Biochemical and functional characterization of Bothropoidin: the first haemorrhagic metalloproteinase from Bothrops pauloensis snake venom.

Journal of Biochemistry Advance Access published September 26, 2014

Biochemical and functional characterization of Bothropoidin: The first hemorrhagic metalloproteinase from Bothrops pauloensis snake venom Mário Sérgio R. Gomesa,b*, Dayane L. Naves de Souzaa,e , Denise O. Guimarãesa, Daiana S. Lopesa, Carla C. N. Mamedea,c,e , Sarah Natalie C. Gimenesa, David C. Achêa , Renata S. Rodriguesa , Kelly A. G. Yoneyamaa, Márcia H. Borgesd, Fábio de Oliveirac,e, Veridiana M. Rodriguesa, e* a

Downloaded from http://jb.oxfordjournals.org/ at University of San Diego on September 29, 2014

Instituto de Genética e Bioquímica, Universidade Federal de Uberlândia, UFU, Uberlândia-MG, Brazil; b Departamento de Química e Exatas, Universidade Estadual do Sudoeste da Bahia (UESB), BA, Brazil, c Instituto de Ciências Biomédicas, Universidade Federal de Uberlândia (UFU), Brazil, d Fundação Ezequiel Dias, FUNED, Belo Horizonte-MG, Brazil, e INCT, Instituto Nacional de Ciência e Tecnologia em Nano-Biofarmacêutica.

*Corresponding author: [email protected] (Instituto de Genética e Bioquímica, Universidade Federal de Uberlândia (UFU), 38400-902 Uberlândia-MG, Brazil. Tel.: +55 34 32182203x22; fax: +55 34 32182203x24 and [email protected] (Departamento de Química e Exatas, Universidade Estadual do Sudoeste da Bahia (UESB), 45506-210 Jequié-BA, Brazil).

Abstract We present the biochemical and functional characterization of Bothropoidin, the first hemorrhagic metalloproteinase isolated from Bothrops pauloensis snake venom. This protein was purified after three chromatographic steps on cation exchange CM-

Sepharose fast flow, size-exclusion column Sephacryl S-300 and anion exchange Capto Q. Bothropoidin was homogeneous by SDS-PAGE under reducing and non-reducing conditions, and comprised a single chain of 49,558 Da according to MALDI TOF analysis. The protein presented an isoelectric point of 3.76, and the sequence of six fragments obtained by MS (MALDI TOF\TOF) showed a significant score when compared to other PIII SVMPs. Bothropoidin showed proteolytic activity on azocasein,

© The Authors 2014. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved. 1

Aα-chain of fibrinogen, fibrin, collagen and fibronectin. The enzyme was stable at pH 6–9 and at lower temperatures when assayed on azocasein. Moreover, its activity was inhibited by EDTA, 1.10-phenanthroline and β-mercaptoethanol. Bothropoidin induced hemorrhage (MHD=0.75 µg), inhibited platelet aggregation induced by collagen and ADP, and interfered with viability and cell adhesion when incubated with endothelial cells in a dose and time dependent manner. Our results showed that Bothropoidin is a hemorrhagic metalloproteinase that can play an important role in the toxicity of

of SVMPs on hemostatic disorders and tumor metastasis.

Keywords: Bothrops pauloensis; Fibrinogenolytic activity; Hemorrhagic activity; Platelet aggregation; Snake venom metalloproteinase

1. Introduction

Snake venom metalloproteinases (SVMPs) are the most representative toxins in viperidic snakes venom [1, 2, 3, 4] and are responsible for the majority of local and systemic effects observed after envenomation [5, 6,]. They degrade extracellular matrix components [7], plasmatic proteins [8, 6], cell membrane proteins, as well as interact with specific receptors [9, 10, 11] on endothelial cells [12, 13] and fibroblasts [14] resulting in various pathophysiological effects such as bleeding, inflammation, platelet aggregation inhibition and apoptosis [7, 15, 16, 17, 6]. SVMPs comprise an important class of zinc-dependent enzymes of varying molecular mass and were originally classified in four distinct classes (PI to PIV) according to their structural diversity [18]. In 2008, Fox and Serrano [19] proposed 2


Bothrops pauloensis envenomation and might be used as a tool for studying the effects

changes in this classification based on studies of the precursors of SVMPs, as well as the products generated by post-translational modifications. According to this proposal several subclasses were established (P-Ia, P-IIa, P-IIb, P-IIc, P-IId, D-I, P-IIe, P-IIIa, PIIIb, P-IIIc and P-IIId). SVMPs from P-I class are generally fibrinogenolytic and weakly hemorrhagic composed in the mature form only of the catalytic domain. The PII SVMPs are synthesized with a metalloproteinase plus a disintegrin domain, but are frequently found in venoms as a processed form containing only a disintegrin domain.

domain followed by disintegrin-like and cysteine-rich domains, linked via the carboxyterminal to the catalytic domain. P-I and P-III SVMPs are the most abundant group in viper venoms which conserve the catalytic domain in the mature form [19]. During past few years, different SVMPs have been isolated from snake venoms and their structure and mechanisms of action were described [20, 21, 22, 23]. Moreover, these toxins have become important targets of research, as new models for the development of antitumor, antiparasitic, antimicrobial and thrombolytic agents [24, 6, 25, 26]. In this context, we have exploited the Bothrops pauloensis venom, a species abundant in central region of Brazil [27, 28], to search new compounds that can interfere on hemostasis [20, 29, 30] or interfere in different cellular organisms [31, 32, 33]. Besides, our studies about gland transcriptome and proteome of Bothrops pauloensis venom demonstrated the presence of many peptides and proteins that had not previously been isolated or characterized in this species, including SVMPs [4]. Therefore, in the present work, we aimed to isolate and characterize biochemically and functionally the first hemorrhagic metalloproteinase from Bothrops pauloensis snake venom. 3


SVMPs from P-III class are frequently potent hemorrhagins that consist of a catalytic

2. Material and methods 2.1 Venom and animals Bothrops pauloensis snake venom was purchased from Serpentarium Bioagents, Batatais, São Paulo, Brazil. This serpentarium is registered in the Brazilian Institute of

obtained from Center for Animal Experimentation of the Federal University of Uberlândia. Animal experimental procedures were approved by Ethics Committee for Animal utilization of Federal University of Uberlândia (nº 046/09).

2.2. Purification of Bothropoidin The enzyme was purified according to methodology previously described by our research group [20, 30] with some modifications. B. pauloensis crude venom (125 mg) was resuspended in 50 mM ammonium bicarbonate pH 7.8 (AMBIC) buffer, and clarified by centrifugation at 1000g for 10 min. The supernatant solution was fractionated on a cation exchange CM-Sepharose fast flow column (1.4x26cm), previously equilibrated with the same buffer. A linear gradient was then applied up to 500mM AMBIC buffer at a flow rate of 1.0 mL/min and fractions were monitored at 280 nm. CM1 fraction which showed azocaseinolytic and hemorrhagic activities was chromatographed on a size-exclusion column Sephacryl S-300 HR HiPrep 26/60 column. The fractions were eluted with 50 mM ammonium bicarbonate pH 7.8 (AMBIC) buffer at a flow rate of 0.2 mL/min using AKTA prime plus equipment (Amersham Biosciences). 4


Environment and Renewable Natural Resources (nº 471301). BALB/c mice were

Hemorrhagic fraction (S1) was further applied to an anion exchange HiTrep

Capto Q column (three columns of 1mL each) previously equilibrated with 50 mM ammonium bicarbonate pH 7.8 (AMBIC) buffer. Elution was performed by a linear gradient (50–500mM AMBIC) at a flow rate of 6 mL/hour resulting in two fraction (Q1 and Q2). Q2 fraction, named Bothropoidin was submitted to reverse-phase chromatography in an HPLC system using a C2C18 (4.6x150mm) column (GE Healthcare Bio-Sciences) previously equilibrated with solvent A (0.1% trifluoroacetic

solvent B (80% acetonitrile, 0.1% trifluoroacetic acid) from 0 to 100% at a flow rate of 0.5 mL/min at room temperature. The protein contents were monitored at 280 nm and the single peak was separated and lyophilized to determine the partial amino acid sequence. Protein quantification was performed according to Bradford [34].

2.3 Biochemical Characterization 2.3.1. Determination of Mr Molecular mass of Bothropoidin was estimated by 12% SDS-PAGE (w/v) under reducing and non-reducing conditions [35]. Mr was obtained by interpolation from a linear logarithmic plot of relative molecular mass versus distance of migration. Bovine serum

albumin

(66 kDa),

ovalbumin (45 kDa),

glyceraldehyde-3-phosphate

dehydrogenase (36 kDa), carbonic anhydrase (29 kDa), trypsinogen (24 kDa), trypsin inhibitor (20 kDa) and α-lactalbumin (14.2 kDa) (Amersham Biosciences) were used as molecular mass markers.

2.3.2. MALDI TOF Mass Spectrometry

5


acid and 5% acetonitrile). The protein elution was conducted using a linear gradient of

Bothropoidin was also submitted to mass spectrometric analysis, using an AutoFlex III (Bruker Daltonics, Bremen, Germany) controlled by the software FlexControl 3.0 (Bruker Daltonics, Bremen, Germany). The sample was mixed with sinapinic acid matrix solution (1:1, v/v) directly onto a target plate (Bruker Daltonics, Bremen, Germany) and dried at room temperature. The mean mass of the protein was obtained in linear mode with external calibration, using a Protein Calibration Standard (Bruker Daltonics, Bremen, Germany). The software Flex Analysis 3.0 (Bruker

2.3.3. Isoelectric focusing Bothropoidin (20µg) was precipitated using the 2-D Clean-up Kit (GE Healthcare) as instructed by the manufacturer. After 5 minutes of drying, the sample was dissolved in 200 µL rehydration buffer (6 M urea, 2 M thiourea, 2% ASB-14 (w/v), 15 mM DTT, 0.002% blue bromophenol (w/v) and IPG Buffer, pH 3–10 0.5% (v/v) (GE Healthcare). Rehydration was performed in the IPGPhor III system (GE Healthcare), at 40 volts, for 14 hours. After rehydration, polyacrylamide strips (11 cm) with an immobilized pH gradient (pH 3 to 10) were subjected to isoelectric focusing in the same system at a total of 34636 V/h. Strips were treated with 10 mL of equilibrium solution (1.5 M Tris- HCl pH 8.8, 6 M urea, 30% glycerol (w/v), 2% SDS (w/v), 0.002% bromophenol blue (w/v)) containing 10 mg/mL of dithiothreitol (DTT) for 15 min. and after treated with 25 mg/mL of iodoacetamide. Then, the strips were subjected to 12% SDS-PAGE (w/v) in the Multiphor II system, according to the manufacturer’s instructions, and the gel was stained with 2% (w/v) Coomassie G-250. After 2D electrophoresis, the gel was scanned in Image Scanner (GE Healthcare), using Image

6


Daltonics, Bremen, Germany) was used for analysis of mass spectrometric data.

Master LabScanTM v 5.0, with a resolution of 300 dpi. Analysis was performed using Image Master 2D Platinum v 6.0 (GE Healthcare).

2.3.4. MALDI-TOF/TOF mass spectrometer analysis Bothropoidin (80 µg) was digested with trypsin and subjected to MALDITOF/TOF mass spectrometer (Autoflex III MALDI-TOF-TOF, Bruker Daltonics,

reflector mode with external calibration, using a standard mixture of peptides (Bruker Daltonics, Billerica, EUA). Six selected peaks were subjected to MS/MS fragmentation using the LIFT technique and mass spectra were analyzed using Flex Analysis and Biotools software (Bruker Daltonics). The Mascot software [36] was utilized to identify the sequence, and searches were performed against the NCBI database. The peptide fragments generated were aligned using the program ClustalW [37].

2.4. Enzymatic characterization 2.4.1. Azocaseinolytic activity Azocasein (1.5 mg/mL) was dissolved in 20mM Tris–HCl (pH 7.4) containing 5mM CaCl2 and incubated with 5 µg of enzyme at 37 °C for 60 min. After incubation, the reaction was interrupted by the addition of 100µl of 20% (w/v) trichloroacetic acid (TCA). The plate was incubated at room temperature for 20 min, centrifuged at 5000 g for 15 min and absorbance of the supernatant was determined at 405 nm. One unit (U) of azocaseinolytic activity was defined as an increase of 0.01 absorbance units at 405 nm under standard assay conditions. All assays were performed in triplicate. The

7


Billerica, EUA). The monoisotopic peptide masses were obtained by positive and

stability of the enzyme at various pH intervals (3 to 11) and temperatures (20 to 80ºC) was examined by incubating 5µg protein for 30 min at 37 °C. The azocaseinolytic activity was assayed as described above. The effect of protease inhibitors and divalent metals was also examined. The enzyme (5 µg) was preincubated with 10µL of βmercaptoethanol (10mM), EDTA (10mM), 1,10-phenanthroline (10mM), PMSF (10mM), aprotinin (10mM) or divalent metals (CaCl2, BaCl2, MgCl2, ZnCl2 and HgCl2) (10mM) for 1 h at 37 °C. Then the azocaseinolytic activity was assayed as described

2.4.2. Fibrinogenolytic activity Fibrinogenolytic activity was determined according to [38] Gomes et al., (2011) with some modifications. Briefly, samples of 50 µL of fibrinogen (1.5 mg/mL) in 50mM Tris-HCl, pH7.4 and 5mM CaCl2 were incubated with 5 µg of Bothropoidin for different periods of time (5, 10, 15, 30, 60 or 120 min) at 37 °C. The reaction was stopped with 25 µL 50mM Tris-HCl, pH 8.8, containing 10% (v/v) glycerol, 5% (v/v) βmercaptoethanol, 2% (w/v) SDS, and 0.05% (w/v) bromophenol blue. The samples were then analyzed by 12% SDS-PAGE (w/v).

2.4.3. Proteolytic activity upon fibrin Bovine fibrinogen (2.0 mg/mL) was dissolved in 50mM Tris-HCl, pH 7.4 containing 5mM CaCl2 and incubated with 10 NIH units/mL human thrombin (Sigma) at room temperature for 2 hours for polymerization. After 2 h, the solution was treated with 5µg of enzyme in the same buffer and incubated at various time intervals (2, 4, 6, 12 hours) at 37 °C, followed by the addition of denaturing solution containing 2% SDS (w/v), 5% β-mercaptoethanol, 10% glycerol and 0.005% bromophenol blue. The fibrin digestion was analyzed by 12% SDS-PAGE (w/v). 8


above.

2.4.4. Degradation of extracellular matrix components Proteolytic activity on extracellular matrix components was assayed as previously described by [39] Kumar et al., (2010), with modifications. Fibronectin (1mg/mL) or Type IV collagen solution (2.5 mg/mL) in 50mM Tris–HCl pH 7.4 and 5mM CaCl2 was incubated with 5µg Bothropoidin at 37 ºC for 2, 4 and 6 h. The reaction was stopped by adding 25 µL of 0.05 M Tris–HCl, pH 8.8, containing 10% glycerol (v/v), 2% SDS (w/v), 0.005% bromophenol blue (v/v) and 5% β-

(w/v).

2.4.5. Hydrolytic activity upon chromogenic substrates The proteolytic activity of Bothropoidin was also evaluated using different

substrates S-2238 (thrombin-like substrate), S-2222 (Factor Xa), S-2251 (plasmin) and S-2302 (plasmatic kallikreins). Briefly, a mixture of 200µL of substrate (50mM TrisHCl and 5mM CaCl2, pH 7.4) was incubated with 50µL Bothropoidin (1 or 2µg) for 20 min at 37 °C. Proteolytic activity was monitored in polystyrene 96-well plates at 405 nm in a multi-well scanning spectrophotometer (ELISA reader). One unit (U) of activity was defined as an increase of 0.01 absorbance units at 405 nm under standard assay conditions. All assays were performed in triplicate.

2.5. Biological characterization 2.5.1. Hemorrhagic activity The hemorrhagic activity was measured in the mouse skin as described by [40] Nikai et al., (1984) with some modifications. Groups of five Balb-C mice (18–20 g) were intradermally (i.d.) injected Bothropoidin (0.5–4 µg) or crude venom (2–6 µg), 9


mercaptoethanol (v/v). The substrate digestion was analyzed by 7.5% SDS-PAGE

previously diluted in 50 µL of PBS. Control animals received only 50µL of PBS. After 3 h, the animals were anesthetized (ketamin® 10% (0.05ml/kg) + xilasin® 2% (0.025ml/kg) and sacrificed. The skin was removed and the diameters of the hemorrhagic halos were measured. The minimum hemorrhagic dose (MHD) was defined as the dose of enzyme that results in a hemorrhagic lesion of 10 mm2 diameter after 3 h. Hemorrhagic activity was also evaluated after incubation with the toxin pretreated with 1 mM and 10 mM EDTA (1 h at 37ºC).

Defibrinating activity was assayed according to [41] Gene et al., (1989), with some modifications. Groups of five Balb-C mice (18–20 g) were intraperitoneally injected with 5µg of Bothropoidin previously diluted in 50 µL of PBS. Control animals received only 50µL of PBS. After 3 h, the animals were anesthetized (ketamin® 10% (0.05ml/kg) + xilasin® 2% (0.025ml/kg) and sacrificed and bled by cardiac puncture. Whole blood was placed in tubes and kept at 25°C for observation of coagulation until clotting occurred. 2.5.3. Coagulant activity The coagulant effect was tested by adding 5µg of Bothropoidin in 50mM TrisHCl pH 7.4 to 200µL of citrated bovine plasma at 37°C. The tests were performed in a micro-processor Quick-Timer analyzer (DRAKE LTDA).

2.5.4. Activated partial thromboplastin time (aPTT) and prothrombin time (PT) For evaluation of APTT and PT groups of five Balb-C mice (18–20 g) were intraperitoneally injected with 5µg of Bothropoidin previously diluted in 50 µL of PBS. Control animals received only 50µL of PBS. After 3 h, the animals were anesthetized 10


2.5.2. Defibrinating activity

(ketamin® 10% (0.05ml/kg) + xilasin® 2% (0.025ml/kg), sacrificed and bled by cardiac puncture. Blood samples were centrifuged (360g for 15 min at 4°C) to obtain the plasma. APTT and PT were measured on a micro-processor Quick-Timer analyzer (DRAKE LTDA) according to instructions supplied by the Bioclin Kit manufacturer.

2.5.5. Platelet aggregation Platelet aggregation was assayed as previously described by [42] Moura-da-

presence of citrate and centrifuged at 360g for 15 minutes at room temperature to obtain platelet-rich plasma (PRP). PRP was diluted in 0.1% EDTA and centrifuged for 20 min at 850 g. The pellets containing the platelets were washed twice with Tyrode’s solution (0.35% (w/v) bovine serum albumin (BSA) and 0.1 mM EGTA, pH=6.5) and the platelet concentration was adjusted to 3–4 x 105platelets/mL for the aggregation assay with a Agg RAM (Remote Aggregation Analyzer). Samples of 200 µL washed platelets were incubated for 15 min with 5µg of Bothropoidin and challenged with 100µg collagen or 5mM ADP. The extent of aggregation was calculated for 5 min after the addition of collagen or ADP.

2.5.6. Cell viability and adhesion Murine endothelial cell line derived from thymus hemangioma (tEnd) [43] was cultivated in RPMI1640 medium containing 10% (v/v) FBS, 2 mM L-glutamine, 1 mM sodium pyruvate, 1 mM non-essential amino acid, 100 UI/mL penicillin, 100 mg/mL streptomycin and incubated at 37 ºC and 5% CO2. Cell viability of cultures treated with Bothropoidin was evaluated by MTT assay. Cells were seeded at 2 × 104 cells/well in 11


Silva et al., (2008), with some modifications. Bovine blood was collected in the

96-well microplates. After 24 h, the medium was changed and the samples containing Bothropoidin (40, 20, 10, 5, 2.5, 1.25 and 0.625 µg/mL) or control (culture medium) were added. After 6 and 24h, cells were incubated with 20 µL/well of MTT (5 mg/mL diluted in PBS) kept 3 h at 37 ºC. Formazan crystals resulting from MTT reduction were dissolved by adding 100 µL of PBS containing 10% SDS and 0.01 M HCl (18 h, 37 ºC and 5% CO2). The absorbance was read on a multi-well scanning spectrophotometer (ELISA reader) at 570 nm. To quantify the number of adherent cells, the detached cells

remaining cells were stained by MTT assay as described above. Morphological changes in endothelial cells induced by Bothropoidin was observed in culture of cells incubated with catalytically active Bothropoidin (20 µg/ml) or Bothropoidin inactivated by EDTA (1 mM) for 24 hours by phase contrast under a magnification of X200.

2.5.7. Statistical Analysis The results were presented as means ± standard deviation (S.D.). The statistical significance of the results was evaluated using Student's t-test and the Graphpad Prism 5 Project program.

3. Results and discussion In the present work we report the purification and characterization of the first hemorrhagic SVMP from Bothrops pauloensis snake venom. The protein was purified using three chromatographic steps. Initially, crude venom (125 mg) was fractionated on a CM-Sepharose fast flow column resulting in six fractions (CM1 to CM6) (Fig. 1A). The fraction with azocaseinolytic and hemorrhagic activity (CM1) was further 12


were removed after treatment with Bothropoidin by two washes with PBS, and the

chromatographed on a HiPrep Sephacryl S-300 column and resolved into seven fractions (S1 to S7) (Fig. 1B). S1 showed azocaseinolytic and hemorrhagic activity (Table I and Fig. 1B). Subsequently, S1 fraction was applied to a HiTrep Capto Q column resulting in two fractions (Q1 and Q2) (Fig. 1C). Q2 fraction (with hemorrhagic activity) (Fig. 1C) was analyzed by reverse-phase HPLC (C2C18/RP-HPLC) (Fig. 2B) and consisted of a single polypeptide chain of approximately 52,000 and 49,800 by SDS-PAGE under reducing and non-reducing

(Fig. 2D). This fraction named Bothropoidin represented 0.34% of B. pauloensis venom and showed proteolytic activity upon azocasein (Table I). The partial sequence of Bothropoidin was determined by mass spectrometry. Protein hydrolyses with trypsin resulted in six peptides with masses of 727, 1109, 1669, 2020, 2145 and 2675 Da (data not shown), which were fragmented by the LIFT method (Bruker Daltonics) (data not shown). MS/MS spectrum were analyzed by Flex Analysis 3.0 (Bruker Daltonics) and submitted to Biotools (Bruker Daltonics) and Matrix Science (Mascot Search) software for protein identification. Results revealed a significant score (scores > 49 and p

Molecular cloning and pharmacological properties of an acidic PLA2 from Bothrops pauloensis snake venom.

Isolation and biochemical characterization of a new thrombin-like serine protease from Bothrops pirajai snake venom.

Molecular characterization of metalloproteases from Bothrops alternatus snake venom.

Molecular cloning of a hyaluronidase from Bothrops pauloensis venom gland.

Bp-13 PLA2: Purification and Neuromuscular Activity of a New Asp49 Toxin Isolated from Bothrops pauloensis Snake Venom.

Purification and biochemical characterization of three myotoxins from Bothrops mattogrossensis snake venom with toxicity against Leishmania and tumor cells.

Isolation and characterization of basic myotoxic phospholipases A2 from Bothrops godmani (Godman's pit viper) snake venom.

Heterologous expression and biochemical and functional characterization of a recombinant alpha-type myotoxin inhibitor from Bothrops alternatus snake.

CcMP-II, a new hemorrhagic metalloproteinase from Cerastes cerastes snake venom: purification, biochemical characterization and amino acid sequence analysis.

Data for a direct fibrinolytic metalloproteinase, barnettlysin-I from Bothrops barnetti (barnett(,)s pitviper) snake venom with anti-thrombotic effect.

A neutralizing recombinant single chain antibody, scFv, against BaP1, A P-I hemorrhagic metalloproteinase from Bothrops asper snake venom.

Neuromuscular activity of Bothrops fonsecai snake venom in vertebrate preparations.

Neutralization of myotoxic phospholipases A2 from the venom of the snake Bothrops asper by monoclonal antibodies.

Rat atrial responses to Bothrops jararacussu (jararacuçu) snake venom.

Biochemical and pharmacological characterization of three toxic phospholipase A2s from Daboia russelii snake venom.

Activation of Bothrops jararaca snake venom gland and venom production: a proteomic approach.

Purification and characterization of BmooAi: a new toxin from Bothrops moojeni snake venom that inhibits platelet aggregation.

BaltDC: purification, characterization and infrared spectroscopy of an antiplatelet DC protein isolated from Bothrops alternatus snake venom.

Systemic haemorrhage in rats induced by a haemorrhagic fraction from Bothrops jararaca venom.

Activation of J77A.1 macrophages by three phospholipases A2 isolated from Bothrops atrox snake venom.

Rapid purification of a new P-I class metalloproteinase from Bothrops moojeni venom with antiplatelet activity.

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

Molecular cloning, expression and characterization of albolamin: a type P-IIa snake venom metalloproteinase from green pit viper (Cryptelytrops albolabris).

Pathological and biochemical changes induced in mice after intramuscular injection of venom from newborn specimens of the snake Bothrops asper (Terciopelo).