Batroxase, a new metalloproteinase from B. atrox snake venom with strong fibrinolytic activity

The structures and functional activities of metalloproteinases from snake venoms have been widely studied because of the importance of these molecules in envenomation. Batroxase, which is a metalloproteinase isolated from Bothrops atrox (Pará) snake venom, was obtained by gel filtration and anion exchange chromatography. The enzyme is a single protein chain composed of 202 amino acid residues with a molecular mass of 22.9 kDa, as determined by mass spectrometry analysis, showing an isoelectric point of 7.5. The primary sequence analysis indicates that the proteinase contains a zinc ligand motif (HELGHNLGISH) and a sequence C₁₆₄I₁₆₅M₁₆₆ motif that is associated with a “Met-turn” structure. The protein lacks N-glycosylation sites and contains seven half cystine residues, six of which are conserved as pairs to form disulfide bridges. The three-dimensional structure of Batroxase was modeled based on the crystal structure of BmooMPα-I from Bothrops moojeni. The model revealed that the zinc binding site has a high structural similarity to the binding site of other metalloproteinases. Batroxase presented weak hemorrhagic activity, with a MHD of 10 μg, and was able to hydrolyze extracellular matrix components, such as type IV collagen and fibronectin. The toxin cleaves both α and β-chains of the fibrinogen molecule, and it can be inhibited by EDTA, EGTA and β-mercaptoethanol. Batroxase was able to dissolve fibrin clots independently of plasminogen activation. These results demonstrate that Batroxase is a zinc-dependent hemorrhagic metalloproteinase with fibrin(ogen)olytic and thrombolytic activity.     

Purification and characterization of a hemorrhagic metalloproteinase from Bothrops lanceolatus (Fer-de-lance) snake venom.

Bothrops snake venoms contain metalloproteinases that contribute to the local effects seen after envenoming. In this work, a hemorrhagic metalloproteinase (BlaH1) was purified from the venom of the snake Bothrops lanceolatus by a combination of gel filtration, affinity (metal chelating) and hydrophobic interaction chromatographies. The hemorrhagin was homogeneous by SDS-PAGE and had a molecular mass of 28 kDa that was unaltered by treatment with beta-mercaptoethanol. BlaH1 gave a single band in immunoelectrophoresis and immunoblotting using commercial bothropic antivenom. BlaH1 had hemorrhagic, caseinolytic, fibrinogenolytic, collagenolytic and elastinolytic activities, but no phospholipase A(2) activity. The hemorrhagic and caseinolytic activities were inhibited by EDTA, indicating that they were metal ion-dependent. In contrast, aprotinin, benzamidine and PMSF did not affect these activities. The caseinolytic activity of BlaH1 had a pH optimum of 8.0 and was stable in solution at up to 40 degrees C; activity was completely lost at > or =70 degrees C. The hemorrhagic activity was neutralized by commercial bothropic antivenom. These properties suggest that this new hemorrhagin belongs to class P-I snake venom metalloproteinases.     

Albocollagenase, a novel recombinant P-III snake venom metalloproteinase from green pit viper (Cryptelytrops albolabris), digests collagen and inhibits platelet aggregation

Molecular cloning and functional characterization of P-III snake venom metalloproteinases (SVMPs) will give us deeper insights in the pathogenesis of viper bites. This may lead to novel therapy for venom-induced local tissue damages, the complication refractory to current antivenom. The aim of this study was to elucidate the in vitro activities of a new SVMP from the green pit viper (GPV) using recombinant DNA technology. We report, here, a new cDNA clone from GPV (Cryptelytrops albolabris) venom glands encoding 614 amino acid residues P-III SVMP, termed albocollagenase. The conceptually translated protein comprised a signal peptide and prodomain, followed by a metalloproteinase domain containing a zinc-binding motifs, HEXGHXXGXXH-CIM and 9 cysteine residues. The disintegrin-like and cysteine-rich domains possessed 24 cysteines and a DCD (Asp-Cys-Asp) motif. The albocollagenase deduced amino acid sequence alignments showed approximately 70% identity with other P-III SVMPs. Notably, the prodomain was highly conserved, while the metalloproteinase, disintegrin-like and cysteine-rich domains contained several differences. Albocollagenase without the signal peptide and prodomain was expressed in Pichia pastoris with an N-terminal six-histidine tag. After affinity purification from the supernatant of methanol-induced media, SDS-PAGE and Western blot analysis in both reducing and non-reducing conditions showed a protein band of approximately 62 kDa. The recombinant albocollagenase could digest human type IV collagen from human placenta basement membrane within 1 min. After 10-min incubation, it also inhibited collagen-induced platelet aggregation with 50% inhibitory concentration (IC₅₀) of 70 nM. This is the first report of the active recombinant SVMP enzymes expressed in P. pastoris. The results suggest the significant roles of P-III SVMP in local and systemic pathology of envenomated patients. Inhibitors of this SVMP will be investigated in further studies to find a better treatment for viper bites.     

Purification and functional characterization of bothrojaractivase, a prothrombin-activating metalloproteinase isolated from Bothrops jararaca snake venom.

Bleeding at the site of bite and/or systemic hemorrhage are symptoms frequently observed in envenomation by Bothrops jararaca snakes. In this study, we purified and characterized a prothrombin activator from B. jararaca that is probably involved in these clinical manifestations. The enzyme was isolated by a combination of gel filtration and ion exchange chromatographies and named bothrojaractivase. It has a single polypeptide chain with a molecular weight of 22,829 Da as measured by mass spectroscopy. Bothrojaractivase generates active thrombin from prothrombin, independently of cofactors. SDS-PAGE analysis of the prothrombin activation products shows that bothrojaractivase converts prothrombin into meizothrombin producing similar fragments to those generated by group A prothrombin's activators. In addition, bothrojaractivase degraded fibrinogen and fibrin. Chelating agents completely inhibited the enzymatic activity, whereas inhibitors of serine and cysteine proteinases had no effect. Amino acid sequence of four peptides demonstrated high similarity of bothrojaractivase with P-I class of snake venom metalloproteinases. Thus, our results indicate that bothrojaractivase is a new metalloproteinase that acts on different protein factors of the clotting cascade especially displaying a key and most relevant functional action in the generation of thrombin through prothrombin activation in a similar mode of action as that of group A activators.     

Isolation and characterization of a myotoxin from the venom of Macrovipera lebetina transmediterranea

The Macrovipera lebetina venom consists of a complex mixture of proteins belonging to a few main families according to their enzymatic and pharmacological activity. Given the serious pathophysiological effects caused by M. lebetina bites mainly induced by muscle degeneration, we decided to investigate the myotoxic activity of some venom fractions. In the present study we describe the purification and characterization of a 22.600 kDa protein, named in the following Mlp4.2, that shares myotoxic but not haemorrhagic activity in vivo. Herein we report that Mlp4.2 is a metalloproteinase belonging to the PI-SVMPS family able, in vitro, to proteolyse extracellular matrix proteins as laminin and fibronectin. Histological observations of mouse anterior tibialis Mlp4.2-treated muscle, demonstrate that this protein induces a massive degeneration of myofibers but not haemorrhage. The immunofluorescence analysis of protein-treated anterior tibialis, demonstrates that Mlp4.2 is able to disarray the laminin network surrounding muscle fibers. Finally Mlp4.2 did not show any direct cytolytic activity towards the myogenic cell line C2C12 in culture. The data reported herein suggest that the myotoxicity of Mlp4.2 is primarily linked to the disruption of the muscle fibers interaction with extracellular matrix proteins.     

Isolation and characterization of a presynaptic neurotoxin,P-elapitoxin-Bf1a from Malaysian Bungarus fasciatus venom

Presynaptic neurotoxins are one of the major components in Bungarus venom. Unlike other Bungarus species that have been studied, β-bungarotoxin has never been isolated from Bungarus fasciatus venom. It was hypothesized that the absence of β-bungarotoxin in this species was due to divergence during evolution prior to evolution of β-bungarotoxin. In this study, we have isolated a β-bungarotoxin isoform we named P-elapitoxin-Bf1a by using gel filtration, cation-exchange and reverse-phase chromatography from Malaysian B. fasciatus venom. The toxin consists of two heterogeneous subunits, subunit A and subunit B. LCMS/MS data showed that subunit A was homologous to acidic phospholipase A₂ subunit A3 from Bungarus candidus and B. multicinctus venoms, whereas subunit B was homologous with subunit B1 from B. fasciatus venom that was previously detected by cDNA cloning. The toxin showed concentration- and time-dependent reduction of indirect-twitches without affecting contractile responses to ACh, CCh or KCl at the end of experiment in the chick biventer preparation. Toxin modification with 4-BPB inhibited the neurotoxic effect suggesting the importance of His-48. Tissue pre-incubation with monovalent B. fasciatus (BFAV) or neuro-polyvalent antivenom (NPV), at the recommended titer, was unable to inhibit the twitch reduction induced by the toxin. This study indicates that Malaysian B. fasciatus venom has a unique β-bungarotoxin isoform which was not neutralized by antivenoms. This suggests that there might be other presynaptic neurotoxins present in the venom and there is a variation in the enzymatic neurotoxin composition in venoms from different localities.     

BthMP: a new weakly hemorrhagic metalloproteinase from Bothrops moojeni snake venom

In this work, a new weakly hemorrhagic metalloproteinase (BthMP) was purified from Bothrops moojeni snake venom. This enzyme was homogeneous by native and SDS-PAGE. It showed a polypeptide chain of 23.5 kDa, pI = 7.1, and N-terminal blocked. BthMP is comprised of high proteolytic activity on casein, fibrin and bovine fibrinogen, with no coagulating, esterase or phospholipase A₂ activities; it was inhibited by EDTA, EGTA and 1,10-phenanthroline and maintained its activity on pH from 7.0 to 9.0 and temperature from 5-40 °C. Assays with metal ions showed that Ca²⁺ is an activator, whereas Zn²⁺ and Hg²⁺ inhibited about 50 and 80% of its activity, respectively. The edema evidenced the important role of the toxin in the inflammatory activity of the venom. BthMP also caused unclotting, and provoked histological alterations in the gastrocnemius muscle of mice inducing hemorrhage, necrosis and leukocytic infiltrate. The molecular mass and the inhibition assays suggest that the metalloproteinase BthMP belongs to class P-I of SVMPs.     

Isolation and structural characterization of a new fibrin(ogen)olytic metalloproteinase from Bothrops moojeni snake venom.

A proteinase, named BmooMPalpha-I, from the venom of Bothrops moojeni, was purified by DEAE-Sephacel, Sephadex G-75 and heparin-agarose column chromatography. The enzyme was purified to homogeneity as judged by its migration profile in SDS-PAGE stained with coomassie blue, and showed a molecular mass of about 24.5 kDa. Its complete cDNA was obtained by RT-PCR and the 615 bp codified for a mature protein of 205 amino acid residues. The multiple alignment of its deduced amino acid sequence and those of other snake venom metalloproteinases showed a high structural similarly, mainly among class P-IB proteases. The enzyme cleaves the Aalpha-chain of fibrinogen first, followed by the Bbeta-chain, and shows no effects on the gamma-chain. On fibrin, the enzyme hydrolyzed only the beta-chain, leaving the gamma-dimer apparently untouched. It was devoid of phospholipase A(2), hemorrhagic and thrombin-like activities. Like many venom enzymes, it is stable at pH values between 4 and 10 and stable at 70 degrees C for 15 min. The inhibitory effects of EDTA on the fibrinogenolytic activity suggest that BmooMPalpha-I is a metalloproteinase and inhibition by beta-mercaptoethanol revealed the important role of the disulfide bonds in the stabilization of the native structure. Aprotinin and benzamidine, specific serine proteinase inhibitors, had no effect on BmooMPalpha-I activity. Since the BmooMPalpha-I enzyme was found to cause defibrinogenation when administered i.p. on mice, it is expected that it may be of medical interest as a therapeutic agent in the treatment and prevention of arterial thrombosis.     

Purification, characterization, and cDNA cloning of acidic platelet aggregation inhibiting phospholipases A2 from the snake venom of Vipera lebetina (Levantine viper)

Two novel acidic phospholipase A₂s (PLA₂) were isolated by size exclusion chromatography and reversed-phase chromatography from the crude Vipera lebetina venom. The molecular masses of VLPLA₂-1 (13,704 Da) and VLPLA₂-2 (13,683 Da) and their internal tryptic peptides were determined by MALDI-TOF mass-spectrometry. When tested in human platelet-rich plasma, both enzymes showed a potent inhibitory effect on aggregation induced by ADP and collagen. Chemical modification with p-bromophenacylbromide abolished the enzymatic activity of PLA₂; its anti-platelet activity was fully inhibited in case of collagen as inducer and partially inhibited in case of ADP as inducer. The complete cDNAs encoding PLA₂ were cloned from a single venom gland cDNA library. Complete amino acid sequences of the VLPLA₂ were deduced from the cDNA sequences. The full-length cDNA sequences of the VLPLA₂ possess 615 bp and encode an open reading frame of 138 amino acids that include signal peptide (16 amino acids) and mature enzyme (122 amino acids). The VLPLA₂s have significant sequence similarity to many other phospholipase A₂s from snake venoms. The phylogenetic analysis on the basis of the amino acid sequence homology demonstrates that VLPLA₂s grouped with other Asp49 PLA₂s and they appear to share a close evolutionary relationship with the European vipers.     

Hemostatic properties of Venezuelan Bothrops snake venoms with special reference to Bothrops isabelae venom

In Venezuela, Bothrops snakes are responsible for more than 80% of all recorded snakebites. This study focuses on the biological and hemostatic characteristics of Bothrops isabelae venom along with its comparative characteristics with two other closely related Bothrops venoms, Bothrops atrox and Bothrops colombiensis. Electrophoretic profiles of crude B. isabelae venom showed protein bands between 14 and 100 kDa with the majority in the range of 14-31 kDa. The molecular exclusion chromatographic profile of this venom contains five fractions (F1-F5). Amidolytic activity evaluation evidenced strong thrombin-like followed by kallikrein-like activities in crude venom and in fractions F1 and F2. The fibrinogenolytic activity of B. isabelae venom at a ratio of 100:1 (fibrinogen/venom) induced a degradation of Aα and Bβ chains at 15 min and 2 h, respectively. At a ratio of 100:10, a total degradation of Aα and Bβ chains at 5 min and of γ chains at 24 h was apparent. This current study evidences one of rarely reported for Bothrops venoms, which resembles the physiologic effect of plasmin. B. isabelae venom as well as F2 and F3 fractions, contain fibrinolytic activity on fibrin plate of 36, 23.5 and 9.45 mm²/μg, respectively using 25 μg of protein. Crude venom and F1 fraction showed gelatinolytic activity. Comparative analysis amongst Venezuelan bothropoid venoms, evidenced that the LD₅₀ of B. isabelae (5.9 mg/kg) was similar to B. atrox-Puerto Ayacucho 1 (6.1 mg/kg) and B. colombiensis-Caucagua (5.8 mg/kg). B. isabelae venom showed minor hemorrhagic activity, whereas B. atrox-Parguasa (Bolivar state) was the most hemorrhagic. In this study, a relative high thrombin-like activity was observed in B. colombiensis venoms (502-568 mUA/min/mg), and a relative high factor Xa-like activity was found in B. atrox venoms (126-294 mUA/min/mg). Fibrinolytic activity evaluated with 10 μg protein, showed that B. isabelae venom contained higher specific activity (50 mm²/μg) than B. colombiensis and B. atrox venoms, which should encourage the isolation of these fibrinolytic molecules to improve the quality of immunotherapy.     

Biochemical characterization of the Micrurus pyrrhocryptus venom

Snake venom toxicity is the consequence of a combination of peptides and proteins whose identification and characterization are of great importance to understand envenomation and develop new clinical treatments. The Elapinae subfamily includes coral snakes whose bite causes mainly neurotoxic effects which disable muscle contraction and paralyse the heart as well as inhibit respiration. However, the structure-function relationship of venom toxins has been investigated only for a few species. We herein study biological aspects of the Micrurus pyrrhocryptus venom such as LD₅₀, hemorrhagic, necrotic, coagulant, myotoxic and hemolytic activity as well as the ability of venom components to compete with alpha-Bungarotoxin for the ligand-binding site of the nicotinic acetylcholine receptor. Besides, we report the determination of the molecular mass and N-terminal sequence of toxins including PLA2s, short, long and weak neurotoxins. The complete sequence of one of the short neurotoxins has also been obtained, this being the first sequence of an alpha-neurotoxin determined in the M. pyrrhocryptus venom and one of the few fully determined in members of the Micrurus genus.     

A lethal cardiotoxic-cytotoxic protein from the Indian monocellate cobra (Naja kaouthia) venom

A lethal cardiotoxic-cytotoxic protein (mol. wt. 6.76 kDa) has been purified from the Indian monocellate cobra (Naja kaouthia) venom by ion-exchange chromatography and HPLC. CD spectra indicated the presence of 23% α helix, 19% β sheets and 35% coil. Complete amino acid sequence was determined by MALDI, which showed similar homology with cardiotoxins/cytotoxins isolated from venom of other Naja species. Intraperitoneal LD₅₀ was 2.5 mg kg⁻¹ in BalbC male mice. In vitro cardiotoxicity studies on isolated guinea pig auricle showed that the molecule produced auricular blockade that was abolished after trypsin treatment. Cytotoxicity studies on human leukemic U937 and K562 cells showed that it significantly inhibited cell proliferation in a dose and time dependent manner, as observed by trypan blue exclusion method and tetrazolium bromide reduction assay. IC₅₀ on U937 and K562 cells were 3.5 μg/ml and 1.1 μg/ml respectively. Morphometry and cell sorting studies indicated apoptosis induction in toxin treated leukemic cells. Apoptosis was caspase 3 and 9 dependent and the treated leukemic cells were arrested in sub-G1 stage. There was an increase in Bax-Bcl2 ratio, decrease in HSP (Heat shock protein) 70 and HSP90 and induction of PARP cleavage after NK-CT1 treatment. The toxin showed low cytotoxic effect on normal human leukocytes as compared with imatinib mesylate. Further detailed cytotoxic and cardiotoxic effects at the molecular level are in progress.     

Bhalternin: Functional and structural characterization of a new thrombin-like enzyme from Bothrops alternatus snake venom

A serine protease from Bothrops alternatus snake venom was isolated using DEAE-Sephacel, Sephadex G-75 and Benzamidine-Sepharose column chromatography. The purified enzyme, named Bhalternin, ran as a single protein band on analytical polyacrylamide gel electrophoresis (SDS-PAGE) and showed molecular weights of 31,500 and 27,000 under reducing and non-reducing conditions, respectively. Its complete cDNA was obtained by RT-PCR and the 708 bp codified for a mature protein of 236 amino acid residues. The multiple alignment of its deduced amino acid sequence showed a structural similarly with other serine proteases from snake venoms. Bhalternin was proteolytically active against bovine fibrinogen and albumin as substrates. When Bhalternin and bovine fibrinogen were incubated at 37 °C, at a ratio of 1:100 (w/w), the enzyme cleaved preferentially the Aα-chain, apparently not degrading the Bβ and γ-chains. Stability tests showed that the intervals of optimum temperature and pH for the fibrinogenolytic activity were 30-40 °C and 7.0-8.0, respectively. Also, the inhibitory effects of benzamidine on the fibrinogenolytic activity of Bhalternin indicate that it is a serine protease. This enzyme caused morphological alterations in heart, liver, lung and muscle of mice and it was found to cause blood clotting in vitro and defibrinogenation when intraperitoneally administered to mice, suggesting it to be a thrombin-like enzyme. Therefore, Bhaltenin may be of interest as a therapeutic agent in the treatment and prevention of thrombotic disorders.     

Purification and characterization of a novel peptide with antifungal activity from Bothrops jararaca venom.

Different peptides have been isolated from a wide range of animal species. It is has become increasingly clear that due to the development of antibiotic-resistant microbes, antibacterial and antifungal peptides have attracted the attention in recent years, in order to find new therapeutic agents. In this work, a novel peptide with high inhibitory activity against fungi growth have been isolated from the venom of the Brazilian snake Bothrops jararaca. A Sephacryl S-100 gel filtration column was employed for further separation of proteins. The FV fraction with high antifungal activity was named Pep5Bj, pooled and submitted to reverse-phase chromatography in HPLC. The fraction containing the isolated peptide inhibited the growth of different phytopathogenic fungi (Fusarium oxysporum and Colletotrichum lindemuthianum) and yeast (Candida albicans and Saccharomyces cerevisiae). The peptide minimal inhibitory concentration is comparable to other known antifungal peptides, like insect defensins and cecropins, found in the last years in a large diversity of animals. We investigate F. oxysporum cells membrane permeabilization using SYTOX Green uptake, an organic compound that fluoresces upon interaction with nucleic acids after penetration in cell with compromised plasma membranes. When viewed under fluorescence optical microscopy, F. oxysporum cells exposed to Pep5Bj display strong SYTOX Green fluorescence in the cytosol, especially in the nuclei. The SYTOX Green data suggested that this effect is related to membrane permeabilization. The molecular masses of this peptide was obtained by MALDI-TOF spectrometry and corresponded to 1370Da.     

Phylogenetic analysis of serine proteases from Russell’s viper (Daboia russelli siamensis) and Agkistrodon piscivorus leucostoma venom

Serine proteases are widely found in snake venoms. They have variety of functions including contributions to hemostasis. In this study, five serine proteases were cloned and characterized from two different cDNA libraries: factor V activator (RVV-V), alpha fibrinogenase (RVAF) and beta fibrinogenase (RVBF) from Russell’s viper (Daboia russelli siamensis), and plasminogen activator (APL-PA) and protein C activator (APL-C) from Agkistrodon piscivorus leucostoma. The snake venom serine proteases were clustered in phylogenetic tree according to their functions. K_A/K_S values suggested that accelerated evolution has occurred in the mature protein coding regions in cDNAs of snake venom serine proteases.     

Presynaptic action of Bothriopsis bilineata smargadina (forest viper) venom in vitro

In this work, we examined the neuromuscular activity of Bothriopsis bilineata smargadina (forest viper) venom in vertebrate isolated nerve-muscle preparations. In chick biventer cervicis preparations the venom caused concentration-dependent (0.1-30 μg/ml) neuromuscular blockade that was not reversed by washing, with 50% blockade occurring in 15-90 min. Muscle contractures to exogenous acetylcholine and KCl were unaffected by venom, but there was a slight increase in creatine kinase release after 120 min (from 80 ± 15 to 206 ± 25 U/ml, n = 6, p < 0.05). In mouse phrenic nerve-diaphragm preparations, the venom (1, 10 and 30 μg/ml) produced marked facilitation (∼120% increase above basal) at the highest concentration followed by neuromuscular blockade; the effects at lower concentrations were considerably less marked. Venom increased the quantal content values after 15 and 30 min followed by significant inhibition at ≥90 min. However, venom did not alter the muscle membrane resting potential or the response to exogenous carbachol. In both preparations, incubation at 22 °C instead of 37 °C delayed the onset of blockade, as did inhibition of venom PLA₂ activity. In curarized mouse preparations, the venom produced only muscle facilitation. These results indicate that B. b. smargadina venom causes neuromuscular blockade in vitro by a presynaptic mechanism involving PLA₂.     

Isolation and characterization of a new serine protease with thrombin-like activity (TLBm) from the venom of the snake Bothrops marajoensis

The thrombin-like serine protease TLBm from Bothrops marajoensis was isolated in one chromatographic step in reverse phase HPLC. Its molecular mass was 33239.95 Da, as based on the determined primary structure and confirmed experimentally by MALDI-TOF mass spectrometry (33332.5 Da) and it contains 12 half-cysteine residues. This TLBm exhibited high specificity for BAρNA, Michaelis-Menten behavior with K_m 2.3 × 10⁻¹ M and the V_max 0.52 × 10⁻¹ nmoles ρ-NA/lt/min for this substrate. TLBm also showed ability to coagulate bovine fibrinogen and was inhibited by soybean trypsin inhibitor, EDTA and S(Dm) from the serum of the species Didelphis marsupialis. The primary structure of TLBm showed the presence of His(45), Asp(103) and Ser(228) residues in the corresponding positions of the catalytic triad established in the serine proteases and Ser(228) are inhibited by phenylmethylsulfonyl fluoride (PMSF). Amino acid analysis showed a high content of Asp, Glu, Gly, Ser, Ala and Pro as well as 12 half-cysteine residues and calculated pI of 6.47; TLBm presented 285 amino acid residues. In this work, we investigated the ability of TLBm to degrade fibrinogen and we observed that it is able to cause α- and β-chain cleavage. Enzymatic as well as the platelet aggregation activities were strongly inhibited when incubated with PMSF, a specific inhibitor of serine protease. Also, TLBm induced platelet aggregation in washed and platelet-rich plasma, and in both cases, PMSF inhibited its activity.     

Functional and structural characterization of a new serine protease with thrombin-like activity TLBan from Bothrops andianus (Andean Lancehead) snake venom

A new serine protease with thrombin-like activity (TLBan) from Bothrops andianus (Andean Lancehead) was isolated in two chromatographic steps in LC molecular exclusion and reverse phase-HPLC. TLBan is a glycoprotein that contains both N-linked carbohydrates and sialic acid in its structure, with Mr ∼29 kDa under reducing conditions and non-reducing ∼25 kDa conditions and confirmed by MALDI-TOF mass spectrometry (25,835.65 Da) and exhibited high specificity for BAρNA, Michaelis-Menten behavior with Km 5.4 × 10⁻¹ M and the V_max 7.9 × 10⁻¹ nmoles ρ-NA/L/min for this substrate and high stability when was analyzed at different temperatures (25 to 60 °C), pHs (4.0 to 8.0), was inhibited by soybean trypsin inhibitor, EDTA and phenylmethylsulfonyl fluoride (PMSF).The total amino acid sequence was obtained through sequencing of selected tryptic peptides and by inference obtained using SwissProt database http://br.expasy.org/ with the search restricted to serine proteases from Crotalinae snakes and show high amino acid sequence identity with other serine proteases from snake venom.TLBan showed the presence of His(44), Asp(91) residues and Ser was deduced (187) position, in the corresponding positions to the catalytic triad established in the serine proteases and Ser(187) are inhibited by phenylmethylsulfonyl fluoride (PMSF).In this work, we investigated the ability of TLBan to degrade fibrinogen and we observed that it is able to cause α- and β-chain cleavage. Enzymatic activities as well as the platelet aggregation were strongly inhibited when were incubated with PMSF, a specific inhibitor of serine protease. TLBan showed a potential medical-scientific interest to understand the pathophysiological mechanism of the snake venom action and identification of new blood coagulation cascade acting enzymes of natural sources.     

Purification and characterization of an anticoagulant phospholipase A(2) from Indian monocled cobra (Naja kaouthia) venom.

An anticoagulant, non-toxic phospholipase A(2) was isolated from the venom of Indian monocled cobra (Naja kaouthia) by a combination of ion-exchange chromatography on CM-Sephadex C-50 and gel filtration on Sephadex G-50. This purified protein named NK-PLA(2)-I, had a subunit molecular mass of 13.6 kDa and migrated as a dimer under non-reduced condition in SDS-PAGE. NK-PLA(2)-I was a highly thermostable protein requiring basic pH optima for its catalytic activity and showed preferential hydrolysis of phosphotidylcholine. This protein exhibited higher anticoagulant, indirect hemolysis, liver and heart tissue damaging activity but exerted less toxicity, direct hemolysis, edema and lung tissue damaging activity as compared to whole venom. Treatment of NK-PLA(2)-I with rho-BPB, TPCK, PMSF, antivenom and heating had almost equal effect on PLA(2), and other pharmacological properties except in vitro tissue damaging activity. Current investigation provides a fairly good indication that NK-PLA(2)-I induces various pharmacological effects by mechanisms, which are either dependent or independent of its catalytic activity.     

Biochemical and biological activities of the venom of a new species of pitviper from Vietnam, Triceratolepidophis sieversorum.

Biochemical and biological activities of a venom sample from a recently discovered new genus and species of pitviper from Vietnam, Triceratolepidophis sieversorum, were assayed and compared with those of five other viperid snakes (Bothrops asper, Crotalus atrox, Protobothrops flavoviridis, Trimeresurus insularis, and Vipera ammodytes). The venom had high casein hydrolysis, arginine ester hydrolysis and haemorrhagic activities, lacked measurable phosphodiesterase and L-amino acid oxidase activities, and had no procoagulant activity on either bovine fibrinogen or human plasma. Other enzymatic activities (phospholipase A(2), kallikrein) were moderate. The approximate i.p. LD(50) (mice) of the venom is about 5-6 mg/kg.