gy, Part C 145 (2007) 601–609www.elsevier.com/locate/cbpc

Comparative Biochemistry and Physiolo

Variability in expression of Bothrops insularis snake venom proteases:An ontogenetic approach

André Zelanis a,b, Janaina de Souza Ventura c, Ana Marisa Chudzinski-Tavassi c,Maria de Fátima Domingues Furtado b,⁎

a Departamento de Fisiologia, Instituto de Biociências, Universidade de São Paulo, Brazilb Laboratório de Herpetologia, Instituto Butantan, Av. Vital Brazil, 1500, 05503-900, São Paulo-SP, Brazil

c Laboratório de Bioquímica e Biofísica, Instituto Butantan, Av. Vital Brazil, 1500, 05503-900, São Paulo-SP, Brazil

Received 14 September 2006; received in revised form 12 February 2007; accepted 13 February 2007Available online 21 February 2007

Abstract

Bothrops insularis is a threatened snake endemic to Queimada Grande Island, southern coast of São Paulo, Brazil, and the occurrence of sexualabnormalities in males, females and intersexes (females with functional ovaries and rudimentary hemipenis) has been reported in this population.The aim of this study was to identify ontogenetic shifts in protease expression of offspring of captive-bred B. insularis. Three neonates from asingle litter were maintained at the facilities of Laboratory of Herpetology, Institute Butantan, for 41 months. The snakes were individually milkedand venoms were analyzed both by SDS–PAGE, under reducing conditions, and for biochemical activities. The venoms from the mother and froma pool of adult specimens were used as references. In regard to the electrophoretic patterns, common bands were identified mainly between 14 and50 kDa among snakes. The occurrence of proteolytic activity was noticed predominantly between 27 and 45 kDa in zymograms. Inhibitory assayswith 1,10-phenantroline (10 mM) and PMSF (5 mM) showed that venoms possessed both metalloproteases and serine proteases. Venoms of youngspecimens showed a higher coagulant activity than those of adults, especially upon factors X and II. All venoms presented fibrino(geno)lyticactivity, degrading Aα and Bβ chains of fibrinogen, and lysing fibrin plate. These findings can reflect important individual, ontogenetic and sexualdifferences on venom composition and are likely correlated with diet habits of this species.© 2007 Published by Elsevier Inc.

Keywords: Bothrops insularis; Island species; Proteases; Ontogeny

1. Introduction

The composition of snake venoms results from the interaction ofmultiple factors, leading to an efficient biological product that hasas main purposes the immobilization, the death and the promotionof initial digestion of preys (Kochva et al., 1983; Mebs, 1999).Venom variability is associated with several factors, such astaxonomic criteria (within intraspecies, interspecies, intergenus andinterfamily levels), evolutionary processes (geographic isolationand speciation) and ontogenetic features (Chippaux et al., 1991).

Ontogenetic variability has been extensively reported forsnake venoms (Minton and Weinstein, 1986; Gutiérrez et al.,1990; Gutiérrez et al., 1991; Chaves et al., 1992; Mackessy,

⁎ Corresponding author. Tel.: +55 11 3726 7222x2239; fax: +55 11 3726 1505.E-mail address: fatifurtado@butantan.gov.br (M.F.D. Furtado).

1532-0456/$ - see front matter © 2007 Published by Elsevier Inc.doi:10.1016/j.cbpc.2007.02.009

1993; Tan et al., 1993; Andrade and Abe, 1999; López-Lozanoet al., 2002; Saravia et al., 2002;Mackessy et al., 2003; Saldarriagaet al., 2003), and the modification of prey types has been con-sidered an important factor for the ontogenetic variability ofvenom composition (Gutiérrez et al., 1990; Mackessy, 1993;Daltry et al., 1996; Mackessy et al., 2003).

Bothrops envenomation causes several pathophysiologicaldisturbances, including systemic hemostatic disorders related toactivation of factors X and II, fibrinogen consumption andinhibition of platelet aggregation, leading to characteristicsystemic symptoms (Nahas et al., 1979; Kamiguti and Cardoso,1989; Sano-Martins and Santoro, 2003). Proteolytic enzymesare intrinsically involved in the pathogenesis of a wide range ofbiological effects induced by Bothrops venoms. Expressivedifferences have been reported, frequently related to hemostaticdisturbances, on clinical manifestations of envenomations

Fig. 1. SDS–PAGE (7.5–17.5% gradient) of reduced samples of Bothropsinsularis venoms (120 μg/lane) during its ontogeny. Lanes: A — intersex 1;B — intersex 2; C — male; M — mother: PB — pool of B. insularis venom.Molecular markers are represented at left. The arrow at the right indicates the 24-kDa protein band.

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caused by newborn and adult Bothrops specimens (Kamigutiet al., 1986; Ribeiro and Jorge, 1989). In fact, Furtado et al.(1991) showed significant ontogenetic venom variability in thecoagulant activity of adult Bothrops sp. female snakes and theiroffspring, both maintained in captivity.

Bothrops insularis is a snake endemic to Queimada GrandeIsland, located on the southern coastline of São Paulo, Brazil(24°30′S 43°42′W), which was isolated from the continentabout 11,000 years ago (Vanzolini, 1973). The B. insularispopulation presents sexual abnormalities, which are possiblycaused by inbreeding or eventual mutations. Such populationpresents males, females and intersexes, which are functionaland genetic females presenting a rudimentary bilateral orunilateral non-functional hemipenis (Hoge et al., 1959; Beçaket al., 1990). This arboreal species (Amaral, 1921; Amaral,1927; Duarte et al., 1995; Wüster et al., 2005) shows animportant ontogenetic shift in prey types, so that juveniles feedon ectothermic preys (centipedes, frogs and lizards), whereasadults feed only on birds (Martins et al., 2002).

In comparison with other Bothrops species, few studies havebeen carried out about B. insularis venom, and none has reportedthe ontogenetic variability of this unique species. Junqueira-de-Azevedo and Ho (2002) showed the transcript diversity ofB. insularis venom gland through the generation of expressedsequence tags (ESTs), and metalloproteases were found to be themajor components among toxin sequences. Some toxins isolatedfrom B. insularis venom have important biological activity, suchas the thrombin-like enzyme (Selistre and Giglio, 1987),bradykinin potentiating peptides (Cintra et al., 1990), and aphospholipase A2 containing fraction, which seems to be in-volved in the prominent presynaptic neurotoxicity of this venom(Cogo et al., 1993; Cogo et al., 1998; Rodrigues-Simioni et al.,2004). Besides, a functional snake venom vascular endotheliumgrowth factor (svVEGF) was cloned and expressed from a libraryof B. insularis venom gland (Junqueira-de-Azevedo et al., 2001).Guimarães-Gomes et al. (2004) have identified and characterizeda 32-kDa dimeric C-type lectin, named BiL, which presentshemagglutinating activity. Recently, a prothrombin activator,named insularinase Awas characterized from B. insularis venomby Modesto et al. (2005). Insularinase A belongs to the snakevenom metalloproteases (SVMP) family and is expressed invenom as a PI-class metalloprotease, although its precursor is aPII-class metalloprotease (Modesto et al., 2005).

Since proteolytic enzymes are one of the most importantcomponents of viperid venoms, the aim of this study was toidentify ontogenetic shifts in protease expression of offspring ofcaptive-bred B. insularis.

2. Materials and methods

2.1. B. insularis venoms

One pregnant specimen of B. insularis (an intersex) wascaptured on Queimada Grande Island and brought to thefacilities of Laboratory of Herpetology, Butantan Institute, SãoPaulo. After parturition, neonates (one male and two intersexes)were maintained for 41 months and the snakes were indi-

vidually milked. The venom obtained from the mother and avenom pool of several B. insularis specimens were used ascontrols. Venoms were milked from neonates at 4, 10, 15, 20,24, 33 and 41 months old and used for assays.

2.2. Sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS–PAGE)

SDS–PAGE was carried out according to Laemmli (1970).Venom samples were resuspended on SDS–PAGE sample bufferunder reducing conditions, applied to a 4% polyacrylamidestacking gel and run on gradient gels (7.5 to 17.5%) at 10 °C. Gelswere stained with Coomassie Brilliant Blue R-250 (Sigma) anddestained on methanol/acetic acid solution in water (30/10, v/v).

Fig. 2. Gelatinolytic activity of venoms (40 μg/lane) under non-reducing conditions, evaluated by zymography in 10% polyacrylamide gels. The second and thirdcolumns illustrate previous incubation of samples with 10 mM 1,10-phenantroline and 5 mM PMSF, respectively. Lanes (numbers indicate the animal age in months):(A) intersex 1; (B) intersex 2; (C) male; (D) mother and pool of B. insularis. Molecular markers are represented on the left.

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Proteins standards were: phosphorylase b (94.0 kDa), albumin(67.0 kDa), ovalbumin (43.0 kDa), carbonic anhydrase (30.0 kDa),trypsin inhibitor (20.1 kDa) and α-lactalbumin (14.4 kDa)(Pharmacia, USA).

2.3. SDS–PAGE zymograms

Detection of venom proteases was performed as described byHeussen and Dowdle (1980) using 10% polyacrylamide gels,which were copolymerized with gelatin at a final concentrationof 1 mg/mL. Samples of 40 μg of venom were dissolved inSDS–PAGE sample buffer, in the absence of reducing agents,and electrophoresed at 80 mA at 4 °C. After electrophoresis,gels were washed for 30 min in 50 mM Tris–HCl buffer solu-

tion containing 2.5% Triton X-100, pH 7.5, to remove traces ofSDS and incubated for 3 h in 50 mM Tris–HCl buffer solutioncontaining 200 mM NaCl and 10 mM CaCl2, pH 7.5. Gels werestained by Coomassie Brilliant Blue R-250 (Sigma). Clearzones of substrate lyses against a blue stained backgroundindicated the presence of degrading enzymes. To verify thepresence of metalloproteases and serine proteases, venomsamples were previously incubated with 10 mM 1,10-phenantroline or 5 mM PMSF (final concentrations).

2.4. Coagulation

Minimum coagulant dose (MCD) was used to measurecoagulant activity of venom samples. The minimum coagulant

Table 1Comparative analysis of coagulant and hemorrhagic activities of B. insularisvenom

Coagulant activity(mg/L) a

Thrombinunits (TU) b

Hemorrhagicactivity (MHD) c

Intersex 115 6.2±0.35 0.15±0.00 2.0±0.3541 9.5±3.11 0.20±0.09 1.3±0.05

Intersex 215 11.2±0.35 0.21±0.03 2.0±0.2641 14.5±1.5 0.13±0.09 1.66±0.20

Male15 8.3±0.5 0.08±0.007 1.4±0.0541 9.1±0.1 0.17±0.01 0.6±0.4

Mother 11.9±0.07 0.09±0.01 1.4±0.1Pool ofB. insularis

15.5±0.7 0.05±0.01 1.2±0.2

For each animal the first and second numbers refer to values (mean±SD)obtained for 15 and 41 months old of animals, respectively.a MCD-P=minimum coagulant dose on human plasma (mg/L) (see

Materials and methods section); results are expressed as mean of threedeterminations±standard deviation.b Thrombin-like activity was expressed as Thrombin Units (TU) (see

Materials and methods section); results are expressed as mean of threedeterminations±standard deviation.c MHD=minimum hemorrhagic dose on mice (μg) (see Materials and

methods section); results are expressed as mean of six determinations±standarddeviation.

Table 2Fibrinolytic activity of venom from young and adults of B. insularis

Animals Age(months)

Fibrin plate lysisarea (mm2)

+10 mM 1,10-phenantroline

+5 mMPMSF

Intersex 1 15 360 0 28941 361 0 323

Intersex 2 15 420 120 38041 462 121 256

Male 15 324 0 48341 460 0 483

Mother 506 210 380Pool of B.insularis

484 156 440

Control a 228a Streptokinase 12.5 U/mL.

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dose (MCD) is defined as theminimum amount of venom resultingin clot formation within 60 s at 37 °C (Theakston and Reid, 1983).Coagulation activity was determined on human citrated plasma(200 μL) using serial dilutions of venom samples in a final volumeof 100 μL (dissolved in 0.85% saline solution).

2.5. Fibrinolysis — fibrin plate

The fibrin plate method was used according to Jespersen andAstrup (1983). A fibrin–agarose gel was prepared by mixinghuman fibrinogen (1 mg/mL clottable protein, Calbiochem)with a preheated solution of 2% agarose in 50 mM Tris–HClbuffer containing 5 mM CaCl2, 200 mM NaCl, pH 7.3 and2 U/mL bovine thrombin. Ten microliters of venom (1 mg/mL)(or venom plus inhibitor) was applied on the plate (9.0×1.5 cm).The system was incubated for 18 h at 37 °C and the fibrinolyticarea was measured and expressed in mm2. Streptokinase(12.5 U/mL) was used as a control. To verify the presence ofmetalloproteases and serine proteases, venom samples werepreviously incubated with 10 mM 1,10-phenantroline or 5 mMPMSF.

2.6. Effect of protease inhibitors on fibrinogenolytic activity ofvenoms

A mixture of human fibrinogen (1 mg/mL of clottableprotein, Calbiochem), and venom samples (1:100 venom:fibrinogen ratio) was incubated at 37 °C for 2 h in the presenceor absence of the inhibitors 1,10-phenantroline (10 mM), EDTA(10mM) or PMSF (5mM). Afterwards, the mixture was reducedby the addition of β-mercaptoethanol and boiled for 5 min.

Fibrinogen degradation profile was recorded on 10% SDS–PAGE. Gels were stained and destained as described above.

2.7. Factors X and II activation

Venoms samples (0.2 μg) were incubated at 37 °C with0.55 μM prothrombin or 0.7 μM factor X (Chromogenix) in20 mM Tris–HCl, pH 8.3, containing 5 mM CaCl2. After10 min of incubation, 400 μM S-2238 (H-D-Phe-PIP-Arg-pNA·2HCl) or S-2765 (N-α-Z-D-Arg-Gly-Arg-pNA·2HCl)(Chromogenix), chromogenic substrates for factors IIa andXa, respectively, was added and the hydrolysis was monitoredat 405 nm for 30 min at 37 °C. Amidolytic activity upon bothsubstrates was monitored at the same conditions in the absenceof factors II or X.

2.8. Thrombin-like activity

One hundred microliters of each venom sample (1 mg/mL)was incubated with human fibrinogen solution (1 mg/mLclottable protein) at 37 °C, and the time necessary to form fibrinclot was measured (in seconds). Thrombin-like activity ofvenom samples was expressed as Thrombin Units (TU), andwas calculated taking a standard curve made with serialdilutions of bovine thrombin (2, 1, 0.5, 0.25, 0.125, and0.0625 Thrombin Units).

2.9. Hemorrhage — minimum hemorrhagic dose (MHD)

The method described by Kondo et al. (1960) and modifiedby Gutiérrez et al. (1985) was used. Groups of male Swiss mice,weighing 18–22 g, were injected intradermically in the abdomenwith several doses of venom samples dissolved in 0.85% salinesolution. The minimum hemorrhagic dose was the venom dosethat induced a hemorrhagic area of 10 mm diameter.

3. Results

The results obtained herein showed an expressive ontoge-netic and individual variation of venoms, especially in theintensity of their activities. For most activities, the venom ofadults was similar to those of the mother and B. insularis pool,

Fig. 3. Degradation profile of fibrinogen in 10% SDS–PAGE after 2 h of incubation with venom samples (in the presence or not of protease inhibitors). Venom:fibrinogen ratio=1:100. A — cleavage of Aα and Bβ chains of fibrinogen by B. insularis venom; B — venoms previously incubated with 10 mM EDTA; C —venoms previously incubated with 10 mM 1,10-phenantroline; D— venoms previously incubated with 5 mM PMSF. Lane numbers: 1, 3, 5=intersex 1, intersex 2 andmale (15 months old), respectively; 2, 4 and 6= intersex 1, intersex 2 and male (41 months old); 7 and 8=offspring mother and pool of B. insularis venom,respectively.

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which were used as controls. Patterns of venom proteinsanalyzed by SDS–PAGE showed a relative similarity amongsamples and it was possible to distinguish proteins distributedover several ranges of molecular mass (Fig. 1). Most bandsbetween 50 and 14 kDa were common to all venom samples andwere present at all ages, but their intensity varied amongindividuals (Fig. 1). A single 24-kDa band seemed to increaseits concentration during offspring aging.

Proteolytic activity on gelatin increased during animal aging.Marked proteolysis of zymograms was observed betweenmolecular masses of 45 and 27 kDa (Fig. 2). All proteolyticpatterns were strongly inhibited by 10 mM 1,10-phenantrolineand partially by 5 mM PMSF. Adult venoms showed higheractivity than juveniles. Interestingly, the venoms of the motherand the pool of B. insularis showed bands with intense activityaround 90 kDa and between 27 and 45 kDa (Fig. 2D).

Marked ontogenetic variability of venoms was observed inregard to the clotting of human plasma. Venoms from youngspecimens were more active than adult ones to clot plasma(Table 1). On other hand, fibrinolytic activity did not showexpressive variability over time (Table 2). Inhibitory assayswith 1,10-phenantroline (10 mM) and PMSF (5 mM) showedthat venoms have both metalloproteases and serine proteases.Nevertheless, metalloproteases seem to have a major participa-tion on venom composition, since previous incubation with of10 mM 1,10-phenantroline reduced significantly the lysing offibrin plates (Table 2). No significant age-dependent variabilitywas noted for thrombin-like activity (Table 1) nor for fibrinogendegradation pattern in SDS–PAGE (Fig. 3).

All the venoms could promote cleavage of Aα and Bβfibrinogen chains, with the exception of the venom sample ofthe male (15 months old), which partially hydrolyzed fibrinogenBβ chain. However, previous incubation with inhibitors showedthat this cleavage was drastically reduced by 10 mM EDTA and10 mM 1,10-phenantroline, while incubation with 5 mM PMSFpartially prevented Bβ chain digestion (Fig. 3B, C and D,respectively).

Activation of factors X and II showed slight ontogeneticvariability. Venoms from young animals (15 months old) weremore active than those of adults (Figs. 4B and 5B, respectively).Intersex venoms, both of young and adults, were similar infactor X activation. The pool of B. insularis venom had thelowest activity followed by the venom of male. No directamidolytic activity was verified upon S-2765 substrate (data notshown). Factor II activation was similar in all samples, with theexception of male and intersex 2 venoms, which were the lowestones. Direct amidolytic activity upon S-2238 substrate washigher for samples of the adults (Fig. 4C and D). Hemorrhagicactivity of venoms showed an expressive ontogenetic variabil-ity, as adult venoms were more hemorrhagic than those ofjuveniles (Table 2). This activity showed no significantvariability between venom of intersexes, but the venom ofmale showed the most intense activity.

4. Discussion

This work demonstrates an ontogenetic variation in theexpression of proteases in B. insularis venom. In fact, B.

Fig. 4. Factor IIa activity upon chromogenic substrate S-2238, monitored at405 nm, 37 °C during 30 min. Venom samples: (A) adult (41 months old) and(B) juvenile (15 months old), respectively; (C and D) direct amidolytic activityupon S-2238 by adult and young venom samples, respectively.

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insularis is not only difficult to obtain, but also to maintain incaptivity. This species is considered critically endangered byIUCN Red List of Threatened Species (www.iucnredlist.org)(Marques et al., 2004) and has small litter sizes, which av-erages 6.5 neonates per litter (Hoge et al., 1959). Despite thesefactors, results demonstrated important ontogenetic andindividual shifts. These results are in agreement with previousinvestigations about Bothrops venoms, which showed age-dependent changes of venom composition and activities(Gutiérrez et al., 1990; Furtado et al., 1991; Andrade andAbe, 1999; López-Lozano et al., 2002, Saldarriaga et al.,2003). In spite of some individual variations, protein patternsseem to be very similar among venoms. The increase in in-tensity of the 24-kDa band (Fig. 1) may be related to on-togenetic regulatory mechanisms, once this pattern wasobserved in all three individual venoms analyzed here, sug-gesting an ontogenetic expression of proteins at this specificmolecular mass, which might be comprised mainly of PI classmetalloproteases. Such enzymes have a broad spectrum ofaction on biological systems, e.g., blood coagulation factors,extracellular matrix components and endothelial cells, induc-ing dermonecrosis, myonecrosis, edema and hemorrhageduring envenomation (Bjarnason and Fox, 1995; Gutiérrezand Rucavado, 2000; Serrano and Fox, 2005). Lomonte andCarmona (1992), using electrophoretic and immunoblottingtechniques, found individual differences in myotoxin expres-sion in Bothrops asper venom. Mackessy (1993) and López-Lozano et al. (2002) showed that metalloproteases seem to be

Fig. 5. Factor Xa activity upon chromogenic substrate S-2765. Monitoredhydrolysis at 405 nm, 37 °C, during 30 min. (A and B) Adult (41 months) andyoung (15 months) venom.

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also under ontogenetic regulation in Crotalus viridis oreganusand Bothrops atrox venoms, respectively.

Diet and feeding habits are well known for Brazilian viperidsnakes, mainly those belonging to Bothrops genus (Martins et al.,2002). There are no mammals at Queimada Grande Island andB. insularis has a well-recorded ontogenetic shift on its diet, withnewborns feeding on ectothermic animals (such as centipedes,lizards and amphibians) and on endothermic ones (migratorybirds) when they become adults (Amaral, 1921; Amaral, 1927;Duarte et al., 1995; Martins et al., 2002; Wüster et al., 2005).Moreover, B. insularis presents an increase in head length inrelation to Bothrops jararaca (its mainland sister taxa), animportant feature of ornitophagous species (Wüster et al., 2005).

The strong proteolytic activity is often correlated to Bo-throps envenomations and has important implications onclinical manifestations (Kamiguti et al., 1986; Ribeiro andJorge, 1989). Proteases have an important role in venomcomposition, since they are related to several physiologicalprocesses that, as main purpose, assist prey digestion (Thomasand Pough, 1979; Kochva et al., 1983; Mackessy, 1993; Daltryet al., 1997; Mebs, 1999). The increase in proteolytic activitywith aging may be due to the necessity of feeding on largerpreys and chiefly to provide a rapid prey immobilization anddeath. Thus, the higher content of proteases in adulthood mightbe associated with digestion of large size preys. In addition,from a library of B. insularis venom gland, Junqueira-de-Azevedo et al. (2001) have cloned and expressed a vascularendothelium growth factor, named svVEGF, that facilitatesvenom spreading by increasing vascular permeability, andtherefore, contributing to systemic action of whole venom.

The great efficacy of B. insularis venom to kill birds waspreviously reported by Afrânio do Amaral, who observed rapiddeath of birds following snakebites (Amaral, 1921; Amaral,1927). According to the zymogram patterns, the clear zonesfound mainly between 45 and 27 kDa are associated with PIIIand PI classes of snake venom metalloproteases (Serrano andFox, 2005), since 1,10-phenantroline strongly inhibited prote-olysis within this range (Fig. 2). This feature was common to allanalyzed samples, suggesting an important role for theseenzymes on venom activity. Interestingly, the venoms of themother and of the pool of B. insularis showed bands of∼90 kDa with gelatinolytic activity, a likely result of non-processed metalloprotease precursors or the presence of dimericPIII class metalloproteases (Serrano and Fox, 2005).

The diversity of transcripts of B. insularis venom glandshows that metalloproteases are the major components amongtoxin sequences (Junqueira-de-Azevedo and Ho, 2002). Metal-loproteases are zinc-containing enzymes which are present inmost viperid venoms and have a paramount role in thepathogenesis of hemorrhage during envenomation (Bjarnasonand Fox, 1995; Gutiérrez and Rucavado, 2000; Serrano andFox, 2005). Thus, the increase in the hemorrhagic activity ofadult venoms may be a consequence of an increase inmetalloprotease expression during B. insularis ontogeny, sincethese enzymes are frequently associated with hemorrhagicdisorders (Bjarnason and Fox, 1995; Gutiérrez and Rucavado,2000; Serrano and Fox, 2005).

Coagulant activities also showed an age-dependent pattern,as shown by previous investigations (Furtado et al., 1991;López-Lozano et al., 2002, Saldarriaga et al., 2003). Theincreased capacity of venoms from young animals (15 monthsold) to promote activation of factors X and II may in partexplain its intense procoagulant activity observed on MCDassay (Table 1). The fibrino(geno)lytic activity of venomsseems to be highly associated with the activity of metallopro-teases, since inhibitors like 1,10-phenantroline and EDTAabolished fibrinogen degradation and partially fibrin degrada-tion. We found that Aα and Bβ chain degradation is remarkablyassociated with metalloprotease activity. Insularinase A, ametalloprotease purified by Modesto et al. (2005) fromB. insularis snake venom, shows hydrolytic activity uponfibrinogen, promoting Aα and Bβ chains degradation. On theother hand, a serine protease isolated from B. insularis venomalso displays degradation of fibrinogen Aα and Bβ chain(Selistre and Giglio, 1987).

Another important factor is that we could follow the age ofsnakes and accomplish venom extractions in defined periods,since animals were maintained in captivity. We chose to workonly with individual venoms, instead of pooling them, in order tofollow individual ontogenetic variations. The variability of resultsobtained for the three specimens studied here, regardless of itsontogeny, may be attributed to many factors, including sexualdifference. B. insularis has a prominent sexual dimorphism inregard to the body weight, length and stoutness, and males areshorter and less stout than females (Hoge et al., 1959, Wüsteret al., 2005). Recently Menezes et al. (2006) showed sexual andindividual variation of the venom proteome of B. jararaca speci-mens, with important differences in the proteolytic profile.

The intense selective pressure upon this snake on its isolatedenvironment is another factor that must be considered.Although on small areas genetic variability tends to be low,selective pressure is considerable and toxin genes are certainlysubjected to it, leading to important molecular events, such asgene duplication and accelerated evolution, in order to diversifythe toxins and, consequently, its actions towards certain preytypes (Neurath, 1984; Nakashima et al., 1995; Zupunski et al.,2003). Biological assays with B. insularis venom showedselectivity for local preys (arthropods and birds), a main factorfor the evolutionary success of this species on the island(Zelanis, A. et al. in preparation). This feature was alsoobserved by Furtado (2005), who studied the venom of anotherinsular species, Bothrops alcatraz, whose high values ofproteolytic and procoagulant activities in venom were correlat-ed to an optimization of venom composition for a specializeddiet. The well-known ontogenetic change of prey types(arthropods to birds) observed in B. insularis has an importantrole for venom variability, since an increase in the size of preysis followed by an increase in the proteolytic activity of venom.

Acknowledgements

We are grateful to Alexandre Zanotti and Silvia R.T. Cardosofor their help in maintaining snakes in captivity. This work wassupported by CNPq (grant no. 133384/2004-7).

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