Comparative Biochemistry and Physiology Part C 135(2003) 469–479

1532-0456/03/$ - see front matter� 2003 Elsevier B.V. All rights reserved.doi:10.1016/S1532-0456(03)00169-8

Hydrolysis of DNA by 17 snake venoms

Adolfo Rafael de Roodt *, Silvana Litwin , Sergio O. Angela, a b

Area Investigacion y DesarrolloySerpentario, Instituto Nacional de Produccion de Biologicos,a ´ ´ ´Administracion Nacional de Laboratorios e Institutos de Salud, ‘Dr. Carlos G. Malbran’, Ministerio de Salud,´ ´

Av. Velez Sarsfield 563, CP 1281 Buenos Aires, Argentina´Departamento de Parasitologıa, Instituto Nacional de Enfermedades Infecciosas,b ´

Administracion Nacional de Laboratorios e Institutos de Salud, ‘Dr. Carlos G. Malbran’, Ministerio de Salud, Buenos Aires,´ ´Argentina

Received 6 February 2003; received in revised form 10 July 2003; accepted 11 July 2003

Abstract

DNA hydrolysis caused by venoms of 17 species of snakes was studied by different methodologies. Endonucleolyticactivity was tested by incubation of the venoms with the plasmid pBluescript and subsequent visualization of theelectrophoretic patterns in 1% agarose gels stained with ethidium bromide. DNA was sequentially degraded, fromsupercoiled to opened circle, to linear form, in a concentration dependent manner. The highest hydrolytic activity wasobserved inBothrops (B.) neuwiedii and Naja (N.) siamensis venoms. Exonucleolytic activity was analyzed onpBluescript digested withSmaI or EcoRI. All venoms caused complete hydrolysis after 2 h of incubation. SDS-PAGEanalysis in gels containing calf thymus DNA showed that the hydrolytic bands were located at approximately 30 kDa.DNA degradation was studied by radial hydrolysis in 1% agarose gels containing calf thymus DNA plus ethidiumbromide and visualized by UV light. Venom ofB. neuwiedii showed the highest activity whereas those ofB.ammodytoides andOvophis okinavensis (P-0.05) showed the lowest activity. Antibodies against venom ofB. neuwiediior N. siamensis neutralized the DNAse activity of both venoms. In conclusion, venom from different snakes showedendo- and exonucleolytic activity on DNA. The inhibition of DNA hydrolysis by EDTA and heterologous antibodiessuggests similarities in the structure of the venom components involved.� 2003 Elsevier B.V. All rights reserved.

Keywords: DNAse activity; Endonucleases; Exonucleases; Neutralization; Nucleases; Snakes; Venoms; Phosphodiesterase

1. Introduction

Snake venom is a complex mixture of proteinsand peptides containing a small proportion of othercomponents like lipids, carbohydrates, nucleicacids and minerals. The primary functions ofanimal venoms are the capture of prey and tofacilitate its digestion(Mebs, 2001). In fact, sev-

*Corresponding author. Tel.:q54-11-4303-1807–11x250;tel.yfax: q54-11-4303-2492.

E-mail address:aderoodt@uolsinectis.com(A.R. de Roodt).

eral snake venom components have a high simi-larity with pancreatic enzymes(Kochva, 1987;Markland, 1998). Many enzymes have beendescribed, including those hydrolyzing nucleicacids. Enzymes that act on nucleic acids degradeor hydrolyze DNA (deoxyribonucleases(DNAse)), RNA (ribonucleases(RNAse)) or both(nucleases) (Aird, 2002; Sittenfeld et al., 1991).

Enzymatic activity from animal venom nucle-ases may be related to hunting, defense and sur-vival. It has been suggested that the DNAseactivity of Vespa orientalis (Hymenoptera) may

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be related to destruction of the genetic material ofother queens(Ring et al., 1978, 1981). In contrast,the alkaline DNAse II ofRadhiantus macrodacty-lus (Cnidaria) and Rana catesbieana (Amphibia)possesses ‘lectin-like’ characteristics that wouldfacilitate the binding of proteins to glycoproteinsthat contain fucose, participating in the binding ofthe surface of plasma membranes(Gaphurov etal., 1999).

In snake venoms this activity could be relatedto the hydrolysis of nucleic acids of the prey’scells that produces adenosine liberation with relax-ant effects over the capillary wall and multipleeffects on different systems participating in theglobal toxic effect of venom by means of purineliberation, as has been suggested by Aird in anexcellent review(Aird, 2002).

The exonucleases or phosphodiesterases(EC3.1.15.1) and endonucleasesyRNAse (EC 3.1.-.-)in snake venoms were described(Aird, 2002;Auron et al., 1982; Sulkowski and Laskowski,1971; Dolapchiev et al., 1974). Among exonucle-ases, phosphodiesterase I(59-exonuclease, EC3.1.41) (Sulkowski and Laskowski, 1971; Dolap-chiev et al., 1974), DNAse I (EC 3.1.21.1) andDNAse II (DNAse 39-oligonucleotide-hydrolase,EC 3.1.22.1) were reported(Auron et al., 1982).DNAse II has an acid pH optimum and divalentcations are not a requirement for its activity,whereas DNAse I and phosphodiesterase areMg dependent and have an optimal neutral or2q

alkaline pH (Haessler and Cunningham, 1957;Elliott, 1969).

Endo- or exonuclease activities have beendescribed in venoms of snakes from differentregions of the world(Elliott, 1969). Studies onvenom nucleases have been undertaken onCrotal-us (C.) adamanteus (‘eastern diamond rattle-snake’) venom, where phosphodiesterase I of 140kDa with exonuclease activity(Dolapchiev et al.,1974; Pritchard et al., 1977) and single chainendonuclease activities(Stoynov et al., 1997) withcharacteristics of glycoprotein(Dolapchiev et al.,1974) was described. Sittenfeld et al.(1991)analyzed venoms from different Crotalids fromCosta Rica(Bothrops (B.) asper, B. godmani, B.schlegelli, B. lateralis, B. nasutus, C. durissus andLachesis muta), and they found that all the venomspresented DNAse I or phosphodiesterase I. Exo-and endonuclease activities were also described insome American snake venoms likeB. atrox (Elli-ott, 1969; Frischauf and Eckstein, 1973; Phillipps,

1976) and exonuclease activity in venoms fromsnakes of the principal groups of venomous snakes:Crotalinae (C. viridis oreganus, C. adamanteus,Agkistrodon (A.) piscivorus, A. halys blomhoffi, A.contortrix mokeson), Viperinae(Vipera (V.) aspis,V. russelli, V. palestinae, V. ursini renardi, Trimer-esurus wagleri, Bitis arietans) and Elapidae(N.naja, N. haje, Hemachatus haemachatus, Notechisscutatus and Bungarus fasciatus) (Elliott, 1969;Tatsuki et al., 1975; Phillipps, 1975, 1976; Levyand Bdolah, 1976; Ballario et al., 1977; Khamud-khanova and Sakhibov, 1984). However, there islittle information available on the activities ofSouth American snake venoms on DNA.

In the present work, we studied the endonucle-ase and exonuclease activity of crude venoms ofeight snake species from Argentina and nine fromother regions of the world. This activity wasstudied by means of the hydrolysis of the plasmidpBluescript(Stoynov et al., 1997) and by enzymediffusion (Sittenfeld et al., 1991). pBluescript is adouble-stranded circular plasmid. However, it con-tains a region downstream of theLactamase gene,where the double helix is unstable in the negativesupercoiled(SC) version. Endonucleolytic activitywas studied using the intact plasmid, exonucleo-lytic activity was studied on pBluescript partiallypre-digested withSmaI or EcoRI. Hydrolytic activ-ity on DNA was compared by studying the abilityof crude venoms to hydrolyze nucleic acids con-tained in agarose plates by radial diffusion and byhydrolysis of calf thymus DNA on polyacrylamidegels after electrophoresis. Additionally, the capac-ity of homologous or heterologous antivenoms toneutralize DNA hydrolytic activity of the wholevenom was studied.

2. Materials and methods

2.1. Venoms

Venoms from B. alternatus, B. neuwiedii, B.moojeni, C. durissus terrificus andM. pyrrhocryp-tus were obtained from snakes of the serpentariumof the Instituto Nacional de Produccion de Biolo-´ ´gicos—A.N.L.I.S. ‘Dr Carlos G. Malbran’(INPB).´B. jararaca and B. jararacussu venoms wereprovided by Dr Alejadro U. Vogt, from the Ser-pentarium of the Centro Zootoxicologico de´Misiones, Argentina;B. ammodytoides venom wasa gift from Dr Eduardo F. Gould of the Serpentar-ium of the Fundacion de Estudios Biologicos,´

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Fig. 1. Establishment of optimal conditions of the snake venomendonuclease activity. Two hundred nanogram of pBluescriptdiluted in incubation buffer were treated with 0 ng(1), 10 ng(2), 100 ng(3) and 1mg (4) of B. neuwiedii venom at 378Cduring 30 and 60 min. C , pBluescript; C , pBluescript digest-1 2

ed withEcoRI. OC, open circle; L, linear; and SC, super coiledDNA.

Presidente Peron, Buenos Aires, Argentina and the´venoms fromB. asper, Athropoides (At.) nummi-fer, C. d. durissus, C. basilicus, A. bilineatus andBitis gabonica venoms were a gift of Dr AlejandroAlagon of Instituto de Biotecnologıa de la Univ-´ ´ersidad Autonoma de Mexico, Cuernavaca, More-´ ´los, Mexico. Venoms were obtained by manual´milking, followed by vacuum drying and thenstored aty20 8C. N. siamensis venom was pur-chased from the Miami Serpentarium andOvophisokinavensis (ex T. okinavensis) venom was pur-chased from Sigma Chemicals. All the venomswere diluted in 0.15 M NaCl, filtered through0.22-mm membranes and stored aty20 8C untiluse.

2.2. Detection of endo- and exonucleolytic activity

Plasmid Bluescript II SK was extracted fromEscherichia coli cultures using the Flexiprep kit(Pharmacia). Elution of DNA from Sephaglassparticles was done with bi-distilled water(Sam-brook et al., 1989). To detect endonucleolyticactivity, 200 ng of SC DNA were incubated withcrude venom in MgCl 5 mM, Tris–HCl 0.1 M2

pH 8.0 at 378C. After digestion the DNA waselectrophoresed in 1% agarose gel containing 0.5mgyml of ethidium bromide. To detect the exonu-cleolytic activity, the plasmid was first digestedwith EcoRI or SmaI endonuclease, purified fromgels by Qiaex II(Qiagen) and resuspended in bi-distilled water.

To determine the optimal conditions for endon-uclease activity,B. neuwiedii venom was chosenas the reference venom. Different amounts of thisvenom as well as two different incubation timeswere assayed to hydrolyze 200 ng of SC p-Bluescript(Fig. 1). Ten nanograms of crude venomproved to be insufficient to detect endonucleaseactivity at either 30- or 60-min incubation, whereas1.0 mg showed optimal results. In fact, with 1.0mg of crude venom endonucleolytic activity wasreadily detected at 30 min, since the ratio of SCyopen circle(OC) DNA decreased(Fig. 1, 30 minlane 4). Extending the incubation time to 60 min,the SC form of DNA disappeared with 1.0mg ofsnake venom and OC as well as linear(L) formsappeared(Fig. 1, 60 min lane 4). Thus, 1.0mgsnake venom and an incubation time of 60 minwere employed. Fourteen venom samples werestudied under the conditions described above

(Table 1). At least three independent experimentswere performed by triplicate.

2.3. Endonucleolytic activity of crude venom fromdifferent snakes with and without EDTA

To detect the activity on DNA in the presenceof an inhibitor, EDTA was added to venoms thatshowed the highest endonuclease activity. Onemicrogram of crude venom fromB. neuwiedii, B.moojeni, B. jararaca, B. jararacussu, B. asper, A.bilineatus, N. siamensis or B. gabonica was incu-bated in incubation buffer in with or without 10mM EDTA and the endonucleolytic activity ofboth ways was compared. At least three independ-ent experiments were performed by triplicate.

2.4. DNAse activity in gel

DNAse activity was also measured by electro-phoresis in DNA containing sodium dodecyl sul-fate polyacrylamide 10% gels(SDS-PAGE) undernon-reducing conditions as described by Laemmli(1970). Five micrograms of crude venom weremixed with loading buffer and electrophoresed inSDS gels containing 0.15 mgyml of double-strand-ed calf thymus DNA Type V(Sigma) in a Mini-protean II system(Bio-Rad). To remove SDS, gelswere washed for 8=15 min in 50 ml of incubationbuffer (Tris–HCl 10 mM pH 8, CaCl 10 mM,2

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Table 1Radial hydrolysis, hydrolysis in zymogram of calf thymus DNA and hydrolysis of plasmid pBluescript(endonucleolytic activity) orpBluescript digested by restriction enzymes(exonucleolytic activity)

Venoms Diameter Zymogram Endonucleolytic Exonucleolytic(cm)a activity activity

B. alternatus 1.45"0.04 y q qB. neuwiedii 2.05"0.06 q qqq qB. ammodytoides 0.98"0.06 y y qB. jararacussu 1.71"0.06 q qq qB. jararaca 1.75"0.02 q qq qB. moojeni 1.72"0.06 q qq qB. asper 1.73"0.04 q qq qC. d. terrificus 1.44"0.03 y q qC. d. durissus 1.42"0.07 q ND NDC. basiliscus 1.74"0.04 q q qC. scutulatus 1.43"0.04 yb ND NDAt. nummifer 1.51"0.04 q q qA. bilineatus 1.44"0.05 y qq qO. okinavensis 0.82"0.06 ND ND NDB. gabonica 1.55"0.08 q qq qN. siamensis 1.66"0.07 q qqq qM. pyrrhocryptus ND ND qq q

Results of radial hydrolysis are expressed as mean"S.D. (cm). The absence of hydrolysis is expressed as(y), the hydrolysis witha

high venom doses(q) and the apparent higher activity as(qq) or (qqq). ND, not determined.In C. scutulatus hydrolysis was observed after 48 h of incubation.b

MgCl 10 mM) at room temperature. Then, gels2

were incubated in the aforementioned buffer for16 h at 378C, stained with ethidium bromide(5mgyml) and UV illuminated. Finally, gels werestained with Coomassie blue to develop the wholeprotein profile. This assay was also done using thevenoms previously incubated with 10 mM EDTA.At least three independent experiments were per-formed by triplicate.

2.5. Radial hydrolysis of nucleic acids

Hydrolytic activity on DNA was studied asdescribed by Sittenfeld et al.(1991) with somemodifications. Petri dishes were covered with 20ml of 2% agarose of electrophoresis grade(Gibco)in incubation buffer containing 100mgyml ofdouble-stranded calf thymus DNA Type V(Sigma)and 1mgyml of ethidium bromide. Thirty microl-iters of 2mgyml of each venom diluted in 0.15 MNaCl were loaded in wells of 0.5 mm of diameterpunched in the agarose gel and incubated for 48 hat 37 8C. The activity was visualized under UVlight. Two major perpendicular diameters ofhydrolysis were measured with a caliper, and theresults were expressed in centimeter. All experi-ments were performed by triplicate and at leastfour times. Data were analyzed by Student’st-test

with the PRISMA software version 3(GraphPadSoftware, San Diego, CA).

2.6. Chromatography of B. neuwiedii venom

B. neuwiedii venom showed the highest activityon agarose plates by enzyme diffusion. To observethe chromatographic fractions of the venom withDNA hydrolytic activity, 30 mg ofB. neuwiediivenom were run in a Sephadex G-75 column(1m=1.5 cm) equilibrated in 0.2 M ammoniumacetate pH 5.1, 0.3 mlymin and the DNAse activityof the fractions was studied by radial hydrolysis.After that, fractions were lyophilized. Fractionswith DNAse activity were reconstituted in distilledwater and studied by 10% SDS-PAGE in non-reducing conditions, using as molecular weightmarker the prestained broad range standard(Bio-Rad). Gels were stained using Coomassie BrilliantBlue (Bio-Rad) and Silver Staining(Silver Stain-ing Protein Kit, Pharmacia) in order to detectminimal amount of proteins not visualized usingCoomassie.

In addition, the procoagulant(Theakston andReid, 1983) and indirect hemolytic activities(Al-Abdulla et al., 1991) were studied in the chromat-ographic fractions.

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2.7. Antivenoms

The antivenoms used in this study were theBivalente antivenom of the INPB Batch 246, Exp.Date: 10y1997 and an experimental Anti-N. sia-mensis antivenom. Bivalente antivenom was pro-duced by the immunization of horses withB.alternatus and B. neuwiedii venoms. Plasma fromimmunized horses was fractionated by doubleammonium sulfate precipitation, thermocoagula-tion and digestion with pepsin to obtain F(ab9)2fragments(Pope, 1939; Christensen, 1966). Antiv-enom was sterilized by filtration and fractionated.The F(ab9) preparation had a protein content of2

90"15 mgyml and thimerosal(1 gy20 l)–phenol(2.5 mlyl) were added as preservatives. The Anti-N. siamensis antivenom was obtained by immuni-zation of horses withN. siamensis venom (thesame batch of venom used for the hydrolysisassay). Briefly, a horse was immunized with 0.5,1, 2.5, 4 and 5 mg of venom at days 1, 8, 15, 30and 45, respectively. The horse was partially bledby jugular puncture. The plasma was treated withcaprylic acid 5% vyv to separate the immunoglob-ulin fraction (Rojas et al., 1994), then dialyzed,filtered, treated with thimerosalyphenol asdescribed and stored at 48C. This antivenom hada final protein content of 39"7 mgyml. Antibo-tulinum A antitoxin from INPB Batch 001y98(Exp. Date: 12y2002), processed as described forthe Bivalente and, an Antibotulinum A antitoxinprocessed as described for the Anti-N. siamensisantivenom, were used as negative controls.

2.8. Inhibition of hydrolysis by antivenoms

Neutralization assays of the hydrolytic activityof B. neuwiedii and N. siamensis venoms wereperformed using the antivenoms mentioned above.To determine the homologous and heterologousinhibition of the DNAse hydrolytic activity, 400mg of B. neuwiedii or N. siamensis venoms werepreincubated for 30 min at 378C with differentamounts of each antivenom(150–0.5 ml) in afinal volume of 190ml in 0.15 M NaCl. For thedetermination of radial hydrolysis the venom–antivenom mixtures were placed in wells locatedin Petri dishes containing 40 ml of 2% agarose(Gibco, electrophoresis grade) in the conditionsdescribed above. The samples were incubated for24 h at 378C, and the diameters were measuredwith a caliper under UV light. Four hundred

micrograms of each venom diluted in 190ml of0.15 M NaCl were used as the positive control.As negative controls different dilutions of eachantivenom(150–0.5ml, diluted in 0.15 M NaCl)or 0.15 M NaCl alone in a final volume of 190mlywell with 0.15 M NaCl were included. Inaddition, as a control of inespecific neutralization,a seroneutralization assay was performed underthe conditions described above, using both prepa-rations of antibotulinum antitoxin. The inhibitionwas estimated as a percentage of decrease of theradial hydrolysis when compared with the positivecontrols. The values were analyzed by non-linearregression using thePRISMA software(GraphPadSoftware). The results were expressed as effectivedose 50%(ED ) representing the antivenom dose50

that reduced 50% the diameter of the hydrolytichaloes when compared with the positive controls.The experiment was performed by quadruplicate.

3. Results

3.1. Endo- and exonucleolytic activity of crudevenom from different snakes

Endonucleolytic activity of different crude ven-oms was measured using a preparation of theplasmid pBluescript enriched in the SC version.Almost all venoms showed this activity(Fig. 2).However, differences in the level of endonucleaseactivity were observed.B. neuwiedii and N. sia-mensis venoms seemed to have the highest activity,whereasB. jararaca, B. asper, B. jararacussu, B.moojeni, A. bilineatus and B. gabonica venomsshowed low endonucleolytic activity levels underthe same condition.C. d. terrificus, C. basiliscusand B. alternatus venoms showed plasmid linear-ization only when 10mg of venom were used(Table 1), andB. ammodytoides crude venom didnot show any single-strand endonucleolytic activ-ity. M. pyrrhocryptus venom also showed highendonucleolytic activity(data not shown).

When plasmid DNA was incubated with venomin the presence of EDTA, endonucleolytic activitywas completely abolished(data not shown), sug-gesting that the hydrolytic activity was restrictedto DNase I-like or phosphodiesterase I-likeenzymes.

After 2 h of incubation of theEcoRI or SmaI-linearized plasmid with 1.0mg of each crudevenom, exonucleolytic degradation was observed,

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Fig. 2. Endonucleolytic activity of different snake venoms. Twohundred nanograms of pBluescript diluted in incubation bufferwere incubated for 60 and 120 min at 378C with 1 mg ofsnake venoms fromB. neuwiedii (1), B. jararacussu (2), B.jararaca (3), B. ammodytoides (4), B. alternatus (5), B. moo-jeni (6), C. d. terrificus (7), C. basiliscus (8), B. asper (9),A. nummifer (10), A. bilineatus (11), B. gabonica (12) andN.siamensis (13). OC, open circle; L, lineal and SC, super coiledDNA.

which was abolished in the presence of EDTA(data not shown).

3.2. Detection of DNAse activity from differentsnake venoms by SDS-PAGE

Venom from B. neuwiedii and N. siamensis(venoms with high endo- and exonucleolytic activ-ities) analyzed by zymogram showed hydrolyticbands at approximately 30 kDa(Fig. 3a and b).

When the zymogram was repeated with othervenoms, only 10 out of 15 venoms showed DNAseactivity (Fig. 3c, Table 1). These wereB. neuwie-dii, B. jararaca, B. jararacussu, B. moojeni. B.asper, C. d. durissus, C. basiliscus, At. nummifer,B. gabonica andN. siamensis venoms. The venomsthat showed this activity were those that showedhigher activity in the radial hydrolysis assay. Inaddition, all these venoms showed DNAse activity

associated with a similar migration pattern andmolecular mass close with that ofB. neuwiediiandN. siamensis (Fig. 3c). The activity of all thevenoms was completely inhibited in the presenceof EDTA (data not shown).

3.3. Radial hydrolysis of nucleic acids

Radial hydrolysis was observed with all venomsstudied (Table 1). Hydrolysis increased linearlywith incubation time. Venom ofB. ammodytoidesand O. okinavensis showed the lowest activity,while B. neuwiedii venom was the most active(P-0.05).

Activities of B. jararaca, B. moojeni, B. jarar-acussu, B. asper, C. basiliscus and N. siamensisvenoms were very similar, producing very closehydrolytic haloes. Venoms ofC. d. terrificus, C.d. durissus, C. scutulatus, A. nummifer, B. alter-natus, A. bilineatus and B. gabonica displayedvery low activity. Hydrolysis rings generated byBothrops species andCrotalus species venomswere very similar. Values obtained were1.63"0.32 cm (95% confidence interval 1.48–1.77) for Bothrops species and 1.51"0.15 cm(CI1.42–1.60) for Crotalus species. However, whenthe activity of Bothrops venoms was consideredexcludingB. ammodytoides venom, higher differ-ences were observed between the hydrolytic activ-ity of Bothrops venoms (which increases to1.73"0.18 cm, CI 1.64–1.83) and Crotalusvenoms.

3.4. Analysis of the activity of the chromatographicfractions of B. neuwiedii venom

The study of the fraction samples showed thatDNAse activity started to elute at the end of thefirst chromatographic peak(see A in Fig. 4a)together with procoagulant activity(data notshown). The highest DNAse activity was shownbetween both major chromatographic peaks(seeB in Fig. 4a). The DNAse activity could not bedetected when the presence of indirect hemolyticactivity could be observed(data not shown). TheSDS-PAGE of these fractions showed slightlystained bands under 60 kDa in fractions BI, BIIand BIII (Fig. 4b).

3.5. Inhibition of DNA-hydrolysis by antivenoms

Both antivenoms produced homologous and het-erologous inhibition of the DNAse activity ofB.

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Fig. 3. Zymogram analysis of snake venom DNAses. Snake venoms were electrophoresed in a SDS-polyacrilamide gel containing calfthymus DNA, incubated in incubation buffer at 378C overnight. Gel was stained with Coomassie blue(a) after ethidium bromide-gelstaining(b). M, molecular mass marker(Sigma Wide Molecular Range); IgG, horse immunoglobulin G as a;150 kDa mass marker(Sugiura et al., 2000); F(ab9) , F(ab9) fragment of horse IgG as a 90–100 kDa mass marker;Bn, B. neuwiedii crude venom(20 mg);2 2

Ns, N. siamensis crude venom(20 mg). (C) DNAse activity of several snake venoms revealed by ultraviolet exposure after ethidiumbromide-gel staining.B. moojeni (1), C. d. terrificus (2), B. ammodytoides (3), B. moojeni (4), B. jararacussu (5), B. jararaca (6),B. neuwiedii (7), B. alternatus (8), A. bilineatus (9), N. siamensis (10), B. asper (11), C. basiliscus (12), C. d. durissus (13), C.scutulatus (14), B. nummifer (15), Agk. bilineatus (16) andB. gabonica (17).

neuwiedii andN. siamensis venoms(Fig. 5). WhenAnti-Naja antivenom was used, the ED against50

N. siamensis and B. neuwiedii venoms were 15.5ml (8.7–27.9) and 28.3(16.7–48.0), respectively,showing a better neutralization with each homol-ogous venom. The same phenomenon wasobserved with the Bivalente. The ED againstB.50

neuwiedii venom was 77.9ml (36.5–166.2) andagainstN. siamensis venom was 104.2ml (49.5–219.3). In both the cases, Anti-N. siamensis anti-venom seemed to have higher neutralizingcapacity. When assays were performed preincubat-ing the venoms with the negative controls(bothAnti-Botulinum antitoxins) no neutralization wasobserved.

4. Discussion

All venoms showed hydrolytic activity on DNAwhen tested on the intact pBluescript plasmid,indicating endonucleolytic andyor exonucleolyticactivities. On calf thymus DNA,B. neuwiediivenom showed the highest activity when tested byradial diffusion, in agreement with the observationson the activity analyzed by hydrolysis of p-Bluescript and in zymograms.

When venoms were electrophoresed in gel con-taining double-stranded calf thymus DNA, hydrol-ysis was observed only with venoms fromB.neuwiedii, B. jararaca, B. jararacussu, B. moojeni,B. asper, C. d. durissus, C. basiliscus, At. num-

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Fig. 4. (a) Chromatography of 30 mg ofB. neuwiedii venomon Sephadex G-75(column 1 m=1.5 cm, Buffer Ammoniumacetate 0.2 M pH 5.1, 0.3 mlymin). The X-axis indicates theelution volume andY-axis the absorbance of eluted. Arabicnumbers in figure indicate the peaks. The elution volume inwhich the DNAse activity was detected is indicated as A. Bindicates the elution volume with the highest DNAse activity.(b) SDS-PAGE of wholeB. neuwiedii venom and chromato-graphic fractions with DNAse activity. M, molecular weightmarker(Bio-Rad, Broad range, prestained); CV, crude venom;A1, Fraction with DNAse activity from chromatographic peak1; I, II and III, fractions with DNAse activity corresponding toelution volume B.

Fig. 5. Inhibition of DNAse activity by anti-venom sera. Fourhundred micrograms ofN. siamensis or B. neuwiedii crude ven-om were incubated for 30 min at 378C with different dosesof Anti-N. siamensis or Bivalente antivenom(Anti-B. neuwie-dii and B. alternatus serum), placed in wells located in Petridishes containing 40 ml of 2% agarose in incubation buffercontaining 100mgyml of DNA from calf thymus and 1mgymlof ethidium bromide for the determination of radial hydrolysis.The samples were incubated for 24 h at 378C, the activity wasvisualized under UV light and the diameters measured with acaliper. The positive controls were 400mg of each venom. Asnegative controls there were used different dilutions of eachantivenom alone or NaCl 0.15 M. Homologous and heterolo-gous DNAse activity neutralization was estimated as inhibitionpercentage by comparison with respective positive controls.(s) AN-N. siamensis (Anti N. siamensis antivenom preincu-bated withN. siamensis venom); (d) AN-B. neuwiedii (AntiN. siamensis antivenom preincubated withB. neuwiedii ven-om); (h) Biv-N. siamensis (Bivalente antivenom preincubatedwith N. siamensis venom); (j) Biv-B. neuwiedii (Bivalenteantivenom preincubated withB. neuwiedii venom).

mifer, B. gabonica andN. siamensis. In all venoms,hydrolysis seemed to result from proteins withclose molecular mass(Fig. 3). No hydrolysis was

observed whenB. alternatus, B. ammodytoides, A.bilineatus, C. d. terrificus and C. scutulatus ven-oms were used although these venoms showedhydrolytic activity on DNA in the radial hydrolysisassay. These differences could be due by theamount of venom used between assays, since inthe zymograms it was used 5mg whereas in theradial hydrolysis it was used 60mg of venom. Itis necessary to point out that the venoms thathydrolyzed the DNA in this assay(exceptingC.d. durissus) were those that showed the highestDNAse activity by radial hydrolysis.

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The requirement of Mg and the inactivating2q

effect of EDTA or Cu on phosphodiesterases I2q

or DNAse I are well known(Elliott, 1969; Prit-chard et al., 1977; Laskowski, 1980). DNAseactivity in the venoms assayed in this study seemsto be due by enzymes with these characteristics,since the activity was abolished by EDTA. Theseresults agree with those published by Sittenfeld etal. (1991), who described this type of activity invenoms from Central American Crotalids. Bothendonucleolytic activity in plasmid and DNAseactivity in zymogram were inhibited in the pres-ence of EDTA.

The chromatographic study of venom fractionswith DNAse activity showed that components thathydrolyze DNA had a molecular mass of approx-imately 20–40 kDa, below the molecular mass ofthe known phosphodiesterases of several snakevenoms(0100 kDa) (Elliott, 1969; Dolapchievet al., 1974; Pritchard et al., 1977; Stoynov et al.,1997). We cannot assure that these components ofapproximately 30 kDa were the only responsiblefor hydrolytic activity on DNA in the wholevenom, since DNAse activity by radial hydrolysiscould be detected in fractions with componentswith higher molecular range(fraction A 1, Fig.4). However, we could observe hydrolytic activityon DNA in venom fractions with molecular massesunder 60 kDa, in concordance with the hydrolyticareas observed in zymograms. In fact, the molec-ular mass of the components of chromatographicfractions with DNAse activity observed here werelocated between the fractions with procoagulantand phospholipase activity. Metalloproteinases andserinoproteases responsible of procoagulant activ-ity of Bothrops venoms from Argentina possessmolecular masses at least over 20–40 kDa(deRoodt, 2002) and the Bothrops phospholipasesrange of approximately 15 kDa(Gutierrez and´Lomonte, 1995; de Roodt, 2002).

Noteworthy, it seems that some similarity existsbetween the structure of these enzymes from dif-ferent venoms, since hydrolysis was inhibited byEDTA and antibodies against one of the venomscould neutralize the activity of an heterologousvenom(Fig. 5).

It is known that exonucleases are strongly anti-genic (Dolapchiev et al., 1981). As mentionedabove, the antivenoms tested here showed homol-ogous and heterologous neutralizing capacityagainst DNA hydrolysis. It is important to pointout that the venoms used in this assay were from

two different families of venomous snakes(B.neuwiedii: Viperidae andN. siamensis: Elapidae).Antibodies againstN. siamensis showed a higherneutralizing capacity against both venoms. We donot have a direct explanation for the higher neu-tralization provided by Anti-N. siamensis antiven-om againstB. neuwiedii venom. One possibilitycould be a higher amount of this type of enzymein N. siamensis venom or that this venom did notsuffer proteolysis while the immunization mixturewas prepared, as observed in previous experiencesin Bothrops venom immunization mixtures(Cari-cati et al., 1993; de Roodt et al., 1996), offering ahigher amount of enzyme to the equine immunesystem. In addition, in the Bivalente that is pro-duced with the mixture ofB. alternatus and B.neuwiedii venoms,B. alternatus venom showedlower DNAse activity, strengthening the theory ofthe possible mentioned deficit of these enzymes inthe immunogenic mixture.

By radial hydrolysis,Bothrops venoms showeda slightly higher activity compared to the others,with the exception ofB. ammodytoides. RegardingB. ammodytoides, despite this venom has activitiesquite similar to other bothropic venoms(de Roodtet al., 2000), we have observed certain differencesin some biological activities(de Roodt, 2002).Those differences were also reflected in the DNAseactivity in comparison with the otherBothropsvenoms studied. This snake is zoologically differ-ent from the otherBothrops species. Morphologi-cally is smaller and with a very different patternof spots on its body when compared with otherBothrops species. On the other hand, its ecologyis very different since it is the Southern viper inthe world and inhabits regions of very low tem-peratures, strong winds and in some regions nearthe see. At present its systematic position is beingreviewed.

The function of enzymes from snake venomsthat hydrolyze nucleic acids is not completelyunderstood, since these components in nature havenot been studied in the same degree as other snakeenzymes like proteinases or phospholipases. Avery possible function of this activity of the snakevenoms could be those described by Aird(2002),participating in the purine liberation contributingin a very important way with the global toxicityof the venom. By this cause, it is possible that thepresence of this type of enzymes in snake venomscan be related with the hunting and defense(Aird,2002), since it has been found in all venoms of

478 A.R. de Roodt et al. / Comparative Biochemistry and Physiology Part C 135 (2003) 469–479

the major groups of venomous snakes: Elapidaeand Viperidae(in both subfamilies: Viperinae andCrotalinae) (Elliott, 1969; Sittenfeld et al., 1991),and it was found in this study in other species ofthis group of venomous snakes(Micrurus andNaja wElapidaex; Bothrops, Agkistrodon, Athropo-ides, Crotalus, Bitis and Ovophis wViperidaex).Differences observed on the level of these activi-ties between the distinct species of snakes have nodirect explanation at present.

As far as we know, this is the first report aboutthe enzymatic activity on nucleic acids of venom-ous snakes from South America, of DNAse activityby components under 100 kDa in snake venoms,and on the immunochemical cross-reactivity ofnucleases from different families of snakes.

Acknowledgments

S.O. Angel is a researcher of the NationalCouncil of Research(CONICET – Argentina). Wethank Dr M Rosenzvit and V. Martin from theDepartamento de Parasitologıa of the ANLIS for´critical reading of the manuscript. The authors aredeeply grateful to Dr Jose Maria Gutierrez from´ ´the Instituto Clodomiro Picado, Costa Rica for hiscritical reading and helpful advice. Authors arevery grateful with the reviewers of the manuscriptby the suggestions made to improve the qualityand understanding of this work.

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