Isolation of a proteinase with plasminogen-activating activity from Lachesis muta muta (bushmaster) snake venom

A plasminogen activator enzyme (LV-PA) from Lachesis muta muta venom was purified to homogeneity using gel filtration and anion exchange chromatography. SDS-PAGE under reducing conditions showed a single protein band with an Mr of 33,000 Da. It is an acidic glycoprotein which activates plasminogen to plasmin indirectly, functioning via prior formation of a molecular complex, known as plasminogen activator. The purified preparation catalyzes the hydrolysis of several p-nitroanilide peptide substrates containing Lys at the scissile bond. In contrast, no hydrolysis was detected on the synthetic substrates TAME and BAPNA, which contain arginine. By the use of the plasmin-specific chromogenic substrate Tos-Gly-Pro-Lys-pNA, the preparation had a plasmin-like activity of 0.68 U/mg, which was 35.8-fold higher than that of the crude venom from which it was prepared. In vitro, fibrin hydrolysis using LV-PA as plasminogen activator displayed more similarity with the effect produced by streptokinase (SK). SDS-PAGE (10%) analysis showed a 115-kDa complex formation after incubation of plasminogen with either LV-PA or SK. At a molar ratio of 50:1 (fibrinogen:enzyme), the preparation exhibited weakly fibrinogenolytic activity. However, LV-PA is distinguished from thrombin in that it does not clot fibrinogen. After incubation of LV-PA with platelet-rich plasma, the enzyme (2 microM) showed no effect on platelet aggregation induced by ADP, epinephrine, or collagen. Comparison of the N-terminal sequence of LV-PA with other snake venom plasminogen activators revealed that LV-PA exhibits a high degree of sequence identity with the TsVPA from Trimeresurus stejnegeri (90%) and with the Haly-PA from Agkistrodon halys (85%). LV-PA also has homology with other snake venom serine proteinases such as the thrombin-like/gyroxin analogue (38%) from bushmaster venom and with other coagulation serine proteases. The proteinase was readily inhibited by treatment with p-nitrophenyl p-guanidinebenzoate, p-aminobenzamidine, and phenylmethanesulfonyl fluoride but was not affected by metal chelators.     

Purification and properties of the thrombin-like enzyme from the venom of Lachesis muta muta

1. The coagulating enzyme of the Lachesis muta muta venom was purified to homogeneity by a combination of a gel filtration in Sephadex G-100 and affinity chromatography on agarose-agmatine resin. 2. Several forms of the enzyme were prepared by isoelectric focusing with pIs ranging from 3.1 to 5.0; the asialoenzyme focused as a narrow band at pH 8.7. SDS-PAGE analysis of the purified enzyme revealed a single broad band with apparent Mr of 41-47 kDa. 3. The enzyme cleaves only fibrinopeptide A from fibrinogen; it does not activate factor XIII and is devoid of kallikrein-like activity. 4. Kinetic properties of the enzyme were determined for representative synthetic chromogenic substrates and inhibitors.     

Isolation and characterization of lactose-binding lectins from the venoms of the snakes Lachesis muta and Dendroaspis jamesonii

Two lectins have been isolated: one from the venom of Lachesis muta (bushmaster lectin) and one from Dendroaspis jamesonii venom (Jameson's mamba lectin). The lectin from bushmaster venom (BML) is similar to the lactose-binding lectins previously isolated from snake venoms (Gartner et al. (1980) FEBS Lett. 117, 13-16; Gartner & Ogilvie (1984) Biochem. J. 224, 301-307) in that it is calcium-dependent, lactose inhibitable, and is a dimer of molecular weight 28,000. In contrast, the lactose-blockable lectin from Jameson's mamba venom (JML) has an apparent molecular weight of 26,000 and agglutinates erythrocytes in the presence of EDTA. The absorption spectra of BML were affected by the binding of calcium, or calcium and lactose to the lectin. However, JML spectra were not affected by these conditions. While the hemagglutination activity of each of the previously described lactose-binding snake venom lectins is inhibited by reducing agent, the activities of BML and JML are not affected by reducing agent. Antiserum against bushmaster lectin cross-reacts with thrombolectin, cottonmouth lectin (CML), rattlesnake lectin (RSL), and copperhead lectin (CuHL) but not lectin from Jameson's mamba venom. This evidence plus a comparison of atomic absorption spectra, isoelectric points and amino acid analyses of the lectins demonstrate that JML and BML are different from thrombolectin, CML, RSL, and CuHL.     

Purification and properties of a coagulant proteinase isolated from bushmaster (Lachesis muta) venom (Serpentes: Viperidae)

The venom of Lachesis muta is a rich source of a thrombin-like enzyme. Its coagulant proteinase was purified by DEAE -Sephadex A -50 followed by agmatine CH -Sepharose and gel filtration on Sephadex G-100. On polyacrylamide gel electrophoresis at pH 8.4 a single band was observed. Its molecular weight by gel filtration was 49,000. The coagulant and esterolytic activities toward human fibrinogen and Tame of the inudasa were 662 NIH units/mg of protein and 4.37 delta OD225/min x 10(-3)/micrograms/ml, respectively. These values represent 23 and 5.7 fold increase over the crude venom. The enzyme mudasa, was evaluated with serum from human patients at Hospital Nacional de Ni?os Dr. Carlos Sáenz Herrera and found to be a valuable reagent for the quantification of fibrinogen on heparinized plasma.     

Purification and some properties of hemagglutinating protein mutina from bushmaster Lachesis muta snake venom

Lachesis muta snake venom induced aggregation of bromelain sensitized human erythrocytes at a concentration of 1 mg/ml. The hemagglutinating protein was purified by DEAE-Sephadex A-50 column chromatography. Polyacrylamide gel electrophoresis revealed at least three bands, whereas SDS electrophoresis in the presence of 2-mercaptoethanol showed a single one. Isoelectric focusing revealed hemagglutinating activity in the range of pH 3-8. The maximum peak (mutina) at pH 5.5. This fraction was active in agglutinating human RBC of types A, B, O Rh (+) and B, O Rh (-). One mM EDTA and 1 mM Ca++ did not alter the agglutinating time significantly. Lactose and inositol inhibited the agglutination of A, B, O Rh (+) and B, O Rh (-) human RBC. The present study showed the non specificity of the hemagglutinating activity of mutina. It was also shown that mutina is a non-mitogenic protein.     

The complete amino acid sequence of the haemorrhagic factor LHFII, a metalloproteinase isolated from the venom of the bushmaster snake (Lachesis muta muta)

The complete amino acid sequence the haemorrhagic agent LHFII, a Zn and Ca containing metalloproteinase isolated from the venom of the Bushmaster snake Lachesis muta muta was determined by automated and DABITC/PITC microsequencing of the intact protein, fragments derived by cleavage with cyanogen bromide, and peptides resulting from enzymatic digestions with trypsin and the protease from S. aureus V8. The protein is composed of 200 residues and exhibits considerable sequence homology with the haemorrhagic toxins from a number of other snake venoms, and some metalloproteinases in the region of the putative Zn-binding sites.     

Changes in the electrophoretic pattern of the venom of the bushmaster (Lachesis muta stenophrys) stored under various conditions

Liquid and lyophilized samples of Lachesis muta venom were stored at different temperatures and for different periods of time, and analyzed by polyacrylamide gel electrophoresis (PAGE) and immunoelectrophoresis. Only slight variations were evident when three pools of freeze-dried venom, that had been kept at -30 degrees C for several months were compared with fresh venom. These results suggest that L. muta venom is not altered drastically when stored under these conditions.     

Purification and characterization of the hemorrhagic factor II from the venom of the Bushmaster snake (Lachesis muta muta)

Hemorrhagic factor II (LHF-II) was isolated from Lachesis muta muta (Bushmaster snake) venom using column chromatographies on Sephadex G-100, CM-Sepharose CL-6B and two cycles on Sephadex G-50. This preparation was devoid of phospholipase A2 as well as of the enzymes active on arginine synthetic substrates (TAME and BAPNA) which are present in the crude venom. LHF-II was homogeneous by SDS-polyacrylamide gel electrophoresis, immunodiffusion and immunoelectrophoresis. Also, a single symmetrical boundary with a value of 2.59 S was obtained by ultracentrifugation. LHF-II contains 180 amino acid residues, has a molecular weight of 22,300, and an isoelectric point of 6.6. It contains one gatom zinc and two gatoms calcium per mol protein. The hemorrhagic factor possesses proteolytic activity toward various substrates such as, casein, dimethylcasein, hide powder azure, fibrinogen and fibrin. It hydrolyzes selectively the A alpha-chain of fibrinogen, leaving the B beta- and gamma-chains unaffected. LHF-II is activated by Ca2+ and inhibited by Zn2+. The hemorrhagic as well as the proteinase activity is inhibited by cysteine and by metal chelators such as EDTA, EGTA and 1,10-phenanthroline. Inhibitors of serine proteinases such as phenylmethanesulfonyl fluoride (PMSF) and soybean trypsin inhibitor (SBTI) have no effect on the hemorrhagic factor.     

Biochemical and enzymatic characterization of two basic Asp49 phospholipase A2 isoforms from Lachesis muta muta (Surucucu) venom

Two basic phospholipase A₂ (PLA₂) isoforms were isolated from Lachesis muta muta snake venom and partially characterized. The venom was fractionated by molecular exclusion chromatography in ammonium bicarbonate buffer followed by reverse-phase HPLC on a C-18 μ-Bondapack column and RP-HPLC on a C-8 column. From liquid chromatography-electrospray ionization/mass spectrometry, the molecular mass of the two isoforms LmTX-I and LmTX-II was respectively measured as 14,245.4 and 14,186.2 Da. The pI was respectively estimated to be 8.7 and 8.6 for LmTX-I and LmTX-II, as determined by two-dimensional electrophoresis. The two proteins were sequenced and differentiated from each other by a single amino acid substitution, Arg₆₅ (LmTX-I) → Pro₆₅ (LmTX-II). The amino acid sequence showed a high degree of homology between PLA₂ isoforms from Lachesis muta muta and other PLA₂ snake venoms. LmTX-I and LmTX-II had PLA₂ activity in the presence of a synthetic substrate and showed a minimum sigmoidal behaviour; with maximal activity at pH 8.0 and 35–45 °C. Full PLA₂ activity required Ca²⁺ and was respectively inhibited by Cu²⁺ and Zn²⁺ in the presence and absence of Ca²⁺. Crotapotin from Crotalus durissus cascavella rattlesnake venom significantly inhibited (P < 0.05) the enzymatic activity of LmTX-I, suggesting that the binding site for crotapotin in this PLA₂ was similar to another in the basic PLA₂ of the crotoxin complex from C. durissus cascavella venom.     

Lachesis muta (Viperidae) cDNAs reveal diverging pit viper molecules and scaffolds typical of cobra (Elapidae) venoms: implications for snake toxin repertoire evolution

Efforts to describe toxins from the two major families of venomous snakes (Viperidae and Elapidae) usually reveal proteins belonging to few structural types, particular of each family. Here we carried on an effort to determine uncommon cDNAs that represent possible new toxins from Lachesis muta (Viperidae). In addition to nine classes of typical toxins, atypical molecules never observed in the hundreds of Viperidae snakes studied so far are highly expressed: a diverging C-type lectin that is related to Viperidae toxins but appears to be independently originated; an ohanin-like toxin, which would be the third member of the most recently described class of Elapidae toxins, related to human butyrophilin and B30.2 proteins; and a 3FTx-like toxin, a new member of the widely studied three-finger family of proteins, which includes major Elapidae neurotoxins and CD59 antigen. The presence of these common and uncommon molecules suggests that the repertoire of toxins could be more conserved between families than has been considered, and their features indicate a dynamic process of venom evolution through molecular mechanisms, such as multiple recruitments of important scaffolds and domain exchange between paralogs, always keeping a minimalist nature in most toxin structures in opposition to their nontoxin counterparts.     

Hemolytic activity of venoms from snakes of the genera Bothrop, Lachesis, Crotalus, and Micrurus (Serpentes: Viperidae and Elapidae]

Hemolytic activity of eight Peruvian snake venoms from the families Viperidae and Elapidae (Bothrops atrox, B. pictus, B. hyoprorus, B. bilineatus, B. neuwedii, Lachesis m. muta, Crotalus d. terrificus, Micrurus tschudi), and three Brazilian viperids (B. jararacussu, B. alternatus and C. d. collilineatus) is described. None of the venoms caused direct lysis on washed human erythrocytes. However, all of them caused indirect hemolysis provided that the incubation medium contains an exogenous source of lecithin. Venom of Micrurus tschudi was the most hemolytic (HD50 2.8 ug/ml) while that of B. bilineatus was the least (HD50 681.3 ug/ml). Only six of eleven venoms showed parallel curves of hemolytic activity, and the HD50 varied from 198 to 681 ug/ml and the following decreasing order of hemolytic activity was obtained: L. muta, C. d. terrificus, C. d. collilineatus, B. hyoprorus, B. bilineatus, B. alternatus.     

Characterization of a proteolytic enzyme from Lachesis muta venom (Serpentes: Viperidae).

A proteolytic enzyme from L. muta stenophrys was isolated by gel filtration on Bio Gel P-100 followed by FPLC on MONO S column. The enzyme exhibited proteolytic activity toward casein, hemoglobin and fibrinogen with a pH optimum around 10. The activity was inhibited by EDTA while trypsin inhibitors were not inhibitory. It is a glycoprotein, Mr 14 kDa with a high content of Asp, Glu, and Leu residues and a low content of Cys and Trp. The protease is devoid of myotoxic, hemorrhagic, esterolytic and amidolytic activities. It lyses the alfa and beta chains of human fibrinogen and releases kinin from L.M.W. kininogen. No release of histamine was observed upon incubation with mast cells.     

Cardiovascular alterations induced by the venom of Lachesis muta (serpentes: Viperidae) and its fibrinogenolitic enzyme

The fibrinogenolytic activity of Lachesis muta stenophyrs venom was studied. Wistar rats catheterized at carotid artery and jugular vein were inoculated with crude venom or enzyme and changes in arterial pressure, cardiac frequency and electrocardiogram were monitored. The enzyme induced a greater fibrinogen reduction than the crude venom without any cardiovascular or histological alteration. In vitro crude venom coagulated blood whereas the enzyme reduced fibrinogen in 23%. Results suggest the potential use of the fibrinogenolytic enzyme as antithrombotic agent.     

The biological properties of venoms from juvenile and adult taipan (Oxyuranus scutellatus) snakes.

1. The biological properties of four venom pooled samples from adult taipan (Oxyuranus scutellatus) snakes and one pooled venom sample from six juvenile taipan snakes (11 months old) were compared. 2. The intravenous LD50 (median lethal dose), procoagulant activity and enzymatic activities of the juvenile venom were not significantly different from those of the adult venoms. 3. The juvenile and adult venoms exhibited similar polyacrylamide gel electrophoretic (PAGE) and SDS-PAGE patterns, indicating that they possessed a similar protein composition. 4. The results suggest that there is no significant age-dependency in the biological properties of taipan venom.     

Purification, Properties, and N-Terminal Amino Acid Sequence of a Kallikrein-like Enzyme from the Venom of Lachesis muta rhombeata (Bushmaster)

Pit viper venoms contain multiple proteinases which cause considerable damage in tissues and systemic effects after envenomation. A proteinase, kallikrein-like enzyme, belonging to the serine group must play a very important role on systemic effects. The corresponding enzyme from Lachesis muta rhombeata venom was purified to homogeneity by a combination of isoelectrofocusing fractionation followed by one step of gel filtration HPLC. The enzyme focused with pI 5.0–6.5, it had a molecular mass of 32 kDa by gel filtration HPLC, had edematogenic activity, and induced a hypotensic effect in anesthetized rats. It exhibited strong N-α-tosyl-L-Arg methyl esterase (955.38 units/mg) and N-BZ-DL-Arg-pNA amidolytic (233.02 units/mg) activities, hydrolyzed tripeptide nitroanilide derivatives weakly or not at all, and cleaved selectively the A-α and B-β chains of fibrinogen, apparently leaving the Y-chain unaffected. The 30 N-terminal amino acid sequence of the L. m. rhombeata protein showed greatest identity (74% in 26 amino acids) with Crotalus viridis kallikrein-like protein, but significant similarities in sequence were observed with enzymes from other snake venoms and pig pancreatic kallikrein.     

A comparative study of the biological activities of venoms from snakes of the genus Agkistrodon (moccasins and copperheads)

1. The hemorrhagic, procoagulant, anticoagulant, phosphodiesterase, alkaline phosphomonoesterase, 5'-nucleotidase, hyaluronidase, arginine ester hydrolase, phospholipase A, L-amino acid oxidase and protease activities of 31 samples of venom from three species of Agkistrodon (A. bilineatus, A. contortrix and A. piscivorus) and 10 venom samples from five other related species belonging to the same tribe of Agkistrodontini were examined. 2. The results indicate that interspecific differences in certain biological activities of the Agkistrodon venoms are more marked than individual variations of the activities, and that these differences can be used for differentiation of the species. Particularly useful for this purpose are the phosphodiesterase, arginine ester hydrolase and anticoagulant activities of the venoms. 3. Venoms of the subspecies of A. contortrix and A. piscivorus do not differ significantly in their biological activities.     

A comparative study of the biological properties of venoms from snakes of the genus Vipera (true adders)

1. The hemorrhagic, procoagulant, anticoagulant, phosphodiesterase, hyaluronidase, alkaline phosphomonoesterase, 5'-nucleotidase, arginine ester hydrolase, phospholipase A, L-amino acid oxidase and protease activities of 26 samples of venoms of 13 taxa of Vipera were determined and the Sephadex G-75 gel filtration patterns for some of the venoms were also examined. 2. The results indicate the presence of certain common characteristics among the venoms, particularly if V. russelli is excluded from the comparison. The results also support the recently proposed reassignment of V. russelli to a separate genus. 3. The data show that information on venom biological properties can be used for differentiation of venoms of many species of Vipera. Particularly useful for this purpose are the protease, phosphodiesterase, phospholipase A and the procoagulant activities and the Sephadex G-75 gel filtration patterns of the venoms.     

Comparative chromatography of Brazilian coral snake (Micrurus) venoms.

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

Coagulant thrombin-like enzyme (barnettobin) from Bothrops barnetti venom: Molecular sequence analysis of its cDNA and biochemical properties

The thrombin-like enzyme from Bothrops barnetti named barnettobin was purified. We report some biochemical features of barnettobin including the complete amino acid sequence that was deduced from the cDNA. Snake venom serine proteases affect several steps of human hemostasis ranging from the blood coagulation cascade to platelet function. Barnettobin is a monomeric glycoprotein of 52 kDa as shown by reducing SDS-PAGE, and contains approx. 52% carbohydrate by mass which could be removed by N-glycosidase. The complete amino acid sequence was deduced from the cDNA sequence. Its sequence contains a single chain of 233 amino acid including three N-glycosylation sites. The sequence exhibits significant homology with those of mammalian serine proteases e.g. thrombin and with homologous TLEs. Its specific coagulant activity was 251.7 NIH thrombin units/mg, releasing fibrinopeptide A from human fibrinogen and showed defibrinogenating effect in mouse. Both coagulant and amidolytic activities were inhibited by PMSF. N-deglycosylation impaired its temperature and pH stability. Its cDNA sequence with 750 bp encodes a protein of 233 residues. Indications that carbohydrate moieties may play a role in the interaction with substrates are presented. Barnettobin is a new defibrinogenating agent which may provide an opportunity for the development of new types of anti-thrombotic drugs.