The isolation of an easily reversible postsynaptic toxin from the venom of a sea snake, Laticauda semifasciata

A weakly neurotoxic component (Ls-III) was isolated by CM-cellulose column chromatography from the venom of a sea snake Laticauda semifasciata. The content of component LsIII was about 10-20% of the venom as determined by u.v. absorption at 280nm. Component LsIII was homogeneous on rechromatography and disc electrophoresis, and its molecular weight was shown to be 7100 by ultracentrifugation and 7300 by sodium dodecyl sulphate-polyacrylamide-gel disc electrophoresis. The isoelectric point of component LsIII was pH7.2. Component LsIII consisted of 66 amino acid residues including 10 half-cystine residues. The LD(50) of component LsIII by intramuscular injection was 1.24mug/g body wt. for mice and 0.45mug/g for baby chicks, which is about eight to ten times less toxic than erabutoxins a, b and c, all of which are contained in the same venom. Experiments with three isolated muscle preparations from different species indicated that component LsIII was a post-synaptically acting toxin, the action of which was easily reversed by washing.     

The isolation, properties and amino acid sequence of erabutoxin c, a minor neurotoxic component of the venom of a sea snake Laticauda semifasciata

Erabutoxin c, a minor neurotoxic component of the venom of a sea snake Laticauda semifasciata, was isolated in pure form by repeated column chromatography on CM-cellulose columns. The toxin was crystallizable and monodisperse in rechromatography, disc electrophoresis and isoelectric focusing (isoelectric point, pH9.23-9.25). The molecular weight of the toxin, as estimated by gel filtration, was 7000. The toxin showed the same lethal activity to mice (0.13mug/g body wt., intramuscular injection) and the same effect on isolated frog muscle as erabutoxins a and b, the main toxic components of the venom. The toxin inhibited the acetylcholine contracture but not the potassium chloride contracture of muscle. Erabutoxin c consisted of 62 amino acid residues, containing one fewer lysine and one more histidine than erabutoxin a and one fewer lysine and one more aspartic acid (or asparagine) than erabutoxin b. Erabutoxin c was reduced, S-carboxymethylated and hydrolysed with trypsin. The only fragment different from the corresponding fragments from erabutoxin b was hydrolysed further with pepsin. One of the peptic fragments, which was assumed to have the aspartic acid (or asparagine) residue in question at the C-terminal end, was treated with carboxypeptidase A. The C-terminal residue was found to be an asparagine. It was therefore concluded that erabutoxin c was [51-asparagine]-erabutoxin b.     

Preparation and chemical characterization of the three chains of the major hemoglobin of the sea snake, Pelamis platurus.

One of the 2 main hemoglobins of the sea snake Pelamis platurus, (the Yellow-bellied sea snake) comprising about 70% of the total hemoglobin, was separated by DEAE-Sephadex column chromatography. From results of gel filtration and iron content determination, both intact sea snake hemoglobin, and the isolated major hemoglobin, were concluded to be composed of 4 subunits with a molecular weight of 66,000-67,000 daltons. Separation of the chains of globin of the major hemoglobin by CM 52 column chromatography gave 3 peaks, named, chains a, b, and c. The approximate molecular weights of chains a, b, and c were deduced by SDS gel electrophoresis to be 14,000, 16,000, and 20,000 daltons, respectively. The peptide maps and amino acid compositions of the three chains were distinctly different. N-Terminal and C-terminal amino acid sequence studies reveal that chains a, b, and c represented the alpha-chain, beta-chain, and beta'-chain differing from the normal beta-chain in having a C-terminal sequence of -Arg-Leu-His-Tyr. From the peak areas of the 3 chains obtained by CM 52 column chromatography, and the peak sizes of the 3 bands separated by SDS gel electrophoresis, it was concluded that the sea snake hemoglobin is composed of a hybrid tetramer, alpha2betabeta'.     

Dehydration and drinking responses in a pelagic sea snake

Recent investigations of water balance in sea snakes demonstrated that amphibious sea kraits (Laticauda spp.) dehydrate in seawater and require fresh water to restore deficits in body water. Here, we report similar findings for Pelamis platurus, a viviparous, pelagic, entirely marine species of hydrophiine ("true") sea snake. We sampled snakes at Golfo de Papagayo, Guanacaste, Costa Rica and demonstrated they do not drink seawater but fresh water at variable deficits of body water incurred by dehydration. The threshold dehydration at which snakes first drink fresh water is -18.3 ± 1.1 % (mean ± SE) loss of body mass, which is roughly twice the magnitude of mass deficit at which sea kraits drink fresh water. Compared to sea kraits, Pelamis drink relatively larger volumes of water and make up a larger percentage of the dehydration deficit. Some dehydrated Pelamis also were shown to drink brackish water up to 50% seawater, but most drank at lower brackish values and 20% of the snakes tested did not drink at all. Like sea kraits, Pelamis dehydrate when kept in seawater in the laboratory. Moreover, some individuals drank fresh water immediately following capture, providing preliminary evidence that Pelamis dehydrate at sea. Thus, this widely distributed pelagic species remains subject to dehydration in marine environments where it retains a capacity to sense and to drink fresh water. In comparison with sea kraits, however, Pelamis represents a more advanced stage in the evolutionary transition to a fully marine life and appears to be less dependent on fresh water.     

Studies of an acidic cardiotoxin isolated from the venom of Mojave rattlesnake (Crotalus scutulatus).

A major lethal protein was isolated from the venom of Mojave rattlesnake (Crotalus scutulatus) by successive purification in DEAE column chromatography and isoelectric focusing. This homogeneous and monomeric form of toxin is designated as "Mojave toxin". Unlike basic neurotoxins or cytotoxins isolated from venoms of cobras, kraits and sea snakes, the Mojave toxin is an acidic protein with an isoelectric point of 4.7. The toxin is also different from crotoxin (from Crotalus durissus terrificus) which consists of both acidic and basic components. The molecular weight determined by Sephadex G-75 column chromatography resulted in a value of about 22 000. A singel protein band with a molecular weight of about 12 000, was observed after sodium dodecyl sulfate gel electrophoresis of the reduced Mojave toxin. Isoelectric focusing gel in the presence of 8 M urea also showed a single protein band, suggesting that the toxin is composed of subunits. Unlike the neurotoxic nature of the basic proteins from the venoms of Elapidae and sea snakes (Hydrophiidae) and crotoxin, Mojave toxin is cardiotoxic rather than neurotoxic. It is very likely that venoms of all rattlesnakes from North and Central America contain Mojave toxin as the common toxin.     

Structural properties of mojave toxin of crotalus scutulatus (Mojave rattlesnake) determined by laser Raman spectroscopy.

Laser Raman Spectra were obtained on aqueous and solid samples of Mojave toxin isolated from the venom of the Mojave rattlesnake ($\text{Crotalus}\text{scutulatus}$). The Raman spectra reveal that the Mojave toxin, an acidic protein of molecular weight about 22,000, contains a predominantly α-helical secondary structure and that the tyrosyl residues, on the basis of the Raman frequencies and intensities, are exposed to the solvent. These features of the Mojave toxin distinguish it structurally from the neurotoxins of sea snake venoms. However, like the sea snake venom toxins, Mojave toxin contains four disulfide bridges and is not greatly altered in structure by removal of the aqueous solvent.     

Fingerprint correspondence of hemoglobins and the relationships of sea snakes.

1. Peptide fingerprints of tryptic digests of the globins of sea snake species of Hydrophis, Pelamis, Aipysurus, Laticauda and the terrestrial elapid Naja were compared. 2. Globin divergence, as estimated from peptide fingerprints, paralleled closely transferrin divergence, as measured immunologically. 3. Taxonomic affinities, suggested by the fingerprint data, are concordant with McDowell's taxonomic system for sea snakes with the following exceptions: (a) Laticauda shows a closer affinity to the true sea snakes than to the terrestrial elapid Naja. (b) Sea snakes appear to be more widely divergent from terrestrial elapids than his scheme suggests.     

Purification from black widow spider venom of a protein factor causing the depletion of synaptic vesicles at neuromuscular junctions

The aqueous extract of the venom glands of black widow spiders was fractionated on a column of Sephadex G-200 and then on a column of DEAE-Sephadex A-50 pH 8.2. A protein fraction was obtained that caused a great increase in the frequency of occurrence of miniature end plate potentials at the frog neuromuscular junction, and caused swelling of the nerve terminals and depleted them of their vesicles. The fraction consists of a least four protein components that are similar in their molecular weights (about 130,000) and isoelectric points (ranging from pH 5.2 to 5.5) and are immunologically indistinguishable. It contains no sugar residues and has little or no lipolytic or proteolytic activity. The fraction is toxic to mice and is different from the fractions that act on houseflies, the crayfish stretch receptor and the cockroach heart. It seems pure enough to warrant a detailed study of its site and mode of action.     

Assignment of the three disulfide bonds in ShK toxin: A potent potassium channel inhibitor from the sea anemone Stichodactyla helianthus

Summary ShK toxin, a 35-residue peptide isolated from the Caribbean sea anemone Stichodactyla helianthus, is a potent inhibitor of the Kv 1.3 potassium channel in lymphocytes. The natural toxin contains three disulfide bonds. The disulfide pairings of the synthetic ShK toxin were elucidated as a prerequisite for studies on its structure-function relationships. The toxin was fragmented at pH 6.5 using either thermolysin or a mixture of trypsin and chymotrypsin followed by thermolysin. The fragments were isolated by RP-HPLC and were identified by sequence analysis and MALDI-TOF mass spectrometry. The three disulfides were unambiguously identified in either proteolytic digest: Cys³ to Cys³⁵, Cys¹² to Cys²⁸ and Cys¹⁷ to Cys³². The Cys³-Cys³⁵ disulfide, linking the amino- and carboxyl-termini, defines the characteristic cyclic structure of the molecule. A similar disulfide pairing motif is found in the snake venom-derived potassium channel blocker dendrotoxin and the mammalian antibiotic peptide defensins.     

Preliminary insights into the phylogeography of the yellow-bellied sea snake, Pelamis platurus

The yellow-bellied sea snake, Pelamis platurus (Elapidae, Hydrophiinae), has the largest distribution of any snake species, and patterns related to its distribution and regional color variation suggest there is population structuring in this species. Here, we use mitochondrial (ND4, Cyt-b) and nuclear (RAG-1) DNA to (1) test whether genetic variation is associated with local variation in color pattern, and (2) assess whether large-scale patterns of genetic variation are correlated with geographic distribution across the Pacific Ocean. We found low levels of genetic variation and shallow population structure that are correlated with local variation in color pattern and with geographic distribution. The low levels of genetic divergence indicate a relatively high rate of gene flow throughout the Pacific region and/or a recent expansion of range, both of which could be attributable to the passive drifting of these snakes on oceanic surface currents. The mtDNA data conform closely to a model of past exponential population growth, and this may have been associated with the species' large eastward and westward expansion of range. The pattern of low nucleotide and high haplotype diversity suggests that this population growth occurred in the relatively recent past. Data from drifting buoys can potentially act as informative models for predicting patterns of drifting in Pelamis and for generating additional testable hypotheses relating to its population structure and biogeography. Future studies should employ nuclear microsatellite markers to investigate population structure in this species at a finer scale. The exploitation of oceanic currents as a novel and highly efficient dispersal mechanism has likely facilitated gene flow throughout the Pacific Ocean in this uniquely pelagic species of sea snake, resulting in a distribution spanning over half of the earth's circumference.     

Proteins toxic to arthropods in the venom of elapid snakes.

It has been found that the lethal action of elapid snake venoms to arthropods (fly larvae and isopods) is due to proteic factors differing from the toxins which are strongly and specifically active on mammals.This conclusion was based on the following: (1) Lack of any correlation between the toxic activity on larvae, isopods, and mice of ten elapid snake venoms. (2) Absence of any toxicity to arthropods in pure toxins isolated and purified from several elapid snake venoms according to their lethality. (3) Electrophoretical separation of the venom of the snake Naja mossambica mossambica (= N. nigricollis mossambica) resulted in fractions active either to arthropods and/or to mice. (4) Separation of the above venom by gel filtration on Sephadex G-50 enabled the isolation of fractions highly toxic to arthropods. (5) The above fractions demonstrated a high phospholipase activity corresponding to about 80 per cent of the total activity of the whole venom. The link between phospholipase and toxicity to arthropods will serve as a target for further investigation.It appears that the phenomenon of diversity in toxic activities of different proteins to different groups of organism, as previously demonstrated in scorpion venoms, is equally shared by elapid snake venoms.     

New method for isolation of venom from the sea anemone Actinia cari. Purification and characterization of cytolytic toxins

1. Venom from the sea anemone Actinia cari was obtained by the "milking" method. Two lethal and hemolytic polypeptide toxins, caritoxins I (CTX I) and II (CTX II), were isolated with gel and ion exchange chromatography. 2. The mol. wt of the pure toxin was 19,800. The isoelectric points of CTX I and II were 9.45 and 10.0, respectively. The toxins had similar amino acid compositions lacking cysteine. 3. The intravenous CTX I and CTX II lethal dose (LD50) in mice was found to be 54 +/- 25 and 90 +/- 1 micrograms/kg, respectively. Their hemolytic activity was inhibited by sphingomyelin.     

Chemical properties and amino acid composition of beta1-bungarotoxin from the venom of Bungarus multicinctus (Formosan banded krait)

beta-Bungarotoxin purified from the venom of Bungarus multicinctus (Formosan banded krait) contained no carbohydrate and behaved as a homogeneous protein on polyacrylamide gel electrophoresis at pH 4.1 and SDS-polyacrylamide gel electrophoresis without 2-mercaptoethanol treatment. Its molecular weight and isoelectric point were estimated to be about 21,000 by gel filtration and about 9.5 by isoelectric focusing, respectively. The toxin treated with the reducing agent was split into two polypeptide chains as revealed by SDS-polyacrylamide gel electrophoresis and their molecular weights were calculated to be about 13,000 and 7,000. The two polypeptide chains (the large one named the A chain and the small one the B chain) were isolated by gel filtration after reduction of disulfide bonds in the toxin followed by alkylation. The A chain contained 120 amino acid residues including 13 half-cystines and the B chain 60 residues including 7 half-cystines. The two chains were supposed to link by disulfide bond(s) in the intact toxin which contained no free sulfhydryl groups. The N-terminal residues of the A and B chains were asparagine and arginine and the C-terminal ones were glutamine and proline, respectively, in accordance with the results of the terminal analyses of the intact toxin.     

Isolation and amino acid sequence of a short-chain neurotoxin from an Australian elapid snake, Pseudechis australis.

A short-chain neurotoxin Pseudechis australis a (toxin Pa a) was isolated from the venom of an Australian elapid snake Pseudechis australis (king brown snake) by sequential chromatography on CM-cellulose, Sephadex G-50 and CM-cellulose columns. Toxin Pa a has an LD50 (intravenous) value of 76 micrograms/kg body wt. in mice and consists of 62 amino acid residues. The amino acid sequence of Pa a shows considerable homology with those of short-chain neurotoxins of elapid snakes, especially of true sea snakes.     

Staphylococcal toxic shock exotoxin (its isolation and characteristics)

The method for obtaining the preparation of toxic shock exotoxin (TSE) has been developed. This method comprises the following operations: the sorption of the toxin from the culture fluid on Amberlite CG-50, elution, dialysis, gel chromatography in a column with biogel P-2, isoelectric focusing, and gel chromatography in a column with Sephadex G-75. TSE is a relatively thermostable protein with a molecular weight of 24,000. Its isoelectric point is 7.2. Monospecific antiserum to TSE with precipitating antibody titer equal to 1:16, identical to the reference serum (M. S. Bergdoll), has been prepared. This antiserum has shown no cross reactions with the homogeneous preparations of staphylococcal enterotoxins.     

Reproduction in the yellow-bellied sea snake (Pelamis platurus) from Panama: Field and laboratory observations

Little is known of the reproductive biology of the yellow-bellied sea snake (Pelamis platurus), a species widely distributed in the Indo-Pacific and eastern Pacific Oceans. We observed mating, birth, and free-ranging neonates of P. platurus while collecting this snake once a month over 19 months in the Gulf of Chiriquí, Panama. A pair of copulating snakes was netted on the water surface during February. Neonates, which were identified by size, were observed from September to December. Captive females gave birth during September. Neonates born in captivity emerged head- or tailfirst, shed the remnants of the fetal membranes by coiling their body in a circular loop, and then surfaced to breathe. © 1996 Wiley-Liss, Inc.     

A cysteine-linkable,short cleavable photoprobe with dual functionality to explore protein-protein interfaces

We developed a bifunctional photoprobe with dual functionality, that can be specifically tethered to cysteinyl residues of peptides and proteins through a short cleavable disulfide bond. Thus, an aryldiazonium moiety is positioned at approximately 8.5 A from the modified cysteinyl alpha-carbon, leading to one of the shortest cleavable linkages. In a sodium azide-containing buffer, the aryldiazonium moiety is transformed into an aryl azide. Therefore, with one bifunctional photoprobe two types of photogenerated species can be obtained: a hydrophilic and positively charged arylcation or a hydrophobic nitrene. We coupled the aryldiazonium probe, in a site-directed manner, to a nicotinic acetylcholine receptor competitive antagonist, obtained by chemical engineering of an analogue of a snake alpha-neurotoxin. In this molecule, Arg33, which is known to interact with the receptor, was replaced by a cysteine residue, where the photoprobe could be attached. Under inactinic light, this novel photosensitive snake toxin behaved as a reversible ligand on the Torpedo acetylcholine receptor. However, when irradiated at 391 nm, it generated a highly reactive arylcation which labeled mostly the receptor alpha-subunit, confirming the location of the tip of the second toxic loop near this receptor subunit. Finally, we showed that reduction of the disulfide bond, linking the ligand to the photocoupled receptor, allowed introduction of radioactivity on the labeled residue(s), opening the way to further characterization and avoiding the synthesis of a radioactive bifunctional photoprobe.     

Simplified purification method for Clostridium botulinum type E toxin.

Clostridium botulinum type E toxin was purified in three chromatography steps. Toxin extracted from cells was concentrated by precipitation and dissolving in a small volume of citrate buffer. When the extract was chromatographed on DEAE-Sephadex without RNase or protamine treatment, the first protein peak had most of the toxin but little nucleic acid. When the toxic pool was applied to a carboxymethyl Sepharose column, toxin was recovered in the first protein peak in its bimolecular complex form. The final chromatography step at 4 degrees C on a DEAE-Sephacel column at a slightly alkaline pH purified the toxin (Mr, 145,000) by separating the nontoxic protein from the complex. At least 1.5 mg of pure toxin was obtained from each liter of culture, and the toxicity was 6 X 10(7) 50% lethal doses per mg of protein. These values are significantly higher than those previously reported.     

Chemical synthesis of a neurotoxic polypeptide from the sea anemone Stichodactyla helianthus

The sea anemone Stichodactyla helianthus neurotoxin I, a 48-residue polypeptide, was synthesized by automated solid phase methodology. The fully reduced polypeptide was subsequently refolded in the presence of a glutathione oxidoreduction buffer to the biologically active species containing three disulfide bonds. The overall yield after rigorous purification was 12.5%. The circular dichroism (CD), and proton nuclear magnetic resonance (1H NMR) spectra of the HPLC-purified synthetic toxin were indistinguishable from those obtained concurrently with the natural toxin. A subtilisin digest of the synthetic neurotoxin generated peptide fragments identical to that of a sample of the natural toxin subjected to the same treatment. The toxicity of the synthetic polypeptide was identical to that of the natural toxin (crab LD50, 3.1 micrograms/kg). The equilibrium dissociation constant (28 nM) for interaction of the synthetic toxin with crab axolemma vesicles was nearly identical to that of the natural toxin (25 nM).     

Simplified purification method for Clostridium botulinum type E toxin

Clostridium botulinum type E toxin was purified in three chromatography steps. Toxin extracted from cells was concentrated by precipitation and dissolving in a small volume of citrate buffer. When the extract was chromatographed on DEAE-Sephadex without RNase or protamine treatment, the first protein peak had most of the toxin but little nucleic acid. When the toxic pool was applied to a carboxymethyl Sepharose column, toxin was recovered in the first protein peak in its bimolecular complex form. The final chromatography step at 4 degrees C on a DEAE-Sephacel column at a slightly alkaline pH purified the toxin (Mr, 145,000) by separating the nontoxic protein from the complex. At least 1.5 mg of pure toxin was obtained from each liter of culture, and the toxicity was 6 X 10(7) 50% lethal doses per mg of protein. These values are significantly higher than those previously reported.