Purification and characterization of a neurotoxic phospholipase A2 from Indian cobra (Naja naja naja) venom.

Snake venoms contain multimolecular forms of phospholipase A2 which are diverse with respect to their pharmacological properties. A neurotoxic PLA2 from Naja naja naja venom has been purified in two steps. (1) The whole venom was fractionated on CM-Sephadex C-25 column; 4.6% of the total PLA2 activity recovered was found in the NN-V fraction. (2) The NN-Vb-PLA2 fraction was purified to homogeneity by gel filtration of fraction NN-V on Sephadex G-50. It is a basic protein with a mol. wt between 10,500 and 11,000, and is more toxic than other basic PLA2s purified from Naja naja naja venom. The LD50 of NN-Vb-PLA2 is 0.27 mg/kg body wt. It induced neurotoxic symptoms in experimental mice and is devoid of myotoxic, anticoagulant and edema-inducing activities.     

Comparison of two Elapidae venoms: Naja naja and Naja nigricollis

In our study on the identification of snake venoms by electrofocusing technics, the protein profiles of Naja naja naja, Naja nigricollis pallida and Naja nigricollis West Africa were compared. The typical profile of venoms of Elapidae was easily identified in this comparison. The two species could easily be differentiated, whereas the differences between the two sub-species were more difficult to evidence, since they are geographic variants of the same type.     

In vitro procoagulant and anticoagulant properties of Naja naja naja venom.

Bites by the Indian cobra (Naja naja naja) are common in India and Sri Lanka because of its close association with humans. Cobra venoms are complex and contain several toxic components, including neurotoxins that cause post-synaptic neuromuscular blockade with respiratory paralysis and even death. Bites may also cause extensive local necrosis by mechanisms not fully elucidated. Although no significant coagulopathy has been reported, N.n. naja venom can form blood clots in vitro by activating prothrombin as demonstrated by thrombin-specific chromogenic substrate. Scanning electron microscopy demonstrates that the clots formed by venom lack the thin fibrin strands of normal blood clots formed by thromboplastin or glass contact. Rheometry shows that clots formed by venom have abnormally low elasticity over an extended period and then, as the platelets contract, a retarded and more feeble increase in elasticity. Purified N.n. naja venom PLA2 inhibits platelet aggregation in PRP and explains the decreased clot retraction and retarded and compromised elasticity build up. The present study shows that the PLA2 and the prothrombin activator from N.n. naja venom have effects on haemostasis and blood clotting, although such effects are not observed systemically in envenomed humans.     

眼镜蛇毒细胞毒素CTX-d的分离纯化及抗肿瘤活性

目的 分离纯化眼镜蛇毒细胞毒素,测定其体内外抗癌作用.方法 应用柱层析及RP-HPLC,从眼镜蛇毒粗毒中分离纯化细胞素素(CTX).体内外抗癌作用利用噻唑兰(MTT)法及对荷瘤小鼠U14瘤的抑瘤作用.结果 分离纯化获得的CTX-d不混有PLA2,在体内外实验中显示明显的抗肿瘤作用.结论 结合阳离子交换层析和RP-HPLC可从眼镜蛇毒中高效分离获得不含PLA2的CTX.     

Hemolytic activity of thionin from Pyrularia pubera nuts and snake venom toxins of Naja naja species: Pyrularia thionin and snake venom cardiotoxin compete for the same membrane site

Pyrularia thionin (P. thionin) is a strongly basic peptide of 47 amino acids which is hemolytic, cytotoxic and neurotoxic. It shows the greatest hemolytic activity toward human erythrocytes. Rabbit, guinea pig and pig erythrocytes show decreasing activity in that order, and little or no activity is shown with sheep, horse, cow or mouse erythrocytes. Crotalus venoms are inactive, but the venoms from Naja naja atra, Naja naja ceylonicus and Naja naja melanoleuca and, more specifically, cardiotoxin from Naja naja kaouthia have significant hemolytic activities toward human erythrocytes. The cardiotoxin preparation used had no phospholipase activity, and was less active than P. thionin (23% compared to 35% hemolysis by P. thionin in 60 min at 10 micrograms/ml toxin). Since iodinated P. thionin is inactive, it was used as an inhibitor of hemolysis catalyzed by native P. thionin, N. ceylonicus venom and by cardiotoxin. Examination of the kinetics of the reactions catalyzed by N. ceylonicus venom and cardiotoxin in the absence and presence of iodinated P. thionin shows that both N. ceylonicus venom and cardiotoxin exhibit Michaelis-Menten kinetics, yielding apparent Km values of 7.4 micrograms/ml and 0.69 microM, respectively. These values compare to an apparent Km for P. thionin of 1.6 microM for erythrocyte hemolysis and a binding constant of 2.1 microM (Osorio e Castro, V. R. Van Kuiken, B. A. and Vernon, L. P. (1989) Action of a thionin isolated from nuts of Pyrularia pubera on human erythrocytes. Toxicon 27, 501). The inhibition constants Ki for iodinated P. thionin in the reactions with N. ceylonicus venom and cardiotoxin are 3.8 and 5.3 microM, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of cardiotoxin D from Naja naja siamensis snake venom upon murine splenic lymphocytes

Cardiotoxin D from Naja naja siamensis is cytotoxic to T-lymphocytes above 150 femtomoles/10(6) cells. Equivalent lysis of macrophages or B-lymphocytes requires at least 1000 times more toxin. Reduction and carboxamidomethylation of cardiotoxin D does not effect T cell lysis. At higher toxin concentrations, 50% T-cell lysis occurs within 10 min. Splenocytes cultured with mitogens are up to five times more susceptible to toxin than unstimulated cells. Cardiotoxin D may directly disrupt the plasma membrane, since lysis is unaltered at 4 degrees C.     

Factors in snake venoms that increase capillary permeability

Capillary permeability increasing (CPI) activity is a phenomenon of the microvasculature caused by many agents such as snake venoms, histamine, 5-hydroxytryptamine (5-HT), prostaglandins and leukotrienes. Since no systematic study has been done to determine what components of snake venom cause CPI activity, a CPI factor from Naja naja atra (Taiwan cobra) venom was isolated using intravenous injections of Evan's blue dye as the indicator of increased permeability and the factor's properties were extensively studied. Cardiotoxin from Naja naja kaouthia (Thailand cobra) and Mojave toxin from Crotalus scutulatus scutulatus (Mojave rattlesnake) venoms demonstrated CPI activity. Postsynaptic neurotoxins from an elapid and a hydrophid and myotoxin a from Crotalus viridis viridis (prairie rattlesnake) showed no CPI activity at the dose studied. The purified CPI active component from Naja naja atra venom was found to have cardiotoxic activity. Therefore, Elapidae cardiotoxins are CPI active factors. However, CPI activity is not due to cardiotoxins alone as the presynaptic neurotoxin, Mojave toxin, also showed CPI activity. Selective inhibitors were used to indicate possible mechanisms of action on the capillaries by Naja naja atra venom and Crotalus scutulatus scutulatus venom. The histamine H1-receptor blockers diphenhydramine, promethazine, and cyproheptadine were effective against both venoms in preventing increased capillary permeability. These results suggested that histamine release activity is the most likely mechanism resulting in CPI activity from these venoms.     

Purification and characterization of a myotoxic phospholipase A2 from Indian cobra (Naja naja naja) venom

A major phospholipase A2 (NN-XIII-PLA2) which constitutes 20% of the whole Naja naja naja venom was purified to homogeneity on CM-Sephadex C-25 column chromatography. NN-XIII-PLA2 is a basic protein with a mol. wt of 11,200 by SDS-PAGE. This enzyme has low enzymatic activity but is more toxic to mice than the whole venom. The LD50 value (i.p.) of NN-XIII-PLA2 is 2.4 mg/kg body weight (whole venoms LD50 is 2.8 mg/kg body weight). It induces neurotoxic-like signs in experimental animals. It induces myotoxicity when injected i.m. into the thigh muscle of mice and edema when injected into the foot pads of mice. This enzyme has a fluorescence maxima between 310-316 nm which is typical of tyrosine residues.     

Biochemical characterization of a toxin from Indian cobra (Naja naja naja) venom

A major toxic component was isolated from the venom of Indian cobra (Naja naja naja) by ammonium sulfate fractional precipitation followed by carboxymethyl cellulose column chromatography and Sephadex gel filtration. This component constituted 2% of the venom and produced a block of neuromuscular transmission in nerve muscle preparations. Three other toxic fractions comprising 3% of the venom were also detected. The major toxic component was homogeneous on starch and polyacrylamide gel electrophoresis and on rechromatography on CM-cellulose. Its molecular weight was approximately 6300. This toxin contained 61 amino acid residues including 8 half-cystine residues whereas alanine, methionine and phenylalanine were totally absent. Its ld₅₀, as determined by i.p. injection in mice, was 0·2 mg per kg body weight. The fraction did not possess any enzymatic, hemolytic or hemagglutinin activities of crude venom but showed a close resemblance to the major neurotoxin of Formosan cobra venom.     

Long-lasting convulsant effect on the cerebral cortex of Naja naja venom

1. A neurotoxic fraction has been prepared from Indian cobra venom (Naja naja) by column chromatography on CM Sephadex.2. When assayed for lethality in mice, or for neuromuscular blocking potency in the rat phrenic nerve-diaphragm preparation, this fraction was 2.4-2.9 times as potent as whole venom.3. Application of either whole venom (0.5-1.0 mg/ml) or the neurotoxic fraction (0.25-1.0 mg/ml) to the exposed cerebral cortex of the rat led to the appearance in the somatosensory evoked potential of abnormal negative waves, resembling in amplitude and latency those produced by the cortical application of strychnine or curare.4. This effect differed from that produced by strychnine or curare in that after washing off the toxin the abnormal responses persisted for as long as the experiment continued (8-9 hours).     

Region-specific neutralization of Indian cobra (Naja naja) venom by polyclonal antibody raised against the eastern regional venom: A comparative study of the venoms from three different geographical distributions

Indian cobra (Naja naja) venoms from different geographical locations vary in their composition, biochemical, and pharmacological properties. Venom samples from eastern, western and southern India are compared in this study. The venom from eastern region was found to be the most lethal of the three regional venoms. Monovalent antivenom (NNEV-IgG) prepared against the eastern venom was found to cross-react with the other two regional venoms. NNEV-IgG at an Ag:Ab ratio of 1:25 completely neutralized the lethality of eastern venom. At this ratio, it did not neutralize the other two venoms, but the survival time of experimental mice was extended significantly. Commercially available polyvalent antivenom neutralized the lethality of western venom at an Ag:Ab ratio of 1:60 and increased the survival time of experimental mice injected with eastern and southern venoms marginally. Further, NNEV-IgG neutralized the tested pharmacological and enzymatic activities of all the three venom samples dose dependently, with neutralization potency varying with the geographic origin of the tested venoms. Thus, the present study demonstrates the diversity in the immunological properties of venom from different geographical regions and underscores the importance of developing region-specific antivenoms for therapeutic purpose.     

Classification of myonecrosis induced by snake venoms: venoms from the prairie rattlesnake (Crotalus viridis viridis), western diamondback rattlesnake (Crotalus atrox) and the Indian cobra (Naja naja naja)

The pathogenesis of myonecrosis induced by three different snake venoms was studied by light microscopic examination of skeletal muscle tissue taken at time periods ranging from 0.25 hr to 4 weeks after an intramuscular injection of the venom into mice. It was possible to identify different types of myonecrosis based on the abnormal morphologic states of the damaged cells. The types of myonecrosis observed correlated with the types of components present in the venom injected. Venoms containing direct acting toxins such as myotoxin a or phospholipase A2 induced specific types of myonecrosis. Also, venoms containing hemorrhagic toxins produced a type of myonecrosis similar to that induced by pure hemorrhagic toxins. The pathogenesis of each type of myonecrosis could be divided into the same four phases based on the pathologic states of the affected cells and the time after injection. During the 'early phase' (0.25-3 hr) affected muscle cells were in several different pathologic states reflecting the types of components present in the venom injected. During the 'intermediate phase' (6-24 hr) the pathologic state of the damaged cells had changed and depending on the venom new states might be present. By the 'late phase' (48-96 hr) all damaged cells have reached a common pathologic state of necrosis. The 'final phase' (1-4 weeks) is characterized by regeneration (partial or complete) of muscle cells. Although the number of different types of myonecrosis depended on the type of venom injected, i.e. Naja naja naja venom produced only two different types whereas Crotalus atrox venom produced at least four different types, cells of each tpe of myonecrosis progressed through the same four phases. In studies of the myotoxicity of snake venoms it is important to examine tissues taken during the early and intermediate phases to obtain accurate and useful information on the types of myonecrosis caused by the venom.     

Affinity chromatography of phospholipase A2 from Naja naja naja (Indian cobra) venom

A rapid and improved purification procedure is described for phospholipase A2 from the Indian cobra, Naja naja naja. The procedure is based on affinity chromatography of the venom through Affi-Gel Blue to obtain a 9-fold purification in one step. However, as there are multiple forms of the enzyme in the venom and other proteins do bind to Affi-Gel Blue, further purification is achieved by DE-11 cellulose. Finally, a minor contaminant is removed by Sulfopropyl Sephadex C-25 chromatography. The resulting product was found to be pure by analytical isoelectric focusing and double diffusion precipitin tests. An isoelectric focusing column run with venom collected from a single snake gave a similar profile to that obtained with venom pooled from several snakes.     

Development of reversed passive latex agglutination for detection of Thai cobra (Naja kaouthia) venom.

A simple, rapid, and sensitive diagnostic kit for detecting Thai cobra (Naja kaouthia) venom was developed using latex particles sensitized with venom specific immunoglobulin. The kit is capable of detecting 25-50 ng/ml of Thai cobra venom. The capability was not affected by human plasma. Specificity of the kit was proven using snake venoms from Vipera russelli, Calloselasma rhodostoma, Trimeresurus albolabris, Naja siamensis, Ophiophagus hannah, and Bungarus fasciatus. The diagnostic kit does not lose its capability under refrigeration for two months and by lyophilization.     

Sensitivity of cultured embryonic heart cells to cardiotoxin obtained from Naja naja siamensis venom.

Clumps of embryonic chick heart cells in culture were exposed to cardiotoxin prepared by Sephadex CM-50 fractionation from snake (Naja naja siamensis) venom. Cardiotoxin irreversibly depolarized these cells. More of the cells cultured from 4-day embryos, than from 12–13-day embryos, continued to beat spontaneously after 30 min exposure to the toxin at a concentration of 15–20 μg per ml.     

Effect of crotapotin and heparin on the rat paw oedema induced by different secretory phospholipases A2.

The effects of crotapotin (a non-toxic and non-enzymatic acid polypeptide naturally complexed with phospholipase A2) and heparin on rat paw edema induced by different secretory phospholipases A2 (sPLA2) have been investigated. The ability of crotapotin to affect the enzymatic activity of the sPLA2(s) have also been evaluated. Secretory PLA2(s) obtained from both snake (Naja naja, Naja mocambique mocambique, Crotalus adamanteus and Crotalus durissus terrificus) and bee (Apis mellifera) venoms as well as that from bovine pancreas were used in this study. Rat paw oedema was induced by a single subplantar injection of the sPLA2s (5-30 microg/paw) in absence and presence of either crotapotin (10-100 microg/paw) or heparin (50 U/paw). Paw volume was measured using a hydroplethysmometer. Phospholipase A2 from Naja naja, Naja mocambique mocambique, Apis mellifera venoms and the basic component of Crotalus durissus terrificus venom all induced dose-dependent rat paw oedema whereas those from Crotalus adamanteus venom and bovine pancreas were ineffective. Paw oedema induced by PLA2(s) from both Naja naja and Apis mellifera venoms was significantly (P < 0.05) inhibited by crotapotin (0.1-100 microg/site) whereas the Naja mocambique mocambique venom PLA2-induced oedema was significantly potentiated (P < 0.05) by this polypeptide (40 microg/site). On the other hand, heparin (50 U/paw) had no effect on the paw oedema induced by PLA2 from Naja naja and Apis mellifera venoms but significantly inhibited the Naja mocambique mocambique venom PLA2-induced oedema. The measurement of the in vitro phospholipasic activity revealed that crotapotin inhibited by 60-70% the enzymatic activities of PLA2(s) from Crotalus adamanteus, Naja mocambique mocambique, Apis mellifera venoms and bovine pancreas. Our results suggest that despite the great homology between the various types of sPLA2 they interact with crotapotin on cell surfaces in different ways leading to either inhibition or potentiation of the paw oedema by a mechanism unrelated to their enzymatic activities. Since heparin reduced paw oedema induced by PLA2 from Naja mocambique mocambique venom it is likely that this sPLA2 is similar to the novel heparin-sensitive PLA2 found in mast cells.     

The multiplicity of cardiotoxins from Naja naja atra (Taiwan cobra) venom.

Four novel cardiotoxins were isolated from Naja naja atra (Taiwan cobra) venom by successive separation on a SP-Sephadex C-25 column and a reverse phase column. Amino acid sequences of the cardiotoxins were determined by Edman degradation and carboxypeptidase digestion. It shows that these cardiotoxins comprise 60 amino acid residues. Comparative analyses on the amino acid sequences of cardiotoxins from the venoms of N. naja atra and other Naja species indicated that amino acid substitutions of cardiotoxin isoforms frequently occurred at positions 7-11, 27-32 and 45-47. The hypervariable segments encoded by the second and third exon of cardiotoxin genes are located at or near the tips of loop structure of cardiotoxin molecules. These results, together with the suggestions that the residues at the tips of cardiotoxins' loop structure were involved in the manifestation of the biological activities of cardiotoxins, reflect that the preferential mutations may contribute to alterations in the function of cardiotoxin molecules. Analysis on the secondary structure of pre-mRNAs of N. naja atra cardiotoxin 4 gene and N. naja sputatrix cardiotoxin 3 gene has shown that the hypervariable regions of the exon 2 pertain to form intra-exon pairings and are not involved in the formation of intron-exon pairings. Since the pairings of splice sites and gene architecture were supposed to be associated with intron-exon recognition, it is likely that the preferred loci of mutations occurring with the evolution of cardiotoxin genes would not affect the processing of cardiotoxin precursors.     

中华眼镜蛇毒组分F的体外抗血管生成活性研究

目的研究中华眼镜蛇毒组分F对血管内皮细胞黏附功能的抑制作用和体外的抗血管生成活性。方法MTT快速测定法测定组分F对原代培养的血管内皮细胞对纤维蛋白原(Fg)、纤连蛋白(Fn)、Matrigel的黏附作用的影响;采用平面拟毛细血管生成培养法,观察组分F对血管生成的抑制效应。结果组分F可抑制血管内皮细胞的黏附功能,对细胞黏附Fn、Fg和Matrigel 3种物质的作用从1.2μg/ml浓度起即有抑制效应(P<0.05),且均呈浓度依赖性,组分F对细胞黏附Fg和Matrigel的抑制作用较其对Fn的作用强(P<0.05);组分F可抑制内皮细胞在培养基质中生成血管样网状结构的反应,并且随浓度的不同抑制程度有明显不同。结论中华眼镜蛇毒组分F具有体外抗血管生成活性。     

Factors influencing the hemolysis of human erythrocytes by cardiotoxins from Naja naja kaouthia and Naja naja atra venoms and a phospholipase A2 with cardiotoxin-like activities from Bungarus fasciatus venom

The effects of red blood cell age and incubation conditions (temperature, divalent cation type and concentration, pH and glucose) on hemolysis induced by cardiotoxin fractions from Naja naja atra and Naja naja kaouthia venoms, a phospholipase A2 with cardiotoxin-like activities from Bungarus fasciatus venom and bee venom phospholipase A2 were examined. Hemolysis by the snake venom toxins was dependent on red blood cell age (aged more susceptible than fresh) and the temperature of incubation (37 degrees C greater than 20 degrees C). Divalent cations at 0.5-2.0 mM enhanced (Ca2+) or slightly decreased (Sr2+, Ba2+) hemolysis due to N. n. kaouthia and N. n. atra toxins, and greatly decreased (Ca2+, Sr2+, Ba2+) hemolysis by these toxins at higher concentrations (5-40 mM). For the B. fasciatus phospholipase A2, Ba2+ and Sr2+ could not fully support hemolysis in any concentration while both low (less than 0.5 mM) and high (greater than 40 mM) Ca2+ enhanced hemolysis. Bee venom phospholipase A2 only induced hemolysis (greater than 10% at greater than 40 mM) at high concentrations of Ca2+. Increasing the pH from 7.5 to 8.5 greatly increased the levels of hemolysis by the snake venom toxins and enzyme. Glucose (5.3 mM) increased hemolysis by the snake venom components at low concentrations of divalent cations (2 mM) and slightly decreased hemolysis at high concentrations (40 mM). Treatment with p-bromophenacyl bromide abolished phospholipase A2 activity of bee venom and B. fasciatus phospholipases, but did not affect hemolytic potency of N. n. kaouthia or B. fasciatus toxins. A similar mechanism, which is independent of phospholipase A2 activity, may be involved in hemolysis by the N. n. kaouthia and N. n. atra cardiotoxins. The B. fasciatus cardiotoxin-like phospholipase A2 appears to have two mechanisms of hemolysis; the first is similar to that of the two typical cardiotoxins and the second appears dependent on phospholipase A2 activity and is only evident at high Ca2+ concentrations.     

一种水解血小板膜糖蛋白GPIb的眼镜蛇毒组分的分离纯化与鉴定

目的　建立从中华眼镜蛇毒中分离、纯化可水解血小板膜糖蛋白Ｉｂ（ｐｌａｔｅｌｅｔｍｅｍｂｒａｎｅｇｌｙｃｏｐｒｏｔｅｉｎＩｂ,ＧＰＩｂ）组分的方法。方法　肝素亲和层析,分子筛层析,ＳＤＳ聚丙烯酰胺凝胶电泳（ＳＤＳ ＰＡＧＥ）。结果　经Ｈｅｐａｒｉｎ ＳｅｈｐａｒｏｓｅＣＬ ６Ｂ亲和层析及ＳｅｐｈａｄｅｘＧ １５０分子筛层析,从中华眼镜蛇毒纯化出可降解纤维蛋白原和ＧＰＩｂ的活性组分,回收率为０－８８％,ＳＤＳ ＰＡＧＥ证实此组分为相对分子量约４７１００的单肽链蛋白。结论　此方法成功地从中华眼镜蛇毒中纯化出了水解ＧＰＩｂ的组分,具有高效、简便的特点。