced RNase Gene and Protein expression. AIDS Res Hum Retrovir. 2006;22(9):897-907. doi:10.1089/aid.2006.22.897.
  21. Domachowske JB, Bonville CA, Dyer KD, Rosenberg HF. Evolution of antiviral activity in the ribonuclease a gene superfamily: evidence for a specific interaction between eosinophil-derived neurotoxin (EDN/RNase 2) and respiratory syncytial virus. Nucleic Acids Res. 1998;26(23):5327-32.
  22. Domachowske JB, Dyer KD, Adams AG, Leto TL, Rosenberg HF. Eosinophil cationic protein/RNase 3 is another RNase A-family ribonuclease with direct antiviral activity. Nucleic Acids Res. 1998;26:3358-63.
  23. Domachowske JB, Dyer KD, Bonville CA, Rosenberg HF. Recombinant human eosinophil-derived neurotoxin/RNase 2 functions as an effective antiviral agent against respiratory syncytial virus. J Infect Dis. 1998;177:1458-64.
  24. Huang H-C, Wang S-C, Leu Y-J, Lu S-C, Liao Y-D. The Rana catesbeiana rcr gene encoding a cytotoxic ribonuclease. Tissue distribution, cloning, purification, cytotoxicity, and active residues for RNase activity. J Biol Chem. 1998;273:6395-401.
  25. Kim JS, Soucek J, Matousek J, Raines RT. Mechanism of ribonulcease cytotoxicity. J Biol Chem. 1995;270(52):31097-102.
  26. Kim JS, Sousek J, Matousek J, Raines RT. Structural basis for the biological activities of bovine seminal ribonuclease. J Boil Chem. 1995;270(18):10525-30.
  27. Leland PA, Raines RT. Cancer chemotherapy-Rionucleases to the rescue. Biol Chem. 2001;8:405-13.
  28. Raines RT. Ribonuclease a: from model system to cancer chemotherapeutic. In: Frey PA, Northrop DB, editors. Enzymatic mechanism. Washington DC: IOC press; 1999. p. 235-49. Accessed 5 July 2017.
  29. Wu Y, Mikulski SM, Ardelt W, Rybark SM, Youle RJ. A cytotoxic ribonuclease: study of the mechanism of onconase cytotoxicity. J Biol Chem. 1993; 268(14):10686-10693. PMID: 8486718. pubmed/8486718. Accessed 5 July 2017
  30. Shcheglovitova O, Maksyanina E, Ionova I, Rustam'yan YL, Komolova G. Cow milk angiogenin induces cytokine production in human blood leukocytes. Bull Exp Biol Med. 2003;135:158-60.
  31. Tamgurrini M, Scala G, Verde C, Ruocco MR, Parente A, Venuta S, et al. Immunosuppressive activity of bovine seminal RNase on T-cell proliferation. Eur J Biochem. 1990;190(1):145-8.
  32. Yang D, Chen Q, Rosenberg HF, Rybak SM, Newton DL, Wang ZY, et al. Human ribonuclease a superfamily members, eosinophil-derived neurotoxin and pancreatic ribonuclease, induce dendritic cell maturation and activation. J Immunol. 2004;173:6134-42.
  33. Goo SM, Cho S. The expansion and functional diversification of the mammalian Ribonuclease a Superfamily epitomizes the efficiency of Multigene families at generating biological novelty. Genome Biol Evol. 2013;5:2124-40.
  34. Koczera P, Martin L, Marx G, Schuerholz T. The Ribonuclease a Superfamily in humans: canonical RNases as the buttress of innate immunity. Int J Mol Sci. 2016;17(8):1278.
  35. Sorrentino S. The eight human "canonical" ribonucleases: molecular diversity, catalytic properties, and special biological actions of the enzyme proteins. FEBS Lett. 2010;584:2194-200.
  36. Gupta SK, Haigh BJ, Griffin FJ, Wheeler TT. The mammalian secreted RNases: mechanisms of action in host defense. Innate Immun. 2013;19:86-97.
  37. Irie M, Nitta K, Nonaka T. Biochemistry of frog ribonucleases. Cell Mol Life Sci. 1998;54:775-84.
  38. Fry BG, Scheib H, de LM I, Junqueira de Azevedo I, Silva DA, Casewell NR. Novel transcripts in the maxillary venom glands of advanced snakes. Toxicon. 2012;59(7-8):696-708.
  39. Vasilenko SK, Babkina GT. Isolation and properties of ribonuclease isolated from cobra venom. Biokhimiia. 1965;30(4):705-712. [Article in Russian] PMID: 5894094. Accessed 5 July 2017
  40. Mahalakshmi YV, Jagannadham MV, Pandit MW. Ribonuclease from cobra snake venom: purification by affinity chromatography and further characterization. IUBMB Life. 2000;49:309-16. doi/10.1080/15216540050033186/epdf. Accessed 5 July 2017
  41. Mahalakshmi YV, Pandit MW. The new ribonuclease from cobra venom (Naja naja) showing specificity towards cytidylic acid. Biochem Biophys Res Commun. 1987;145(2):740-8. from-cobra-venom-naja-naja-showing-specificity-towards. html#. Accessed 5 July 2017
  42. Babkina GT, Vasilenko SK. Nuclease activity of the venom of central Asian snakes. Biokhimiya. 1964;29(2):268-272. [Article in Russian] PMID: 14207641. Accessed 5 July 2017
  43. Raines RT. Ribonuclease a. Chem Rev. 1998;98:1045-65.
  44. Haigis MC, Kurten EL, Raines RT. Ribonuclease inhibitor as an intracellular sentry. Nucleic Acids Res. 2003;31:31024-32.
  45. Ardelt W, Mikulski SM, Shogen K. Amino acid sequence of an anti-tumor protein from Rana pipiens Oocytes and early embryos. Homology to pancreatic ribonucleases. J Biol Chem 1991;266(1):245-251. PMID:1985896. Accessed 5 July 2017.
  46. Klink TA, Raines RT. Conformational stability is a determinant of ribonuclease cytotoxicity. J Biol Chem. 2000;275(23):17463-7.