Prey specificity, comparative lethality and compositional differences of coral snake venoms.

Toxicities of crude venoms from 49 coral snake (Micrurus sp.) populations, representing 15 nominal taxa, were examined in both laboratory mice and in native prey animals and compared with data gathered from two non-micrurine elapids and a crotalid, which served as outgroups. These venoms were further compared on the basis of 23 enzymatic activities. Both toxicities and enzymatic activities were analyzed with respect to natural prey preferences, as determined from stomach content analyses and literature reports. Venoms of nearly all Micrurus for which prey preferences are known, are more toxic to natural prey than to non-prey species. Except for amphisbaenians, prey are more susceptible to venoms of Micrurus that feed upon them, than to venoms of those that eat other organisms. All venoms were more toxic i.v.>i.p.>i.m. Route-specific differences in toxicity are generally greatest for preferred prey species. Cluster analyses of venom enzymatic activities resulted in five clusters, with the fish-eating M. surinamensis more distant from other Micrurus than even the crotalid, Bothrops moojeni. Ophiophagous and amphisbaenian-eating Micrurus formed two close subclusters, one allied to the outgroup species Naja naja and the other to the fossorial, ophiophagous Bungarus multicinctus. Prey preference is shown to be the most important determinant of venom composition in Micrurus.

Primary structure of gamma-bungarotoxin, a new postsynaptic neurotoxin from venom of Bungarus multicinctus.

The primary structure of gamma-bungarotoxin, a new toxin from Bungarus multicinctus venom, was determined using mass spectrometry and Edman degradation. The toxin has a mass of 7524.7 D and consists of 68 residues having the following sequence: MQCKTCSFYT CPNSETCPDG KNICVKRSWT AVRGDGPKRE IRRECAATCP PSKLGLTVFC CTTDNCNH. Gamma-bungarotoxin is structurally similar to both kappa-bungarotoxin and elapid long postsynaptic neurotoxins. Its C-terminal nine residues are identical to those of the kappa-toxins. Its disulfide bond locations appear identical to those of several elapid toxins of unknown pharmacology and its hydrophobicity profile is also strikingly similar. However, with an LD50 of 0.15 microg/g i.v. in mice, gamma-bungarotoxin is 30-150-fold more toxic than other members of this latter class. Its toxicity is comparable to those of alpha-nicotinic acetylcholine receptor antagonists.

A single-step purification of bothropstoxin-1

Bothrops venoms are complex mixtures of components with a wide range of biological activities. Among these substances, myotoxins have been investigated by several groups. Bothropstoxin-1 (Bthtx-1) is a phospholipase A2-like basic myotoxin from Bothrops jararacussu. The purification of this component involves two chromatographic steps. Although providing a pure material, the association of these two steps is time consuming and a single-step method using high performance chromatography media would be useful. In the present study, we describe a single-step purification method for Bthtx-1. Bothrops jararacussu venom was dissolved in 1 ml buffer. After centrifugation, the supernatant was injected into a Resource-S cation exchange column connected to an FPLC system and eluted with a linear salt gradient. The complete procedure took 20 min, representing a considerable time gain when compared to a previously described method (Homsi-Brandenburgo MI et al. (1988) Toxicon, 26: 615-627). Bthtx-1 purity and identity, assessed by SDS-PAGE and N-terminal sequencing, resulted in a single band with a molecular mass of about 14 kDa and the expected sequence of the first 5 residues, S-L-F-E-L. Although the amount of protein purified after each run is lower than in the previously described method, we believe that this method may be useful for small-scale purifications

A single-step purification of bothropstoxin-1.

Bothrops venoms are complex mixtures of components with a wide range of biological activities. Among these substances, myotoxins have been investigated by several groups. Bothropstoxin-1 (Bthtx-1) is a phospholipase A2-like basic myotoxin from Bothrops jararacussu. The purification of this component involves two chromatographic steps. Although providing a pure material, the association of these two steps is time consuming and a single-step method using high performance chromatography media would be useful. In the present study, we describe a single-step purification method for Bthtx-1. Bothrops jararacussu venom was dissolved in 1 ml buffer. After centrifugation, the supernatant was injected into a Resource-S cation exchange column connected to an FPLC system and eluted with a linear salt gradient. The complete procedure took 20 min, representing a considerable time gain when compared to a previously described method (Homsi-Brandenburgo MI et al. (1988) Toxicon, 26: 615-627). Bthtx-1 purity and identity, assessed by SDS-PAGE and N-terminal sequencing, resulted in a single band with a molecular mass of about 14 kDa and the expected sequence of the first 5 residues, S-L-F-E-L. Although the amount of protein purified after each run is lower than in the previously described method, we believe that this method may be useful for small-scale purifications.

Primary structures of two proteins from the venom of the Mexican red knee tarantula (Brachypelma smithii).

Venom of the Mexican red knee tarantula (Brachypelma smithii) was fractionated by gel filtration over Sephadex G-50 Fine. Small polypeptides present in the second and third peaks were subfractionated by cation exchange and reversed-phase FPLC. One major, basic protein was isolated and sequenced from each G-50 fraction using a gas-phase protein sequencer. Primary structures were completed and confirmed using tandem mass spectrometry and carboxypeptidase digestions. Protein 1 contains 39 residues, including six cysteine residues in three disulfide bonds. It is identical to one of the isoforms of ESTX from the venom of the tarantula Eurypelma californicum. Brachypelma smithii Protein 5 contains 34 residues, including six cysteine residues in three disulfide bonds. Disulfide bond assignments for both proteins are provided. Protein 5 shows most similarity with toxin Tx2-9 from the Brazilian 'armed' spider, but only displays 41% sequence identity. Similarities with other proteins are lower. Proteins 1 and 5 appear unrelated to each other.

Multiple myotoxin sequences from the venom of a single prairie rattlesnake (Crotalus viridis viridis).

Multiple myotoxin a sequences have been determined from the venom of a single adult male prairie rattlesnake (Crotalus viridis viridis). This is the first time such individual variation has been reported for this toxin class and the number of isoforms suggest that myotoxin a is the product of a duplicated locus.

Human envenomation by the South American opisthoglyph Clelia clelia plumbea (Wied).

A 4-year-old girl was hospitalized 10 hr after having been envenomated by a 1.4 m Clelia clelia plumbea, a colubrid. Although the patient exhibited pronounced edema and local hemorrhage, she did not manifest systemic symptoms. Because the attending physician viewed the case as a possible Bothrops bite, anti-Bothrops antivenom (FUNED) was administered. All local symptoms disappeared gradually over a period of 3 days.

Comparative enzymatic composition of Brazilian coral snake (Micrurus) venoms.

1. Venoms of 11 coral snake taxa, including Micrurus albicinctus, M. corallinus, M. frontalis altirostris, M. f. brasiliensis, M. f. frontalis, M. fulvius fulvius, M. ibiboboca, M. lemniscatus ssp., M. randonianus, M. spixii spixii, and M. surinamensis surinamensis, were examined for 13 enzymatic activities. 2. These were compared with venoms of three outgroup taxa: Naja naja kaouthia, Bungarus multicinctus, and Bothrops moojeni. 3. Enzyme activity levels in Micrurus venoms were highly variable from species to species. 4. All venoms possessed phospholipase activity. 5. Protease activity against synthetic or dyed natural substrates was generally negligible in all elapid venoms examined. By contrast, most Micrurus venoms displayed ample L-leucine aminopeptidase activity. 6. Venom of M.s. surinamensis was significantly different from those of its congeners in most assays.

Comparative chromatography of Brazilian coral snake (Micrurus) venoms.

1. Elution profiles of 11 coral snake venoms, including those of Micrurus albicinctus, M. corallinus, M. frontalis altirostris, M. f. brasiliensis, M. f. frontalis, M. fulvius fulvius, M. ibiboboca, M. lemniscatus ssp., M. rondonianus, M. spixii spixii and M. surinamensis surinamensis, were compared using high performance gel filtration and reverse phase media. 2. Micrurus venom profiles were compared with those of "outgroup" taxa Bothrops moojeni, Naja naja kaouthia and Bungarus multicinctus. 3. Purified elapid venom constituents were also chromatographed under identical conditions in order to suggest possible identities of Micrurus venom constituents. 4. Masses of various components were confirmed by mass spectrometry. 5. Phospholipase constituents in three venoms were positively identified based on their reverse phase chromatograms. 6. Venoms of M. rondonianus and M. s. surinamensis are shown to be significantly different in their peptide composition from other Micrurus venoms.

A new small myotoxin from the venom of the prairie rattlesnake (Crotalus viridis viridis).

Fast atom bombardment (FAB) mass spectrometry was used to identify a new small myotoxin from the venom of the prairie rattlesnake (Crotalus viridis viridis). FAB mass spectrometry and Edman degradation were used to characterize its structure. This toxin is similar to myotoxin I from C. v. concolor, except that it possesses an additional. C-terminal asparaginyl-alanine. At 45 residues it is the longest known myotoxin a homolog. A myotoxin of 43 residues, identical to myotoxin I from C. v. concolor, was also found. To date no other species has been shown to produce more than one length of myotoxin. The present paper documents 42-, 43-, and 45-residue myotoxins from the venom of a single animal.

The amino acid sequence of the acidic subunit B-chain of crotoxin.

The B-chain of the acidic subunit of crotoxin proved refractory to Edman degradation. When subjected to sequence analysis using tandem mass spectrometry, pyroglutamate was found at the amino-terminal end, even though earlier attempts to de-block with pyroglutamate aminopeptidase were unsuccessful. The B-chain contained 35 amino acids and showed 91% amino acid identity with the corresponding segment from Mojave toxin, a homologous neurotoxin from Crotalus scutulatus scutulatus. The sequence of the last 24 residues of the B-chain is consistent with that previously published (Aird, S.D., Kaiser, I.I., Lewis, R.V. and Kruggel, W.G. (1985) Biochemistry 24, 7054-7058), except at position 20, where Edman degradation gave glycine and mass spectrometry gave glutamic acid.

The amino acid sequence of a myotoxic phospholipase from the venom of Bothrops asper.

A myotoxic, basic phospholipase A2 (pI greater than 9.5) with anticoagulant activity has been purified from the venom of Bothrops asper, and its amino acid sequence determined by automated Edman degradation. It is distinct from the B. asper phospholipase A2 known as myotoxin I [Lomonte, B. and Gutierrez, J. M., 1989, Toxicon 27, 725] but cross-reacts with myotoxin I rabbit antisera, suggesting that the proteins are closely related isoforms. To our knowledge, this is the first myotoxic phospholipase to be sequenced that lacks presynaptic neurotoxicity (iv LD50 approximately equal to 8 micrograms/g in mice). The protein appears to exist as a monomer, contains 122 amino acids, and fits with subgroup IIA of other sequenced phospholipase A2 molecules. Its primary sequence shows greatest identity with ammodytoxin B (67%), a phospholipase A2 presynaptic neurotoxin from Vipera ammodytes ammodytes venom. Hydropathy profiles of B. asper phospholipase and the ammodytoxins also show great similarities. In contrast, even though the amino acid sequence identities between B. asper phospholipase and the basic subunit of crotoxin remain high (64%), their hydropathy profiles differ substantially. Domains and residues that may be responsible for neurotoxicity are discussed.

Amino acid sequence of the basic subunit of Mojave toxin from the venom of the Mojave rattlesnake (Crotalus s. scutulatus).

The complete sequence of the basis subunit of Mojave toxin from the venom of the Mojave rattlesnake (Crotalus s. scutulatus) is presented. It is shown to have great similarity to the basic subunits of related toxins from the venoms of the South American and midget faded rattlesnakes.

Comparative spectroscopic studies of four crotoxin homologs and their subunits.

Structures of four related neurotoxins and their purified subunits from the venoms of Crotalus durissus terrificus, C. vegrandis, C. s. scutulatus and C. viridis concolor were examined by circular dichroism (CD), deconvolution Fourier-transform infrared (FTIR) and fluorescence spectroscopy. CD spectra suggest that in general, the isolated subunits were decreased slightly in alpha-helix, while they were increased in beta-sheet structure, relative to intact toxins. These results were consistent with FTIR results. Fluorescence quenching (50-80%) was also observed in three of the four intact toxins as compared to spectra predicted by summation of free acidic and basic subunit spectra. It was tempting to conclude from these results that major conformational changes occur in individual subunits upon formation of the dimeric toxins. Intact crotoxin, however, when exposed to urea, yields spectra (CD, FTIR and fluorescence) that are virtually identical to control intact crotoxin. These findings suggest that the enhanced fluorescence exhibited by the isolated subunits, as well as the secondary structural changes in alpha-helix and beta-sheet, are artifacts resulting from irreversible structural changes that occur during subunit isolation by urea ion-exchange chromatography. In spite of these structural changes, LD50 values of intact crotoxin reassembled from isolated subunits are unaltered from those of native crotoxin.

Spectroscopic characterization of textilotoxin, a presynaptic neurotoxin from the venom of the Australian eastern brown snake (Pseudonaja t. textilis).

Spectroscopic behavior of textilotoxin, from the venom of Pseudonaja t. textilis, and its subunits were investigated using fluorescence, circular dichroism and Fourier transform infrared spectroscopy. Circular dichroism spectra of the B, C and D subunits indicate considerable similarity in their alpha-helix and beta-sheet contents. By contrast, the A subunit displays significantly more beta-sheet and 'remainder' structure. FTIR spectra confirm conclusions drawn from CD spectra. Fluorescence spectra indicate that, in general, tryptophan residues in the A, B and D subunits are relatively exposed to the solvent. The C subunit exhibits no fluorescence, suggesting a lack of tryptophan. Comparisons of individual subunit spectra with those of the intact toxin suggest that significant changes in secondary structure may occur when the toxin dissociates.

A gyroxin analog from the venom of the bushmaster (Lachesis muta muta).

Clinical observations of possible neurotoxic activity in bushmaster (Lachesis muta muta) envenomations, coupled with the accepted ancestral relationship of Lachesis to other crotalids, suggested that Lachesis venom might contain a crotoxin-like molecule. Crude venom and gel-filtration fractions showed modest reactivity in enzyme-linked immunosorbent assays using rabbit polyclonal antibodies raised against the basic subunit of crotoxin, but no reaction was detected with a murine monoclonal antibody raised against the same antigen. Phospholipase assays, LD50 determinations and SDS-polyacrylamide gel electrophoresis indicated the presence of non-toxic phospholipases, but no crotoxin homologs. A higher mol.wt, toxic protein (60,000) with an LD50 of 0.07 micrograms/g in mice was isolated and purified, which induced gyroxin-like, rapid rolling motions in mice. Its amino terminal sequence shows considerable amino acid sequence identity with gyroxin from the venom of Crotalus durissus terrificus and other serine proteases.

Physiological and immunological properties of small myotoxins from the venom of the midget faded rattlesnake (Crotalus viridis concolor).

Myotoxins from C. v. concolor venom were isolated by gel filtration. This crude myotoxin peak was subfractionated into either two or four subfractions by cation exchange FPLC, depending upon the source of the venom. When injected at 2 micrograms/g, crude concolor myotoxin caused vacuolation of mouse muscle cells typical of myotoxin a from C. v. viridis and crotamine from C. d. terrificus. All four subfractions showed qualitatively identical myotoxin activity. In double immunodiffusion studies, myotoxin a antiserum produced lines of identity when reacted with myotoxin a, crude concolor myotoxin and the four concolor subfractions. A second batch of material showed two major components when subfractionated by cation exchange FPLC. The more basic of these two components displayed approximately twice the i.v. lethality of the more acidic component. The LD50 for the basic component lies between 0.625 and 0.75 microgram/g while that of the acidic component falls between 1.00 and 1.25 micrograms/g.

A crotoxin homolog from the venom of the Uracoan rattlesnake (Crotalus vegrandis).

A major protein toxin from the venom of Crotalus vegrandis was examined by gel filtration, anion-exchange chromatography, and SDS polyacrylamide gel electrophoresis. The toxin was separated into several isoforms by ion-exchange chromatography and spontaneously dissociated into free acidic and basic subunits, mimicking the behavior of crotoxin. Rabbit antisera raised against crotoxin reacted strongly in enzyme-linked immunosorbent assays with the intact C. vegrandis toxin isoforms and their basic subunits, and formed precipitin lines of identity with intact crotoxin in double immunodiffusion gels. These results indicate that vegrandis toxin is strongly homologous with crotoxin from the venom of Crotalus durissus terrificus.

A complete amino acid sequence for the basic subunit of crotoxin.

The complete amino acid sequence of the basic subunit of crotoxin from the venom of Crotalus durissus terrificus has been determined. Fragmentation of the protein was achieved by using cyanogen bromide and arginine- and lysine-specific endoproteases. Sixteen Glx and Asx residues reported by Fraenkel-Conrat et al. (1980) in Natural Toxins (D. Eaker and T. Wadstrom, eds.), pp. 561-567, Pergamon, Oxford.) have been resolved as Glu or Gln and Asp or Asn residues, respectively. Most of the remaining sequence is identical to that reported by the foregoing authors although several significant differences were evident in our protein. Tyr-61 was not present; thus the correct sequence is Lys-60, Trp-61. The latter sequence aligns with sequences of all other known viperid and crotalid phospholipases A2 (S. D. Aird, I. I. Kaiser, R. V. Lewis, and W. G. Kruggel (1985) Biochemistry 24, 7054-7058). Other differences include Asx-99, which is Ser, and Asx-105, which is Tyr. Some positions display allelic variation. In some lots of venom Glx-33 is Gln, while in others it is Arg. Positions 37 and 69 occur as mixtures of both Lys and Arg. Amino acid sequence comparisons between the basic and acidic subunits of crotoxin and between the basic subunit and other phospholipase A2 molecules indicate that the basic subunit is structurally most similar to the monomers of nontoxic, dimeric phospholipases A2 from the venoms of Crotalus adamanteus, Crotalus atrox, and Trimeresurus okinavensis, and to the toxic monomeric phospholipase A2 from the venom of Bitis caudalis.