A new hemorrhagic metalloprotease from Bothrops jararacussu snake venom: isolation and biochemical characterization

Toxicon 44 (2004) 215–223 www.elsevier.com/locate/toxicon

A new hemorrhagic metalloprotease from Bothrops jararacussu snake venom: isolation and biochemical characterization Maurı´cio V. Mazzia, Silvana Marcussib, Guilherme B. Carlosb, Rodrigo G. Sta´belic,d, Joa˜o J. Francoa, Fa´bio K. Ticlia, Ade´lia C.O. Cintraa, Suzelei C. Franc¸ab, Andreimar M. Soaresb,1, Suely V. Sampaioa,* a

Departamento de Ana´lises Clı´nicas, Toxicolo´gicas e Bromatolo´gicas, FCFRP, USP, Ribeirao Preto-SP, Brazil b Unidade de Biotecnologia, UNAERP, Ribeirao Preto-SP, Brazil c Laborato´rio de Bioquı´mica do Instituto de Pesquisas em Patologias Tropicais (IPEPATRO), Porto Velho-RO, Brazil d Departamento de Bioquı´mica e Imunologia, FMRP, USP, Ribeirao Preto-SP, Brazil Received 22 March 2004; revised 27 May 2004; accepted 1 June 2004

Abstract A hemorrhagic metalloprotease, named BjussuMP-I, was isolated from Bothrops jararacussu snake venom by a combination of gel filtration on Sephacryl S-200 (0.01 M Tris – HCl, pH 7.6 buffer) and Phenyl Sepharose CL-4B chromatography (0.01 M Tris – HCl plus 4 M NaCl, pH 8.6 buffer, followed by a concentration gradient from 4 to 0 M NaCl at 25 8C in the same buffer). BjussuMP-I is a 60 kDa protein with a pI , 5.5, which induced hemorrhage after intradermal injection in mice, with a minimum hemorrhagic dose of 4.0 mg. The hemorrhagic activity of BjussuMP-I was totally abolished after incubation with a chelating agent (EDTA), corroborating the metal-dependency of this effect. BjussuMP-I shows proteolytic activity on casein and fibrinogen, although having an activity lower than that of crude B. jararacussu venom and the metalloprotease neuwiedase isolated from Bothrops neuwiedi snake venom. It was recognized by anti-neuwiedase antibodies, with a reaction of partial immunologic identity. BjussuMP-I also shows bactericidal activity against Escherichia coli and Staphylococcus aureus. This is the first report on the isolation and characterization of a high molecular weight hemorrhagic metalloprotease (BjussuMP-I) from B. jararacussu venom, which may play a relevant role in local and systemic bleeding which characterizes Bothrops envenomations. q 2004 Elsevier Ltd. All rights reserved. Keywords: Metalloproteases; Hemorrhagic and proteolytic activity; Snake venom; Bothrops jararacussu

1. Introduction Venomous animals have been evolved in a vast array of proteins and peptides. They comprise a complex mixture of Abbreviations: SVMP, snake venoms metalloproteases; BjussuMP-I, Bothrops jararacussu metalloprotease I; PLA2, phospholipase A2; BthTX-I, bothropstoxin-I from B. jararacussu; MP, metalloprotease. * Corresponding author. Tel.: þ 55-16-633-1936. E-mail addresses: [email protected] (S.V. Sampaio), [email protected] (A.M. Soares). 1 Fax: þ55-16-603-7030. 0041-0101/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.toxicon.2004.06.002

both toxic and nontoxic components for prey capture and defence (Pal et al., 2002; Lewis and Garcia, 2003). Features of Bothrops genus envenomation are characterized by serious muscle damage, hemorrhage and blood coagulation disorders (Bjarnason and Fox, 1994; Kamiguti et al., 1996; Gutie´rrez, 2002). Moreover, it is believed that this effect arises due to synergic action of proteolytic enzymes, such as metalloproteases and serine-proteases (Matsui et al., 2000). Hemorrhage occurs not only locally at the site of the bite, but also systemically, contributing to the cardiovascular shock, characteristic of severe envenomations (Warrell, 1995). Snake venom hemorrhagic components are zincdependent metalloproteases of varying molecular weights

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that hydrolyze proteins of the basal lamina of capillary vessels, inducing extravasation (Sto¨cker et al., 1995; Kamiguti et al., 1996). Snake venoms metalloproteases (SVMP) are members of the Reprolysin subfamily of enzymes, which selectively cleave key peptide bonds of basement membrane components (Gong et al., 1998; Rodrigues et al., 2000), effecting the interactions with endothelial cells (Gutie´rrez and Rucavado, 2000; Kawano et al., 2002). These toxins are synthesized in the venom gland as large multidomain proteins, including a proenzyme domain and a conserved zinc-protease domain (Jia et al., 1996; Matsui et al., 2000). They are secreted as preproenzymes and contain additional regulatory modules, which are responsible for interactions with the extracellular matrix and integrins. The mature P-I class proteins have only a metalloprotease domain, whereas the P-II, P-III, and P-IV classes have disintegrin or disintegrin-like, cysteine-rich, and lectin-like domains found close to the carboxyl end of the protease, respectively (Matsui et al., 2000; Gutie´rrez and Rucavado, 2000). SVMP are able to hydrolyze proteins of the basal membrane including fibronectin, laminin, and type IV collagen (Esteva˜o-Costa et al., 2000; Lorı´a et al., 2003). They also degrade fibrinogen and fibrin. Fibrinogenases with specificity for the Aa chain of fibrinogen contain Zn2þ and lack arginine esterase activity. These enzymes have been purified from the venoms of Bothrops moojeni, Bothrops jararacussu, Bothrops jararaca, Bothrops neuwiedi, Agkistrodon halys brevicadus, and Trimeressurus elegans. The primary and three-dimensional structures of several metalloproteases have been determined, showing that they are structurally related (Matsui et al., 2000). This paper reports the isolation and functional characterization of a new 60 kDa hemorrhagic fibrin(ogen)olytic metalloprotease, BjussuMP-I, from the venom of B. jararacussu, a snake found in Southeastern Brazil.

Fractions of 3 ml/tube were collected at a flow rate of 10 ml/h at 25 8C. The fraction displaying hemorrhagic activity (50 mg) was then dissolved in 2 ml of 0.01 M Tris – HCl buffer, pH 7.6, plus 4 M NaCl and applied to a 1.5 £ 9 cm Phenyl Sepharose CL-4B previously equilibrated with the same buffer. A reverse linear NaCl gradient (4– 0 M) was then applied and fractions of 2.5 ml/tube were collected at a flow rate of 50 ml/h at 25 8C. The fraction displaying hemorrhagic activity was submitted to biochemical, enzymatic and pharmacological characterization. 2.3. SDS-polyacrylamide gel electrophoresis SDS-PAGE was carried out according to previously described methods (Laemmli, 1970). Samples were pretreated in reducing conditions (SDS plus b-mercaptoethanol) at a 100 8C for 5 min. Gels were stained with 0.1% Coomassie brilliant blue R-350 in ethanol:acetic acid (5:1, v/v) for 15 min and distained in 10% acetic acid. The molecular mass was estimated by interpolation from a linear logarithmic plot of relative molecular mass versus distance of migration. Standards molecular weight markers (Merck) were phosphorylase b (94,000), bovine serum albumin (BSA) (67,000), ovoalbumin (43,000), carbonic anhydrase (30,000), soybean trypsin inhibitor (20,100) and a-lactalbumin (14,400). 2.4. Reverse phase HPLC of the purified metalloprotease In order to assay the isolated metalloprotease for purity, a reverse phase HPLC was run on a C-18 column (Supelco 4.6 £ 250 mm) of a Shimadzu SPD MIOA vp, eluting with acetonitrile: 0.1% TFA (5:95 v/v, buffer A) and acetonitrile: 0.1% TFA (60:40 v/v, buffer B), following a linear gradient up to 25% of buffer B after 30 min at a flow rate of 1 ml/min. 2.5. Hemorrhagic activity assay

2. Material and methods 2.1. Materials B. jararacussu venom was purchased from Butantan Institute, Sa˜o Paulo, Brazil. Sephacryl S-200 and Phenyl Sepharose CL-4B were purchased from Amersham Bioscience. Fibrinogen bovine and weight markers were from Sigma Chemical Co. All other reagents were of analytical grade. 2.2. Isolation of metalloprotease Venom (300 mg) was dissolved in 3 ml of 0.01 M Tris – HCl buffer, pH 7.6 and centrifuged at 5000 £ g for 5 min. The supernatant was applied to a 2.3 £ 20 cm Sephacryl S-200 column previously equilibrated with the same buffer.

Hemorrhagic activity was quantitatively estimated by the method of Kondo et al. (1960) with some modifications. Groups of five Swiss mice (18 – 22 g) were shaved on the back and then intradermicaly (i.d.) injected with different doses of crude venom or fractions, in 50 ml of phosphate buffered saline (PBS). After 2 h, the animals were anesthetized and killed. The skin was removed from the back and both diameters of the hemorrhagic halos were then measured. The minimum hemorrhagic dose (MHD) was obtained from the mean of these diameters (mm). The MHD is defined as the dose of venom or fractions which results in a hemorrhagic lesion of 10 mm diameter after 2 h. 2.6. Edema-inducing activity Groups of five Swiss male mice (18– 22 g) were injected in the subplantar paw region with different concentrations

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of crude venom and fractions in 50 ml of PBS. After 0.5, 1 and 3 h, the paw edema was measured with the aid of a low pressure spring caliper (Mitutoyo-Japan) (Soares et al., 2000). Zero time values were then subtracted and the differences reported as median% ^ SD. 2.7. Myotoxic activity The assay for creatine kinase (CK) was carried out using the Merck Diagnostic kit (CK-NAC, Granutest 2.5). Approximately, 30 mg/50 ml PBS of BjussuMP-I or BthTX-I, a myotoxic Lys49-phospholipase A2 (PLA2) isolated from the same venom (Andria˜o-Escarso et al., 2000), were injected (i.m.) in male Swiss mice of 18 – 22 g ðn ¼ 6Þ: After 0.5, 3, 6 and 24 h, the blood from the tail was collected in heparin coated tubes and centrifuged for separation of the plasma. The amount of CK was then determined using 4 ml plasma which were incubated for 3 min at 37 8C with 1.0 ml of the reagent according to the Merck kit protocol. Activity was expressed in IU/l, where one unit is defined as the amount of enzyme that catalyses the transformation of 1 mmol of substrate (NADH) per minute. 2.8. Fibrinogenolytic activity Proteolytic activity upon fibrinogen was measured as described by Rodrigues et al. (2000) with some modifications. Fibrinogen solution (2 mg/ml) in 20 ml (PBS) was incubated with different amounts of MP diluted in 20 ml buffer (pH 7.5) at 37 8C for 2 h. The reaction was stopped with 20 ml of a solution containing 10% (v/v) glycerol, 10% (v/v) b-mercaptoethanol, 2% (v/v) SDS, and 0.05% (w/v) bromophenol blue. Fibrinogen hydrolysis was demonstrated by SDS-PAGE using 12% polyacrylamide gels. Other parameters were observed preincubating the enzyme with equal parts of fibrinogen at different pHs (2.5– 8.0), period of time (15 min to 24 h), and temperatures (4 – 100 8C). Similarly, the effect of inhibitors was assayed by incubating 4 mg of enzyme with 20 ml of 10 mM protease inhibitors and chelating agents (EDTA, EGTA, aprotinin, leupeptin, b-mercaptoethanol and 1,10-phenantroline). Using the same concentration of the enzyme, other parameters were observed after preincubating the enzyme with divalent ions (Caþ þ , Mgþ þ , Mnþ þ , Feþ þ , Znþ þ , Coþ þ and Niþ þ ). 2.9. Proteolytic activity upon collagen Proteolytic activity upon type IV collagen (Sigma Chem. Co.) was assayed as described by Lorı´a et al. (2003) with some modifications. To evaluate the effects of BjussuMP-I on type IV collagen, the enzyme (1– 64 mg) was incubated with 10 ml of collagen (2 mg/ml) for 120 min at 37 8C. After this time, the reaction was stopped by 20 ml of a solution containing 10% (v/v) glycerol, 10% (v/v) b-mercaptoethanol, 2% (v/v) SDS, and 0.05% (w/v) bromophenol blue.

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Aliquots of the incubation mixture were analyzed on 10% polyacrylamide gel using Coomassie Briliant Blue as stain. 2.10. Bactericidal activity The ability of BjussuMP-I to induce bactericidal activity against E. coli (ATCC 29648) and S. aureus (ATCC 25923) was assayed as previously described (Soares et al., 2000). In short, 4 £ 107 cells/ml were incubated with 40 and 80 mg BjussuMP-I for 30 and 60 min at 37 8C and surviving bacteria were counted by the plaque dilution technique. 2.11. Enzyme-immunoassays Microplate wells (Dynatech Lab.) were coated with crude venoms or isolated metalloproteases at 0.2 mg/well by overnight incubation in 0.1 M Tris, 0.15 M NaCl, pH 9.0, buffer (Soares et al., 2004). After five washings with solution A (0.05 M Tris, 0.15 M NaCl, 20 mM ZnCl2, 1 mM MgCl2, pH 7.4, buffer), the plates were air-dried and stored at 4 8C. Purified rabbit antibodies to B. neuwiedi neuwiedase (P-I metalloprotease), diluted in solution A containing 2% BSA, were added to triplicate wells, and after five washings with solution A, were incubated at room temperature for 2 h. Bound antibodies were detected with anti-rabbit immunoglobulin conjugated to alkaline phosphatase (Sigma Chem. Co.), diluted 1:2000 with solution A-BSA and incubated for 90 min. After washing, colour was developed with p-nitrophenylphosphate and absorbances were recorded on a microplate reader at 410 nm. Normal rabbit serum was used as a negative control and crotamine was included as an unrelated antigen.

3. Results and discussion 3.1. Metalloprotease isolation The snake venom hemorrhagic toxins denominated metalloproteases are the main compounds responsible for bleeding, the prominent local and systemic effects that results from Bothrops snake bites (Kamiguti et al., 1996; Gutie´rrez and Rucavado, 2000). These toxins comprise endopeptidases with molecular masses ranging from 15 to 100 kDa divided into four classes (Matsui et al., 2000). In this work, a new hemorrhagic metalloprotease, BjussuMP-I, was purified through two relatively rapid and efficient steps, namely, a combination of gel filtration and hydrophobic interaction chromatography (Fig. 1). After fractionation of the crude venom on Sephacryl S-200, the hemorrhagic pool (BJ-I) was resolved into seven main fractions (BJ-I-SI to BJ-I-SVII) on Phenyl Sepharose. The sixth fraction, named BjussuMP-I, induced hemorrhage in mice and showed a single band on SDS-PAGE (Fig. 1C), as well as a single symmetric peak by HPLC (Fig. 1D). This is the first 60 kDa

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Fig. 1. Isolation, electrophoretic and chromatographic analysis of BjussuMP-I. (A) Gel filtration chromatography of B. jararacussu venom. (300 mg) on a 2.3 £ 120 cm column of Sephacryl S-200 previously equilibrated with 0.05 M, pH 7.6, Tris –HCl buffer at room temperature and eluted with the same buffer. (B) Chromatography of the pooled hemorrhagic fraction S-I (50 mg) on a 1.5 £ 6.0 cm column of Phenyl Sepharose CL-4B previously equilibrated with 0.01 M, pH 7.6, Tris–HCl plus 4 M NaCl at room temperature and eluted with a decreasing concentration gradient from 4 to 0 M NaCl. (C) SDS-PAGE at 12% (w/v), in Tris –glycine buffer, pH 8.4, for 120 min at 10 mA and 50– 100 V. Lanes 1, B. jaracussu snake venom (4 mg/ml); 2, BJ-I fraction (4 mg/ml); 3, BjussuMP-I (4 mg/ml); 4, MW markers (phosphorylase b, serum albumin, ovoalbumin, carbonic anhydrase, trypsin inhibitor, a-lactoalbumin). (D) Reverse phase HPLC run on a C-18 column (Supelco 4.6 £ 250 mm) of a Shimadzu SPD MIOA vp, used for elution, (a) acetonitrile: 0.1% TFA (5:95, buffer A) and (b) 0.1% acetonitrile:TFA (60:40 buffer B). Elution was performed using a linear gradient up to 25% B after 30 min, at a flow rate of 1 ml/min.

hemorrhagic metalloprotease from B. jararacussu snake venom so far purified. 3.2. Biochemical and functional characterization The isolated hemorrhagic and fibrinogenolytic component (BJ-I-SVI or BjussuMP-I) has a molecular mass

close to 60 kDa, as determinated by SDS-PAGE under reducing conditions, and the enzyme showed a high hemorrhagic activity (MHD ¼ 4.0 mg) in comparison with the crude venom and BJ-I fraction (Fig. 2A). Despite inducing hemorrhage and moderate edema (Fig. 2B), BjussuMP-I did not show myotoxic activity (Fig. 2C).

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Fig. 2. Biological activities induced by B. jararacussu venom and isolated metalloprotease. (A) Minimum hemorrhagic dose of crude venom and fractions in mice (18–22 g). Animals were submitted to intradermical injection (i.d.) on the back with different concentrations of venom and fractions in 50 ml of PBS. Fifty microliters of PBS and 50 ml of crude venom were as used negative and positive controls, respectively. (B) Edema-inducing activity of 10 mg BjussuMP-I in mice (18–22 g). After subplantar injection with different sample concentrations of crude venom and fractions, in 50 ml of PBS. 50 ml of PBS and 50 ml of crude venom were used as negative and positive controls, respectively. (C) Myotoxic activity of BjussuMP-I in mice (18–22 g). Animals were injected i.m. with 30 mg MP in 50 ml of PBS. As a positive control a myotoxin from B. jararacussu snake venom (30 mg Lys49-PLA2 BthTX-I) was used. Control mice were injected with PBS alone. Each bar represents the mean ^ SD ðn ¼ 5Þ:

High molecular weight metalloproteases usually display higher hemorrhagic activity than class P-I hemorrhagic toxins (Bjarnason and Fox, 1994). It has been suggested that disintegrin-like and high-cysteine domains present in large metalloproteases contribute to hemorrhagic activity by

directing the toxin to appropriate targets and possibly by affecting platelet function (Kamiguti et al., 1996, 1998; Jia et al., 1996). The precise mechanism of action of hemorrhagic metalloproteases has not been fully elucidated, although it is widely accepted that enzymatic degradation of

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basement membrane components of capillary vessels is a key step (Bjarnason and Fox, 1994). In the case of B. jararacussu metalloprotease BjussuMP-I, it has been suggested that after enzymatic degradation of basement membrane in capillary blood vessels, endothelial cells are affected, becoming thinner and eventually developing gaps in their continuity, leading to extravasation (Moreira et al., 1994). In our studies, we also investigated the toxicity of the enzyme and the results showed that BjussuMP-I, despite

exhibiting hemorrhagic activity, myotoxic activity was null when compared with a myotoxic PLA2 (BthTX-I) isolated from the same venom by Andria˜o-Escarso et al. (2000). In addition, the enzyme was unable to clot plasma (results not shown). The ability of fraction BjussuMP-I to degrade fibrinogen is evident from Fig. 3. The metalloprotease (4 mg) degraded the Aa . Bb chain of fibrinogen in a dose dependent manner (Fig. 3A). A significant degradation of Bb chain was detected, however, at high concentration while the g

Fig. 3. Fibrinogenolytic activity of BjussuMP-I. Reducing SDS-gel electrophoresis (12%) of fibrinogen (2 mg/ml) after incubation with the enzyme. (A) Enzyme in different amounts (0.5, 1.0, 2.0, 4.0, 8, 16 and 32 mg) incubated with fibrinogen for 2 h, pH 7.5 at 37 8C. (B) 4 mg enzyme incubate with fibrinogen at different times (0.25, 0.5, 1.0, 2.0, 6.0, 12 and 24 h), pH 7.5 at 37 8C. Fibrinogen incubated for 24 h was used as a control. (C) 4 mg enzyme incubated with fibrinogen at different temperatures (4.0, 37, 50, 75 and 100 8C) for 15 min, pH 7.5. (D) 4 mg enzyme incubated with fibrinogen at different pHs (2.5, 3.5, 4.5, 5.5, 7.0 and 8.0) for 15 min at 37 8C. (E) 4 mg enzyme incubated with fibrinogen in the presence of inhibitors for 15 min at 37 8C, pH 8.0. (F) 4 mg enzyme incubated with fibrinogen in the presence of ions.

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chain was unchanged. The fibrinogenolytic activity was noticed at 15 min and the Bb chain of fibrinogen was degraded within 6 h (Fig. 3B). The hemorrhagic toxins found in Viperidae and Crotalinae venoms are zinc endopeptidases possessing fibrino(geno)lytic activity upon Aa or both Aa and Bb chains of fibrinogen (Markland, 1991, 1998). Some classes of these enzymes with low and medium molecular mass (20 – 60 kDa) have been reported from B. jararaca (Maruyama et al., 1992), B. moojeni (Serrano et al., 1993), Bothrops asper (Gutie´rrez, 1994), B. neuwiedi (Rodrigues et al., 2000) and Lachesis muta muta (Esteva˜o-Costa et al., 2000; Sanchez et al., 2000). In our studies, the process of fibrinogen hydrolysis by BjussuMP-I was similar to that observed with a metalloprotease neuwiedase isolated by Rodrigues et al. (2000). The optimal temperature and pH of the metalloprotease for degradation of Aa chain was 37 8C and 4.5– 8.0, respectively, when the Bb chain was poorly digested (Fig. 3C and D). Hydrolysis of the Aa chain was completely inhibited by EDTA, EGTA and 1.10 phenanthroline, but partially inhibited by b-mercaptoethanol. No inhibition was noticed with aprotinin and leupeptin (Fig. 3E). These results indicate that BjussuMP-I is a metalloprotease, since it is inhibited by chelating agents such as EDTA and EGTA. In addition, the Aa chain of fibrinogen was completely hydrolyzed in the presence of Caþ þ and Mgþ þ , although some ions (Mnþ þ , Feþ þ , Znþ þ , Coþ þ and Niþ þ ) showed a lower influence on fibrinogen degradation (Fig. 3F). Electrophoretic analysis of the hydrolysis products obtained from incubation of BjussuMP-I with type IV collagen revealed a partial degradation of the g chain of collagen under reducing conditions (results not shown). This possible feature of hemorrhagic metalloproteases action and the probable mechanism of collagen-induced platelet aggregation have been investigated (Kamiguti et al., 1998; Esteva˜o-Costa et al., 2000; Lorı´a et al., 2003). BjussuMP-I also displays dose dependent bactericidal activity against both gram positives and gram negative bacteria (Fig. 4). Bactericidal effects were previously reported for PLA2 and L -amino acid oxidases from snake venoms (Pa´ramo et al., 1998; Soares et al., 2000, 2001; Du and Clemetson, 2002; Sta´beli et al., 2004). Anti-neuwiedase was able to recognize different venoms: Bothrops (90 –100%: B. asper, B. jararacussu, B. moojeni, B. neuwiedi, B. jararaca and Bothrops alternatus) and, to a lesser extent, Crotalus atrox (80%) venoms (Fig. 5). Cross-reactivity of the anti-neuwiedase antibodies was further studied using several venoms and isolated metalloproteases. Franceschi et al. (2000) showed that the high molecular metalloprotease from crude venom of B. asper, BaH4 (P III class) showed a partial identity against anti-BaH1 antibodies, but the cross-reactivity with anti-BaP1 antibodies (class I metalloprotease) was not observed. However, the BjussuMP-I presented an immunological partial reactivity against the anti-neuwiedase

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Fig. 4. Bactericidal activity of BjussuMP-I upon Escherichia coli and Staphyloccocus aureus. Different concentrations (40 and 80 mg) of BjussuMP-I were incubated with 4 £ 105 CFU for 30 min and 1 h. Each bar represents the mean ^ SD ðn ¼ 3Þ:

Fig. 5. Cross-reactivity of antibodies to Bothrops neuwiedi metalloprotease neuwiedase against other snake venoms and isolated protease by enzyme immunoassay. Microplate wells were coated with antigen and the binding of antibodies was detected as described in Section 2. Cross-reactivity was expressed as a percentage of the absorbance signal resulting from the binding of antibodies to the homologous antigen (B. neuwiedi neuwiedase). Crotamine was included as an unrelated, negative control antigen. Each bar represents the mean ^ SD ðn ¼ 3Þ:

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antibodies, a P I class metalloprotease isolated from B. neuwiedi (Fig. 5). In conclusion, the role of BjussuMP-I in the pathophysiology associated with B. jararacussu envenoming was evaluated. This toxin induces hemorrhage and lacks myotoxic and significant edema-inducing effects following local injection. The action of proteases that may interfere with coagulation and the fibrinogenolytic system following envenomation by Bothrops snake bites has been reported (Matsui et al., 2000). A number of these proteins has been used clinically as anticoagulants or in preclinical research to investigate their possible therapeutic potential (Braud et al., 2000; Marsh, 2001; Lewis and Garcia, 2003). Therefore, the hemorrhagic fibrinogenolytic metalloprotease BjussuMP-I stands out as a promising tool for its importance as a thrombolytic enzyme and for a better understanding of the action mechanism of Bothrops venoms. In addition, BjussuMP-I showed potential bactericidal activity, making it important pharmacological probe for study of therapeutic targets and new drugs discovery.

Acknowledgements The authors express their gratitude to Fundac¸a˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo (FAPESP) and Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq) for financial support. We thank Prof. Dr Jose´ R. Giglio (FMRP/USP) for their relevant contribution to revision and academic advise. Grateful to Eliandra G. Silva (UNAERP), Carlos A. Vieira and Odete A.B. Cunha (FMRP-USP) for their helpful technical collaboration. These studies were part of the Doctoral Thesis in FCFRP of M.V. Mazzi presented to the Universidade de Sa˜o Paulo-USP, SP.

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