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Some chemical properties of the venom of the rattlesnake, Crotalus viridis helleri
Abstract
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.
... The N-terminus of F7 was obtained, which eluted in minute 38, with the sequence YKRCHKKGGHCFPKTVICLPPSSDFGKMDCR, matching 100% with a crotamine from C. helleri helleri (AEU60012.1) from a venom gland, Protein Database, NCBI, and 100% with peptide C of C. helleri helleri venom reported by Maeda and Tamiya [44]. ...
... Eighteen of the 73 spots were not assigned to a snake venom family. Using the PDQuest software (Biorad), SVMP, SVSP, and crotamine were found to comprise~65-75% of the proteins separated by two-dimensional SDS-PAGE of C. helleri caliginis venom, with crotamine being the most abundant component (30-35%), similar to what is reported by Maeda et al. [44] for C. helleri helleri. ...
... These fractions represent approximately 30-35% of the total venom ( Figure 3E-F). The peak 7 sequence (YKRCHKKGGHCFPKTVICLPPSSD-FGKMDCR) is 100% identical to the peptide C sequence reported by Maeda et al. [44] in the C. helleri helleri venom. The peak 8 sequence is similar to peak 7, with only changes in the amino acid residues 15, 16, and 19 (YKRCHKKGGHCFPKEKICIPPSSDFG). ...
The Baja California Peninsula has over 250 islands and islets with many endemic species. Among them, rattlesnakes are the most numerous but also one of the least studied groups. The study of island rattlesnake venom could guide us to a better understanding of evolutionary processes and the description of novel toxins. Crotalus helleri caliginis venom samples were analyzed to determine possible ontogenetic variation with SDS-PAGE in one and two dimensions and with RP-HPLC. Western Blot, ELISA, and amino-terminal sequencing were used to determine the main components of the venom. The biological and biochemical activities demonstrate the similarity of C. helleri caliginis venom to the continental species C. helleri helleri, with both having low proteolytic and phospholipase A2 (PLA2) activity but differing due to the absence of neurotoxin (crotoxin-like) in the insular species. The main components of the snake venom were metalloproteases, serine proteases, and crotamine, which was the most abundant toxin group (30–35% of full venom). The crotamine was isolated using size-exclusion chromatography where its functional effects were tested on mouse phrenic nerve–hemidiaphragm preparations in which a significant reduction in muscle twitch contractions were observed. The two Mexican antivenoms could neutralize the lethality of C. helleri caliginis venom but not the crotamine effects.
... The three other peptides are isoforms of myotoxin-3 and differ from myotoxin-3 in the number of residues and in the presence of either Histidine or Leucine at position 5 (45-residue Leu 5 -myotoxin-3, 43-residue His 5 -myotoxin-3 and 43-residue Leu 5 -myotoxin-3). The 43residue His 5 -myotoxin-3 isoform, called peptide C, was isolated in 1978 from Crotalus viridis helleri venom [23]. ...
Microtubule targeting agents (MTA) are anti-cancer molecules that bind tubulin and interfere with the microtubule functions, eventually leading to cell death. In the present study, we used an in vitro microtubule polymerization assay to screen several venom families for the presence of anti-microtubule activity. We isolated myotoxin-3, a peptide of the crotamine family, and three isoforms from the venom of the Northern Pacific rattlesnake Crotalus oreganus oreganus, which was able to increase tubulin polymerization. Myotoxin-3 turned out to be a cell-penetrating peptide that slightly diminished the viability of U87 glioblastoma and MCF7 breast carcinoma cells. Myotoxin 3 also induced remodeling of the U87 microtubule network and decreased MCF-7 microtubule dynamic instability. These effects are likely due to direct interaction with tubulin. Indeed, we showed that myotoxin-3 binds to tubulin heterodimer with a Kd of 5.3 µM and stoichiometry of two molecules of peptide per tubulin dimer. Our results demonstrate that exogenous peptides are good candidates for developing new MTA and highlight the richness of venoms as a source of pharmacologically active molecules.
... Most current knowledge of myotoxin sequence diversity comes from homologous peptides identified from the venom of several rattlesnake species including C. durissus (Laure, 1975), C. viridis (Cameron and Tu, 1977;Griffin and Aird, 1990), C. oreganus (Bieber et al., 1987;Maeda et al., 1978), C. adamanteus (Samejima et al., 1991), and C. horridus (Allen et al., 1996). To date, the only description of the genic architecture of any myotoxin is that of crotamine from C. d. terrificus, which is approximately 1.8 kb in length and contains three exons, separated by a long and a short intron (Oguiura et al., 2005;Rádis-Baptista et al., 2003). ...
Crotamine, myotoxin a and homologs are short peptides that often comprise major fractions of rattlesnake venoms and have been extensively studied for their bioactive properties. These toxins are thought to be important for rapidly immobilizing mammalian prey and are implicated in serious, and sometimes fatal, responses to envenomation in humans. While high quality reference genomes for multiple venomous snakes are available, the loci that encode myotoxins have not been successfully assembled in any existing genome assembly. Here, we integrate new and existing genomic and transcriptomic data from the Prairie Rattlesnake (Crotalus viridis viridis) to reconstruct, characterize, and infer the chromosomal locations of myotoxin-encoding loci. We integrate long-read mRNA (Pacific Bioscience's Iso-Seq) and short-read RNA-seq to infer gene sequence diversity and characterize patterns of myotoxin and paralogous β-defensin expression across multiple tissues. We also identify two long non-coding RNA sequences which both encode functional myotoxins, demonstrating a newly discovered source of venom coding sequence diversity. We also integrate long-range mate-pair chromatin contact data and linked-read sequencing to infer the structure and chromosomal locations of the three myotoxin-like loci. Further, we conclude that the venom-associated myotoxin is located on chromosome 1 and is adjacent to non-venom paralogs. Consistent with this locus contributing to venom composition, we find evidence that the promoter of this gene is selectively open in venom gland tissue and contains transcription factor binding sites implicated in broad trans-regulatory pathways that regulate snake venoms. This study provides the best genomic reconstruction of myotoxin loci to date and raises questions about the physiological roles and interplay between myotoxin and related genes, as well as the genomic origins of snake venom variation.
... o. helleri). Our results can be explained by the high level of identity found in crotamine molecules described in the venoms from these three different species (Borja et al., 2018;Laure, 1975;Maeda et al., 1978, Fig. 1 S). Recently was described a B-cell linear epitope in crotamine from C. d. terrificus (Vaz et al., 2020), which is likely to be shared by crotamine molecules of several other species allowing cross-reactivity and neutralization between them, but this assumption deserves further attention. ...
Crotamine is a paralyzing toxin (MW: ∼ 5 kDa) found in different proportions in some rattlesnake venoms (up to 62%). Mexican pit viper antivenoms have shown low immunoreactivity against crotamine, which is an urgent quality to be improved. The objective of this work was to evaluate the ability of a novel recombinant fusion protein composed of sphingomyelinase D and crotamine, and two whole venoms from Crotalus molossus nigrescens and C. oreganus helleri to produce neutralizing antibodies against crotamine. These immunogens were separately used for immunization procedures in rabbits. Then, we generated three experimental antivenoms to test their cross-reactivity via western-blot against crotamine from 7 species (C. m. nigrescens, C. o. helleri, C. durissus terrificus, C. scutulatus salvini, C. basiliscus, C. culminatus and C. tzabcan). We also performed pre-incubation neutralization experiments in mice to measure the neutralizing potency of each antivenom against crotamine induced hind limb paralysis. Our antivenoms showed broad recognition across crotamine from most of the tested species. Also, neutralization against crotamine paralysis symptom was successfully achieved by our three antivenoms, albeit with different efficiencies. Our results highlight the use of crotamine enriched venoms and our novel recombinant fusion protein as promising immunogens to improve the neutralizing potency against crotamine for the improvement of Mexican antivenoms.
... Crotalus oreganus helleri venom is known to contain a neurotoxin similar to that of mojave toxin (French et al., 2004;Mackessy, 1988Mackessy, , 2008. However, visual observation of mice injected with Riverside 2 and San Bernardino 1 venoms showed the possible presence of a crotamine-like toxin (Maeda et al., 1979) due to the spastic paralysis of the hind limbs (Gonçalves, 1956;Cheymol et al., 1971;Chang and Tseng, 1978) that we observed in the mice. Riverside 1 venom also induced such paralysis but in a milder form, which indicates the quantitative differences in toxins amongst these venoms, which is also apparent in the LD 50 (Table 1). ...
Envenomations by the southern Pacific rattlesnake (Crotalus oreganus helleri) are the most common snakebite accidents in southern California. Intraspecies venom variation may lead to unresponsiveness to antivenom therapy. Even in a known species, venom toxins are recognized as diverse in conformity with interpopulational, seasonal, ontogenetic and individual factors. Five venoms of individual C. oreganus helleri located in Riverside and San Bernardino counties of southern California were studied for their variation in their hemostatic activity. The results demonstrated that Riverside 2 and San Bernardino 1 venoms presented the highest lethal activity without hemorrhagic activity. In contrast, San Bernardino 2 and 3 venoms had the highest hemorrhagic and fibrinolytic activities with low lethal and coagulant activities. Riverside 1, Riverside 2 and San Bernardino 1 venoms presented a significant thrombin-like activity. San Bernardino 2 and 3 venoms presented an insignificant thrombin-like activity. In relation to the fibrinolytic activity, San Bernardino 3 venom was the most active on fibrin plates, which was in turn neutralized by metal chelating inhibitors. These results demonstrate the differences amongst C. oreganus helleri venoms from close localities. A metalloproteinase, hellerase, was purified by anionic and cationic exchange chromatographies from San Bernardino 3 venom. Hellerase exhibited the ability to break fibrin clots in vitro, which can be of biomedically importance in the treatment of heart attacks and strokes.
... Peaks IX and X were partially retained past the total column volume, and may represent basic peptides. Peak VIII is also likely to be a peptide component of the venom, perhaps a myotoxin (Cameron and Tu, 1977;Maeda et al., 1978) since it eluted at a position expected for a molecule of Mr 4000-5000. ...
1. Venoms of Crotalus viridis oreganus show marked ontogenetic variation in protease activity. Adult venoms are approximately five-fold higher in protease (caseinolytic) activity. 2. Of seven potential protease inhibitors, only EDTA and 1,10-phenanthroline caused a significant decrease in protease activity. Responses of juvenile and adult venoms were essentially equivalent, and attempts at recovery of protease activity of EDTA-treated venoms by the addition of Ca2+ or Zn2+ were unsuccessful. 3. Gel filtration resolved two proteases from juvenile and subadult venoms with approximate M(r) of 100,000 and 78,000. Four proteases were resolved from adult venom, and M(r) estimates were 78,000, 61,000, 35,000 and 19,000. 4. Proteases from juvenile and adult venoms showed fibrinogenolytic activity, each producing some unique degradation products. 5. The occurrence of three "new" proteases in adult venom produced the ontogenetic increase in activity seen in the crude venoms.
Crotamine and crotamine-like peptides are non-enzymatic polypeptides, belonging to the family of myotoxins, which are found in high concentration in the venom of the Crotalus genus. Helleramine was isolated and purified from the venom of the Southern Pacific rattlesnake, Crotalus oreganus helleri. This peptide had a similar, but unique, identity to crotamine and crotamine-like proteins isolated from other rattlesnakes species. The variability of crotamine-like protein amino acid sequences may allow different toxic effects on biological targets or optimize the action against the same target of different prey. Helleramine was capable of increasing intracellular Ca2+ in Chinese Hamster Ovary (CHO) cell line. It inhibited cell migration as well as cell viability (IC50 = 11.44 μM) of C2C12, immortalized skeletal myoblasts, in a concentration dependent manner, and promoted early apoptosis and cell death under our experimental conditions. Skeletal muscle harvested from mice 24 h after helleramine injection showed contracted myofibrils and profound vacuolization that enlarged the subsarcolemmal space, along with loss of plasmatic and basal membrane integrity. The effects of helleramine provide further insights and evidence of myotoxic activities of crotamine-like peptides and their possible role in crotalid envenomings.
Snake venom myotoxins can be categorized into three types: small, basic polypeptides such as myotoxin a and crotamine; cardiotoxins from cobra venoms; and phospholipase A(2) toxins such as crotoxin and notexin. All three types of myotoxins induce depolarization and contraction of skeletal muscle cells. However, the myonecrosis induced by the small, basic polypeptide myotoxins is different from that induced by the cardiotoxins and phospholipase A(2) myotoxins in that the former do not appear to lyse the sarcolemma whereas the latter two types cause lysis of the sarcolemma which is of rapid onset. Molecular properties of the toxins are similar in that they are all highly basic proteins, and a large portion of their surface charge is positive. Also, they all have considerable beta-sheet structure which may be involved in interaction with the membrane. The purpose of this review is to describe the structure and function of these myotoxins and to evaluate features they might share which could shed light on their mechanism of myotoxic action.
The interaction of myotoxin α with intact sarcoplasmic reticulum (SR) components was investigated, and two SR proteins were identified that associated with myotoxin α. One of the proteins has an apparent molecular weight similar to the Ca{sup 2+}-ATPase, the major SR protein responsible for calcium loading. Ca{sup 2+}-ATPase was established by isolating chemically cross-linked myotoxin αCa{sup 2+}-ATPase complexes and further delineated by cleaving the protein and a smaller C-terminal fragment of 14 residues. Competition experiments with ¹²âµI-myotoxin α showed that the C-terminal fragment competed better against ¹²âµI-myotoxin α than the N-terminal fragment for SR protein binding. Two overlapping peptides covering the sequence of the N-terminal fragment were synthesized to clarify the interaction of the N-terminal fragment of myotoxin α with SR proteins. A 16-residue peptide corresponding to residues 1-16 competed strongly with ¹²âµI-myotoxin α, while a second peptide (residues 13-28) did not.
The effects of an acidic phospholipase A2 (PLA2) from king cobra (Ophiophagus hannah) venom on mouse soleus muscle were studied by light and electron microscopic examination. Mice were injected intramuscularly with doses of 8 mg/kg of O. hannah venom PLA2 and tissue samples were taken at 4, 6, 24, 48 hr, and 7 days. The PLA2 induced a progressive muscle degeneration and necrosis after injection. At 4 and 6 hr, infrequent damage to the peripheral muscle fibres could be observed. The plasma membranes of affected cells were ruptured in the area of delta lesions, the myofibrils were hypercontracted, eventually forming dumped dense masses. By 24 hr and 48 hr, the affected cells were in an advanced stage of necrosis and showed a more homogeneous appearance. There was a widespread degradation of myofibrillar components in the cellular space. Some mitochondria were swollen and had flocculent electron densities in their matrix spaces, and most of them showed only one membrane. Phagocytosis of muscle fibers started by 4 hr and was very obvious by 48 hr. Regeneration of muscle was rapid; at 7 days many regenerated fibres containing centrally located nuclei were seen. It is concluded that the acidic PLA2 from O. hannah venom causes necrosis of skeletal muscle cells after i.m. injection.
Snake venom myotoxins can be categorized into three types: small, basic polypeptides such as myotoxin a and crotamine; cardiotoxins from cobra venoms; and phospholipase A2, toxins such as crotoxin and notexin. All three types of myotoxins induce depolarization and contraction of skeletal muscle cells. However, the myonecrosis induced by the small, basic polypeptide myotoxins is different from that induced by the cardiotoxins and phospholipase A2 myotoxins in that the former do not appear to lyse the sarcolemma whereas the latter two types cause lysis of the sarcolemma which is of rapid onset. Molecular properties of the toxins are similar in that they are all highly basic proteins, and a large portion of their surface charge is positive Also, they all have considerable β-sheet structure which may be involved in interaction with the membrane The purpose of this review is to describe the structure and function of these myotoxins and to evaluate features they might share which could shed light on their mechanism of myotoxic action
Abstract Myotoxin a and a group of closely related, small, basic toxins cause myonecrotic destruction of muscle tissue upon envenomation. The sarcoplasmic reticulum swells and eventually breaks down to small vesicles. Degeneration of myofibrils and myofilaments ensues and loss of the classic striation pattern is apparent. These toxins exhibit high sequence similarity as well as sequence microheterogeneity. A conformational heterogeneity was recently discovered in myotoxin a. The existence of myotoxin a in two forms in equilibrium in solution hinders the generation of a well defined three-dimensional structure. The difference, if any, in the biological activity of the two forms has not been established yet. Recent biochemical studies indicate that myotoxin a is a potent Ca2+ releasing agent that binds to calsequestrin in the lumen of the sarcoplasmic reticulum.
Abstract The fractionation of the previously unstudied venom for Crotalus viridis concolor (midget faded rattlesnake) provided a protein fraction (FD-1) that generated myotoxic responses from envenomated mice. Highly basic homologous myotoxic proteins have been purified from the protein fraction, FD-1. The two major components, designated myotoxin I and myotoxin II, have considerable sequence homology with crotamine from C. d. terrificus, myotoxin a from C. v. viridis and peptide c from C. v. helleri. The amino acid sequences of the toxins from C. v. concolor contain at least two more residues of amino acid than previously described homologous toxins and myotoxin II contains a sequence region with microheterogeneity. The myotoxins cause extensive damage to the sarcoplasmic reticulum as evidenced by electron microscopic studies of envenomated muscle. The destruction is comparable to that observed with the homologous toxins. Studies with isolated sarcoplasmic reticulum vesicles failed to demonstrate any significant changes in Ca2+-Mg2+-ATPase or in uptake or release of Ca2+. Current evidence indicates that a closely related group of homologous toxins are present in rattlesnake venoms but the biochemical mode of action of these proteins remains obscure.
The 400 MHz 1H-NMR spectrum of myotoxin a from the venom of Crotalus viridis viridis is described. The identification of spin systems in the aromatic region corresponding to the six aromatic residues of myotoxin a was completed using both one- and two-dimensional NMR spectroscopy and the pH dependence of chemical shifts. Assignments of these spin systems to specific residues was possible for the singly occuring amino acids Tyr-1 and Phe-12. Resonances from Tyr-1, His-5 and His-10 were shifted significantly from their random coil values in a pH-dependent manner. These shift perturbations were deemed evidence of a helical arrangement of the amino terminal region which placed these residues in close proximity to each other.
R. M. Kini and S. Iwanaga. Structure - function relationships of phospholipases II: charge density distribution and the myotoxicity of presynaptically neurotoxic phospholipases. Toxicon24, 895 – 905, 1986.—The charge density distribution of 24 phospholipases A2 has been examined to identify the involvement of charged amino acid residues in the determination of the pharmacological properties of these proteins. There is no characteristic difference between the presynaptically neurotoxic and non-neurotoxic phospholipases, however, presynaptically neurotoxic phospholipases which are also myotoxic have a distinct charge distribution pattern. There is a characteristic cationic site around residues 79 – 87. This site has a relatively fixed position with respect to the hydrophobic ‘neurotoxic’ region, on the NH2 terminal side. This cationic region is located on the outer surface in the three-dimensional structure of phospholipase, just before hydrophobic helix E, and is available for interaction with membranes. Such a characteristic region is absent in non-myotoxic phospholipases which are either presynaptically active or inactive. On the other hand, myotoxins, a group of non-enzymatic proteins inducing myotoxicity, also possess such characteristic regions of cationic and hydrophobic sites.
Antiserum to myotoxin a was tested for its ability to prevent local myonecrosis induced by myotoxin a and C. v. viridis venom. Antiserum was injected i.v. either 5 min before or immediately, 15 min, 30 min, 1 hr or 3 hr after i.m. injection of toxin or venom. A light microscopic method was used to measure the effects of myotoxin a, i.e. vacuolation index, and whole venom, i.e. myonecrosis index. The results show that antimyotoxin a serum neutralizes the myotoxicity of a sublethal amount of myotoxin a if injected 5 min before or immediately after toxin, but not if injected 15 min after the toxin. Its neutralizing ability for crude C. v. viridis venom was considerably better, neutralizing a dose of 0.75 μg/g even if injection of antiserum was delayed for 30 min after venom injection.Thus, antimyotoxin a serum might be useful in treating myonecrosis resulting from prairie rattlesnake (C. v. viridis) venom poisoning.
Capillary electrophoresis (CE) was used to study myotoxins, members of a highly homologous family of small, basic, non-enzymatic proteins found in rattlesnake venoms. Several rattlesnake species were investigated and conditions were set to optimize the one-step separation of the myotoxins from the rest of the venom components. Myotoxin containing venoms, when subjected to CE analysis, yielded multiple myotoxin peaks. The myotoxin were collected and further analysed by matrix-assisted laser desorption time-of-flight mass spectrometry. In some instances, multiple mass peaks were observed from the mass spectra, suggesting the existence of myotoxin isoforms. Furthermore, each venom that contained myotoxin yielded fractions with indistinguishable masses, indicating that myotoxin isomers were present. The isomers were readily detected in the electropherograms since they exist in a ca. 1:4 ratio, data already established for myotoxin a from the prairie rattlesnake. An in-column incubation method was used to address the re-equilibration of the separated isomers.
Ninety-five venom samples from eight snake genera (Agkistrodon, Bitis, Bothrops, Calloselasma, Crotalus, Sistrurus, Naja and Vipera) including venoms of Crotalus species of different geographical origin were assayed using imunodiffusion or an ELISA for the presence of the small basic protein, myotoxin a, known to cause muscle necrosis. Of the eight genera investigated, only Crotalus and Sistrurus venoms contained detectable amounts of myotoxin a-like proteins. The venoms of 13 out of 17 rattlesnake species investigated contained proteins immunologically similar to myotoxin a, including 12 Crotalus species and one Sistrurus species. The highest amounts were detected in venoms of C. exsul, C. viridis oreganus and C. v. viridis. Qualitative differences in the presence or absence of myotoxin a-like proteins were observed in the venoms of C. cerastes, C. horridus, C. lepidus, C. mitchelli, C. scutulatus, C. viridis and S. catenatus specimens of different geographic origin. The toxin was not detected in the venoms obtained from C. adamanteus, C. atrox, C. enyo or C. vegrandis specimens. The toxin appears to be widely distributed among rattlesnake species in the new world, but may vary qualitatively by geographical region in several species and subspecies.
We performed the present experiments to study the action of crotamine, a toxin isolated from the venom of the South American rattlesnake, Crotalus durissus terrificus, on macroscopic Na+ currents in frog skeletal muscle by using the loose patch clamp technique. Crotamine at 50 μM increased the peak Na+ current by 50% (P<0.05). In addition, the voltage dependence of inactivation was shifted by +8 mV. Other parameters of Na+ currents (reversal potential, voltage-dependence of activation and time courses of inactivation, of activation and of removal of inactivation) were not significantly affected. We suggest that crotamine inhibits the direct transition of channels from closed to inactivated states, thereby forcing their transition through the open states.
Crotamine, a low molecular weight cationic polypeptide from the venom of the South American rattlesnake Crotalus durissus terrificus is a natural cell-penetrating peptide with functional versatility. The presence of nine lysine residues and three disulfide bonds renders crotamine highly compact, stable and positively charged. Topologically, crotamine adopts an ancient β-defensin fold that is found in diverse families of endogenous and venom polypeptides dedicated to host defense. Crotamine is unique among several classes of bioactive peptides because it possesses both cell penetrating and antimicrobial activities and selective biological action toward some cell types at a given cell cycle phase. Because it can rapidly and efficiently translocate into actively proliferating cells, crotamine is being investigated for labeling highly replicating cells and for use as a chemotherapeutic adjuvant. Peptides derived from crotamine, nucleolar targeting peptides (NrTPs), have been designed and are being studied. NrTPs retain some crotamine properties, such as efficient cellular uptake and preferential nuclear localization whereas they improve upon other properties. For example, NrTPs are smaller than crotamine, show higher preferential nucleolar localization, and better facilitate ZIP-code localization of therapeutic proteins.
Two major crotamine isoforms (III-4 and III-7) were obtained combining two chromatographic steps on molecular exclusion chromatography (Sephadex G-75) and ion-exchange column (Protein Pack SP 5PW) of the rattlesnake Crotalus durissus cumanensis venom. The "in vivo" myotoxic effect of the venom, its "in vitro" cytotoxicity in myoblasts and myotubes (C2C12) and the neurotoxic and edema-forming activity were characterized. The molecular masses of the crotamine isoforms were 4907.94 Da (III-4) and 4985.02 Da (III-7) and, as determined by mass spectrometry, both contained six Cys residues. Enzymatic hydrolysis followed by de novo sequencing through tandem mass spectrometry was used to determine the primary structure of both isoforms. III-4 and III-7 isoforms presented a 42-amino acid residues sequence and showed high molecular amino acid sequence identity with other crotamine-like proteins from Crotalus durissus terrificus. In vivo, both crotamine isoforms induced myotoxicty and a systemic interleukin-6 response upon intramuscular injection. These new crotamine isoforms induced low cytotoxicity in skeletal muscle myoblasts and myotubes (C2C12) and both induced a facilitatory effect on neuromuscular transmission in young chick biventer cervicis preparation. Edema-forming activity was also analyzed by injection of the crotamine isoforms into the right paw, since both crotamine isoforms exert a strong pro-inflammatory effect.
R. C. Schaeffer, Jr., T. R. Pattabhiraman, R. W. Carlson, F. E. Russell and M. H. Weil. Cardiovascular failure produced by a peptide from the venom of the Southern Pacific rattlesnake, Crotalus viridis helleri. Toxicon17, 447–453, 1979.—Hemodynamic, metabolic and respiratory effects of a 30 min i.v. infusion of crude venom (1·4 mg/kg) and three venom components (Peptide I, 0·5 mg/kg; Protein I, 1·2 mg/kg, Protein II, 3·4 mg/kg) were studied in 24 sedated rats (270–309 g). Venom shock, characterized by hypotension, lactacidemia, hemoconcentration, hypoproteinemia and death was observed in animals given the crude venom or Peptide I. Just prior to death, respiratory distress was observed in most animals that died. Hemolysis and hematuria were observed in the animals given Protein I or Protein II. These data suggest that the increase in vascular permeability to protein and red blood cells induced by the crude venom can, for the most part, be attributed to the peptide. In addition, Protein I and Protein II appear to account for the hemolytic activity. The toxic effects of the venom components appear to be synergistic.
Cultured myoblasts and moytubes were used to study the effects of purified myotoxins from rattlesnake venoms. Standard cell culture techniques were used to establish and maintain primary cultures derived from neonatal rat tissue and two clonal cell lines, rat RMo cells and mouse C2 cells. Toxin concentrations, ranging from 0.04 to 1.0 microM, were added to the cultures at various times under distinct, well-defined conditions. Addition of myotoxin alpha to primary myoblast cultures did not appear to affect the fusion process, whereas similar experiments with two clonal cell lines produced larger myotubes when contrasted with untreated control cultures, particularly with RMo cells. The myotubes derived from primary cell cultures twitched spontaneously but the twitching ceased when the medium was replaced with a serum-free chemically defined incubation medium. Addition of myotoxin alpha to the primary myotubes incubated with serum-free defined medium caused the myotubes to twitch again. Derivatives of myotoxin alpha were prepared by reactions with tetranitromethane and with iodoacetic acid, the latter under reducing and non-reducing conditions. The resulting products, purified but not chemically characterized, were nearly devoid of activity when primary cultures were used to test activity.
1. Reverse-phase HPLC and organic solvents were used to isolate small basic peptide (SBP) toxins from the venoms of Crotalus adamanteus, C. durissus terrificus, C. horridus, C. scutulatus scutulatus, C. viridis concolor, C. viridis helleri and C. viridis viridis. 2. Acid-DEP analyses indicated a high degree of toxin purity which was obtained with a single HPLC run. 3. The combined results of HPLC, immunodiffusion and electrophoresis analyses of venoms from different geographical regions indicate that the SBP toxin content in the venoms of Crotalus adamanteus, Crotalus horridus, Crotalus scutulatus and Crotalus viridis viridis may vary regionally.
The complete amino acid sequence of a myotoxin isolated from Crotalus adamanteus venom (CAM-toxin) was determined. The total number of amino acid residues was 45, giving a mol. wt of 5202. The amino acid sequence was compared with those of previously determined Crotalus myotoxins. The CAM-toxin shows a surprisingly close relationship to those of peptide c (C. v. helleri), myotoxin a (C. v. viridis), crotamine (C. d. terrificus), myotoxin I and II (C. v. concolor) with homologies of approximately 98, 83, 90, 95 and 91%, respectively. The hydropathy profile of the CAM-toxin is almost identical to peptide c and myotoxin I, as can be assumed from the sequence homology. Despite the absence of any close sequence homology with the myotoxic phospholipase A2 enzymes, CAM-toxin contains a similar cationic myotoxic region at residues 2-10 as found in the other five myotoxic peptides.
A basic, dimeric myotoxic protein, myotoxin II, purified from Bothrops asper venom has a similar molecular weight and is immunologically cross-reactive with antibodies raised to previously isolated B. asper phospholipases A2, except that it shows only 0.1% of the phospholipase activity against L-alpha-phosphatidylcholine in the presence of Triton X-100. Its 121 amino acid sequence, determined by automated Edman degradation, clearly identifies it as a Lys-49 phospholipase A2. Key amino acid differences between myotoxin II and phospholipase active proteins in the Ca2(+)-binding loop region, include Lys for Asp-49, Asn for Tyr-28, and Leu for Gly-32. The latter substitution has not previously been seen in Lys-49 proteins. Other substitutions near the amino terminus (Leu for Phe-5 and Gln for several different amino acids at position 11) may prove useful for identifying other Lys-49 proteins in viperid and crotalid venoms. Myotoxin II shows greater sequence identity with other Lys-49 proteins from different snake venoms (Agkistrodon piscivorus piscivorus, Bothrops atrox, and Trimeresurus flavoviridis) than with another phospholipase A2 active Asp-49 molecule isolated from the same B. asper venom. This work demonstrates that phospholipase activity per se, is not required in phospholipase molecules for either myotoxicity or edema inducing activities.
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.
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.
Necrosis of skeletal muscle is produced by two types of snake venom components: single chain peptides consisting of 42-44 amino acid residues and phospholipases A2 representing either single chain proteins or existing as complexes of several enzyme subunits or combined with other nonenzymatic proteins. Vacuolation, lysis and necrosis of skeletal muscle cells are the major pathological effects of these myotoxins. Although the exact mode of action of these toxins is not clear, interactions with the plasma membrane leading to permeability changes for ions and to their complete destruction is evident. The high specificities of some venom phospholipases A2 for skeletal muscle cells suggest a specific binding to certain membrane receptors; however, an enzymatic action on membranes may also be involved.
A cDNA library containing snake toxin genes was constructed in bacteriophage lambda by using mRNA isolated from the glands of the South American rattlesnake, Crotalus durissus terrificus. The first high-density screening of 400,000 plaques for crotamine-containing genes yielded over 800 positives when a labeled cDNA probe with sequence homology to crotamine was used. Four of these clones with insert sizes from 270 to 400 base pairs were chosen and their inserts subcloned into pGEM-3Z and sequenced. Nucleotide sequence analysis of the cloned cDNAs predicted the existence of multiple variants of the crotamine toxin. The different forms, identified from the DNA sequences, displayed discrepancies in amino acid sequence for crotamine when compared with previously published reports. Direct amino acid sequencing of commercially purified crotamine and CNBr fragments thereof confirmed the structures predicted by the nucleic acid sequences.
Several proteins and polypeptides of reptilian, amphibian, insect, and microbial origin share a common cytolytic property. However, these cytolysins fulfill different objectives. They provide offensive armament in the case of toxins, but defensive systems in the case of antibacterial peptides. The sequences of several nonenzymatic cytolysins and their analogues were compared to identify the structural requirements for cytolytic activity. These cytolysins, although isolated from phylogenetically unrelated organisms, possess the common sequence features of a cationic site flanked by a hydrophobic surface. The presence of such a region apparently confers the cytolytic activity of various cytolysins. The concept of a cytolytic region is strongly supported by the existence of several natural and synthetic analogues of cytolysins and by chemical modification studies of these cytolysins. This prediction provides a new focus for cytolysin research. The understanding of this structure-function relationship should facilitate the design, synthesis, and development of better antibacterial and anticancer peptides.
Proton nuclear magnetic resonance (NMR) spectra of crotamine, a myotoxic protein from a Brazilian rattlesnake (Crotalus durissus terrificus), have been analyzed. All the aromatic proton resonances have been assigned to amino acid types, and those from Tyr-1, Phe-12, and Phe-25 to the individual residues. The pH dependence of the chemical shifts of the aromatic proton resonances indicates that Tyr-1 and one of the two histidines (His-5 or His-10) are in close proximity. A conformational transition takes place at acidic pH, together with immobilization of Met-28 and His-5 or His-10. Two sets of proton resonances have been observed for Ile-17 and His-5 or His-10, which suggests the presence of two structural states for the crotamine molecule in solution.
Biochemical differences in white and yellow venoms produced in the separate venom glands of an individual southern Pacific rattlesnake (Crotalus viridis helleri) were investigated. Compared to the yellow venom, the white venom contained fewer low molecular weight components and was considerably less toxic. Although the exact LD50 was not determined, the white venom did not produce toxic effects in mice when injected i.v. at concentrations up to 10 mg/kg. The i.v. LD50 of the yellow venom was approximately 1.6 mg/kg. Both white and yellow venoms had hemorrhagic activity, but the white venom caused less intradermal hemorrhage in mice. No L-amino acid oxidase activity was measured in the white venom and protease and phospholipase A2 activities of the white venom were much less than in the yellow venom. The white and yellow venoms both produced myonecrosis at 1, 3 and 24 hr after i.m. injection into mice, however, there were some qualitative differences in the myonecrosis produced. When the venom samples were reacted against Wyeth's polyvalent (Crotalidae) antivenom using immunodiffusion, three precipitin bands formed against the yellow venom, whereas only one formed against the white venom. When reacted against an antiserum to myotoxin alpha from C. viridis viridis venom, both the white and yellow venoms produced one precipitin band each.
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.
Rattlesnake bite is most common in young men who often are intoxicated and have purposely handled a venomous snake. The incidence of bites is highest in the spring and early summer months, and they most often occur in the afternoon. The hands and feet only are involved in 95% of all bites. First-aid therapy should be limited to splinting the extremity and transporting the victim to a medical facility. Definitive therapy is administering antivenin (Crotalidae) polyvalent intravenously in adequate initial doses and repeating every two hours until the venom is completely neutralized. Serum sickness usually follows all doses of more than five vials but is readily controlled by giving corticosteroids. Bites are avoided by protecting the hands and feet, not handling venomous snakes, and using utmost caution while in the snakes' habitat.
Myotoxins I and II were isolated from the venom of Crotalus viridis concolor. Complete sequences were derived for each reduced, alkylated toxin with data obtained by a single run on a gas phase sequencer and from fragments derived by cyanogen bromide cleavage. The results demonstrate that microheterogeneity is present in myotoxin II. The newly established sequences were compared with 3447 protein sequences in the Protein Information Resource database. The only homologous proteins found were other known myotoxins from rattlesnake venoms, namely myotoxin a, crotamine and peptide C.
Neonatal rat myoblasts were isolated and grown in culture until they fused into multinucleated myotubes. A small percentage of the myotubes showed spontaneous contractions when maintained in Dulbecco's Modified Eagle's Medium with 10% fetal bovine serum. Incubation of mature myotubes (at least 3 days after fusion) with myotoxin II from Crotalus viridis concolor venom at a concentration as low as 18.5 nM caused a marked increase in the number of myotubes demonstrating contractile activity. The increase was apparent within 24 hr of myotoxin application. The response of the myotubes appeared to be specific since, of the proteins tested, only native myotoxins caused the increase in contractile activity. This tissue culture system offers a rapid screening assay that requires less time and fewer animals than the assays currently in use for determining myotoxic activity.
The potentiating effect of sodium acetate on the toxicity of crotamine from Crotalus durissus terrificus venom, E toxin from Crotalus horridus horridus venom, and myotoxin a from Crotalus viridus viridis venom was examined. Subcutaneous injection of 6.3 mg/kg body weight of either crotamine or E toxin in 0.6 ml of water or myotoxin a in 0.6 ml of 0.05 M Tris/0.1 M NaCl buffer, pH 9.0, failed to produce lethality in mice. Injection of either E toxin or crotamine at doses of 4.0 mg/kg in 0.6 ml of 20 mM phosphate, pH 7.2, containing 1 M sodium chloride also failed to produce lethality. However, when any of the toxins were injected in 0.4 ml of 1 M sodium acetate, pH 7.0, lethality was observed. LD50 values of 1.43 mg/kg for E toxin, 1.39 mg/kg for crotamine and 0.56 mg/kg for myotoxin a were determined under these conditions. Lethality was also observed when either sodium propionate or sodium butyrate was used as a carrier for E toxin. The effect of these two buffers on crotamine and myotoxin a was not examined. Injection of E toxin s.c. in water followed at various time intervals with i.p. injections of 1 M sodium acetate produced lethality, even when the acetate was injected up to 4 hr after the toxin challenge.
Purified phospholipase A2 from Bothrops alternatus venom is one single protein species with a molecular weight of 15,000 and isoelectric point 5.08. When injected i.p. or i.v. at a dose of 0.7 microgram/g body weight it is lethal to mice, eliciting a typical syndrome of dyspnea, tachycardia, arrhythmia and irreversible shock. Post mortem and histopathologic studies have demonstrated that the lungs (massive pulmonary hemorrhage), heart (foci of myocardial and endocardial necrosis with interfibrillar hemorrhage), liver (congestion, hepatocytic microvacuolization with zones of massive necrosis) and kidneys (foci of tubular and glomerular necrosis) were severely injured. Except for the less extensive hemorrhages and the significantly longer survival time, the observed lesions are similar to those observed after the injection of lethal doses of whole venom. The lethal potency of the purified enzyme (LD50 i.p. 0.14 microgram/g body weight) is 46-fold greater than that of the whole venom (LD50 i.p. 6.4 micrograms/g body weight). The contribution of phospholipase A2 to the overall lethal effect of B. alternatus venom is suggested by the decreased lethal potency of a venom sample in which a significant amount of phospholipase A2 has been removed and the full restoration of the lethal potency upon supplementation of the depleted sample with purified enzyme. It is concluded that phospholipase A2 is a major component responsible for lethality of the whole B. alternatus venom, while the contribution of other venom components appears to be significant mainly in reducing the time of survival.
Gel filtration profiles of crude snake venoms from 38 Crotalus viridis, representing the nominal subspecies concolor, viridis and lutosus, were compared. Mean protein concentration is greatest in C. v. concolor. Individual fractions show up to 9-fold variation in protein content within taxa, and 23-fold with all specimens considered together. Small myotoxins are the major components in C. v. concolor and C. v. viridis venoms, comprising an average of 37% and 44% of total venom protein, respectively. In C. v. lutosus, fractions 1 and 2 each account for 26% of total protein. The three taxa are readily distinguished on the basis of quantitative and qualitative differences in gel filtration elution profiles.
The Gaboon viper has acquired an impressive reputation which is at least partly unfounded. This handsome animal with such striking features is undoubtedly docile which accounts for the very low incidence of bite amongst humans. There are only six detailed clinical reports on the effect of bite and these are summarized in the review. The viper does indeed produce prodigious amounts of venom, but the toxicity, weight for weight, is rather low compared to other poisonous snakes. Venom extractions have been carried out on four snakes over a 13-year-period and the effects of this venom have been studied in a variety of experimental animals. Systemic envenomation is characterized by immediate abrupt hypotension, subsequent cardiac damage and dyspnoea. The individual venom components responsible for these effects have not been isolated but it seems likely that the two enzymes which have been studied extensively (phospholipase A2 and the thrombin-like enzyme, gabonase) do not contribute significantly to lethality. We propose three principal activities which give rise to the major signs of systemic envenomation. Haemorrhagin; causing widespread damage to microvasculature which leads to the pulmonary oedema and hence dyspnoea, and locally causes blistering. Cardiotoxin; a long-acting material causing cardiac muscle damage, arrhythmia and ultimately cardiac failure. Peripheral vasodilator; a short acting effect, operating either locally via bradykinin formation and/or unknown peptides or centrally on the vasomotor centre.
Twenty-five snake venoms were tested for their ability to induce an increase of serum creatine kinase (CK) level after i.m. injection (0.125-1.0 mg/kg) into rates. Of six Australian elapid venoms only those from Pseudechis colletti guttatus and P. australis produced a steep rise of CK-activity (30-70 times the normal value) 4 and 16 hr after injection (0.5 mg/kg). Viperid and crotalid venoms had only slight effects (2-5 times the normal value) even in doses of 1.0 mg/kg except for a sample of Crotalus adamanteus venom which caused a 20 fold increase in CK-level. From this venom a toxin of 5800 mol. wt. consisting of 50 amino acid residues was isolated. This toxin exhibited similarities in amino acid composition and in lethality to crotamine from Crotalus durissus terrificus and to a toxin from C. horridus horridus. The toxin from C. adamanteus induced some increase of CK-level in rats, but this does not account entirely for the activity of the crude venom, whereas crotamine and the toxin from C. horridus horridus were ineffective. Phospholipase A2 (fraction II) from Pseudechis colletti guttatus venom caused a dose-dependent increase of CK-level and myoglobinuria. Intradermal injection of snake venoms into mice is useful for testing hemorrhagic activity, but is too insensitive to measure necrotizing effects. Venom induced myonecrosis can be evaluated by assaying the CK-serum level and by histological examination.
CIDNP and COSY measurements were applied to study aromatic side chain interactions and conformations in myotoxin alpha, a Crotalus venom toxin which acts as blocker of the Ca2+ influx in the sarcoplasmic reticulum calcium pump. New evidence for the existence of a hydrophobic aromatic cluster at the amino terminus was obtained. This cluster consists of Tyr1, His5, His10, and (possibly) F12. The CIDNP data clearly establish that the usual order of the tyrosine 2, 6 and 3, 5 proton signals of Tyr1 is inverted, because of the large diamagnetic shielding effects of one ring on the other. The lines of the 2, 6 ring protons of Tyr1 and proton 4 in each of His5 and His10 are significantly broadened, an outcome of the side-chain hydrophobic interaction. The aromatic cluster could possibly present a hydrophobic sticky patch for binding of toxin by Ca2+ ATPase.
Many rattlesnake venoms (family Crotalidae) contain small, highly basic toxins which cause contracture and necrosis of skeletal muscle in experimental animals. Isolation of these myotoxins requires several chromatographic steps taking several days to complete. We report the isolation of crotamine, myotoxin a, and myotoxin a-like molecules from whole venom of three species of rattlesnakes using a Hydrazide Avidchrom Cartridge. Milligram amounts of highly purified and biologically active myotoxin can be obtained in 30 min to 2 hr.
The binding of radiolabeled myotoxin a to various cultured cell lines was evaluated. One rat skeletal muscle-derived cell line, L8, bound substantially more myotoxin a than did all all other cell lines examined. Several biophysical parameters of myotoxin a-L8 binding were determined. Binding was saturable with a moderate binding affinity. Scatchard analysis and Hill plots indicated a single class of binding sites. The binding was reversible, as demonstrated by chase experiments. Radiolabeled myotoxin a bound to the cell surface at a site inaccessible to the general protease, pronase. Specificity and biological relevance of the binding was suggested by competition with unlabeled toxin and various peptides derived from the toxin. Biologically active peptides, corresponding to the N- and C-terminal sequence of myotoxin a, competed with radiolabeled toxin for L8 binding. It was concluded that the L8 system is a suitable cell model to study myotoxin a mechanism of action.
A myotoxin was isolated from the venom of the broad-banded copperhead (Agkistrodon contortrix laticinctus) by HPLC using anion and cation exchange chromatography. The toxin has a mol. wt of approximately 14,000 and has a pI greater than 9. It does not have phospholipase A activity, but does induce myonecrosis of skeletal muscle cells characterized by a hypercontraction of myofilaments. Electron microscopic analysis showed that the myotoxin appears to disrupt the sarcolemma of skeletal muscle cells. ACL myotoxin is very similar in mol. wt, amino acid composition, and myotoxic activity to myotoxins isolated from the venoms of Bothrops asper and Bothrops nummifer from Central America, suggesting that homologs of this toxin may be found in other crotaline snake venoms.
An acidic phospholipase A2 (OHV A-PLA2) isolated from the venom of the king cobra (Ophiophagus hannah) was tested for its ability to cause pathological changes to myocardium, skeletal muscle and cardiac ganglia. White mice were injected intravenously with dose of 8 mg/kg or 4 mg/kg of OHV A-PLA2 and tissue samples were taken at 6 or 24 hr. Light microscopic examination failed to show significant changes in cardiac muscle and ganglia. Skeletal muscle showed myofibre degeneration and necrosis. Electron microscopic study revealed myodegeneration in cardiac and skeletal muscles, and reduction in synaptic vesicle population of preganglionic nerve terminals in cardiac ganglia. Ultrastructural changes in tissues were dose related. The lower dose (4 mg/kg) of OHV A-PLA2 produced mild myocardial changes, the myofilaments were intact but contracted, and the A band and I band were skewed. OHV A-PLA2 caused myocardial degeneration at a higher dose of 8 mg/kg. The changes included dissolution of actin and myosin filaments, dilatation and disorganization of sarcoplasmic reticulum and degeneration of mitochondria. The skeletal muscle lesions were more severe than the myocardial changes. Some of the myofibrils were severely disorganized and lack typical striated appearance, sarcomeres disrupted, most of mitochondria were vesiculated and destroyed.
Myotoxin a, from the venom of the prairie rattlesnake, Crotalus viridis viridis, exists as a temperature-dependent equilibrium of two interconverting forms. Reverse-phase high-performance liquid chromatography (RP-HPLC) shows that the two forms interconvert slowly enough at 25 degrees C to be seen as two separate peaks with a molar ratio of c. 1:4. Each peak can be isolated and individually injected to give the same two peaks in the same ratio of areas. The two peaks merge during chromatography at elevated temperatures, indicating an increase in the rate of interconversion. At low temperature, c. 5 degrees C, the individual peaks can be isolated and maintained for several days without reaching equilibrium. Mass analysis by matrix-assisted laser desorption ionization (MALDI) time-of-flight mass spectrometry shows that myotoxin a is present in both RP-HPLC peaks, suggesting that the two resolved forms are conformational isomers. Capillary zone electrophoresis (CZE) also shows two resolved, but interconvertible peaks over a range of pH values. Furthermore, RP-HPLC chromatograms of myotoxin a at concentrations from 0.013 mM to 0.41 mM maintain a consistent ratio of peak areas, without evidence of dimerization. Two-dimensional 1H-NMR nuclear Overhauser enhancement spectroscopy indicates the presence of a cis-proline peptide bond, consistent with an equilibrium mixture of cis-trans isomers; however, addition of peptidyl-prolyl cis-trans isomerase (PPI) does not enhance the rate of equilibration of the RP-HPLC peaks isolated at c. 5 degrees C.
Forty proteins with polypeptide chains of well characterized molecular weights have been studied by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate following the procedure of Shapiro, Viñuela, and Maizel (Biochem. Biophys. Res. Commun., 28, 815 (1967)). When the electrophoretic mobilities were plotted against the logarithm of the known polypeptide chain molecular weights, a smooth curve was obtained. The results show that the method can be used with great confidence to determine the molecular weights of polypeptide chains for a wide variety of proteins.
Conditions for regeneration of amino acids from their thiazolinone derivatives formed in the cleavage step of the Edman degradation procedure have been investigated. Highest yields of most amino acids including methionine were obtained simply by hydrolysis in 5.7n HCl containing 0.1% SnCl2 for 4 hr at 150°C. Results from other methods of hydrolysis are provided for comparison. The hydrolytic yield of amino acids from the anilinothiazolinones determined in this study may be used to estimate the cleavage yield at each step of the Edman degradation.
The animal venoms are the most complex o f all the poisons, The study o f their physiopharmacological activities and the autopharmacological responses they produce is one of the most important new fields in toxicology. The value o f venoms as tools in biochemistry and perhaps their use in the treatment o f disease is also becoming apparent. Finally, the injuries produced by venomous animals are of considerable medical importance, Some 35,000 to 50,000 persons a year die from the bites or stings of these animals, and perhaps some 10 million persons are poisoned by them each year. With these things in mind, the author has attempted to review some of the more important chemical and pharmacological properties of the toxins of representative poisonous species from the marine fauna, terrestrial snakes and arthropods. Some consideration has also been given to the venom apparatus o f several species and to the medical problem of enve-nomation.
A weak and reversibly acting neurotoxic protein of Laticauda semifasciata venom, Laticauda semifasciata III (component LsIII), was sequenced. Component LsIII consists of 66 amino acid residues and has five disulphide bridges, one of which was located between residues 26 and 30. The weak and reversible neurotoxicity of component LsIII is discussed in relation to its structure, which falls between those of the neuro- and cardiotoxins of sea snakes and Elapidae snakes isolated and sequenced so far.
The highly basic proteins from three North American rattlesnake venoms have been isolated by a simple combination of Bio-Gel P-2 recycling adsorption chromatography and ion exchange on carboxymethyl-cellulose. These basic proteins constitute less than 10% of the total protein in the crude venoms and are characterized by having small molecular weights, isoelectric points above ≈ pH 10.8 and pharmacological activities (in vivo and in vitro) resembling those of crotamine, the neurotoxin from Crotalus durrisus terrificus, the South American tropical rattlesnake.
Two N-terminal fragments of (A)-chain and β(B)-chain in human fibrinogen have been isolated from a plasmic hydrolyzate. The fragment from the (A)-chain consisted of 43 amino acid residues including two half-cystine residues. On treatment with thrombin, this fragment produced two other peptides in addition to fibrinopeptide A and its analogues. One was a tripeptide, Gly-Pro-Arg, and the other a peptide containing 24 amino acid residues having N-terminal valine. The partial amino acid sequence of the (A)-chain has been found to be: Ala-Asp-Ser-Gly-Glu-Gly-Asp-Phe-Leu-Ala-Glu-Gly-Gly-Gly-Val-Arg-Gly-Pro-Arg-Val-Val-Glu-Arg-His-Gln-Ser-Ala-Cys-Lys-Asp-Ser-Asp-Trp-Pro-Phe-(Cys-Ser-Asp-Glu-Trp-Asn-Tyr)-Lys.The fragment from the β(B)-chain consisted of 21 amino acid residues. This fragment released fibrinopeptide B on treatment with thrombin. The amino acid sequence of the β(B)-chain fragment is: Pyr-Gly-Val-Asn-Asp-Asn-Glu-Glu-Gly-Phe-Phe-Ser-Ala-Arg-Gly-His-Arg-Pro-Leu-Asp-Lys. The linkages between fibrinopeptides and fibrin, which are rapidly hydrolyzed by thrombin, are very resistant towards plasmin.
IN a recent communication by Hulme and Arthington1, writing of the isatin test for proline, they state that ``it is surprising that this useful test has not been more widely used''. This prompts a report of further advantages of this reagent and a description of a general technique which has been in use in this laboratory for some months.
Snake Venoms--Chemistry I Isolation of a lethal protein and peptide from Crotalus viridis helleri venom Separation and purification of Crotalus venom fractions Separation and purification of Crotalus venom fractions
- J W Dubnoff
- F E Dubnoff
- F E Dubnoff
- F E Abst
- J W Dtmnoff
DUBNOFF, J. W. and RUSSELL, F. E. (1969) Snake Venoms--Chemistry I. Grand Rounds, LAC/USC Medical Center, 17 January 1969. DUBNOFF, J. W. and RUSSELL, F. E. (1970a) Isolation of a lethal protein and peptide from Crotalus viridis helleri venom. Proc. west. Pharmac. Soc. 13, 98. DUBNOFF, J. W. and RUSSELL, F. E. (1970b) Separation and purification of Crotalus venom fractions. 2nd lnternat. Syrup. Animal Toxins. Tel Aviv, February, 1970. Toxicon 8, 130, 1970 (Abst.) DtmNOFF, J. W. and RUSSELL, F. E. (1971) Separation and purification of Crotalus venom fractions. In: Toxins of Animal and Plant Origin, Vol. 1, p. 361, (DE VRIES, A. and KOCHVA, E., Eds.). London : Gordon and Breach.
Separation and purification of Crotalus venom fractions
- Dubnoff
A lethal protein from the venom of the Southern Pacific rattlesnake Crotalus viridis helleri
- Pattabhiraman
Die primarstructur des Crotamins
- Laure