Isolation and partial characterisation of a lethal neurotoxin from the venom of the Australian death adder (Acanthophis antarcticus)

The venom of the Australian terrestrial snake Acanthophis antarcticus was subjected to gel permeation and ion exchange chromatography and to isoelectric focusing, and five different lethal fractions were separated. The application of preparative isoelectric focusing to one of these fractions yielded a lethal peptide toxin named acanthophin a, which has been partially characterised. Our experiments confirm earlier work that the crude venom causes death by blocking neuromuscular transmission. Acanthophin a also has this action. It consists of a single chain of 63 amino acid residues crosslinked by four disulphide bridges. It has an approximate i.p. ld₅₀ value in mice of 0·16 mg/kg body weight.     

Isolation and purification of the toxic fractions of Mojave rattlesnake venom.

Fractions of Crotalus scutulatus scutulatus venom were separated by gel filtration and ion-exchange chromatography, and monitored at 280 nM on a spectrophotometer. Peak tubes were pooled, concentrated and the protein concentrations determined. Homogeneity was demonstrated by bipolar strip electrophoresis or by polyacrylamide gel electrophoresis. Further purification, when necessary, was carried out in a Buchler Fractophorator.On Sephadex G-100, five lethal fractions were obtained. Peak B having an approximate molecular weight of 30,000, ld₅₀-0·04 mg per kg, and Peak C, having a molecular weight of 9000, highly basic, ld₅₀-0·05 mg per kg, were the most toxic. The ld₅₀ of the crude venom was 0·14 mg per kg. When peak B was chromatographed on DEAE-Sephadex, three lethal fractions were obtained. The ld₅₀ for the peaks were 2·3, 1·7 and 0·03 mg per kg, respectively.     

Isolation and characterization of a toxic protein from Canavalia ensiformis (jack bean) seeds,distinct from concanavalin A

A toxic protein present in the crude extract of Canavalia ensiformis seeds induces within 24 hr dyspnoea, ataxia, hypothermia, coma, tonic convulsions and death in mice injected i.p. with 100–200 mg of protein per kg. The toxin was separated from concanavalin A by affinity adsorption of the latter on Sephadex G-100, and further purified by sequential fractionation by (a) removal of polysaccharides with 30% v/v ethanol; (b) ammonium sulfate precipitation at 0.35–0.55 saturation and (c) DEAE-cellulose chromatography. The purified toxin, with an ld₅₀ of 2–5 mg of protein/kg mouse, is unstable. Gel-filtration of the purified toxin on Bio-Gel P-200 showed a single protein peak with a molecular weight corresponding to 88,000 daltons. Polyacrylamide gel electrophoresis of this material indicated the presence of two small contaminants. A central convulsive effect of the toxin can be proposed since no effects were found on isolated pharmacological preparations. The name CANATOXIN is suggested for this new toxic protein.     

Preliminary fractionation of tiger rattlesnake (Crotalus tigris) venom

S. A. Weinstein and L. A. Smith. Preliminary fractionation of tiger rattlesnake (Crotalus tigris) venom. Toxicon28, 1447–1455, 1990.—Tiger rattlesnake (Crotalus tigris) venom was fractioned by using fast protein liquid chromatography (FPLC). The crude venom had low protease activity, lacked hemolytic activity and had an i.p. ld₅₀ of 0.070 mg/kg for mice. Lethal fractions obtained by anion and cation exchange were examined for antigenic identity with crotoxin and Mojave toxin. Four toxins were obtained by anion exchange chromatography which showed immunoidentity with these toxins, and one fraction caused rear limb paresis in mice. A lethal toxin (about 10% of total venom protein) purified further with Superose-12 FPLC (molecular sieve) had an i.p. ld₅₀ of 0.050 mg/kg for mice, reacted strongly with anti-crotoxin and anti-Mojave toxin antiserum in ELISA and immunoelectrophoresis. This toxin also showed complete immunoidentity with crotoxin and Mojave toxin in immunodiffusion assays with anti-crotoxin antiserum. The results indicated the presence of crotoxin and/or Mojave toxin isoforms in this venom. Although this species has a low venom yield (average 10 mg per snake), the venom is highly toxic and contains high concentrations of several neurotoxic isotoxins.     

Partial purification of a toxin from tentacles of the sea anemone Anemonia sulcata

A toxin of low molecular weight was isolated from tentacles of the sea anemone Anemonia sulcata P. Partially purified toxin was obtained by precipitation with ammonium sulphate, ion exchange chromatography on DEAE cellulose and chromatography on Sephadex G-50. The molecular weight of a partially purified toxin, probably a basic protein was approximately 6000 as determined by gel filtration. Disc electrophoresis of the partially purified toxin revealed four proteins. Heating for 15 min at 90°C inactivated the toxin. The toxic activity remained unchanged over 12 months when stored in frozen solution at ?26°C. The ld₁₀₀ is 6 mg of the toxin per kg (rat) by i.v. injection. The toxin showed a cardiotropic action on the rat heart. When injected i.v. the toxin caused cardiac arrhythmia and fall of the arterial blood pressure.     

Purification and pharmacological characterization of a cardiotoxin-like protein from Formosan banded krait (Bungarus multicinctus) venom

By ion exchange chromatography followed by gel filtration, a polypeptide toxin was purified from Formosan banded krait (Bungarus multicinctus) venom. The toxin had a molecular weight of 7000 ± 300 and showed an amino acid composition characteristic of cardiotoxin from cobra venom. The i.p. ld₅₀ value of the toxin was 2.5 (1.9–3.2) mg per kg mouse. Pharmacological studies showed that the toxin (10 μg/ml) induced contracture in chick and mouse skeletal muscles, depolarized the cell membrane of the mouse diaphragm, arrested the contraction of spontaneously beating atria and the electrically driven ventricle strip of the rat, and caused direct hemolysis of guinea-pig erythrocytes. From these chemical and pharmacological characterizations it was concluded that this toxin has characteristics similar to those of cobra venom cardiotoxins.     

Geographical variation in Crotalus scutulatus scutulatus (Mojave rattlesnake) venom properties

Individual venom samples were analyzed from 12 specimens of Crotalus scutulatus scutulatus, from north of Tucson to the extreme southeastern region of Arizona. Six of the specimens, from north of Tucson, produced venom lethal toxicity (i.p. ld₅₀) values in mice of 2.0 – 6.0 mg/kg. These coincided with the values previously reported for C. s. scutulatus in the Phoenix, Arizona, region and designated as type B venom (Glenn and Straight, 1978). In contrast, the venom ld₅₀ of six individuals from extreme southeastern Arizona, including one individual near Tucson, ranged from 0.22 – 0.46 mg/kg. This corresponds to the values for C. s. scutulatus venom previously reported and designated as type A venom (Glenn and Straight, 1978). Specimens with type A venom have been collected in California, Nevada, Utah and regions of Arizona. In addition to differences in lethal toxicity, the type B venom consistently exhibits a different protein profile, greater proteolytic activity, greater hemorrhagic activity and contains little or none of the major lethal toxin, Mojave toxin, compared to the type A venom. No external morphological characteristic could be found differentiating the type A venom specimens from the type B venom specimens. These findings further confirm the geographical variation of C. s. scutulatus venom in Arizona.     

Immunosorbent chromatography of sea nettle (Chrysaora quinquecirrha) venom and characterization of toxins

C. S. Cobbs, P. K. Gaur, A. J. Russo, J. E. Warnick, G. J. Calton and J. W. Burnett. Immunosorbent chromatography of sea nettle (Chrysaora quinquecirrha) venom and characterization of toxins. Toxicon21, 385 – 391 1983. — A lethal toxic fraction from nematocysts of the sea nettle (Chrysaora quinquecirrha) fishing tentacle was partially purified by immunochromatography using an immobilized monoclonal antibody column. Elution from the immunosorbent was accomplished under mild conditions which conserved the biological activity of the toxin. The isolated fraction, which contained two purified protein bands with molecular weights of 100,000 and 190,000 daltons on SDS polyacrylamide gels, was both cardiotoxic and neurotoxic and exhibited an intravenous lethal activity (ld₅₀) of 0.37 μg/g in mice.     

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.     

Isolation and partial purification of a toxin from Millepora alcicornis

A toxin from the Hydrozoan coral, Millepora alcicornis, has been partially purified by ammonium sulfate precipitation, anion-exchange chromatography, gel-filtration, and adsorption chromatography. The water soluble toxin is thermolabile, non-dialyzable, and pH sensitive. The ld₅₀ for 20 g mice is 40 μg protein per kg body wt.     

Some chemical properties of the venom of the rattlesnake,Crotalus viridis helleri

The venom of the Southern Pacific Rattlesnake, Crotalus viridis helleri, was separated into three lethal and several non-lethal peaks by gel filtration. Peak I was a protein having a mol. wt of ca. 100,000 and an intravenous ld₅₀ of 0.58 mg/kg. Peak II had a mol. wt of ca. 30,000 and a ld₅₀ of 1·7 mg/kg. Peak III, the peptide, had a mol. wt of ca. 6000 and a ld₅₀ of 1·96 mg/kg and moved as a cation on strip and polyacrylamide gel electrophoresis. On ion exchange chromatography the peptide was resolved into three lethal fractions. The major fraction, C, was a basic polypeptide containing 43 amino acid residues with six half cystine residues. Its mol. wt was 4990, as calculated from its sequence, 7600 as estimated from Sephadex G-50 gel filtration and 5200 by SDS- disc gel electrophoresis. The differences are being studied. Analysis showed the peptide contained almost 20% lysine. On sequencing, the most basic amino acid residues were distributed in the N-terminal and C-terminal parts. The middle part was rather hydrophobic.     

Isolation and partial characterization of a toxin from the venom of the East African orthognath spider Pterinochilus spec.

A minor basic polypeptide in the venom of the East African orthognath spider Pterinochilus spec. was the most toxic of 16 basic components in this venom. This toxin A4/4 was purified by gel permeation on Sephadex G-50 and ion exchange chromatography on CM-Sephadex C-25 with a yield of 1.3% of the total protein content of the crude venom. The polypeptide has an isoelectric point of 9.39 and molecular weight of 10,500. The s.c. ld₅₀ in mice is 0.1 mg/kg. Death occurred due to respiratory paralysis. After administration of sublethal doses rapid reversibility of the symptoms and total recovery of the animals was observed.     

The toxic Duvernoy''s secretion of the wandering garter snake, Thamnophis elegans vagrans

The Duvernoy's secretion of the wandering garter snake (Thamnophis elegans vagrans) is highly toxic to mice, causing marked hemorrhaging in the lungs, diaphragm, mesentery and stomach lining, as well as mild local hemorrhaging. Systemic hemorrhaging was most pronounced in mice receiving doses approximating the p. ld₅₀, while doses two times the ld₅₀ or greater produced massive hermorrhaging in the lungs and diaphragm only. Local extravasations were directly proportional to dose. Oral secretions other than Duvernoy's secretion failed to produce lethal effects in mice challenged with doses up to 7 times the ld₅₀ of Duvernoy's secretion. A micro-aspiration techniques for the collection of Duvernoy's secretion from colubrid snakes is described, and liquid as well as dried secretion yields for Thamnophis elegans vagrans are presented.     

Occurrence of a ciguatoxin-like substance in the Spanish mackerel (Scomberomorus commersoni)

A lipid-soluble toxin, similar to ciguatoxin as isolated by Scheueret al. (1967), has been found in the flesh of the Spanish mackerel, Scomberomorus commersoni, caught in Queensland. The ciguatoxin-like substance was experimentally characterized by examination of specific biological and chromatographic properties of the lipid-soluble extract from a pooled sample of flesh from Spanish mackerel. Flesh from specimens known to have caused S. commersoni poisoning in humans was confirmed as toxic by cat bioassay. A toxin was extracted from S. commersoni which yielded, on partial purification, a clear, oily substance with an ld₅₀ i.p. to mice of 0.72 mg/kg, and which had chromatographic properties similar to those of classical ciguatoxin. However, the R_f value on thin-layer chromatography plates was lower for S. commersoni toxin than for classical ciguatoxin. This is the first record of a ciguatoxin-like substance experimentally identified in S. commersoni, a pelagic fish that occurs throughout Queensland coastal waters. The majority of toxic S. commersoni are caught between latitudes 24° and 26°S.     

A lethal myotoxin isolated from the venom of the Australian king brown snake (Pseudechis australis)

A lethal myotoxin, named mulgotoxin a, was isolated from the venom of the Australian king brown snake (Pseudechis australis) by chromatography on Biorex 70 and SP-Sephadex ion exchange resins. Mulgotoxin a was obtained from one of four lethal fractions which were present in the venom. Mulgotoxin a is a basic toxin consisting of a single polypeptide chain of 122 amino acid residues cross-linked by seven disulphide bridges. Its N-and C-terminal residues are aspartic acid and leucine respectively. Mulgotoxin a causes myoglobinuria in mice and has an approximate ld₅₀ of 200 μg/kg body weight (mice). Its activity appears to be specific for skeletal muscle, causing massive cell damage both in vivo and in vitro.     

Purification of a cytotoxic protein from Pseudomonas aeruginosa.

F. Lutz. Purification of a cytotoxic protein from Pseudomonas aeruginosa. Toxicon17, 467–475, 1979.—A method for preparation of the cytotoxin from Pseudomonas aeruginosa, strain 158, is presented. The procedure described by Scharmann (1976b) was improved by a precipitation step with ammonium sulphate and by subsequent chromatography of the dissolved precipitate on acrylamide-agarose. The recovery of purified toxin was 1 mg from 100 mg protein in supernatant (autolysate). The purity of the preparations was tested by sodium dodecyl sulphate gel electrophoresis, immunodiffusion and enzymatic methods for the presence of other known pseudomonal components.The molecular weight of the toxin was estimated by sodium dodecyl sulphate gel electrophoresis (25,100) and by gel chromatography (22,300–22,800). Amino acid analysis revealed a ratio of acidic to basic amino acids of 4:1. Cysteine was absent. The protein contains neither carbohydrate nor lipids. By disc electrophoresis the toxin separated into 4–6 fractions with the same toxicity and with isoelectric points between 5·0 and 6·4. In a slide adhesion test with human granulocytes the ed₁₀₀ of cell swelling was 9 μg/ml. The ed₅₀ for growth inibition of chicken embryo fibroblasts was 11 μg/ml. At 44 μg/ml the toxin liberates half of the hemoglobin in rabbit red cells, but did not hemolyse erythrocytes of sheep, horse and cow. The ld₅₀ on mice was 25 ng/g body weight. In postmortem histological examination of mice, fatty liver necrosis was observed.     

Purification and characterization of Clostridium perfringens beta toxin

J. and Y. . Purification and characterization of Clostridium perfringens beta toxin. Toxicon 25, 1301 – 1310, 1987.—A new procedure for the purification of beta toxin from culture supernatant fluid of Clostridium perfringens was established. The procedure consists of ammonium sulfate fractionation, affinity chromatography on zinc-chelate Sepharose and gel filtration on Toyopearl HW 60. Beta toxin was purified about 460-fold from the ammonium sulfate fraction with a yield of about 60% in terms of lethality of the toxin. The molecular weight of the toxin, determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and sucrose gradient centrifugation, was approximately 40,000. The isoelectric point was 5.6. The minimal necrotic dose for guinea pigs was approximately 2 ng. The 50% lethal doses for adult mice were 310 ng/kg and 4.5 μg/kg, when injected i.v. and i.p., respectively.     

Acute toxicity of PR toxin,a mycotoxin from Penicillium roqueforti

The toxic effects of PR toxin were observed in mice, rats, anesthetized cats and isolated rat auricle preparations. In mice and rats the toxic effects included abdominal writhing, decrease of motor activity and respiratory rate, weakness of hindleg and ataxia. In mice, the i.p. ld₅₀ was 5.8 mg/kg. In mice, rats and cats PR toxin given i.p. caused ascites fluid and edema in the scrotum and lungs, and i.v. injection caused edema in the lungs, giving rise to a large volume of pleural and pericardial fluid. In rats, at the ld₅₀ dose level (11.6 mg/kg, i.p. and 8.2 mg/kg, i.v.), the water content in the lungs was increased, but in the skin it was decreased. Blood K⁺, hematocrit, red blood cell, white blood cell, hemoglobin, uric acid, cholesterol, blood urea nitrogen and alkaline phosphatase concentrations were all increased, while the total protein and albumin contents were decreased after i.p. injection of PR toxin. High content of protein was found in the pleural fluid and fluid due to ascites. In anesthetized cats the blood pressure and respiratory rate were progressively decreased and the heart rate was reflexly increased after i.p. injection. The i.v. injection produced a multiple response on the arterial blood pressure, but with a progressively decreasing heart rate. Arrhythmias were observed in the late shock stage in the case of i.p. or i.v. injection. In the isolated rat auricle preparations contractile force was more affected than heart rate. We conclude that PR toxin produced acute toxic effects in animals via an increase of capillary permeability and a direct damage to the lungs, heart, liver and kidneys.     

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.     

Isolation and characterization of two toxins from the venom of the Malayan cobra (Naja naja sputatrix)

Two principal toxins of the Malayan cobra (Naja naja sputatrix) venom have been purified to electrophoretic homogeneity by successive SP-Sephadex C-25 ion exchange chromatography and Sephadex G-50 gel filtration chromatography. They are designated as sputa-neurotoxin 1 (SN1) and sputa-neurotoxin 2 (SN2), respectively. Both toxins belong to the group of short neurotoxins which are composed of approximately 60 amino acid residues. The ld₅₀ values following i.p. injection of the purified toxins are 91 (SN1) and 71 (SN2) μg/kg mouse. Amino acid compositions of the two toxins show close similarities to other postsynaptically acting curaremimetic cobra neurotoxins.