- Introduction
- Materials and Methods
- Venom and antivenoms.
- Animals and ethics clearance.
- Chemicals and materials.
- Isolation of the major toxins from Naja naja venom.
- Fractionation of venom using Resource S ion-exchange chromatography.
- Purification of venom toxins using reverse-phase chromatography.
- Sodium dodecyl sulfate polyacrylamide gel electrophoresis.
- In-gel tryptic digestion of protein.
- MS analysis.
- Estimation of the relative protein abundances of venom toxins.
- Enzymatic assay for phospholipases A2.
- Determination of lethality of N. naja venom and its purified toxins.
- Neutralization of N. naja venom and its purified toxins through incubation with antivenoms.
- In vivo challenge-rescue experiment in mice.
- Results
- Discussion
- Conclusion
ProCite
RefWorks
Reference Manager
BibTeX
Zotero
EndNote
-
1.
Alirol E, Sharma SK, Bawaskar HS, Kuch U, Chappuis F, 2010. Snake bite in south Asia: a review. PLoS Negl Trop Dis 4: e603.
-
2.
Tan KY, Tan CH, Fung SY, Tan NH, 2015. Venomics, lethality and neutralization of Naja kaouthia (monocled cobra) venoms from three different geographical regions of southeast Asia. J Proteomics 120: 105–125.
-
3.
Kasturiratne A, Wickremasinghe AR, de Silva N, Gunawardena NK, Pathmeswaran A, Premaratna R, Savioli L, Lalloo DG, de Silva HJ, 2008. The global burden of snakebite: a literature analysis and modelling based on regional estimates of envenoming and deaths. PLoS Med 5: e218.
-
4.
Warrell DA, Gutierrez JM, Calvete JJ, Williams D, 2013. New approaches and technologies of venomics to meet the challenge of human envenoming by snakebites in India. Indian J Med Res 138: 38–59.
-
5.
World Health Organization, 2010. Guidelines for the Management of Snake-bites. Geneva, Switzerland: World Health Organization, Regional Office for South-East Asia.
-
6.
Quraishi NA, Qureshi HI, Simpson ID, 2008. A contextual approach to managing snake bite in Pakistan: snake bite treatment with particular reference to neurotoxieity and the ideal hospital snake bite kit. J Pak Med Assoc 58: 325–331.
-
7.
Williams DJ, Gutierrez JM, Calvete JJ, Wuster W, Ratanabanangkoon K, Paiva O, Brown NI, Casewell NR, Harrison RA, Rowley PD, O'Shea M, Jensen SD, Winkel KD, Warrell DA, 2011. Ending the drought: new strategies for improving the flow of affordable, effective antivenoms in Asia and Africa. J Proteomics 74: 1735–1767.
-
8.
Mackessy SP, 2009. The field of reptile toxinology: snakes, lizards, and their venoms. Mackessy SP, ed. Handbook of Venoms and Toxins of Reptiles. Boca Raton, FL: Taylor and Francis Group, CRC Press, 3–23
-
9.
Alape-Giron A, Sanz L, Escolano J, Flores-Diaz M, Madrigal M, Sasa M, Calvete JJ, 2008. Snake venomics of the lancehead pitviper Bothrops asper: geographic, individual, and ontogenetic variations. J Proteome Res 7: 3556–3571.
-
10.
Daltry JC, Ponnudurai G, Shin CK, Tan NH, Thorpe RS, Wuster W, 1996. Electrophoretic profiles and biological activities: intraspecific variation in the venom of the Malayan pit viper (Calloselasma rhodostoma). Toxicon 34: 67–79.
-
11.
Sintiprungrat K, Watcharatanyatip K, Senevirathne WD, Chaisuriya P, Chokchaichamnankit D, Srisomsap C, Ratanabanangkoon K, 2015. A comparative study of venomics of Naja naja from India and Sri Lanka, clinical manifestations and antivenomics of an Indian polyspecific antivenom. J Proteomics 132: 131–143
-
12.
Ali SA, Yang DC, Jackson TN, Undheim EA, Koludarov I, Wood K, Jones A, Hodgson WC, McCarthy S, Ruder T, Fry BG, 2013. Venom proteomic characterization and relative antivenom neutralization of two medically important Pakistani elapid snakes (Bungarus sindanus and Naja naja). J Proteomics 89: 15–23.
-
13.
Khan MS, 1999. Herpetology of habitat types of Pakistan. Pak J Zool 31: 275–289.
-
14.
Khan MS, 2002. A Guide to the Snakes of Pakistan. Frankfurt am Main, Germany: Edition Chimaira.
-
15.
Khan MS, 2014. The snakebite problem in Pakistan. Bull Chicago Herp Soc 49: 165–167.
-
16.
Wuster W, 1996. Taxonomic changes and toxinology: systematic revisions of the Asiatic cobras (Naja naja species complex). Toxicon 34: 399–406.
-
17.
World Health Organization, 2010. WHO Guidelines for the Production, Control and Regulation of Snake Antivenom Immunoglobulins. Geneva, Switzerland: World Health Organization.
-
18.
Huang HW, Liu BS, Chien KY, Chiang LC, Huang SY, Sung WC, Wu WG, 2015. Cobra venom proteome and glycome determined from individual snakes of Naja atra reveal medically important dynamic range and systematic geographic variation. J Proteomics 128: 92–104.
-
19.
Leong PK, Fung SY, Tan CH, Sim SM, Tan NH, 2015. Immunological cross-reactivity and neutralization of the principal toxins of Naja sumatrana and related cobra venoms by a Thai polyvalent antivenom (Neuro Polyvalent Snake Antivenom). Acta Trop 149: 86–93.
-
20.
Howard-Jones N, 1985. A CIOMS ethical code for animal experimentation. WHO Chron 39: 51–56.
-
21.
Laemmli UK, 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685.
-
22.
Tan NH, Tan CS, 1988. Acidimetric assay for phospholipase A using egg yolk suspension as substrate. Anal Biochem 170: 282–288.
-
23.
Tan CH, Tan NH, Tan KY, Kwong KO, 2015. Antivenom cross-neutralization of the venoms of Hydrophis schistosus and Hydrophis curtus, two common sea snakes in Malaysian waters. Toxins (Basel) 7: 572–581.
-
24.
Finney DJ, 1952. Probit Analysis. England, United Kingdom: Cambridge University Press.
-
25.
Tan CH, Tan KY, Lim SE, Tan NH, 2015. Venomics of the beaked sea snake, Hydrophis schistosus: a minimalist toxin arsenal and its cross-neutralization by heterologous antivenoms. J Proteomics 126: 121–130.
-
26.
Morais V, Ifran S, Berasain P, Massaldi H, 2010. Antivenoms: potency or median effective dose, which to use? J Venom Anim Toxins Incl Trop Dis 16: 191–193.
-
27.
Rodriguez-Acosta A, Lemoine K, Navarrete L, Giron ME, Aguilar I, 2006. Experimental ophitoxemia produced by the opisthoglyphous lora snake (Philodryas olfersii) venom. Rev Soc Bras Med Trop 39: 193–197.
-
28.
Tan CH, Tan NH, 2015. Toxinology of snake venoms: the Malaysian context. Gopalakrishnakone P, Inagaki H, Mukherjee AK, Rahmy TR, Vogel C-W, eds. Snake Venoms. The Netherlands: Springer, 1–37.
-
29.
Petras D, Sanz L, Segura A, Herrera M, Villalta M, Solano D, Vargas M, Leon G, Warrell DA, Theakston RD, Harrison RA, Durfa N, Nasidi A, Gutierrez JM, Calvete JJ, 2011. Snake venomics of African spitting cobras: toxin composition and assessment of congeneric cross-reactivity of the pan-African EchiTAb-Plus-ICP antivenom by antivenomics and neutralization approaches. J Proteome Res 10: 1266–1280.
-
30.
Whitaker RCA, 2004. Snakes of India: The Field Guide. Chennai, India: Draco Books.
-
31.
Leong PK, Sim SM, Fung SY, Sumana K, Sitprija V, Tan NH, 2012. Cross neutralization of Afro-Asian cobra and Asian krait venoms by a Thai polyvalent snake antivenom (neuro polyvalent snake antivenom). PLoS Negl Trop Dis 6: e1672.
-
32.
Dayananda KS, Vishwanath BS, Sharath BK, Gopinath SM, 2013. Purification of non-toxic acidic phospholipase A2 from Indian cobra (Naja naja) venom. Int J Pharma Bio Sci 4: 408–415.
-
33.
Barber CM, Isbister GK, Hodgson WC, 2013. Alpha neurotoxins. Toxicon 66: 47–58.
-
34.
Yap MK, Tan NH, Sim SM, Fung SY, Tan CH, 2014. Pharmacokinetics of Naja sumatrana (equatorial spitting cobra) venom and its major toxins in experimentally envenomed rabbits. PLoS Negl Trop Dis 8: e2890.
-
35.
Malasit P, Warrell DA, Chanthavanich P, Viravan C, Mongkolsapaya J, Singhthong B, Supich C, 1986. Prediction, prevention, and mechanism of early (anaphylactic) antivenom reactions in victims of snake bites. Br Med J (Clin Res Ed) 292: 17–20.
-
36.
Gutierrez JM, Leon G, Lomonte B, 2003. Pharmacokinetic-pharmacodynamic relationships of immunoglobulin therapy for envenomation. Clin Pharmacokinet 42: 721–741.
-
37.
Sriprapat S, Aeksowan S, Sapsutthipas S, Chotwiwatthanakun C, Suttijitpaisal P, Pratanaphon R, Khow O, Sitprija V, Ratanabanangkoon K, 2003. The impact of a low dose, low volume, multi-site immunization on the production of therapeutic antivenoms in Thailand. Toxicon 41: 57–64.
-
38.
Tan NH, 1983. Improvement of Malayan cobra (Naja naja sputatrix) antivenin. Toxicon 21: 75–79.
| Past two years | Past Year | Past 30 Days | |
|---|---|---|---|
| Abstract Views | 708 | 489 | 23 |
| Full Text Views | 858 | 15 | 0 |
| PDF Downloads | 511 | 18 | 0 |
Venom and Purified Toxins of the Spectacled Cobra (Naja naja) from Pakistan: Insights into Toxicity and Antivenom Neutralization
Kin Ying Wong
Kin Ying Wong
Department of Pharmacology, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia; Department of Molecular Medicine, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia
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Choo Hock Tan
Choo Hock Tan
Department of Pharmacology, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia; Department of Molecular Medicine, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia
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Nget Hong Tan
Nget Hong Tan
Department of Pharmacology, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia; Department of Molecular Medicine, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia
Search for other papers by Nget Hong Tan in
Current site
Google Scholar
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Current site
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PubMed
Geographical variations of snake venoms can result in suboptimal effectiveness of Indian antivenoms that are currently used in most South Asian countries. This study investigated the toxicity and neutralization profile of the venom and toxins from Pakistani spectacled cobra, Naja naja, using VINS polyvalent antivenom (VPAV, India), Naja kaouthia monovalent antivenom (NKMAV, Thailand), and neuro bivalent antivenom (NBAV, Taiwan). Cation-exchange and reverse-phase high-performance liquid chromatography fractionations followed by toxin identification through liquid chromatography–mass spectrometry (MS)/MS indicated that the venom comprised mainly of postsynaptic neurotoxins (NTXs) (long neurotoxins [LNTXs], 28.3%; short neurotoxins [SNTXs], 8%), cytotoxins (CTXs) (31.2%), and acidic phospholipases A2 (12.3%). NKMAV is the most effective in neutralizing the lethal effect of the venom (potency = 1.1 mg venom/mL) and its LNTX (potency = 0.5 mg toxin/mL), consistent with the high content of LNTX in N. kaouthia venom. VPAV was effective in neutralizing the CTX (potency = 0.4 mg toxin/mL), in agreement with the higher CTX abundance in Indian cobra venom. All the three antivenoms were weak in neutralizing the SNTX (potency = 0.03–0.04 mg toxin/mL), including NBAV that was raised from the SNTX-rich Taiwanese cobra venom. In a challenge-rescue experiment, envenomed mice were prevented from death by a maximal dose of VPAV (intravenous 200 μL) but the recovery from paralysis was slow, indicating the need for higher or repeated doses of VPAV. Our results suggest that optimal neutralization for Pakistani N. naja venom may be achieved by improving the formulation of antivenom production to enhance antivenom immunoreactivity against long and SNTXs.
Author Notes
Financial support: This work was supported by Fundamental Research Grant (FP028-2014A) and the University of Malaya Research Grant (RG282-14AFR) from the Ministry of Higher Education, Government of Malaysia.
Authors' addresses: Kin Ying Wong and Choo Hock Tan, Department of Pharmacology, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia, E-mails: kinying12@gmail.com and tanchoohock@gmail.com. Nget Hong Tan, Department of Molecular Medicine, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia, E-mail: tanngethong@yahoo.com.sg.
- Introduction
- Materials and Methods
- Venom and antivenoms.
- Animals and ethics clearance.
- Chemicals and materials.
- Isolation of the major toxins from Naja naja venom.
- Fractionation of venom using Resource S ion-exchange chromatography.
- Purification of venom toxins using reverse-phase chromatography.
- Sodium dodecyl sulfate polyacrylamide gel electrophoresis.
- In-gel tryptic digestion of protein.
- MS analysis.
- Estimation of the relative protein abundances of venom toxins.
- Enzymatic assay for phospholipases A2.
- Determination of lethality of N. naja venom and its purified toxins.
- Neutralization of N. naja venom and its purified toxins through incubation with antivenoms.
- In vivo challenge-rescue experiment in mice.
- Results
- Discussion
- Conclusion
ProCite
RefWorks
Reference Manager
BibTeX
Zotero
EndNote
-
1.
Alirol E, Sharma SK, Bawaskar HS, Kuch U, Chappuis F, 2010. Snake bite in south Asia: a review. PLoS Negl Trop Dis 4: e603.
-
2.
Tan KY, Tan CH, Fung SY, Tan NH, 2015. Venomics, lethality and neutralization of Naja kaouthia (monocled cobra) venoms from three different geographical regions of southeast Asia. J Proteomics 120: 105–125.
-
3.
Kasturiratne A, Wickremasinghe AR, de Silva N, Gunawardena NK, Pathmeswaran A, Premaratna R, Savioli L, Lalloo DG, de Silva HJ, 2008. The global burden of snakebite: a literature analysis and modelling based on regional estimates of envenoming and deaths. PLoS Med 5: e218.
-
4.
Warrell DA, Gutierrez JM, Calvete JJ, Williams D, 2013. New approaches and technologies of venomics to meet the challenge of human envenoming by snakebites in India. Indian J Med Res 138: 38–59.
-
5.
World Health Organization, 2010. Guidelines for the Management of Snake-bites. Geneva, Switzerland: World Health Organization, Regional Office for South-East Asia.
-
6.
Quraishi NA, Qureshi HI, Simpson ID, 2008. A contextual approach to managing snake bite in Pakistan: snake bite treatment with particular reference to neurotoxieity and the ideal hospital snake bite kit. J Pak Med Assoc 58: 325–331.
-
7.
Williams DJ, Gutierrez JM, Calvete JJ, Wuster W, Ratanabanangkoon K, Paiva O, Brown NI, Casewell NR, Harrison RA, Rowley PD, O'Shea M, Jensen SD, Winkel KD, Warrell DA, 2011. Ending the drought: new strategies for improving the flow of affordable, effective antivenoms in Asia and Africa. J Proteomics 74: 1735–1767.
-
8.
Mackessy SP, 2009. The field of reptile toxinology: snakes, lizards, and their venoms. Mackessy SP, ed. Handbook of Venoms and Toxins of Reptiles. Boca Raton, FL: Taylor and Francis Group, CRC Press, 3–23
-
9.
Alape-Giron A, Sanz L, Escolano J, Flores-Diaz M, Madrigal M, Sasa M, Calvete JJ, 2008. Snake venomics of the lancehead pitviper Bothrops asper: geographic, individual, and ontogenetic variations. J Proteome Res 7: 3556–3571.
-
10.
Daltry JC, Ponnudurai G, Shin CK, Tan NH, Thorpe RS, Wuster W, 1996. Electrophoretic profiles and biological activities: intraspecific variation in the venom of the Malayan pit viper (Calloselasma rhodostoma). Toxicon 34: 67–79.
-
11.
Sintiprungrat K, Watcharatanyatip K, Senevirathne WD, Chaisuriya P, Chokchaichamnankit D, Srisomsap C, Ratanabanangkoon K, 2015. A comparative study of venomics of Naja naja from India and Sri Lanka, clinical manifestations and antivenomics of an Indian polyspecific antivenom. J Proteomics 132: 131–143
-
12.
Ali SA, Yang DC, Jackson TN, Undheim EA, Koludarov I, Wood K, Jones A, Hodgson WC, McCarthy S, Ruder T, Fry BG, 2013. Venom proteomic characterization and relative antivenom neutralization of two medically important Pakistani elapid snakes (Bungarus sindanus and Naja naja). J Proteomics 89: 15–23.
-
13.
Khan MS, 1999. Herpetology of habitat types of Pakistan. Pak J Zool 31: 275–289.
-
14.
Khan MS, 2002. A Guide to the Snakes of Pakistan. Frankfurt am Main, Germany: Edition Chimaira.
-
15.
Khan MS, 2014. The snakebite problem in Pakistan. Bull Chicago Herp Soc 49: 165–167.
-
16.
Wuster W, 1996. Taxonomic changes and toxinology: systematic revisions of the Asiatic cobras (Naja naja species complex). Toxicon 34: 399–406.
-
17.
World Health Organization, 2010. WHO Guidelines for the Production, Control and Regulation of Snake Antivenom Immunoglobulins. Geneva, Switzerland: World Health Organization.
-
18.
Huang HW, Liu BS, Chien KY, Chiang LC, Huang SY, Sung WC, Wu WG, 2015. Cobra venom proteome and glycome determined from individual snakes of Naja atra reveal medically important dynamic range and systematic geographic variation. J Proteomics 128: 92–104.
-
19.
Leong PK, Fung SY, Tan CH, Sim SM, Tan NH, 2015. Immunological cross-reactivity and neutralization of the principal toxins of Naja sumatrana and related cobra venoms by a Thai polyvalent antivenom (Neuro Polyvalent Snake Antivenom). Acta Trop 149: 86–93.
-
20.
Howard-Jones N, 1985. A CIOMS ethical code for animal experimentation. WHO Chron 39: 51–56.
-
21.
Laemmli UK, 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685.
-
22.
Tan NH, Tan CS, 1988. Acidimetric assay for phospholipase A using egg yolk suspension as substrate. Anal Biochem 170: 282–288.
-
23.
Tan CH, Tan NH, Tan KY, Kwong KO, 2015. Antivenom cross-neutralization of the venoms of Hydrophis schistosus and Hydrophis curtus, two common sea snakes in Malaysian waters. Toxins (Basel) 7: 572–581.
-
24.
Finney DJ, 1952. Probit Analysis. England, United Kingdom: Cambridge University Press.
-
25.
Tan CH, Tan KY, Lim SE, Tan NH, 2015. Venomics of the beaked sea snake, Hydrophis schistosus: a minimalist toxin arsenal and its cross-neutralization by heterologous antivenoms. J Proteomics 126: 121–130.
-
26.
Morais V, Ifran S, Berasain P, Massaldi H, 2010. Antivenoms: potency or median effective dose, which to use? J Venom Anim Toxins Incl Trop Dis 16: 191–193.
-
27.
Rodriguez-Acosta A, Lemoine K, Navarrete L, Giron ME, Aguilar I, 2006. Experimental ophitoxemia produced by the opisthoglyphous lora snake (Philodryas olfersii) venom. Rev Soc Bras Med Trop 39: 193–197.
-
28.
Tan CH, Tan NH, 2015. Toxinology of snake venoms: the Malaysian context. Gopalakrishnakone P, Inagaki H, Mukherjee AK, Rahmy TR, Vogel C-W, eds. Snake Venoms. The Netherlands: Springer, 1–37.
-
29.
Petras D, Sanz L, Segura A, Herrera M, Villalta M, Solano D, Vargas M, Leon G, Warrell DA, Theakston RD, Harrison RA, Durfa N, Nasidi A, Gutierrez JM, Calvete JJ, 2011. Snake venomics of African spitting cobras: toxin composition and assessment of congeneric cross-reactivity of the pan-African EchiTAb-Plus-ICP antivenom by antivenomics and neutralization approaches. J Proteome Res 10: 1266–1280.
-
30.
Whitaker RCA, 2004. Snakes of India: The Field Guide. Chennai, India: Draco Books.
-
31.
Leong PK, Sim SM, Fung SY, Sumana K, Sitprija V, Tan NH, 2012. Cross neutralization of Afro-Asian cobra and Asian krait venoms by a Thai polyvalent snake antivenom (neuro polyvalent snake antivenom). PLoS Negl Trop Dis 6: e1672.
-
32.
Dayananda KS, Vishwanath BS, Sharath BK, Gopinath SM, 2013. Purification of non-toxic acidic phospholipase A2 from Indian cobra (Naja naja) venom. Int J Pharma Bio Sci 4: 408–415.
-
33.
Barber CM, Isbister GK, Hodgson WC, 2013. Alpha neurotoxins. Toxicon 66: 47–58.
-
34.
Yap MK, Tan NH, Sim SM, Fung SY, Tan CH, 2014. Pharmacokinetics of Naja sumatrana (equatorial spitting cobra) venom and its major toxins in experimentally envenomed rabbits. PLoS Negl Trop Dis 8: e2890.
-
35.
Malasit P, Warrell DA, Chanthavanich P, Viravan C, Mongkolsapaya J, Singhthong B, Supich C, 1986. Prediction, prevention, and mechanism of early (anaphylactic) antivenom reactions in victims of snake bites. Br Med J (Clin Res Ed) 292: 17–20.
-
36.
Gutierrez JM, Leon G, Lomonte B, 2003. Pharmacokinetic-pharmacodynamic relationships of immunoglobulin therapy for envenomation. Clin Pharmacokinet 42: 721–741.
-
37.
Sriprapat S, Aeksowan S, Sapsutthipas S, Chotwiwatthanakun C, Suttijitpaisal P, Pratanaphon R, Khow O, Sitprija V, Ratanabanangkoon K, 2003. The impact of a low dose, low volume, multi-site immunization on the production of therapeutic antivenoms in Thailand. Toxicon 41: 57–64.
-
38.
Tan NH, 1983. Improvement of Malayan cobra (Naja naja sputatrix) antivenin. Toxicon 21: 75–79.
| Past two years | Past Year | Past 30 Days | |
|---|---|---|---|
| Abstract Views | 708 | 489 | 23 |
| Full Text Views | 858 | 15 | 0 |
| PDF Downloads | 511 | 18 | 0 |