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Toxicon 76 (2013) 1–10

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Toxicon

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In vitro comparison of enzymatic effects among BrazilianBothrops spp. venoms

Lucas B. Campos 1, Manuela B. Pucca 1, Eduardo. C. Roncolato, Thaís B. Bertolini,Joaquim C. Netto, José E. Barbosa*

Department of Biochemistry and Immunology, Faculty of Medicine of Ribeirão Preto, University of São Paulo, Av. Bandeirantes, 3900,14049-900 Ribeirão Preto, SP, Brazil

a r t i c l e i n f o

Article history:Received 12 April 2013Received in revised form 1 August 2013Accepted 13 August 2013Available online 30 August 2013

This work is dedicated to the memory of Dr.Joaquim Coutinho Netto in gratitude for hismentorship and LBC guidance.

Keywords:BothropsPhospholipase A2ProteinaseL-amino acid oxidaseVenom

* Corresponding author. Tel.: þ55 16 3602 4563,E-mail address: jebarbos@fmrp.usp.br (J.E. Barbo

1 Both authors contributed equally.

0041-0101/$ – see front matter � 2013 Elsevier Ltdhttp://dx.doi.org/10.1016/j.toxicon.2013.08.063

a b s t r a c t

In various types of snake venom, the major toxic components are proteinases and mem-bers of the phospholipase A2 family, although other enzymes also contribute to thetoxicity. In this study, we evaluated the proteolytic, phospholipase, and L-Amino acid ox-idase activities in the venom of five Bothrops speciesdBothrops jararaca, Bothrops jarar-acussu, Bothrops moojeni, Bothrops neuwiedi, and Bothrops alternatusdall of which are usedin the production of commercial antivenom, prepared in horses. The enzymatic activitiesof each species’ venom were classified as high, moderate, or low. B. moojeni venomdemonstrated the highest enzymatic activity profile, followed by the venom of B. neuwiedi,B. jararacussu, B. jararaca, and B. alternatus. To our knowledge, this is the first study tocompare all of these enzymes from multiple species, which is significant in view of theactivity of L-amino acid oxidase across Bothrops species.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction

In Brazil, snakes of the genusBothrops are responsible formore than 70% of all reported snake bites (Bochner andStruchiner, 2003; Saúde, 2010). There are approximatelythirty species in this genus, phylogenetically distributedacross seven groups named after the representative speciesBothrops alternatus; Bothrops atrox; Bothrops jararaca;Bothrops jararacussu; B.microphthalmus; Bothrops neuwiedi;and B. taeniatus. However, some researchers consider thegroups B. microphtalmus and B. taeniatus to be members ofthe Bothrocophias and Bothriopsis genera, respectively(Campbell and Lamar, 2004; Gutberlet and Campbell, 2001).The species Bothrops moojeni belongs to the B. atrox group,

þ55 16 3602 3315.sa).

. All rights reserved.

together with the species Bothrops asper; Bothrops leucurusand Bothrops marajoensis (Furtado et al., 2010).

Various components have been isolated from Bothropsvenom, including enzymes such as serine proteinases,metalloproteinases, phospholipases A2 (PLA2), L-aminoacid oxidases (LAAO), nucleotidases, and hyaluronidases;and proteins with other activities, such as disintegrins,members of the C-type lectin family, and others (Braudet al., 2000; Chaves et al., 1995; Kini, 2006; Nunes et al.,2011; Sant’Ana et al., 2011; Toyama et al., 2011).

Serine proteinases contain a reactive serine residue atthe active site, which is stabilized by the presence of his-tidine and aspartic acid residues (Serrano and Maroun,2005). Although homogenous in their structural charac-teristics, snake venom serine proteinases can presenthighly diverse pharmacological profiles, acting on variouscomponents of the coagulation cascade and the fibrinolyticand kallikrein-kinin systems (Serrano and Maroun, 2005;

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Vilca-Quispe et al., 2010). Most of the enzymes are likely tobe glycoproteins with the number and position of N- or O-glycosylation sites differing from one enzyme to another(Serrano and Maroun, 2005).

Metalloproteinases are enzymes that depend on metalions to be active. Snake venom metalloproteinases areassociatedwithhemorrhage,myonecrosis, skindamage, andreactions manifesting as inflammation or edema (Gomeset al., 2011; Gutierrez et al., 2009; Teixeira et al., 2005).

Members of the PLA2 family are calcium-dependentenzymes that catalyze the hydrolysis of the sn-2 esterbond of phosphoglycerides, leading to the formation of freefatty acids and lysophospholipids (da Silva Cunha et al.,2011; Fuly et al., 2000). In many types of snake venom,the majority of the toxic components are composed of PLA2isoforms. In addition to their role in prey digestion, theyimpair certain major physiological functions and can causepresynaptic neurotoxicity and myotoxicity, as well asinhibit coagulation and platelet aggregation. They are alsoinvolved in the development of convulsions, inflammation,hypotension, hemolysis, and hemorrhage, potentiallycontributing to the development of edema (Campos et al.,2009; Fortes-Dias et al., 1999; Fuly et al., 2007; Huanget al., 1997; Leiguez et al.; Moreira et al.).

In the venom of various snakes, members of the LAAOfamily also contribute to toxicity. The LAAOs catalyze theoxidative deamination of specific L-amino acids to producethe corresponding alpha-keto acid, hydrogen peroxide, andammonia. An LAAO typically presents as a homodimericacidic glycoprotein with a flavin cofactor. Studies haveshown that snake venom LAAOs are involved in theapoptosis of various cell lines, such as vascular endothelialcells, which could contribute to prolonged bleeding after asnake bite (Alves et al., 2008; Suhr and Kim, 1996). Inaddition, LAAOs can inhibit platelet aggregation, therebyhaving an anticoagulant effect (Sakurai et al., 2003).

Bothrops envenomation is characterized by cardiovas-cular effects, proteolytic activity with a pronounced localeffect, myonecrosis, hemorrhage, and edema, all of whichare attributable to the synergism of these enzymes,together with the effects of other components (Gutierrezet al., 2009; Machado et al., 2010; Mebs and Ownby, 1990).

In Brazil, Bothrops antivenom is currently produced atthe Butantan Institute in Sao Paulo. The antivenom is pre-pared by hyper-immunizing healthy horses using thevenom of five species: B. jararaca; B. jararacussu; B. alter-natus; B. moojeni; and B. neuwiedi (Furtado et al., 2010).Multiple species are used because there are differencesamong the species regarding the components of the venom(Furtado et al., 2010; Neiva et al., 2009; Nunez et al., 2009).The goal of the present study is to demonstrate the quali-tative and quantitative differences among these species interms of the complexity and diversity of proteinases, PLA2s,and LAAOs found in the venom of these five species.

2. Materials and methods

2.1. Biological and chemical reagents

Aliquots of B. jararaca, B. jararacussu, B. moojeni, B.neuwiedi, and B. alternatus venom were obtained from the

Serpentarium at the Faculty of Medicine of Ribeirão Preto,University of São Paulo (Faculdade de Medicina de RibeirãoPreto, Universidade de São Paulo), Brazil. For each species,venom was extracted from three snakes captured indifferent regions of São Paulo state and pooled. The venomaliquots were stored at �20 �C until analysis. Protein con-tent was measured by the Lowry method, modified byHartree (1972), using bovine serum albumin as the stan-dard. Sheep erythrocytes (Newprov, Pinhais, Brazil) werecollected in heparinized tubes (Becton Dickinson, FranklinLakes, NJ, USA), centrifuged for 20 min at 300 � g at 4 �C,washed 3 times with PBS, pH 7.4, and centrifuged again.Chemicals and reagents were purchased from Sigma(Sigma Chemical Co., St. Louis, MO, USA).

2.2. Hemolytic activity

A modified kinetic indirect hemolytic assay, standard-ized in our laboratory (Tamarozzi et al., 2006), was per-formed to measure PLA2 activity. We used egg yolk as asubstrate to develop a turbidimetric assay based on thecapacity of snake venom PLA2s to hydrolyze egg phospha-tidylcholine, producing lysophosphatidylcholine and fattyacids. The lysophosphatidylcholine accumulates on eryth-rocyte membranes, promoting their disruption and initi-ating the hemolysis process (Bierbaum et al., 1979). Thisassay was conducted in triplicate in a 96-well microplate(Costar, Corning Incorporated, NY, USA) and samples wereanalyzed at 650 nm, considering that this is well outside ofthe hemoglobin absorption wavelength and that theabsorbance is proportional to the cell concentration.

The hemolysis solution consisted of 10 ml of Tris–HClbuffer, pH 7.4, containing 140mMNaCl, 2.5 mM CaCl2, 25 mlof egg yolk diluted 1:4 (v/v) in sterile saline solution (NaCl0.9%), and 1.5 ml of erythrocytes. The reaction mixture wasprepared by adding 175 ml of hemolysis solution and 75 mlof venom samples (20 mg/ml). The microplate was incu-bated at 37 �C and the decrease in absorbancewas recordedat intervals of 5,10, and 15min. A saline solution containingno venom was used as a negative control. The blank solu-tion was prepared by adding venom to the hemolysis so-lution to achieve a final concentration of 80 mg/ml. After 1 hof incubation at 37 �C, this solution was used to calibratethe microplate reader (Molecular Devices, Sunnyvale, CA,USA). Statistical analyses were based on the absorbanceafter 90 min of reaction.

2.3. Proteolytic activity

Proteolytic activity was assayed by a modified casein-olytic method (Sanchez et al., 1992). The reaction mixtureconsisted of venom (50 and 100 mg/ml) diluted in 500 ml of50 mM Tris–HCl, pH 7.4, 2.0 mM CaCl2, and 0.5% (w/v)denatured casein. After 3 h of incubation at 37 �C, the re-action was terminated by adding 500 ml of 10% (w/v) tri-chloroacetic acid (TCA). Non-digested proteins wereremoved by centrifugation at 11,600 � g for 5 min, and thepeptide content in the clear supernatant was measured at280 nm with a spectrophotometer (DU 640; BeckmanCoulter, Palo Alto, CA, USA). As a negative control, we usedwater instead of venom. To determine whether the protein

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contents of the venom samples contributed to increases inabsorbance, we prepared a control with each venom sam-ple previously denatured with TCA before the addition ofthe substrate solution. As expected, the results were similarto those obtained with the negative control (data notshown).

2.4. LAAO activity

LAAO activity was measured by a colorimetric methodadapted from Costa Torres et al. (2010). The method wasbased on the oxidative deamination of the substrate (L-leucine), generating hydrogen peroxide. Adding horse-radish peroxidase (HRP) to the reaction media reduces thehydrogen peroxide in the presence of o-phenylenediamine(OPD) to form a yellowish product, which can be measuredspectrophotometrically. Reactions were conducted in trip-licate in a 96-well microplate. Two different concentrations(5.0 and 10.0 mg/ml) of each venomwere incubated for 1 hat 37 �C, in 150 ml of 100 mM Tris–HCl, pH 7.4, containing2.0 mM L-leucine, 0.8 U/ml HRP (horseradish peroxidase),and OPD (o-Phenylenediamine dihydrochloride), diluted asindicated by the manufacturer Sigma�. The reaction wasstopped by adding 75 ml of 2.0 M sulfuric acid and theabsorbance was measured at 490 nm using a microplatereader. As a negative control (and blank), we used waterinstead of venom. As a positive control, we used hydrogenperoxide diluted 1:6000.

2.5. Protein analysis and molecular weight assays

2.5.1. Sodium dodecyl sulfate-polyacrylamide gelelectrophoresis

Venom samples were compared by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on a12% polyacrylamide gel under non-reducing conditionswith silver staining, as described by Sambrook (Sambrookand Russell, 2001).

2.5.2. Zymography of PLA2sIn order to identify and estimate the molecular weights

of PLA2s, we analyzed the venom samples using zymog-raphy, incorporating modifications described previously(Rossignol et al., 2008). Samples of venom (2.5 mg each)were electrophoresed at 60 V and 4 �C on a 12% poly-acrylamide gel under non-reducing conditions. The gel waswashed for 1 h in 500mM Tris–HCl, pH 7.4, containing 2.0%(v/v) Triton X-100 and for another 1 h in 100 mM Tris–HCl,pH 7.4, containing 1.0% (v/v) Triton X-100. After SDS resi-dues had been removed, the gel was washed a third timefor 30 min in 50 mM Tris–HCl, pH 7.4, containing 140 mMNaCl and 2.5 mM CaCl2. It was then incubated for 14 h atroom temperature over a 1.0% (w/v) agarose gel prepared in50 mM Tris–HCl, pH 7.4, 140 mM NaCl, 2.5 mM CaCl2, and2.0% egg yolk. Clear zones indicated the presence of PLA2.

2.5.3. Zymography of proteinasesWe identified proteinases using a modified form of the

zymography technique described previously (Heussen andDowdle, 1980), as cited by Zelanis et al. (2007). Samples ofeach venom (40 mg each) were applied to a 12%

polyacrylamide gel, which was copolymerized with 0.07%(w/v) denatured casein. Samples were prepared in thepresence of SDS and in the absence of reducing agents, afterwhich they were electrophoresed at 60 V and 4 �C. Afterelectrophoresis, the gel was washed for 30 min in 50 mMTris–HCl, pH 7.4, containing 2.0% (w/v) Triton X-100, atroom temperature. Next, the buffer was replaced and thegel was washed again. After removal of all residues of SDS,the gel was incubated for 16 h in 50 mM Tris–HCl, pH 7.4,containing 150 mM NaCl and 2.5 mM CaCl2, at 37 �C. It wasthen stained with 0.5% Coomassie Brilliant Blue R-250 in40% (v/v) methanol and 10% (v/v) acetic acid for 1 h.Destaining was performed in 40% (v/v) methanol and 10%(v/v) acetic acid. Venom proteinases hydrolyzed the caseindissolved in the gel, presenting clear zones against the bluebackground.

2.5.4. Zymography of LAAOsThe molecular weights of LAAOs were estimated by

zymography, as described by Campos et al. (2012). Briefly,40 mg of each venom sample was electrophoresed undernon-reducing conditions and the gel was treated with150mM Tris–HCl, pH 7.2, supplemented with 1.0% Triton X-100 for removing SDS residues. The gel was then overlaidon another gel prepared in 150 mM Tris–HCl buffer, pH 7.4,supplemented with 1.0% agarose, 0.8 U/mL HRP, 2.5 mM L-leucine, and OPD diluted as indicated by the manufacturer.The presence of the enzymes was confirmed by theappearance of yellowish bands.

2.6. Enzymatic activity classification

For elucidating the activity of venom enzymes (PLA2,proteinases, and LAAO), we sorted enzymatic activities inBothrops venom into different levels, namely, low, moder-ate and high. The classification was performed using theresults of the hemolytic, proteolytic and LAAO activity as-says. The results of the zymograms were excluded, sincethey do not necessarily reflect the activity of all proteins.This classification is restricted to venom from the speciesincluded in this study and the specific methods used.

2.7. Statistical analysis

Results are indicated as means and standard deviations.Data were submitted to analysis of variance followed byTukey’s post-hoc test. P values < 0.05 were consideredstatistically significant.

3. Results

3.1. Enzymatic activities

In order to compare the Bothrops venoms, in terms oftheir biological activity, we tested the PLA2, proteinase, andLAAO groups of enzymes.

All venoms demonstrated PLA2 activity, as evidenced bytheir hemolytic effects (Fig. 1). Although B. moojeni venomdisplayed amore rapid decrease in absorbance, followed bythe venoms of B. neuwiedi and B. jararacussu, these threevenoms showed no significant differences after 90 min of

Fig. 1. Phospholipase A2 activity. Venoms were incubated with the hemo-lysis solution (250 ml) to a final concentration of 20.0 mg/ml for 90 min at37 �C, and the decrease in absorbance was measured at the intervals of 5, 10,or 15 min at 650 nm. The negative control was prepared by replacing thevenom with saline solution (NaCl 0.9%). To calibrate the plate reader, a re-action mixture, previously incubated with 80 mg/ml of venom for 1 h at37 �C, was used. Statistical analysis was set considering the absorbance after90 min of reaction. Data were collected in triplicate and submitted toanalysis of variance followed by Tukey’s post hoc test. Values of p < 0.05were considered statistically significant. Different letters indicate statisticaldifference.

Fig. 3. L-amino acid oxidase activity. Different concentrations (5.0 and10.0 mg/ml) of each venom were incubated in the presence of L-leucine, o-phenylenediamine, horseradish peroxidase, and Tris–HCl pH 7.4 buffer for1 h at 37 �C. The reaction was stopped with H2SO4, and absorbance wasmeasured at 490 nm. As a negative control (also used as a blank), we usedwater instead of venom, and, as a positive control, we used H2O2 diluted1:6000. Results are expressed as means and standard deviations. Data werecollected in triplicate and submitted to analysis of variance followed byTukey’s post hoc test. Values of p < 0.05 were considered statistically sig-nificant. Different letters indicate statistical difference.

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reaction time had elapsed. B. alternatus venom showedsignificantly lower PLA2 activity while the venom of B.jararaca displayed an intermediate activity level.

Bothrops venoms also demonstrated proteinase activitybut with significant quantitative differences (Fig. 2). B.moojeni venom showed significantly higher proteinase ac-tivity, followed by B. neuwiedi venom. B. jararaca, B. jarar-acussu, and B. alternatus venoms showed significantlylower proteinase activity (Fig. 8).

Significant LAAO activity was also observed in all thetested venoms (Fig. 3). The venom of B. neuwiedi and B.moojeni showed significantly higher LAAO activities,

Fig. 2. Proteinase activity. Different concentrations (50.0 and 100.0 mg/ml) ofeach venom were incubated in the presence of casein and Tris–HCl buffer,pH 7.4, for 3 h at 37 �C. The reaction was stopped with 10% trichloroaceticacid, and non-digested proteins were removed by centrifugation at11,600 � g for 5 min. The peptide content of the supernatant was measuredat 280 nm. The negative control (also used as a blank) was prepared byreplacing the venom with water. Results are expressed as means and stan-dard deviations. Data were collected in triplicate and submitted to analysisof variance followed by Tukey’s post hoc test. Values of p < 0.05 wereconsidered statistically significant. Different letters indicate statisticaldifference.

followed by that of B. jararaca and B. jararacussu. B. alter-natus venom showed significantly lower LAAO activity.

3.2. SDS-PAGE and zymography

In order to compare the various Bothrops venoms, interms of their protein profiles, the venoms were submittedto electrophoresis under non-reducing conditions. The re-sults of the SDS-PAGE analysis are shown in Fig. 4. Despite

Fig. 4. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Theprotein profile of each venom (1.0 mg/lane) were analyzed on a silver-stained12.0% polyacrylamide gel. 1, B. jararaca; 2, B. jararacussu; 3, B. moojeni; 4, B.neuwiedi; 5, B. alternatus and M, protein ladder.

Fig. 5. Phospholipase A2 zymogram. [A] Sodium dodecyl sulfate-polyacrylamide gel electrophoresis. [B] 1.0% agarose gel containing 2.0%egg yolk after overnight incubation on a 12.0% polyacrylamide gel electro-phoresis containing 2.5 mg of each venom per lane. M, molecular marker; 1,B. jararaca; 2, B. jararacussu; 3, B. moojeni; 4, B. neuwiedi and 5, B. alternatus.

L.B. Campos et al. / Toxicon 76 (2013) 1–10 5

the fact that several venoms had some bands in common,their overall profiles showed substantial differences, exceptin the case of B. moojeni versus B. neuwiedi.

The presence of PLA2 in the venoms was analyzed by anegg yolk zymogram. All venoms displayed a clear zone atapproximately 15 kDa, which corresponds to PLA2 activityagainst lecithin on the gel (Fig. 5). Although equal amountsof venomwere used, different patterns of clear zones wereobserved. This observation can be explained by differencesin the activity level of each enzyme and its concentration inthe venom. These findings are in accordance with the re-sults obtained in the hemolytic assay.

Fig. 6. Proteinase zymogram. Caseinolytic activity of proteinases (40.0 mg/lane) on a 12.0% polyacrylamide gel copolymerized with 0.07% casein. M,molecular marker; 1, B. jararaca; 2, B. jararacussu; 3, B. moojeni; 4, B. neu-wiedi and 5, B. alternatus.

The presence of proteinases in the venoms wasconfirmed by the appearance of clear zones against theblue background on the casein zymogram (Fig. 6). B. jar-araca venom showed intense casein degradation in the 25–28 kDa range, while B. neuwiedi venom showed intensedegradation at 28 to 30 kDA. The venoms of B. jararacussuand B. moojeni showed a lower degradation profile atapproximately 30 kDa, while no clear zone was observedfor B. alternatus venom. Although all of the venoms showedproteinase activity, as indicated in Fig. 2, only the venoms ofB. jararacussu and B. moojeni were similar in their patternsof casein degradation.

The venoms also showed LAAO activity, as confirmed bythe presence of yellowish bands in the OPD zymogram(Fig. 7), nevertheless, their molecular mass was variable. B.jararaca venom showed the most intense yellowish band,around 97 kDa. While B. jararacussu and B. moojeni venomsshowed similar band profiles at approximately 84 and82 kDa, respectively. B. neuwiedi venomwas unique in thatit displayed two yellow bands. One intense band of 75 kDaand another, less intense band of 119 kDa were detected. B.alternatus venom displayed the enzyme at approximately107 kDa.

4. Discussion

Proteinases and PLA2s are considered the major toxiccompounds in almost all snake venoms, although otherenzymes also contribute to the toxicity (Correa-Netto et al.,

Fig. 7. L-amino acid oxidase zymogram. A sodium dodecyl sulfate-polyacrylamide gel electrophoresis containing each venom (40 mg/lane)was laid over another gel supplemented with 1.0% agarose, 0.8 U/mL HRP,2.5 mM L-leucine, and OPD. The LAAOs activity was observed by theappearance of yellowish bands. M, molecular marker; 1, B. jararaca; 2, B.jararacussu; 3, B. moojeni; 4, B. neuwiedi and 5, B. alternatus.

Fig. 8. Overall relative comparison of venoms regarding their enzymaticactivities, which were classified as low (white), moderate (gray), or high(black).

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2010; Serrano et al., 2005). LAAO is also an importantenzyme present in the venom of pit vipers, however, itaccounts for about 0.5% of the total toxin transcripts fromthe venom glands, a small percentagewhen comparedwiththe 53.1% and 28.5% reported for metalloproteinases andserine proteinases, respectively (Cidade et al., 2006).

In the present study, we evaluated the PLA2, proteolytic,and LAAO activities of the venoms of five different Bothropsspecies: B. jararaca, B. jararacussu, B. moojeni, B. neuwiedi,and B. alternatus. Such enzymatic variations are highlyrelevant, given that the venom of these species is used inthe production of bothropic antivenom in Brazil (Furtadoet al., 2010). It is noteworthy that, with the exception ofB. neuwiedi, all of the snakes evaluated are on the list ofvenomous snakes of highest medical significance in theAmericas (World Health Organization, 2010).

In the southeast of Brazil, B. jararaca is the most com-mon snake species and it is responsible for most of thesnake bites in the region, although it is not responsible forthe most severe cases of envenomation (Cruz et al., 2009).With regards to PLA2, it comprises a small percentage of thevenom (0.7%), which may explain the relatively low degreeof myonecrosis in victims compared to other Bothropsspecies (Cidade et al., 2006). In agreement with this, ourresults showed that B. jararaca presents moderate PLA2activity, as previously described (Serrano et al., 1999). Thevenom also displayed moderate proteolytic activity. B. jar-araca venom contains several well-described proteinases,such as jararagin (a 52 kDa hemorrhagic metal-loproteinase), two fibrinolytic metalloproteinases (21 and47 kDa, respectively), a 67-kDa trypsin-like serine pro-teinase, small hemorrhagins (w25 kDa), and others(Maruyama et al., 1992; Murayama et al., 2003; Paine et al.,1992). In our zymography analysis, we found that B. jar-araca venom effected intense casein hydrolysis with bandsranging in size from 25 to 28 kDa. Two other disconnectedclear zones were also visible, one atw24 kDa (intense) and

the other at w20 kDa (less intense). In relation to LAAO, B.jararaca venom again displayed moderate enzyme activity.A study comparing the microbicidal activity of severalvenoms found that the venom of B. jararaca was the mostactive and that this was related to its LAAO activity(Ciscotto et al., 2009).

B. jararacussu is found in the southeastern region ofBrazil (FUNASA, 2001). Although the local effects of B. jar-aracussu venom are similar to other Bothrops venoms, someof the systemic effects resemble those of Crotalus spp. en-venomation. This could explain the greater clinical effec-tiveness of Crotalus antivenom over Bothrops antivenom incases of B. jararacussu snake bites (Milani Jr. et al., 1997). Inthe present study, B. jararacussu venom showed high he-molytic activity, which is likely attributable to the biolog-ical activity of several PLA2 enzymes that have beenidentified in the venom, such as SIIISPIIB (Ketelhut et al.,2003), Bothropstoxin-I (Cintra et al., 1993), BothropstoxinII (Pereira et al., 1998) and Bj IV (Bonfim et al., 2001). ThePLA2 zymogram showed an intense band at around 15 kDa,similar to the enzymes previously described (about 13–15 kDa). However, B. jararacussu venom showed moderateproteolytic activity. The zymogram revealed caseinolyticactivity at 28–30 kDa, which could be explained by theactivity of multiple proteinases, such as a 30 kDa P-I met-alloproteinase (Correa-Netto et al., 2010) and a 28-kDaserine proteinase (Bortoleto et al., 2002). The gel alsorevealed proteolytic activity at w34 kDa and a slight clearzone at 24 kDa, which could be explained by the presenceof a 34 kDa serine proteinase and a 24 kDa P-I metal-loproteinase (Correa-Netto et al., 2010). However, otherknown proteinases were not observed (Correa-Netto et al.,2010). B. jararacussu venom also showed moderate LAAOactivity. Proteomic studies have revealed that B. jararacussuvenom contains LAAO isoforms, with molecular massesranging from 47 to 78 kDa (Correa-Netto et al., 2010), as canbe confirmed by the LAAO zymogram results. Recently anisoform of 65 kDa was purified and crystalized (Ullah et al.,2012).

B. moojeni is commonly found in central and south-eastern Brazil, being most prolific in the savanna(‘Cerrado’)(Borges and Araujo, 1998; FUNASA, 2001). Studies haverevealed that B. moojeni venom exhibits high proteolyticactivity and low hemorrhagic action, with high PLA2 levelsand coagulant properties (Assakura et al., 1985). In thepresent study, B. moojeni venom showed the highest ac-tivity among all the enzymes tested. The high PLA2 activitymight be explained by the presence of two acidic phos-pholipases, the 19 kDa BM-PLA2 and the 15 kDa BmooTX-I(Nonato et al., 2001; Santos-Filho et al., 2008). These dataare in accordance with those obtained in the PLA2 zymo-gram. B. moojeni venom also showed high proteolytic ac-tivity, although the zymogram did not indicate intensecasein hydrolysis. It has been reported that B. moojenivenom contains multiple proteinases, including serineproteinases and metalloproteinases, with molecularmasses ranging from 22 to 34 kDa (Assakura et al., 1985;Bernardes et al., 2008; Serrano et al., 1993a). Those re-ports are in accordance with our zymography findings(proteinases of w30 kDa). It has also been reported that B.moojeni venom contains a metalloproteinase composed of

L.B. Campos et al. / Toxicon 76 (2013) 1–10 7

two polypeptide chains of 65-kDa and 55-kDa (Serranoet al., 1993b). However, we were unable to observe thatmetalloproteinase in our zymogram, which may be due tothe fact that it does not renature correctly after the removalof SDS residues. The high phospholipase and proteinaseactivities of this venom might be responsible for theseverity of local damage, as well as for the deleterious ef-fects that it has on renal epithelia in snake bite victims(Assakura et al., 1985; Boer-Lima et al., 1999).We also foundhigh LAAO activity levels, however, the correspondingyellowish band in our zymogram was smaller than the130.8 kDa LAAO enzyme previously reported by other au-thors (Stabeli et al., 2007). This LAAO has already beendescribed to have a potent killing effect in vitro againstLeishmania spp. (Tempone et al., 2001). The highest enzy-matic activities of B. moojeni is reflected in other speciesbelonging to the B. atrox group, which is responsible for alarge proportion of the fatalities related to snake bites inSouth and Central America (Gutierrez et al., 2006).

Unlike the other species evaluated in the present study,B. neuwiedi is not on the World Health Organization list ofmedically important venomous snakes in the Americas(World Health Organization, 2010). The species is foundthroughout southern, southeastern, central, and north-eastern Brazil (FUNASA, 2001). In the present study, the B.neuwiedi venom presented high PLA2 activity as well as themost intense band in the zymography assay. In an earlierstudy on B. neuwiedi venom, two PLA2 isoforms (15 and16 kDa, respectively) were purified; these presentedmarked edema-inducing activity (Daniele et al., 1995).Another 15-kDa PLA2 isoform, with a different N-terminalsequence, was also found to possess edema-inducing ac-tivity (Daniele et al., 1997). On the other hand, B. neuwiedivenom showed low proteinase activity in this study. Thezymogram showed intense caseinolytic activity over therange of 26–28 kDa and a slight clear zone at 24 kDa. Thisvenom presents awell-described 22 kDametalloproteinasecalled neuwiedase (Lopes et al., 2009; Rodrigues et al.,2001); two other metalloproteinases, both of w24 kDaand with similar electrophoretic profiles but different iso-electric properties; and two additional metalloproteinases,of 46- and 58-kDa, respectively, bothwith hemorrhagic andcaseinolytic properties (Mandelbaum et al., 1984). How-ever, not all of these were observed in the zymogram. Inaddition, B. neuwiedi venom showed high LAAO activity,similar to that observed for B. moojeni venom. This activitymight be explained by the presence of a 65 kDa homodi-meric protein capable of inducing platelet activation, aswell as having bactericidal, leishmanicidal, and antitumorproperties (Rodrigues et al., 2009).

The species B. alternatus is widely distributedthroughout southern and south-central Brazil, being pri-marily responsible for cases of snake bites in those re-gions (FUNASA, 2001). Our results demonstrated that B.alternatus venom has low PLA2 activity. However, an acidicPLA2 identified in B. alternatus venom was found to be themajor compound responsible for the lethality of this venomin mice, producing cardiovascular alterations such as dys-pnea, tachycardia, arrhythmia, and circulatory shock, aswell as tissue damage, including hemorrhage and necrosis(Nisenbom et al., 1986a, 1986b). Nevertheless, it is known

that the protein content of B. alternatus venom comprisesmostly metalloproteinases and serine proteinases, ac-counting for 43.1% and 24.1%, respectively (Ohler et al.,2010). The various metalloproteinases identified in B.alternatus venom have molecular masses ranging from 22to 100 kDa, and are capable of causing hemorrhage, edema,myonecrosis, and coagulation disorders. The venom of B.alternatus also contains many serine proteinases that pre-sent coagulant and fibrinogenolytic activities; these en-zymes are considered less toxic than metalloproteinases(Ohler et al., 2010). Although venomics studies haverevealed that metalloproteinases and serine proteinasesare considered the most toxic components (Cardoso et al.,2010), we found that B. alternatus venom showed onlymoderate proteolytic activity. Souza et al. (2000) found thatB. alternatus venom contains a 55 kDa metalloproteinase,designated alternagin (Souza et al., 2000), which has beenshown to be the major component responsible for thehemorrhagic effect of this venom, despite the fact that itdisplayed low proteolytic activity on casein (Gay et al.,2005). This could explain the moderate activity shown inthe liquid assay and the absence of activity on the zymo-gram. B. alternatus showed the lowest LAAO activity. Ven-omics studies have demonstrated that B. alternatus venomcontains five LAAO isoforms, with molecular massesranging from 50 to 57 kDa (monomeric form), collectivelyaccounting for 6.9% of the crude venom, and that there is ahigh homology between these LAAOs and those found in B.moojeni venom (Ohler et al., 2010). Nevertheless, in thepresent study, the activity levels differed between thosetwo species, a fact that might be attributable to the use ofcrude venom rather than purified enzymes. Despite therelatively low overall enzymatic activity observed in ourstudy, B. alternatus bites have often been reported to causelocal tissue damage, hemorrhage, coagulation disorders,respiratory failure, renal failure, and shock (Gay et al.,2009).

On the basis of our results, we classified the enzymaticactivity in the venom of the five species evaluated as low,moderate or high (Fig. 8). Other authors have reported thatvenom components are not homogeneously distributedamong the various Bothrops species (Ferreira et al., 1992;Francischetti et al., 1998; Hodgson and Wickramaratna,2002; Leite et al., 1992; Moura-da-Silva et al., 1990;Moura-da-Silva et al., 1991; Zamuner et al., 2004). However,to our knowledge, this is the first study to compare thesethree enzyme classes. In particular, we found few studiesexamining LAAO activity in Bothrops species.

5. Conclusion

We have demonstrated significant variation amongBothrops species in terms of the enzymes present in thevenom. According to our classification, B. moojeni venomshowed the highest enzyme activity, followed by thevenoms of B. neuwiedi, B jararacussu, B. jararaca, and B.alternatus. Knowledge of such differences is of great rele-vance to the understanding of the effects of snake biteenvenomation, antiserum production, taxonomy, andvenom toxicity, as well as being essential to the study ofvenom components as potential therapeutic targets.

L.B. Campos et al. / Toxicon 76 (2013) 1–108

Declaration of interest

The authors report no declarations of interest. Theauthors alone are responsible for the content of thismanuscript.

Ethical statement

This work do not has an Ethical Statement because allthe assays were done in vitro without animal use.

Acknowledgments

This study received financial support from the Fundaçãode Amparo à Pesquisa do Estado de São Paulo and InstitutoNacional de Ciência e Tecnologia em Toxinas (FAPESP andINCTTOX, São Paulo Research Foundation and NationalInstitute of Toxin Science and Technology; Grant no.573790/2008-6), the Fundação de Apoio ao Ensino, Pesquisa eAssistência (FAEPA, Foundation for the Support of Instruc-tion, Research, and Treatment), the Fundação WaldemarBarnsley Pessoa (Waldemar Barnsley Pessoa Foundation),and the Coordenação de Aperfeiçoamento de Pessoal de NívelSuperior (CAPES, Office for the Advancement of Higher Ed-ucation; scholarships to LBC and MBP).

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