Biochemical characterization of Walterinnesia morgani (desert black cobra) venom (Serpentes: Elapidae)

Year 2023, Volume: 32 Issue: 2, 49 - 58, 31.12.2023

Naşit İğci

https://doi.org/10.38042/biotechstudies.1310004

Abstract

Snake venom contains various bioactive proteins and peptides, of which enzymes make up a significant portion. Desert Black Cobra (Walterinnesia morgani) is a venomous snake distributed mainly in the Middle East including southeastern Türkiye. The aim of the present study is to investigate the key enzyme activities and protein profile of W. morgani venom originating from Sanliurfa province. After the determination of the protein content, the venom sample was subjected to enzymatic activity assays to assess phospholipase A2, protease, ʟ-amino acid oxidase, hyaluronidase, 5′-nucleotidase and, phosphodiesterase activities by a spectrophotometry-based method. Protease activity was also assessed by gelatin zymography. Additionally, the fibrinogenolytic activity of the venom was evaluated using fibrinogen zymography and SDS-PAGE methods. The protein profile was obtained by SDS-PAGE (both reduced and non-reduced) and reversed-phase HPLC methods. According to the results, 11 protein bands between approximately 12-240 kDa were observed on non-reduced SDS-PAGE gel while there were nine bands between 12-140 kDa on the reduced gel. Venom proteins of W. morgani were found predominantly between 25-12 kDa. Proteins were separated into at least 19 major and minor protein groups (peaks) by HPLC analysis. The venom of W. morgani showed all enzyme activities at varying levels.

Keywords

Chromatography, Enzyme activity, SDS-PAGE, Snake Venom, Zymography

References

• Abd El Aziz, T. M., Bourgoin-Voillard, S., Combemale, S., Beroud, R., Fadl, M., Seve, M., & De Waard, M. (2015). Fractionation and proteomic analysis of the Walterinnesia aegyptia snake venom using OFFGEL and MALDI-TOF-MS techniques. Electrophoresis, 36(20), 2594–2605. https://doi.org/10.1002/elps.201500207
• Abd El-Aziz, T. M., Khoury, S. A., Jaquillard, L., Triquigneaux, M., Martinez, G., Bourgoin-Voillard, S., Séve, M., Arnoult, C., Beroud, R., & De Waard, M. (2018). Actiflagelin, a new sperm activator isolated from Walterinnesia aegyptia venom using phenotypic screening. Journal of Venomous Animals and Toxins including Tropical Diseases, 24, 2. https://doi.org/10.1186/s40409-018-0140-4
• Abid, I, Jemel, I., Alonazi, M., & Bacha, A. B. (2020). A new group II phospholipase A2 from Walterinnesia aegyptia venom with antimicrobial, antifungal, and cytotoxic potential. Processes, 8(12), 1560. https://doi.org/10.3390/pr8121560
• Al-Asmari, A. K., Al-Abdulla, I. H., Grouch, R. G., Smith, D. C., & Sjostrom, L. (1997). Assessment of an ovine antivenom raised against venom from the desert black cobra (Walterinnesia aegyptia). Toxicon, 35(1), 141–145. https://doi.org/10.1016/s0041-0101(96)00068-2
• Al-Sadoon, M. K., Abdel-Maksout, M. A., Rabah, D. M., & Badr, G. (2012). Induction of apoptosis and growth arrest in human breast carcinoma cells by a snake (Walterinnesia aegyptia) venom combined with silica nanoparticles: Crosstalk between Bcl2 and Caspase 3. Cellular Physiology and Biochemistry, 30(3), 653–665. https://doi.org/10.1159/000341446
• Al-Saleh, S. S. M., & Khan, S. (2011). Purification and characterization of phosphodiesterase I from Walterinnesia aegyptia Venom. Preparative Biochemistry & Biotechnology, 41(3), 262–277., https://doi.org/10.1080/10826068.2011.575319
• Alshammari, A. M., Badry, A., Aloufi, B. H., & El-Abd, E. (2022). Molecular phylogeny of Walterinnesia aegyptia (Reptilia, Elapidae) isolated from Ha’il province, Saudi Arabia. Open Journal of Applied Sciences, 12(10), 1661–1672. https://doi.org/10.4236/ojapps.2022.1210113
• Amr, Z. S., Abu Baker, M. A. & Warrell, D. A. (2020). Terrestrial venomous snakes and snakebites in the Arab countries of the Middle East. Toxicon, 177, 1–15. https://doi.org/10.1016/j.toxicon.2020.01.012
• Arıkan, H., Göçmen, B., Kumlutaş, Y., Alpagut-Keskin, N., Ilgaz, Ç., & Yıldız, M. Z. (2008). Electrophoretic characterisation of the venom samples obtained from various Anatolian snakes (Serpentes: Colubridae, Viperidae, Elapidae). North-Western Journal of Zoology, 4(1), 16–28.
• Arıkan, H., Göçmen, B., İğci, N., & Akman, B. (2014). Age-dependent variations in the venom proteins of Vipera kaznakovi Nikolsky, 1909 and Vipera ammodytes (Linnaeus, 1758) (Ophidia: Viperidae). Turkish Journal of Zoology, 38, 216–221. https://doi.org/10.3906/zoo-1303-17
• Babkina, G. T., & Vasilenko, S. K. (1964). Nuclease activity of the venoms of mid-Asia snakes. Biokhimiya, 29, 268–272.
• Bacha, A. B., Alonazi, M. A., Elshikh, M. S., & Karray, A. (2018). A novel bactericidal homodimeric PLA2 group-I from Walterinnesia aegyptia venom. International Journal of Biological Macromolecules, 117, 1140–1146. https://doi.org/10.1016/j.ijbiomac.2018.06.024
• Badr, G., Al-Sadoon, M. K., & Rabah, D. M. (2013). Therapeutic efficacy and molecular mechanisms of snake (Walterinnesia aegyptia) venom-loaded silica nanoparticles in the treatment of breast cancer- and prostate cancer-bearing experimental mouse models. Free Radical Biology and Medicine, 65, 175–189. https://doi.org/10.1016/j.freeradbiomed.2013.06.018
• Bergmeyer, H. U., Bergmeyer, J., & Grassl, M. (1983). L-amino acid oxidase. In H. U. Bergmeyer (Ed.), Methods of Enymatic Analysis Vol. II (3rd ed., pp. 149–151). Verlag Chemie GmbH.
• Birrell, G. W., Earl, S. T. H., Wallis, T. P., Masci, P. P., de Jersey, J., Gorman, J. J., & Lavin, M. E. (2007). The diversity of bioactive proteins in Australian snake venoms. Molecular & Cellular Proteomics, 6(6), 973–986. https://doi.org/10.1074/mcp.M600419-MCP200
• Bradford, M. M. (1976). Rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 72(1-2), 248–254. https://doi.org/10.1016/0003-2697(76)90527-3
• Calvete, J. J., Pla, D., Els, J., Carranza, S., Damm, M., Hempel, B.-F., et al. (2021). Combined molecular and elemental mass spectrometry approaches for absolute quantification of proteomes: Application to the venomics characterization of the two species of desert black cobras, Walterinnesia aegyptia and Walterinnesia morgani. Journal of Proteome Research, 20(11), 5064–5078. https://doi.org/10.1021/acs.jproteome.1c00608
• Candiano, G., Bruschi, M., Musante, L., Santucci, L., Ghiggeri, G. M., Carnemolla, B., et al. (2004). Blue silver: a very sensitive colloidal Coomassie G-250 staining for proteome analysis. Electrophoresis, 25(9), 1327–1333. https://doi.org/10.1002/elps.200305844
• Chippaux, J.-P. (2006). Snake venoms and envenomations (1st ed.). Krieger Publishing Company.
• Dhananjaya, B. L., Vishwanath, B. S., & DʼSouza, C. J. M. (2010). Snake venom nucleases, nucleotidases and phosphomonoesterases. In S. P. Mackessy (Ed.), Venoms and Toxins of Reptiles (1st ed., pp. 155-171). CRC Press.
• Du, X.-Y., & Clemetson, K. J. (2002). Snake venom L-amino acid oxidases. Toxicon, 40(6), 659–665. https://doi.org/10.1016/s0041-0101(02)00102-2
• Duhaiman, A. S., Alhomida, A. S., Rabbani, N., Kamal, M. A., & Al-Jafari, A. A. (1996). Purification and characterization of acetylcholinesterase from desert cobra (Walterinnesia aegyptia) venom. Biochimie, 78(1), 46–50. https://doi.org/10.1016/0300-9084(96)81328-9
• Edgar, W., & Prentice, C. R. M. (1973). The proteolytic action of ancrod on human fibrinogen and its polypeptide chains. Thrombosis Research, 2(1), 85–95. https://doi.org/10.1016/0049-3848(73)90082-0
• Eibl, H., & Lands, W. E. M. (1969). A new, sensitive determination of phosphate. Analytical Biochemistry, 30, 51–57. https://doi.org/10.1016/0003-2697(69)90372-8
• Ferrante, N. D. (1956). Turbudimetric measurement of acid mucopolysaccharides and hyaluronidase Activity. Journal of Biological Chemistry, 220, 303–306. https://doi.org/10.1016/S0021-9258(18)65354-2
• Haidar, N. A., & Deitch, E. (2015). Snake bites in the Arabian Peninsula, a review article. Journal of Arid Environments, 112(B), 159–164. https://doi.org/10.1016/j.jaridenv.2014.04.010
• Igci, N., & Ozel Demiralp, D. (2012). A preliminary investigation into the venom proteome of Macrovipera lebetina obtusa (Dwigubsky, 1832) from Southeastern Anatolia by MALDI-TOF mass spectrometry and comparison of venom protein profiles with Macrovipera lebetina lebetina (Linnaeus, 1758) from Cyprus by 2D-PAGE. Archives of Toxicology, 86, 441–451. https://doi.org/10.1007/s00204-011-0763-5
• Igci, N., & Ozel Demiralp, F. D. (2020). A Fourier transform infrared spectroscopic investigation of Macrovipera lebetina lebetina and M. l. obtusa crude venoms. European Journal of Biology, 79(1), 14–22. https://doi.org/10.26650/EurJBiol.2020.0039
• Kasturiratne, A., Wickremasinghe, A. R., de Silva, Gunawardena, N. K., Pathmeswaran, A., Premaratna, R., Savioli, L., Galloo, D. G., & de Silva, H. J. (2008). The global burden of snakebite: A literature analysis and modelling based on regional estimates of envenoming and deaths. PloS Medicine, 5(11), e218.https://doi.org/10.1371/journal.pmed.0050218
• Khan, S. U., & Al-Saleh, S. S. (2015). Biochemical characterization of a factor X activator protein purified from Walterinnesia aegyptia venom. Blood Coagulatin & Fibrinolysis, 26(7), 772–777.
• Kini, R. M. (2003). Excitement ahead: structure, function and mechanism of snake venom phospholipase A2 enzymes. Toxicon, 42(8), 827–840. https://doi.org/10.1016/j.toxicon.2003.11.002
• Keogh, J. S. (1998): Molecular phylogeny of elapid snakes and a consideration of their biogeographic history. Biological Journal of the Linnean Society, 63(2), 177–203. https://doi.org/10.1006/bijl.1997.0178
• Lauer, C., Zickgraf, T. L., & Weisse, M. E. (2011). Case report of probable desert black snake envenomation in 22-year-old male causing profound weakness and respiratory distress. Wilderness & Environmental Medicine, 22(3), 246–249. https://doi.org/10.1016/j.wem.2011.04.003
• Lazar Jr., I., Lazar Sr., I. (2022). GelAnalyzer [Internet]. Version 19.1. 2022. Available from: http://www.gelanalyzer.com/
• Lee, C. Y., Chen, Y. M., & Mebs, D. (1976). Chromatographic separation of the venom of Egyptian black snake (Walterinnesia aegyptia) and pharmacological characterization of its components. Toxicon, 14(4), 275–278. https://doi.org/10.1016/0041-0101(76)90023-4
• Nawarak, J., Sinchaikul, S., Wu, C.-Y., Liau, M.-Y., Phutrakul, S., & Chen, S.-T. (2003). Proteomics of snake venoms from Elapidae and Viperidae families by multidimensional chromatographic methods. Electrophoresis, 24, 2838–2854. https://doi.org/10.1002/elps.200305552
• Nilson, G., & Rastegar-Pouyani, N. (2007). Walterinnesia aegyptia Lataste, 1887 (Ophidia: Elapidae) and the status of Naja morgani Mocquard, 1905. Russian Journal of Herpetology, 14(1), 7–14. https://doi.org/10.30906/1026-2296-2007-14-1-7-14
• Özel Demiralp, F. D., İğci, N., Peker, S., & Ayhan, B. (2014). Temel Proteomik Stratejiler (1st ed.). Ankara Üniversitesi Yayınevi.
• Samejima, Y., Aoki-Tomomatsu, Y., Yanagisawa, M., & Mebs, D. (1997). Amino acid sequence of two neurotoxins from the venom of the Egyptian black snake (Walterinnesia aegyptia). Toxicon, 35(2), 151–157. https://doi.org/10.1016/S0041-0101(96)00138-9
• Simon, T., & Bdolah, A. (1980). Isolation of phospholipase A from the venom of the desert black snake Walterinnesia aegyptia. Toxicon, 18, 369–373. https://doi.org/10.1016/0041-0101(80)90020-3
• Tan, C. H., Wong, K. Y., Tan, N. H., Ng, T. S., & Tan, K. Y. (2019). Distinctive distribution of secretory phospholipases A2 in the venoms of Afro-Asian cobras (Subgenus: Naja, Afronaja, Boulengerina and Uraeus). Toxins, 11(2), 116. https://doi.org/10.3390/toxins11020116
• Trummal, K., Aaspollu, A., Tonismagi, K., Samel, M., Subbi, J., Siigur, J., & Siigur, E. (2014). Phosphodiesterase from Vipera lebetina venom – Structure and characterization. Biochimie, 106, 48–55. https://doi.org/10.1016/j.biochi.2014.07.020
• Tsai, H.-Y., Wang, Y. M., & Tsai, I.-H. (2008). Cloning, characterization and phylogenetic analyses of members of three major venom families from a single specimen of Walterinnesia aegyptia. Toxicon, 51(7), 1245–1254. https://doi.org/10.1016/j.toxicon.2008.02.012
• Üçeş, F., & Yıldız, M. Z. (2020). Snakes of Şanlıurfa province. Commagene Journal of Biology, 4(1), 36–61. https://doi.org/10.31594/commagene.725036
• Von Reumont, B. M., Anderluh, G., Antunes, A., Ayvazyan, N., Beis, D., Caliskan, F., et al. (2022). Modern venomics—Current insights, novel methods, and future perspectives in biological and applied animal venom research. GigaScience, 11, 1–27. https://doi.org/10.1093/gigascience/giac048
• Wüster, W., Crookes, S., Ineich, I., Mané, Y., Pook, C. E., Trape, J.-F., & Broadley, D. G. (2007). The phylogeny of cobras inferred from mitochondrial DNA sequences: Evolution of venom spitting and the phylogeography of the African spitting cobras (Serpentes: Elapidae: Naja nigricollis complex). Molecular Phylogenetics and Evolution, 45(2), 437–453. https://doi.org/10.1016/j.ympev.2007.07.021
• Yıldız, M. Z. (2020). Herpetofauna of Kilis province (Southeast Anatolia, Turkey). Amphibian & Reptile Conservation, 14(2), 145–156.