The properties of the purified fibrinolytic principle from Agkistrodon acutus snake venom.

The properties of the purified fibrinolytic principle from Agkistrodon acutus snake venom. Toxicon15, 161–167, 1977.—In addition to fibrinolytic, fibrinogenolytic and caseinolytic activities, the purified fibrinolytic principle of Agkistrodon acutus venom possessed hemorrhagic activity. Trasylol had a much higher inhibitory action on the fibrinolytic activity of the fibrinolytic principle of the venom than did ε-aminocaproic acid. Thus, the fibrinolytic action of the fibrinolytic principle was chiefly due-to a direct action on fibrin. Both EDTA (5 × 10^-4 M) and cysteine (5 × 10^-3 M) completely inhibited the fibrinolytic, fibrinogenolytic, hemorrhagic and caseinolytic activities of the fibrinolytic principle of the venom. Disulfide bonds might be essential for the biological activities of the fibrinolytic principle.     

Estudio comparativo de los venenos de serpiente Cascabel (Crotalus durissus durissus) de ejemplares adultos y recien nacidos

B. Lomonte, J. A. Gené, J. M. Gutiérrez and L. Cerdas. Estudio comparativo de los venenos de serpiente Cascabel (Crotalus durissus durissus) de ejemplares adultos y recién nacidos. Toxicon21, 379 – 384, 1983. — Venoms from adult and newborn Central American rattlesnakes (Crotalus durissus durissus) were compared for lethal, proteolytic, hemorrhagic, myonecrotic, edematigenous and in vitro hemolytic activities. Electrophoretic and immunoelectrophoretic patterns showed some differences between these venoms. Venom from newborn snakes was devoid of hemorrhagic and edematigenous activities, whereas the venom from adult specimens induced these effects. On the other hand, newborn snake venom showed higher lethality and indirect hemolytic activity, and lower proteolytic activity, than venom from adult specimens. Both types of venoms induced only slight myonecrosis in mice, as judged by histological observation. The ed₅₀ of an antivenom, in terms of absolute weight neutralized per ml of serum, was lower for the newborn specimens venom than for adult's venom, however, for each venom the number of ld₅₀ neutralized was similar.     

Neutralization of proteolytic and hemorrhagic activities of Costa Rican snake venoms by a polyvalent antivenom

J. M. Gutiérrez, J.A. Gpené, G. Rojas and L. Cerdas. Neutralization of proteolytic and hemorrhagic activities of Costa Rican snake venoms by a polyvalent antivenom. Toxicon23, 887–893, 1985. — The polyvalent antivenom produced at the Instituto Clodomiro Picado, Costa Rica, was tested for its capacity to neutralize proteolytic and hemorrhagic activities of ten Costa Rican crotaline venoms. In experiments with preincubation of venom and antivenom, the latter efficiently neutralized proteolytic activities of nine venoms, with ed₅₀ ranging from 50 to 300 μl antivenom/mg venom. The venom of Bothrops nummifer was neutralized less efficiently ($\text{ed}{}_{50}\text{= 760}\text{μl/mg}$). Antivenom was also very effective in neutralizing hemorrhagic activity, having its lowest neutralizing ability against the venom of B. picadoi ($\text{ed}{}_{50}\text{= 430}\text{μl/mg}$ and its highest towards the venom of B. asper (Pacific region) ($\text{ed}{}_{50}\text{= 47 μl/mg}$). There was a significant correlation between the ability of antivenom to neutralize proteolytic and hemorrhagic effects. In spite of the ability of antivenom to neutralize hemorrhage when incubated with venom prior to injection, hemorrhage was only partially neutralized when antivenom was administered i.v. at different time periods after envenomation. This suggests that the rapid development of local hemorrhage, instead of the absence of antivenom antibodies, is the explanation for the poor neutralization observed in these types of experiments.     

Partial amino acid sequence and biological characterization of elegatoxin, hemorrhagic toxin from Trimeresurus elegans (Sakishimahabu) venom.

A hemorrhagic toxin, designated Elegatoxin, was isolated from the venom of Trimeresurus elegans using HW-55, DEAE-Sephacel, CM-Cellulose and Mono S column chromatographies. The purified toxin was shown to be homogeneous by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, isoelectric electrophoresis, and Ouchterlony immunodiffusion. Elegatoxin has a molecular weight of 26,000 with an isoelectric point of 8.6. The toxin demonstrated both hemorrhagic and proteolytic activities. Hemorrhagic activity was inhibited by ethylenediaminetetraacetic acid (EDTA), ethyleneglycol-bis-(2-amino-ethylether)N,N'-tetraacetic acid (EGTA), o-phenanthroline, and N-bromosuccinimide, but not by amidinophenylmethanesulfonyl fluoride hydrochloride (APMSF). The minimum hemorrhagic dose was found to be 0.8 microgram/mouse. Elegatoxin possesses proteolytic activity as evidenced by hydrolyzing type IV collagen, actin and the A alpha, B beta, and gamma chains of bovine fibrinogen. This purified toxin contains 1 mol of zinc and 2 mols of calcium per mol of protein and a partial amino acid sequence was determined. The pathological and biochemical properties of Elegatoxin were investigated, and these results are reported in this paper.     

Isolation and partial characterization of a myotoxin from the venom of the snake Bothrops nummifer

J. M. Gutiérrez, B. Lomonte and L. Cerdas. Isolation and partial characterization of a myotoxin from the venom of the snake Bothrops nummifer. Toxicon24, 885 – 894, 1986. — A myotoxin from the venom of the snake Bothrops nummifer was purified to homogeneity by ionexchange chromatography on CM-Sephadex. The toxin is a basic dimer with a subunit molecular weight of 16,000, as estimated by SDS-polyacrylamide gel electrophoresis. The toxin lacks phospholipase A₂ activity when tested on egg yolk lecithin and skeletal muscle homogenates. It induces skeletal muscle damage both in vivo and in vitro. When injected i.m. it promotes a drastic increase in serum creatine kinase levels; the isozyme CK-MM is responsible for this increment. A rapid release of creatine kinase was observed when mouse gastrocnemius muscle was incubated with the toxin, suggesting that it induces the formation of relatively large ‘lesions’ in the plasma membrane of muscle cells. Moreover, analysis of the dose - response data indicated that the myotoxin affects muscle sarcolemma by a ‘one hit’ mechanism. Skeletal muscle cells are affected by the toxin when calcium is eliminated from the medium. The myotoxin has an i.v. ld₅₀ of 3.9 mg/kg body weight in mice, and induces edema when injected in the foot pad. On the other hand, it is not directly hemolytic, anticoagulant, hemorrhagic nor cytotoxic for lymphocytes. The myotoxin shows partial immunologic identity with a myotoxic phospholipase A₂ isolated from Bothrops asper venom. The polyvalent antivenom produced in Costa Rica forms a precipitation arc against B. nummifer myotoxin on immunoelectrophoresis.     

Intergradation of two different venom populations of the Mojave rattlesnake (Crotalus scutulatus scutulatus) in Arizona

Two distinct venom populations of Crotalus scutulatus scutulatus exist in Arizona. The venom of one population (venom A) contains the toxin 'Mojave toxin' and is lacking in hemorrhagic and specific proteolytic activities. The other population (venom B) does not contain Mojave toxin but does produce hemorrhagic and proteolytic activities. The venoms of 15 Crotalus scutulatus scutulatus from regions between the venom A and venom B populations in Arizona were examined for the presence of Mojave toxin by immunochemical assay, lethality by mouse i.p. LD50, proteolytic activity and hemorrhagic activity in mice. Venom protein constituents were analyzed using reverse-phase HPLC. Seven venoms contained both the Mojave toxin of venom A and the proteolytic and hemorrhagic activities of venom B. The i.p. LD50 values of the A + B venoms were 0.4-2.6 mg/kg, compared to 0.2-0.5 mg/kg for venom A individuals and 2.1-5.3 mg/kg for the venom B individuals. HPLC illustrated that the A + B venoms exhibited a combined protein profile of venom A and venom B. These data indicate that an intergrade zone exists between the two venom types which arcs around the western and southern regions of the venom B population. Within these regions, three major venom types can occur in Crotalus s. scutulatus.     

THE FUMONISIN PARADOX: A REVIEW OF RESEARCH ON ORAL BIOAVAILABILITY OF FUMONISIN B1, A MYCOTOXIN PRODUCED BY FUSARIUM MONILIFORME

Abstract

Rattlesnake envenomation gives rise to many dramatic symptoms in the victim. Included among these are hypotension and hemorrhage. In this paper we present the results of our investigations on several proteases isolated from the venom of Crotalus atror (Western diamondback rattlesnake) which are probably involved in crotalid venom caused hypotension and hemorrhage.

We have isolated two proteases (EI and EII) from C. atrox venom which have molecular weights of 27,500 and 29,200 and isoelectric points of 4.7 and 4.3 respectively. Both enzymes did not demonstrate any proteolytic activity on the oxidized chains of insulin and glucagon. Neither enzyme had any plasmin or fibrinolytic activity but both could release bradykinin from a kininogen analog. This proteolytic activity was inhibited by aprotinin and PMSF (phenylmethylsulfonyl fluoride) but not by EDTA (ethylenediamine tetraacetate). The enzymes were demonstrated to be structurally similar but not identical from spectroscopic, peptide mapping, and sequence analysis. The in vivo role of these kalli-krein-like proteases is uncertain; however, they may be involved in the initial symptoms of hypotension and shock.

The other protease examined was hemorrhagic toxin e (Ht-e), a zinc protease from C. atrox venom. The proteolytic specificity of this protease was examined using oxidized A and B chains of insulin. The cleavage of the insulin chains was inhibited by EDTA but not aprotinin. Ht-e containing cobalt II rather than zinc cleaved the insulin chains at the same sites and at similar rates as the zinc enzyme. No structural differences between the native and the cobalt enzyme were observed using spectroscopic probes. Furthermore the cobalt protease was both hemorrhagic and proteolytic to a similar extent as the native toxin.     

Comparative study on hemorrhagic and proteolytic activities of snake venoms

The hemorrhagic and proteolytic titers of nine snake venoms were measured. Suitable venom dilutions were selected to study the antihemorrhagic and antiproteolytic activities of three warm-blooded animal sera — gray woodrat (Neotoma micropus), hispidus cottonrat (Sigmodon hispidus) and Virginia opossum (Didelphis virginiana). It was demonstrated that all hemorrhagic venoms are proteolytic. The results show that the hemorrhagic activity of the venoms is readily neutralized by the sera; however, the proteolytic activity is not neutralized. We suggest that the mechanisms involved in neutralizing the two activities are not closely related.     

Isolation and characterization of a hemorrhagin from the venom of Ophiophagus hannah (king cobra)

The major hemorrhagin (termed hannahtoxin) of the venom of Ophiophagus hannah (king cobra) was purified to electrophoretic homogeneity by DEAE-Sephacel ion exchange chromatography, Sephadex G-200 gel filtration followed by a second DEAE-Sephacel chromatography. Proteolytic activity was associated with the hemorrhagic activity throughout the purification procedures. Hannahtoxin constituted approximately 2% of the crude venom. It had an isoelectric point of 5.3, a carbohydrate content of 12%, a mol. wt of 66,000 as determined by SDS-polyacrylamide gel electrophoresis and 63,000 as determined by gel filtration. It contains 1 mole of Zn per mole of protein. The minimum hemorrhage doses for hannahtoxin are 0.7 microgram and 75 micrograms, respectively, in rabbits and in mice. Hannahtoxin was not lethal to mice at a dose of 2 mg/kg (i.v.) but killed rabbits at doses above 0.18 mg/kg (i.v.). It liberated protein from rabbit glomerular basement membrane but not rat glomerular basement membrane. Treatment of the hemorrhagin with EDTA and 1,10-phenanthroline eliminated both the proteolytic and hemorrhagic activities completely.     

Purification and some characteristics of a zinc metalloprotease from the venom of Bothrops jararaca (jararaca)

A metalloprotease from Bothrops jararaca venom (J protease) was purified by DEAE-Sephacel, CM-cellulose, Sephacryl S-200 and Sephadex G-75 chromatograph. The proteolytic activity was inactivated by EDTA, o-phenanthroline and DTNB. Phosphoramidon and cysteine protease inhibitors (leupeptin, E64 and its derivatives) were inactive on this enzyme. J protease was activated by calcium and the metal content analysis showed the presence of one mole each of tightly bond zinc and calcium per mole of this J protease. The amino acid composition, N-terminal amino acid sequence (29 residues) and the cleavage sites on the oxidized insulin B chain and angiotensin I were determined.     

Isolation of a myotoxin from Bothrops asper venom: partial characterization and action on skeletal muscle

A myotoxic phospholipase has been isolated from Bothrops asper venom by ion-exchange chromatography on CM-Sephadex followed by gel filtration on Sephadex G-75. The toxin is a basic polypeptide with an estimated molecular weight of 10,700. It has both phospholipase A and indirect hemolytic activities, but is devoid of proteolytic, direct hemolytic and hemorrhagic effects. When injected i.m. into mice the toxin induces a rapid increase in plasma creatine kinase levels and a series of degenerative events in skeletal muscle which lead to myonecrosis. The toxin induces an increase in intracellular calcium levels and is able to hydrolyze muscle phospholipids in vivo. Pretreatment with the calcium antagonist verapamil failed to prevent the myotoxic activity. It is proposed that B. asper myotoxin causes cell injury by disrupting the integrity of skeletal muscle plasma membrane and that myotoxicity is at least partially due to the phospholipase A activity of the toxin.     

Factor X converting and thrombin-like activities of Bothrops Jararaca snake venom

Factor X converting and thrombin-like activities of Bothrops jararaca venom. Toxicon15, 107–114, 1977.—Bothrops jararaca venom is able to activate factor X and to convert fibrinogen to fibrin by a thrombin-like activity. The two activities behaved as if they were due to distinct enzyme proteins as determined by chromatography on Sephadex G-100, isoelectric focusing, disc gel electrophoresis and heat-treatment.     

Effect of Clostridium perfringens alpha toxin on the cardiovascular system of rats

J. Sakurai, Y. Oshita and Y. Fujii. Effect of Clostridium perfringens alpha toxin on the cardiovascular system of rats. Toxicon23, 905–912, 1985. — Clostridium perfringens alpha toxin decreased heart rate, then elevated blood pressure, and finally caused some changes of electrocardiogram readings. The toxin decreased peripheral blood flow before blood pressure started to increase and the blood flow continued to decrease, without any affect on electrocardiogram readings, until the maximal pressure rise caused by the toxin. The toxin caused a rise in blood pressure in a dose-dependent manner. On the other hand, anti-alpha toxin antiserum inhibited both phospholipase C activity and pressor activity. When the toxin was pretreated with cysteine, calcium disodium ethylenediamine tetraacetate and calcium trisodium ethylenetriamine pentaacetate, pressor activity decreased, as well as phospholipase C activity. The results indicate that alpha toxin possesses pressor activity as well as phospholipase C activity.     

尖吻蝮蛇毒研究进展

尖吻蝮(又名五步蛇,中药材名为蕲蛇)蛇毒,是从尖吻蝮毒腺中分泌的毒液,内含磷脂酶A₂、丝氨酸蛋白酶、金属蛋白酶、C型凝集素、L-氨基酸氧化酶等多种蛋白类成分和肽类成分,具有多种生物活性,在抗肿瘤、抗血栓、抗炎、抗菌等方面发挥着重要作用。近年来,蛇毒研究日益广泛,但目前仍缺乏对尖吻蝮蛇毒全面系统的研究。本文通过检索尖吻蝮蛇毒的相关研究进展,在其来源、鉴别、活性成分、毒性研究及质量研究等方面进行总结与分析,以期为尖吻蝮蛇毒进一步开发利用提供参考。     

Production of a monoclonal antibody against hemorrhagic activity of Crotalus atrox (western diamondback rattlesnake) venom

J. C. Perez, V. E. Garcia and S. Y. Huang. Production of a monoclonal antibody against hemorrhagic activity of Crotalus atrox (western diamondback rattlesnake) venom. Toxicon22, 967 – 973, 1984. — Crotalid venoms have cytotoxic properties which could be useful in medical research. Crotalus atrox venom-hyperimmunized mouse spleen cells were fused with SP2/0 myeloma cells. Forty-one wells containing the hybridoma cells were positive for C. atrox venom, as determined by the enzyme linked immunosorbent assay (ELISA). Cell line 1-e12 was cloned and used to produce ascites tumors in BALB/c mice. The monoclonal antibody produced by cloned and subcultured 1-e12 cells reacted with both C. atrox venom and six other venoms in the ELISA and neutralized the hemorrhagic activity of crude C. atrox venom. A series of monoclonal antibodies could be used in studying the nature of snake venoms.     

Characterization of a hemorrhagic factor, LHF-I, isolated from the bushmaster snake (Lachesis muta muta) venom

Hemorrhagic factor I (LHF-I) was previously purified from the venom of the bushmaster snake (Lachesis muta muta). In terms of biochemical and immunological properties, LHF-I is a glycoprotein (mol. wt 100,000, pI 4.7) consisting of two subunits; it loses its activity following mercaptoethanol treatment. LHF-I contains 0.7 g-atom zinc and 1.2 g-atom calcium per mole protein. The hemorrhagic and the proteinase activities are inhibited by EDTA; subsequent addition of Ca²⁺ or Mg²⁺ does not reverse the EDTA-induced inhibition of the hemorrhagic activity. The metalloenzyme does not hydrolyze arginine esters and is devoid of phospholipase A₂ activity. It hydrolyzes the A- > Bβ-chain of fibrinogen without clot formation and hydrolyzes selectively the -chain of fibrin, leaving the Bβ- and τ-chains unaffected. Antibodies to the hemorrhagic factor in bushmaster venom were produced by immunizing rabbits with the purified protein. The antibody was purified by protein-A affinity chromatography. This antibody was also used to screen other Crotalinae venom samples for immunologically similar epitopes by ELISA assay. The purified antibody reacted only with LHF-I and two samples of bushmaster venom from different geographical locations.     

Purification and properties of a lethal,hemorrhagic protein, “Mucrotoxin A”, from the venom of the Chinese habu snake (Trimeresurus mucrosquamatus)

Mucrotoxin A was purified from the lyophilized venom of Trimeresurus mucrosquamatus using gel filtration on a Sephadex G-100 column, followed by chromatography on CM-Sephadex C-50 and DEAE-Sephadex A-50. By these procedures, 14 mg of purified preparation could be obtained from 1 g of crude venom. The purified preparation was homogeneous by disc electrophoresis on polyacrylamide gel at pH 8.3, isoelectric focusing and by the presence of one precipitin line on immunodiffusion. Mucrotoxin A possessed both lethal and hemorrhagic activities, but it did not show caseinolytic activity. Its molecular weight was approximately 94,000 and the isoelectric point was 4.3. Mucrotoxin A contains approximately 3 moles of Ca and 2 moles of Zn per mole of toxin. The amino acid composition of Mucrotoxin A was determined. No carbohydrate was present.     

Neutralising effects of small molecule toxin inhibitors on nanofractionated coagulopathic Crotalinae snake venoms

Repurposing small molecule drugs and drug candidates is considered as a promising approach to revolutionise the treatment of snakebite envenoming. In this study, we investigated the inhibiting effects of the small molecules varespladib (nonspecific phospholipase A₂ inhibitor), marimastat (broad spectrum matrix metalloprotease inhibitor) and dimercaprol (metal ion chelator) against coagulopathic toxins found in Crotalinae (pit vipers) snake venoms. Venoms from Bothrops asper, Bothrops jararaca, Calloselasma rhodostoma and Deinagkistrodon acutus were separated by liquid chromatography, followed by nanofractionation and mass spectrometry identification undertaken in parallel. Nanofractions of the venom toxins were then subjected to a high-throughput coagulation assay in the presence of different concentrations of the small molecules under study. Anticoagulant venom toxins were mostly identified as phospholipases A₂, while procoagulant venom activities were mainly associated with snake venom metalloproteinases and snake venom serine proteases. Varespladib was found to effectively inhibit most anticoagulant venom effects, and also showed some inhibition against procoagulant toxins. Contrastingly, marimastat and dimercaprol were both effective inhibitors of procoagulant venom activities but showed little inhibitory capability against anticoagulant toxins. The information obtained from this study aids our understanding of the mechanisms of action of toxin inhibitor drug candidates, and highlights their potential as future snakebite treatments.     

Purification and characterization of hemorrhagic components from Agkistrodon acutus (hundred pace snake) venom

Three toxic components, designated Aa-hemorrhagin I,II,III, were isolated from the venom of Agkistrodon acutus by DEAE-Sephadex A-50 chromatography and were further purified by gel filtration on Sephadex G-75, and by column chromatography on DE52 cellulose and CM-Sephadex C-25. All of these components show single bands on acrylamide gel electrophoresis and one precipitin line on immunoelectrophoresis. They are distinct from each other immunochemically. Aa-hemorrhagin I and II are acidic proteins, while III is basic. These hemorrhagins have similar mol. wts of about 22,000. In addition to hemorrhagic and lethal activities, these hemorrhagins possess caseinolytic activities. Their hemorrhagic and caseinolytic activities are inhibited by EDTA, cysteine and the serum of homologous snakes. The amino acid composition of the Aa-hemorrhagin I was determined.     

Scyphozoan jellyfish venom metalloproteinases and their role in the cytotoxicity

The present study, for the first time, comparatively investigated the enzymatic activities (proteases and hyaluronidases) in the venoms of four Scyphozoan jellyfish species, including Nemopilema nomurai, Rhopilema esculenta, Cyanea nozakii, and Aurelia aurita. For this, various zymographic analyses were performed using assay specific substrates. Interestingly, all the four jellyfish venoms showed gelatinolytic, caseinolytic, and fibrinolytic activities, each of which contains a multitude of enzyme components with molecular weights between 17 and 130 kDa. These four jellyfish venoms demonstrated a huge variation in their proteolytic activities in quantitative and qualitative manner depending on the species. Most of these enzymatic activities were disappeared by the treatment of 1,10-phenanthroline, suggesting they might be belonged to metalloproteinases. Toxicological significance of these venom proteases was examined by comparing their proteolytic activity and the cytotoxicity in NIH 3T3 cells. The relative cytotoxic potency was C. nozakii > N. nomurai > A. aurita > R. esculenta. The cytotoxicity of jellyfish venom shows a positive correlation with its overall proteolytic activity. The metalloproteinases appear to play an important role in the induction of jellyfish venom toxicities. In conclusion, the present report proposes a novel finding of Scyphozoan jellyfish venom metalloproteinases and their potential role in the cytotoxicity.