Naja Venom Research Articles

Comparison of the primary structures, cytotoxicities, and affinities to phospholipids of five kinds of cytotoxins from the venom of Indian cobra, Naja naja

The molecular mechanism underlying the hemolytic and cytolytic processes of cobra cytotoxins (CTXs) is not yet fully elucidated. To examine this, we analyzed the amino acid sequences, hemolytic and cytotoxic activities, and affinities to phospholipids of the five major CTXs purified from the venom of Indian cobra, Naja naja. CTX2, CTX7, and CTX8 belonged to S-type, and CTX9 and CTX10 to P-type. Comparisons of CTX7 with CTX8 and CTX9 with CTX10 revealed similar primary structures and hemolytic and cytolytic activities. CTX2, whose primary structure was rather different from the others, showed several times weaker hemolytic and cytolytic biological activities than the others. The comparison of CTX2 with CTX7 suggested the importance of Lys30 in loop II for the strong hemolytic and cytolytic activities of S-type CTXs. Cloning of 12 CTX cDNAs from the Naja naja venom cDNA library revealed that 18 out of 23 substitutions found in CTX cDNAs were nonsynonymous. This clearly indicated the accelerated evolution of CTX genes. Multiple sequence alignment of 51 kinds of CTX cDNAs and calculations of nonsynonymous and synonymous substitutions indicated that the codons coding the three loops' regions, which may interact with the hydrophobic tails of phospholipids, have undergone an accelerated evolution. In contrast, the codons coding for amino acid residues considered to participate in the recognition and binding of the hydrophilic head groups of phospholipids, eight Cys residues, and those likely stabilizing β core structure, were all conserved.

Pre-clinical assessment of the effectiveness of modified polyvalent antivenom in the neutralization of Naja naja venom toxicity.

The potency of conventional antivenom (AV) conjugated to soy protein nanoparticles (NPs) (C-AV) was compared with the free AV in neutralizing the systemic toxicity of Naja naja venom. The effective dose (ED50 ) of AV and C-AV to neutralize the venom-induced toxicity in mice was found to be 19.89 and 9.50mg, respectively. The histopathological examination of heart, liver, and kidney indicated that the systemic toxicity induced by the venom was effectively neutralized by lower concentrations of C-AV than compared with AV. In addition, C-AV was found to be more effective in neutralizing the edema forming activity of N. naja venom compared with the AV. Thus, the results of this study indicate that the potency of commercially available AV could be improved by conjugating it to soy protein NPs.

Oxidative stress-induced methemoglobinemia is the silent killer during snakebite: a novel and strategic neutralization by melatonin.

Oxidative stress-induced methemoglobinemia remained an untouched area in venom pharmacology till date. This study for the first time explored the potential of animal venoms to oxidize hemoglobin to methemoglobin. In in vitro whole-blood assay, methemoglobin forming ability of venoms varied as Naja naja > Ophiophagus hannah > Echis carinatus > Daboia russellii > Apis mellifera > Mesobuthus tamulus > Hippasa partita. Being highly potential, N. naja venom was further studied to observe methemoglobin formation in RBCs and in combinations with PMNs and PBMCs, where maximum effect was observed in RBCs + PMNs combination. Naja naja venom/externally added methemoglobin-induced methemoglobin formation was in parallel with ROS generation in whole blood/RBCs/RBCs + PMNs/RBCs + PBMCs. In in vivo studies, the lethal dose (1 mg/kg body weight, i.p.) of N. naja venom readily induced methemoglobin formation, ROS generation, expression of inflammatory markers, and hypoxia-inducible factor-3α. Although the mice administered with three effective doses of antivenom recorded zero mortality; the methemoglobin and ROS levels remained high. However, one effective dose of antivenom when administered along with melatonin (1:50; venom/melatonin, w/w), not only offered 100% survival of experimental mice, but also significantly reduced methemoglobin level, and oxidative stress markers including hypoxia-inducible factor-3α. This study provides strong drive that, complementing melatonin would not only reduce the antivenom load, but for sure greatly increase the success rate of antivenom therapy and drastically minimize the global incidence of snakebite deaths. However, further detailed investigations are needed before translating the combined therapy towards the bed side.

The Antimicrobial Activity of an Acidic Phospholipase A₂ (NN-XIa-PLA₂) from the Venom of Naja naja naja (Indian Cobra).

Microbial resistance against antibiotics is considered as a potentially serious threat to public health. Therefore, there is much interest in developing new molecules with novel modes of action. In this study, when antimicrobial potential of an acidic protein-NN-XIa-PLA2 (Naja naja venom phospholipase A2 fraction-XIa) of N. naja venom was evaluated, it demonstrated potent bactericidal action against the human pathogenic strains. It inhibited more significantly, the gram-positive bacteria, when compared to gram-negative bacteria. The minimum inhibitory concentration (MIC) values ranged from 17 to 20 μg/ml. It was interesting to observe that the NN-XIa-PLA2 showed comparable antibacterial activity to the standard antibiotics used. It was found that there was a strong correlation between phospholipase A2 (PLA2) activities, hemolytic, and antimicrobial activity. Further, it is found that in the presence of p-bromophenacyl bromide (p-BPB), there is a significant decrease in enzymatic activity and associated antimicrobial activities, suggesting that a strong correlation exists between catalytic activity and antimicrobial effects, which thereby destabilize the membrane bilayer. However, other mechanisms cannot be completely ruled out. Thus, these studies encourage further in-depth study on molecular mechanisms of antibacterial properties and thereby help in development of this protein into a possible therapeutic lead molecule for treating bacterial infections.

Protective effect of Euphorbia hirta and its components against snake venom induced lethality

Ethnopharmacological relevanceDespite the use of snake anti-venom therapy, herbal medicine is still in practice to treat snakebites. Euphorbia hirta is traditionally used as antidote for snakebites and also for numerous other ailments. However, the scientific evidence for its anti-snake venom property is still lacking. Materials and methodsMethanolic extract of E. hirta was evaluated for anti-venom activity under in vitro and ex vivo conditions. Histopathological changes in the vital organs of the mice were also monitored. UHPLC-SRM/MS was used to estimate the phenolic constituents whereas GC–MS analysis was performed to analyze the volatile metabolites present. The major compound was further evaluated for its contribution to the overall inhibitory potential of the extract. ResultsMethanolic extract of E. hirta completely inhibited the venom enzymes under in vitro and reduced the edema ratio. The extract increased the survival time (>24h) of mice which was further evidenced by histopathological analysis of vital organs. Phytochemical analysis revealed higher content of phenolic (144mg/g extract) compounds in the extract. UHPLC-SRM/MS demonstrated that ellagic acid, gallic acid and quinic acid are the major phenolics whereas GC–MS analysis revealed pyrogallol as the major constituent (60.07%) among the volatile components of the extract. It was also shown that pyrogallol has the ability to differentially inhibit venom protease but not phospholipase A2. ConclusionThe present study confirmed that E. hirta methanolic extract was able to completely inhibit Naja naja venom induced toxicity under in vitro as well as ex vivo conditions, thus providing scientific evidence to its traditional use.

Neurotoxin II from snake venom Naja Oxiana in solution

Neurotoxin II from snake venom Naja Oxiana in solution

Histopathological Alterations Induced by Naja naja Crude Venom on Renal, Pulmonary and Intestinal Tissues of Mice Model

Aim: Snake bite causes a significant number of mortality and morbidity throughout the world. So, the current study was carried out to estimate the extinct of damage caused by intraperitoneal introduction of cobra venom on kidney, lung and intestinal tissues of mice model using histological technique. Place and Duration of Study: The entire study including the treatment along with preparing histological slide was conducted in protein science laboratory, Department of Genetic Engineering and Biotechnology, University of Rajshahi, Bangladesh between December 2013 to July 2014. Methods: Twenty five mature female albino mice were divided mainly into two groups as control and envenomated group. Lyophilized Naja naja venom was dissolved in 0.9% NaCl solution and injected intraperitoneally into the mice of the envenomated group at dosages equivalent to LD50 Original Research Article Reza et al.; BBJ, 6(3): 119-125, 2015; Article no.BBJ.2015.034 120 (0.25 mg/kg). Whereas the animals from control group were not received any venomous component. Both groups of animal were sacrificed for histological study and visualized under light microscope. Results: Injection of cobra venom induced a range of histological changes in all envenomated mice comparing with their control. Results from the histopathological examination showed mainly inflammatory cellular infiltration, vacuolation in renal tubules, shrinking of glomeruli, raising space between the walls of Bowman’s capsule in renal tissue and alveolar haemorrhage, inflammatory cellular infiltration and edema in pulmonary tissue. No significant histopathological alterations in intestinal tissue were observed without infiltration and mild hemorrhage. Conclusion: The findings from the current study revealed that, cobra venom at lethal dose causes multiple organ failure in experimental animal which could be considered among the factors that lead to death. By observing the site and the mode of action on tissue level, these findings may help to allay the severity of damage by discovering novel anti venom drug.

Anticoagulant mechanism and platelet deaggregation property of a non-cytotoxic, acidic phospholipase A2 purified from Indian cobra (Naja naja) venom: inhibition of anticoagulant activity by low molecular weight heparin.

In the present study, anticoagulant and platelet modulating activities of an acidic phospholipase A2 (NnPLA2-I) purified from Indian cobra Naja naja venom was investigated. The NnPLA2-I displayed a mass of 15.2 kDa and 14,186.0 Da when analyzed by SDS-PAGE and MALDI-TOF-MS, respectively. Peptide mass fingerprinting analysis of the NnPLA2-I showed its significant similarity with phospholipase A2 enzymes purified from cobra venom. BLAST analysis of one tryptic peptide sequence of NnPLA2-I demonstrated putative conserved domains of the PLA2-like superfamily. The Km and Vmax values of NnPLA2-I toward hydrolysis of its most preferred substrate-phosphotidylcholine (PC)-were determined to be 0.72 mM and 29.3 μmol min(-1) mg(-1), respectively. The anticoagulant activity of NnPLA2-I was found to be higher than the anticoagulant activity of heparin/AT-III or warfarin. The histidine modifying reagent, monovalent and polyvalent antivenom differentially inhibited the catalytic and anticoagulant activities of NnPLA2-I. Low molecular weight heparin did not inhibit the catalytic and platelet deaggregation activity of NnPLA2-I, albeit its anticoagulant activity was significantly reduced. The NnPLA2-I showed a non-enzymatic, mixed inhibition of thrombin with a Ki value of 9.3 nM. Heparin significantly decreased, with an IC50 value of 15.23 mIU, the thrombin inhibitory activity of NnPLA2-I. The NnPLA2-I uniquely increased the amidolytic activity of FXa without influencing its prothrombin activating property. NnPLA2-I showed dose-dependent deaggregation of platelet rich plasma (PRP) and inhibited the collagen and thrombin-induced aggregation of PRP. However, deaggregation of washed platelets by NnPLA2-I demonstrated in presence of PC or platelet poor plasma. Alkylation of histidine residue of NnPLA2-I resulted in 95% and 21% reduction of its platelet deaggregation and platelet binding properties, respectively. NnPLA2-I did not show cytotoxicity against human glioblastoma U87MG cells, bactericidal or hemolytic activity. The future therapeutic application of NnPLA2-I for treatment and prevention of cardiovascular disorders is therefore suggested.

PID15, a novel 6 kDa secreted peptide, mediates Naja naja venom phospholipase A₂ induced apoptosis in isolated human peripheral lymphocytes.

BackgroundSnake venoms are a complex mixture of active principles mainly peptides and proteins also including amino acids, nucleotides, free lipids, carbohydrates and metallic elements bound to proteins that interfere in several biological systems. In this study, we aimed to understand the mode of action of the apoptosis inducing ability of Naja naja venom phospholipase A2 (NV-PLA2) using isolated human peripheral lymphocytes.ResultsHuman peripheral lymphocytes when incubated with Naja naja venom phospholipase A2 (NV-PLA2) induced up to 68% DNA fragmentation. The dialysed conditioned media obtained by incubating lymphocytes with NV-PLA2 at 15th min induced 44% DNA fragmentation, referred to as cmlp-active. Cmlp-active showed 20.5% increased protein concentration than the corresponding control condition media cmlp-c-15. Test for creatine kinase activity in cmlp-active proved negative and negligible amount of lactate dehydrogenase did not show significant DNA fragmentation. Fractionation of cmlp-active on Sephadex G-25 showed two peaks, major peak induced 38% DNA fragmentation, which was further rechromatographed on Sephadex G-25. The single peak obtained was named PID15 (Phospholipase A 2 Induced DNA fragmentation factor secreted at 15 th min). Q-Tof MS/MS analysis of PID-15 showed it is a 6 kDa peptide. PID15 sequence analysis gave 40 amino acids in the following order, msilpcknvs iwvikdtaas dkevvlgsdr aikflylatg. The homology search for the sequence revealed it to be an Apoptosis Inducing Factor (AIF).ConclusionResults indicate that the secretion of PID15 is dependent on concentration of NV-PLA2 treatment, incubation time and also on temperature and the probable membrane origin of PID15 and not of cytosolic origin with apoptosis inducing ability.

Vanillin analog--vanillyl mandelic acid, a novel specific inhibitor of snake venom 5'-nucleotidase.

Snake venom 5'-nucleotidase (5'NUC) plays a very important role in envenomation strategies; however, apart from its modulation of hemostatic functions, its other pharmacological effects are not yet well characterized. Several studies have used specific inhibitors of enzyme toxins as a biochemical or pharmacological tool to characterize or establish its mechanism of action. We report here for the first time vanillin mandelic acid (VMA), an analog of vanillin, to potentially, selectively, and specifically inhibit venom 5'NUC activity among other enzymes present in venoms. VMA is much more potent in inhibiting 5'NUC activity than vanillyl acid (VA). The experimental results obtained are in good agreement with the in silico molecular docking interaction data. Both VA and VMA are competitive inhibitors as evident by the inhibition-relieving effect upon increasing the substrate concentration. VMA also dose-dependently inhibited the anticoagulant effect in Naja naja venom. In this study, we report novel non-nucleoside specific inhibitors of snake venom 5'NUC and experimentally demonstrate their involvement in the anticoagulant activity of N. naja venom. Hence, we hypothesize that VMA can be used as a molecular tool to evaluate the role of 5'NUC in snake envenomation and to develop prototypes and lead compounds with potential therapeutic applications against snake bites.

Anti arthritic and anti inflammatory activity of a cytotoxic protein NN-32 from Indian spectacle cobra (Naja naja) venom in male albino rats

The anti arthritic and anti inflammatory activity of NN-32, a cytotoxic protein from Indian spectacle cobra snake (Naja naja) venom has been studied in Freund's complete adjuvant (FCA) induced arthritis and carrageenan induced anti inflammatory model. NN-32 treatment showed significant decrease in physical and urinary parameters, serum enzymes, serum cytokines levels as compared to arthritic control group of rats. NN-32 treatment recovered carrageenan induced inflammation as compared to control group of rats. The findings showed that the cytotoxic protein NN-32 shares anti arthritic and anti inflammatory activity and thus NN-32 may target complex pathophysiological processes like cancer- arthritis-inflammation.

Antivenom potential of ethanolic extract of Cordia macleodii bark against Naja venom

To evaluate the antivenom potential of ethanolic extract of bark of Cordia macleodii against Naja venom induced pharmacological effects such as lethality, hemorrhagic lesion, necrotizing lesion, edema, cardiotoxicity and neurotoxicity. Wistar strain rats were challenged with Naja venom and treated with the ethanolic extract of Cordia macleodii bark. The effectiveness of the extract to neutralize the lethalities of Naja venom was investigated as recommended by WHO. At the dose of 400 and 800 mg/kg ethanolic extract of Cordia macleodii bark significantly inhibited the Naja venom induced lethality, hemorrhagic lesion, necrotizing lesion and edema in rats. Ethanolic extract of Cordia macleodii bark was effective in neutralizing the coagulant and defibrinogenating activity of Naja venom. The cardiotoxic effects in isolated frog heart and neurotoxic activity studies on frog rectus abdominus muscle were also antagonized by ethanolic extract of Cordia macleodii bark. It is concluded that the protective effect of extract of Cordia macleodii against Naja venom poisoning may be mediated by the cardiotonic, proteolysin neutralization, anti-inflammatory, antiserotonic and antihistaminic activity. It is possible that the protective effect may also be due to precipitation of active venom constituents.

Elapid Snake Venom Analyses Show the Specificity of the Peptide Composition at the Level of Genera Naja and Notechis

Elapid snake venom is a highly valuable, but till now mainly unexplored, source of pharmacologically important peptides. We analyzed the peptide fractions with molecular masses up to 10 kDa of two elapid snake venoms—that of the African cobra, N. m. mossambica (genus Naja), and the Peninsula tiger snake, N. scutatus, from Kangaroo Island (genus Notechis). A combination of chromatographic methods was used to isolate the peptides, which were characterized by combining complimentary mass spectrometric techniques. Comparative analysis of the peptide compositions of two venoms showed specificity at the genus level. Three-finger (3-F) cytotoxins, bradykinin-potentiating peptides (BPPs) and a bradykinin inhibitor were isolated from the Naja venom. 3-F neurotoxins, Kunitz/basic pancreatic trypsin inhibitor (BPTI)-type inhibitors and a natriuretic peptide were identified in the N. venom. The inhibiting activity of the peptides was confirmed in vitro with a selected array of proteases. Cytotoxin 1 (P01467) from the Naja venom might be involved in the disturbance of cellular processes by inhibiting the cell 20S-proteasome. A high degree of similarity between BPPs from elapid and viperid snake venoms was observed, suggesting that these molecules play a key role in snake venoms and also indicating that these peptides were recruited into the snake venom prior to the evolutionary divergence of the snakes.

Inhibition of Naja naja venom enzymes by the methanolic extract of Leucas aspera and its chemical profile by GC–MS

PurposeThe present investigation was aimed at evaluating the anti-ophidian properties of ethnomedicinal herb Leucas aspera against Indian cobra, Naja naja venom enzymes. MethodsMethanolic extract of Leucas aspera was evaluated, in vitro, for its ability to inhibit the major enzyme activities of Naja naja venom including protease, phospholipase A2, hyaluronidase and hemolytic factors. The type of phytochemicals present in the extract was analyzed. Also, the major phytoconstituents in the extract was determined by gas chromatography–mass spectrometry (GC–MS). ResultsVenom protease and hyaluronidase activities (two isoforms) were completely (100%) neutralized by the L. aspera methanolic extract at ratio of 1:50 w/w (venom: plant extract) and venom hemolytic activity was also completely neutralized at a ratio of 1:80 w/w by the plant extract. However, the extract failed to neutralize phospholipase A2 activity even at the highest concentration used. Phytochemical analysis revealed the presence of alkaloids, acidic compounds, flavonoids, steroids and cardiac glycosides in the extract. GC–MS analysis indicated that a total of 14 compounds were present in the extract. The major bioactive constituents were found to be 6-octadecenoic acid (32.47%), n-hexadecanoic acid (25.97%), and 17-octadecen-14-yn-1-ol (14.22%) along with the minor constituents, sitosterol (2.45%) and stigmasterol (2%), which was previously reported to exhibit antivenom activity. ConclusionThe results obtained demonstrate for the first time that the methanolic extract of Leucas aspera possesses anti-venom activity and could be considered as a potential source for the anti-ophidian metabolites.

Detection and neutralization of cobra venom using rabbit antiserum in experimental envenomated mice

A sandwich enzyme-linked immunosorbent assay (ELISA) was developed to detect the venom of Indian cobra (Naja naja naja) in various tissues (brain, heart, lungs, liver, spleen, blood, kidneys, and tissue at the site of injection) of mice after cobra venom injected at different time intervals (0, 2, 4, 6, 8, and 12 h intervals up to 24 h). Whole venom antiserum or individual venom protein antiserum (14, 29, 65, 72, and 99 kDa) could recognize N. n. naja venom by Western blotting and ELISA, and antibody titer was also assayed by ELISA. Antiserum raised against cobra venom in rabbit significantly neutralized the toxicity of venom-injected mice at different time intervals after treatment. The assay could detect N. n. naja venom levels up to 2.5 ng/ml of tissue homogenate, and the venom was detected up to 24 h after venom injection. Venom was detected in brain, heart, lungs, liver, spleen, kidneys, tissue at the bite area, and blood. As observed in mice, tissue at the site of bite area showed the highest concentration of venom and the brain showed the least. Moderate amounts of venoms were found in liver, spleen, kidneys, heart, and lungs. Development of a simple, rapid, and species-specific diagnostic kit based on this ELISA technique useful to clinicians is discussed.

Neutralization of cobra venom by cocktail antiserum against venom proteins of cobra (Naja naja naja)

Naja naja venom was characterized by its immunochemical properties and electrophoretic pattern which revealed eight protein bands (14 kDa, 24 kDa, 29 kDa, 45 kDa, 48 kDa, 65 kDa, 72 kDa and 99 kDa) by SDS-PAGE in reducing condition after staining with Coomassie Brilliant Blue. The results showed that Naja venom presented high lethal activity. Whole venom antiserum or individual venom protein antiserum (14 kDa, 29 kDa, 65 kDa, 72 kDa and 99 kDa) of venom could recognize N. naja venom by Western blotting and ELISA, and N. naja venom presented antibody titer when assayed by ELISA. The neutralization tests showed that the polyvalent antiserum neutralized lethal activities by both in vivo and in vitro studies using mice and Vero cells. The antiserum could neutralize the lethal activities in in-vivo and antivenom administered after injection of cobra venom through intraperitoneal route in mice. The cocktail antiserum also could neutralize the cytotoxic activities in Vero cell line by MTT and Neutral red assays. The results of the present study suggest that cocktail antiserum neutralizes the lethal activities in both in vitro and in vivo models using the antiserum against cobra venom and its individual venom proteins serum produced in rabbits.

Cross-Reactivity againstNaja sumatrana(Black Spitting Cobra) Envenoming from the Haffkine Antivenom in a Mouse Model

Naja sumatrana is the dominant cobra species in Malaysia, Singapore, Borneo, and Sumatra, and it does not have specific antivenom. The Haffkine antivenom has been advocated instead. This study aims to determine the efficacy of this antivenom against Naja sumatrana envenoming using a mouse model. Methods. Male Swiss albino mice were used. Intravenous LD50 was first determined separately for Naja naja and Naja sumatrana venom. ED50 was determined by preincubating antivenom with each venom at 2.5 LD50 before administering the mixture into the tail vein. Validation was carried out using a challenge test. Each mouse received 111 µg of Naja sumatrana venom intramuscularly followed by intraperitoneal administration of dilute Haffkine antivenom. Survival was recorded 24 hours after envenoming. Results. The LD50 of Naja naja venom was 78.13 µg, standard error (SE) 13.3 µg. The ED50 of the Haffkine antivenom against Naja naja venom was 45.9 mg, SE 7.5 mg. The LD50 and ED50 of Naja sumatrana venom were 55.5 µg, SE 12.0 µg; and 73.9 mg, SE 12.0 mg, respectively. The intra-peritoneal ED50 against 111 µg intramuscular Naja sumatrana venom was 136.95 mg, SE 36.74 mg. Conclusion. The Haffkine polyvalent antivenom exhibited cross-neutralisation against Naja sumatrana venom when used at a higher dose.

Antivenom activity of triterpenoid (C34H68O2) from Leucas aspera Linn. against Naja naja naja venom induced toxicity

The isolated and identified triterpenoid, 1-hydroxytetratriacontane-4-one (C34H68O2), obtained from the methanolic leaf extract of Leucas aspera Linn. was explored for the first time for antisnake venom activity. The plant (L. aspera Linn.) extract significantly antagonized the spectacled cobra (Naja naja naja) venom induced lethal activity in a mouse model. It was compared with commercial antiserum obtained from King Institute of Preventive Medicine (Chennai, Tamil Nadu, India). N. naja naja venom induced a significant decrease in antioxidant superoxide dismutase, glutathione (GSH) peroxidase, catalase, reduced GSH and glutathione-S-transferase activities and increased lipid peroxidase (LPO) activity in different organs such as heart, liver, kidney and lungs. The histological changes following the antivenom treatment were also evaluated in all these organs. There were significant alterations in the histology. Triterpenoid from methanol extract of L. aspera Linn. at a dose level of 75 mg per mouse significantly attenuated (neutralized) the venom-induced antioxidant status and also the LPO activity in different organs.

Venom proteomic characterization and relative antivenom neutralization of two medically important Pakistani elapid snakes (Bungarus sindanus and Naja naja)

Intra- and interspecific variation in venom composition has been shown to have a major effect upon the efficacy of antivenoms. Due to the absence of domestically produced antivenoms, Pakistan is wholly reliant upon antivenoms produced in other countries, such as India. However, the efficacy of these antivenoms in neutralising the venoms of Pakistani snakes has not been ascertained. This is symptomatic of the general state of toxicological research in this country, which has a myriad of highly toxic and medically important venomous animals. Thus, there is a dire need for knowledge regarding the fundamental proteomics of these venoms and applied knowledge of the relative efficacy of foreign antivenoms. Here we present the results of our proteomic research on two medically important snakes of Pakistan: Bungarus sindanus and Naja naja. Indian Polyvalent Antivenom (Bharat Serums and Vaccines Ltd), which is currently marketed for use in Pakistan, was completely ineffective against either Pakistani species. In addition to the expected pre- and post-synaptic neurotoxic activity, the venom of the Pakistan population of N. naja was shown to be quite divergent from other populations of this species in being potently myotoxic. These results highlight the importance of studying divergent species and isolated populations, where the same data not only elucidates clinical problems in need of immediate attention, but also uncovers sources for novel toxins with potentially useful activities. Pakistan Bungarus sindanus and Naja naja venoms are differentially complex. Naja naja is potently myotoxic. Neither venom is neutralized by Indian antivenom. These results have direct implications for the treatment of envenomed patients in Pakistan. The unusually myotoxic effects of Naja naja demonstrates the value of studying remote populations for biodiscovery.

Detection and neutralization of venom of Indian cobra by rabbit antiserum in experimental envenomation using mice

A sandwich enzyme linked immunosorbent assay (ELISA) was developed to detect Indian cobra ( Naja naja naja ) venom in various organs (brain, heart, lungs, liver, spleen, blood, site of injection and kidneys) as well as tissue at the site of injection of mice, at various time intervals (0, 2, 4, 6, 8 and 12 h intervals up to 24 h) after venom injection. Antiserum significantly neutralized venom levels in serum and tissue samples. Whole venom antiserum or individual venom protein anti serum (14 kDa, 29 kDa, 65 kDa, 72 kDa and 99 kDa) of venom could recognize Naja naja venom by Western blotting and ELISA, and Naja naja naja venom presented antibody titer when assayed by ELISA. The assay could detect Naja naja naja venom levels up to 2.5 ng/ml of tissue homogenate and the venom was detected up to 24 h after venom injection. A highly sensitive and species-specifc microtitre ELISA was also developed to detect venoms of four medically important Indian snakes ( Naja naja naja ) in autopsy specimens of mice. Venoms were detected in brain, heart, lungs, liver, spleen, kidneys, tissue at the bite area and blood. As observed in mice, tissue at the site of bite area showed the highest concentration of venom and the brain showed the least. Moderate amounts of venoms were found in liver, spleen, kidneys, heart and lungs. Development of a simple, rapid and species-specifc diagnostic kit based on this ELISA technique useful to clinicians is discussed.