Neural Computation for Rehabilitation Animal Toxins and Their Advantages in Biotechnology and ...

152 Pages·2014·19.1 MB·English

BioMed Research International
Animal Toxins and Their Advantages
in Biotechnology and Pharmacology
Guest Editors: S. L. Da Silva, E. G. Rowan, F. Albericio, R. G. Stábeli,
Neural Computation for Rehabilitation
L. A. Calderon, and A. M. Soares
Animal Toxins and Their Advantages in
Biotechnology and Pharmacology
BioMed Research International
Animal Toxins and Their Advantages in
Biotechnology and Pharmacology
Guest Editors: S. L. Da Silva, E. G. Rowan, F. Albericio,
R. G. Sta´beli, L. A. Calderon, and A. M. Soares
Copyright©2014HindawiPublishingCorporation.Allrightsreserved.
Thisisaspecialissuepublishedin“BioMedResearchInternational.”AllarticlesareopenaccessarticlesdistributedundertheCreative
CommonsAttributionLicense,whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginal
workisproperlycited.
Contents
AnimalToxinsandTheirAdvantagesinBiotechnologyandPharmacology,S.L.DaSilva,E.G.Rowan,
F.Albericio,R.G.Sta´beli,L.A.Calderon,andA.M.Soares
Volume2014,ArticleID951561,2pages
AlkylationofHistidineResiduesofBothropsjararacussuVenomProteinsandIsolatedPhospholipases
A :ABiotechnologicalTooltoImprovetheProductionofAntibodies,C.L.S.Guimara˜es,
2
S.H.Andria˜o-Escarso,L.S.Moreira-Dill,B.M.A.Carvalho,D.P.Marchi-Salvador,N.A.Santos-Filho,
C.A.H.Fernandes,M.R.M.Fontes,J.R.Giglio,B.Barraviera,J.P.Zuliani,C.F.C.Fernandes,
L.A.Caldero´n,R.G.Sta´beli,F.Albericio,S.L.daSilva,andA.M.Soares
Volume2014,ArticleID981923,12pages
BiochemicalandFunctionalCharacterizationofParawixiabistriataSpiderVenomwithPotential
ProteolyticandLarvicidalActivities,GizeliS.Gimenez,AntonioCoutinho-Neto,AndersonM.Kayano,
RodrigoSimo˜es-Silva,FrancesTrindade,AlexandredeAlmeidaeSilva,SilvanaMarcussi,SauloL.daSilva,
CarlaF.C.Fernandes,JulianaP.Zuliani,LeonardoA.Calderon,AndreimarM.Soares,
andRodrigoG.Sta´beli
Volume2014,ArticleID950538,13pages
SnakeVenomL-AminoAcidOxidases:TrendsinPharmacologyandBiochemistry,
LuizFernandoM.Izidoro,JulianaC.Sobrinho,MirianM.Mendes,Ta´ssiaR.Costa,AmyN.Grabner,
VeridianaM.Rodrigues,SauloL.daSilva,FernandoB.Zanchi,JulianaP.Zuliani,CarlaF.C.Fernandes,
LeonardoA.Calderon,RodrigoG.Sta´beli,andAndreimarM.Soares
Volume2014,ArticleID196754,19pages
PurificationandBiochemicalCharacterizationofThreeMyotoxinsfromBothropsmattogrossensis
SnakeVenomwithToxicityagainstLeishmaniaandTumorCells,Andre´aA.deMoura,
AndersonM.Kayano,GeorgeA.Oliveira,SulamitaS.Setu´bal,Joa˜oG.Ribeiro,NeuzaB.Barros,
RobertoNicolete,LauraA.Moura,AndreL.Fuly,AuroNomizo,SauloL.daSilva,CarlaF.C.Fernandes,
JulianaP.Zuliani,RodrigoG.Sta´beli,AndreimarM.Soares,andLeonardoA.Calderon
Volume2014,ArticleID195356,13pages
IsolationandBiochemicalCharacterizationofaNewThrombin-LikeSerineProteasefromBothrops
pirajaiSnakeVenom,KayenaD.Zaqueo,AndersonM.Kayano,RodrigoSimo˜es-Silva,
LeandroS.Moreira-Dill,CarlaF.C.Fernandes,Andre´L.Fuly,Vin´ıciusG.Maltarollo,KathiaM.Hono´rio,
SauloL.daSilva,GerardoAcosta,MariaAntoniaO.Caballol,EliandredeOliveira,FernandoAlbericio,
LeonardoA.Calderon,AndreimarM.Soares,andRodrigoG.Sta´beli
Volume2014,ArticleID595186,13pages
ANovelPhospholipaseA (D49)fromtheVenomoftheCrotalusoreganusabyssus(NorthAmerican
2
GrandCanyonRattlesnake),W.Martins,P.A.Baldasso,K.M.Hono´rio,V.G.Maltarollo,
R.I.M.A.Ribeiro,B.M.A.Carvalho,A.M.Soares,L.A.Calderon,R.G.Sta´beli,M.A.O.Caballol,
G.Acosta,E.Oliveira,S.Marangoni,F.Albericio,andS.L.DaSilva
Volume2014,ArticleID654170,15pages
AntitumoralActivityofSnakeVenomProteins:NewTrendsinCancerTherapy,LeonardoA.Calderon,
JulianaC.Sobrinho,KayenaD.Zaqueo,AndreaA.deMoura,AmyN.Grabner,Maur´ıcioV.Mazzi,
SilvanaMarcussi,AuroNomizo,CarlaF.C.Fernandes,JulianaP.Zuliani,BrunaM.A.Carvalho,
SauloL.daSilva,RodrigoG.Sta´beli,andAndreimarM.Soares
Volume2014,ArticleID203639,19pages
ActivationofJ77A.1MacrophagesbyThreePhospholipasesA IsolatedfromBothropsatroxSnake
2
Venom,JulianaL.Furtado,GeorgeA.Oliveira,AdrianaS.Pontes,SulamitadaS.Setu´bal,CarolineV.Xavier,
FabianneLacouth-Silva,BeatrizF.Lima,KayenaD.Zaqueo,AndersonM.Kayano,LeonardoA.Calderon,
RodrigoG.Sta´beli,AndreimarM.Soares,andJulianaP.Zuliani
Volume2014,ArticleID683123,13pages
InhibitoryEffectofPlantManilkarasubsericeaagainstBiologicalActivitiesofLachesismutaSnake
Venom,EduardoCoriolanoDeOliveira,CaioPinhoFernandes,EladioFloresSanchez,LeandroRocha,
andAndre´LopesFuly
Volume2014,ArticleID408068,7pages
SnakeVenomPLA2sInhibitorsIsolatedfromBrazilianPlants:SyntheticandNaturalMolecules,
B.M.A.Carvalho,J.D.L.Santos,B.M.Xavier,J.R.Almeida,L.M.Resende,W.Martins,
S.Marcussi,S.Marangoni,R.G.Sta´beli,L.A.Calderon,A.M.Soares,S.L.DaSilva,
andD.P.Marchi-Salvador
Volume2013,ArticleID153045,8pages
RapidPurificationandProcoagulantandPlateletAggregatingActivitiesofRhombeobin:A
Thrombin-Like/Gyroxin-LikeEnzymefromLachesismutarhombeataSnakeVenom,
FrankDenisTorres-Huaco,Cla´udioC.Werneck,CristinaPontesVicente,TalitaVassequi-Silva,
AnaCla´udiaCoelhoNery-Diez,CamilaB.Mendes,EdsonAntunes,Se´rgioMarangoni,
andDanielaC.S.Damico
Volume2013,ArticleID903292,12pages
Hindawi Publishing Corporation
BioMed Research International
Volume 2014, Article ID 951561, 2 pages
http://dx.doi.org/10.1155/2014/951561
Editorial
Animal Toxins and Their Advantages in
Biotechnology and Pharmacology
S.L.DaSilva,1E.G.Rowan,2F.Albericio,3,4,5R.G.Stábeli,6
L.A.Calderon,6andA.M.Soares6
1DepartmentofChemistry,BiochemistryandBioprocessEngineering,FederalUniversityofSa˜oJoa˜odel-Rei,
CampusAltoParaopeba,34420-000OuroBranco,MG,Brazil
2StrathclydeInstituteofPharmacyandBiomedicalSciences,UniversityofStrathclyde,GlasgowG40NR,UK
3InstituteforResearchinBiomedicine,BarcelonaSciencePark,UniversityofBarcelona,08028Barcelona,Spain
4CIBER-BBN,BarcelonaSciencePark,UniversityofBarcelona,08028Barcelona,Spain
5DepartmentofOrganicChemistry,UniversityofBarcelona,08028Barcelona,Spain
6CenterofBiomoleculesStudyAppliedtoHealth,FiocruzRondoˆnia,OswaldoCruzFoundation,MedicineDepartment,
FederalUniversityofRondoˆnia,76812-245PortoVelho,RO,Brazil
CorrespondenceshouldbeaddressedtoS.L.DaSilva;[email protected]
Received13March2014;Accepted13March2014;Published22May2014
Copyright©2014S.L.DaSilvaetal.ThisisanopenaccessarticledistributedundertheCreativeCommonsAttributionLicense,
whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycited.
Biodiversityprovidesahugesourceofnewchemicalentities variation, feeding, and individual intrinsic characteristics
thatcouldbeusefulforthedevelopmentofnewtherapies.An thatproduceintraspecificvariations.
example is venomous animals, which produce venoms that The study of toxins and their isoforms has allowed for
sharecommonfeatures,suchascompositionscharacterized a better understanding of the toxic mechanisms of enveno-
byacomplexcombinationofbioactiveproteinsandpeptides mation. The identification/characterization of different iso-
with wide structural diversity. The biological activities of forms of venom toxins, along with the search for natural
thesecompoundsareselectiveandspecificandarecurrently and synthetic inhibitors, such as monoclonal or polyclonal
dependent on the synergic action of several components. antibodies,andmoleculeswithdifferentchemicalproperties,
Thus, animal venoms are important tools for carrying out such as heparin, clotting factors, and plant extracts, has
biochemical,physiological,andpathologicalstudies,aswell increased the possibility of using these agents as more
asforthedevelopmentofnewbiotechnologicalandpharma- effectivetherapeuticalternatives.
ceuticalproducts. More recently, venomics has focused on better under-
Themodernapproachusedtocharacterizevariouscom- standing of the clinical aspects of human envenomation,
poundsfromanimalvenoms,usingadvancedproteomicand the mechanism of action of venoms and their toxins, the
genomic tools, has been denominated “venomics.” Modern prospectionoftoxinswithbiotechnological/pharmaceutical
technologiescurrentlyavailableinseveralresearchlaborato- potential, and the development of novel antivenom serum
ries have allowed scientists to obtain the identification and andalternativetherapiesforenvenomation.
functional-structuralcharacterizationofhundredsoftoxins Amongvenomtoxiccomponents,phospholipasesA2,L-
fromsnakes,scorpions,spiders,anurans,andmarineinver- amino acid oxidases, and proteolytic enzymes, which are
tebrates,presentingahighdiversityofpharmacologicalactiv- classified as metalloproteases and serine proteases, are able
ities.Venomicsdelineatesascientificareaofhighcomplexity to disrupt the human haemostatic system through different
thatisincreasedbythediversityofvenomproteinsandthe mechanisms. Nonenzymatic proteins, such as disintegrins
influence imposed by age, geographic location, ontogenetic and bradykinin-potentiating peptides (BPPs), and other
2 BioMedResearchInternational
components, such as carbohydrates, lipids, ions, biogenic Researchregardinganimalvenomsgenerallyfocuseson
amines, nucleotides, and free amino acids, are also compo- the clinical features of human envenomation, the relation-
nentsofvenom. ship between structure and function of animal toxins, bio-
This special issue presents three revisions and eight prospectionoftoxinswithpotentialforthedevelopmentof
originalpapersaddressingdifferentaspectsofanimalvenom therapies,diagnostickitsforhumandiseases,andalternative
components, their inhibitors, and possible biotechnological or complementary therapies for envenomation. Therefore,
applications. This is relevant considering that snake enven- research involving the biologically active components of
omation is still an important public health issue in many animal venoms could result in the direct or indirect use of
countries,especiallytropicalones. componentsofvenomsduringthedevelopmentofsynthetic
modelsinordertoobtainnewdrugs.
M.B.A.Carvalhoetal.present,herein,amini-reviewon
Thus, understanding the mechanism of action respon-
phospholipase A2 inhibitors from different sources, such as
sible for venoms’ toxic and pharmacologic effects, along
Brazilianplantsandtheirchemicalsynthesis.Theseauthors
with their biochemical and structural characterizations, is
report on the antivenom effect of different plant extracts
essential in order for them to be used as biotechnological
used by traditional communities. These extracts have been
prototypesinarangeofmedicalandscientificareas.
scientifically validated through the phytochemical identifi-
cationandcharacterizationofsomeisolatedinhibitorsfrom
S.L.DaSilva
these species, leading to the development of new therapeu-
E.G.Rowan
tic alternatives for envenomation. L. F. M. Izidoro et al.
F.Albericio
reviewsL-aminoacidoxidasesisolatedfromsnakevenoms,
R.G.Sta´beli
focusing on their biochemical, biological, and enzymatic
L.A.Calderon
characteristics and discussing the importance of hydrogen
A.M.Soares
peroxide production, antigenicity, molecular and structural
characteristics, and biotechnological and pharmacological
applicationsofLAAOsfocusingontheirantibioticpotential.
Finally, in the third revision paper of this special issue, L.
A. Calderon et al. discussed the continuous search for new
bioactivesinordertodevelopnewanticancerdrugs.Inthis
review, the authors describe the potential of proteins and
peptidesfromsnakevenoms,whichshowinvitroandinvivo
cytotoxicityagainstcancercells,forthedevelopmentofmore
efficientandsafetherapies.
Amongtheeightoriginalpapers,threeofthemdescribe
the isolation and biochemical characterization of phospho-
lipasesA2 andbasicandacidicphospholipaseA2 homologs
from the venoms of Bothrops atrox (J. L. Furtado et al.,
2014),Bothropsmattogrossensis(A.A.deMouraetal.,2014),
and Crotalus oreganus abyssus (W. Martins et al., 2014),
which show inflammatory, leishmanicidal, and antitumoral
activities,respectively.
Withregardtothesearchforcomplementaryalternatives
for conventional serum therapies, E. C. De Oliveira et al.
describe the antivenom potential of Manilkara subsericea
extractagainstLachesismutavenom,andC.L.S.Guimara˜es
et al. relate the biotechnological application of polyclonal
antibodies produced using alkylated myotoxic phospholi-
pases A2 from Bothrops jararacussu venom with reduced
myotoxicactivity.
Finally,threepapersreportonproteasesfromanimalven-
oms, such as snakes and spiders and their biotechnological
potential. F. D. Torres-Huaco et al. and K. D. Zaqueo et al.
describethebiochemicalcharacterizationoftwothrombin-
like enzymes (SVTLE) isolated from Lachesis muta and
Bothrops pirajai venoms, respectively. Both enzymes show
blood-clotting activity, indicating a thrombolytic potential.
In contrast, G. Gimenez et al. demonstrate the proteolytic
activityofvenomfromthesocialspiderParawixiabistriata
anditspossiblebiotechnologicalapplicationasaninsecticide
againstAedesaegypti.
Hindawi Publishing Corporation
BioMed Research International
Volume 2014, Article ID 981923, 12 pages
http://dx.doi.org/10.1155/2014/981923
Research Article
Bothrops jararacussu
Alkylation of Histidine Residues of
Venom Proteins and Isolated Phospholipases A :
2
A Biotechnological Tool to Improve the Production
of Antibodies
C.L.S.Guimarães,1,2,3S.H.Andrião-Escarso,4L.S.Moreira-Dill,1,2
B.M.A.Carvalho,5D.P.Marchi-Salvador,6N.A.Santos-Filho,7C.A.H.Fernandes,8
M.R.M.Fontes,8J.R.Giglio,4B.Barraviera,9J.P.Zuliani,1,2C.F.C.Fernandes,1,2
L.A.Calderón,1,2R.G.Stábeli,1,2F.Albericio,10,11,12S.L.daSilva,5andA.M.Soares1,2
1CenterofAppliedBiomoleculesStudiesinHealth(CEBio),OswaldoCruzFoundation(FiocruzRondoˆnia),
PortoVelho,RO,Brazil
2DepartmentofMedicine,FederalUniversityofRondoˆnia(UNIR),PortoVelho,RO,Brazil
3BrazilianInstituteofEnvironmentandRenewableNaturalResources(IBAMA),PortoVelho,RO,Brazil
4DepartmentofBiochemistryandImmunology,FacultyofMedicineofRibeira˜oPreto,UniversityofSa˜oPaulo(USP),
Ribeira˜oPreto,SP,Brazil
5DepartmentofChemistry,BiotechnologyandBioprocessEngineering,FederalUniversityofSa˜oJoa˜oDelRei(UFSJ),
OuroBranco,MG,Brazil
6DepartmentofMolecularBiology,CenterforScienceandNature,FederalUniversityofPara´ıba(UFPB),Joa˜oPessoa,Brazil
7DepartmentofClinicalAnalysis,FacultyofPharmaceuticalSciencesofRibeira˜oPreto,UniversityofSa˜oPaulo(USP),
Ribeira˜oPreto,SP,Brazil
8DepartmentofPhysicsandBiophysics,StateUniversityPaulista(UNESP),Botucatu,SP,Brazil
9CenterfortheStudyofVenomsandVenomousAnimals(CEVAP),StateUniversityPaulista(UNESP),Botucatu,SP,Brazil
10InstituteforResearchinBiomedicine(IRBBarcelona)andCIBER-BBN,BarcelonaSciencePark,Barcelona,Spain
11DepartmentofOrganicChemistry,UniversityofBarcelona,Barcelona,Spain
12SchoolofChemistryandPhysics,UniversityofKwaZuluNatal,Durban4001,SouthAfrica
CorrespondenceshouldbeaddressedtoS.L.daSilva;[email protected];[email protected]
Received14September2013;Accepted2January2014;Published11May2014
AcademicEditor:EdwardG.Rowan
Copyright©2014C.L.S.Guimara˜esetal. This is an open access article distributed under the Creative Commons Attribution
License,whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperly
cited.
CrudevenomofBothropsjararacussuandisolatedphospholipasesA2(PLA2)ofthistoxin(BthTX-IandBthTX-II)werechemically
modified (alkylation) by p-bromophenacyl bromide (BPB) in order to study antibody production capacity in function of the
structure-functionrelationshipofthesesubstances(crudevenomandPLA2 nativeandalkylated).BthTX-IIshowedenzymatic
activity, while BthTX-I did not. Alkylation reduced BthTX-II activity by 50% while this process abolished the catalytic and
myotoxicactivitiesofBthTX-I,whilereducingitsedema-inducingactivitybyabout50%.Antibodyproductionagainstthenative
and alkylated forms of BthTX-Iand -II and the cross-reactivity of antibodies to native and alkylated toxins did not show any
apparentdifferencesandtheseobservationswerereinforcedbysurfaceplasmonresonance(SPR)data.Histopathologicalanalysis
ofmousegastrocnemiusmusclesectionsafterinjectionofPBS,BthTX-I,BthTX-II,orbothmyotoxinspreviouslyincubatedwith
neutralizingantibodyshowedinhibitionofthetoxin-inducedmyotoxicity.Theseresultsrevealthatthechemicalmodificationofthe
phospholipasesA2(PLA2)diminishedtheirtoxicitybutdidnotaltertheirantigenicity.Thisobservationindicatesthatthemodified
PLA2mayprovideabiotechnologicaltooltoattenuatethetoxicityofthecrudevenom,byimprovingtheproductionofantibodies
anddecreasingthelocaltoxiceffectsofthispoisonoussubstanceinanimalsusedtoproduceantivenom.
2 BioMedResearchInternational
1.Introduction activitiesoftheircorrespondingantigens.Ourresultsidentify
themodifiedtoxinsaspotentialtooltoproduceantivenoms
Snakebitesareapublichealthproblemintropicalcountries andsimultaneouslydiminishthelocaleffectsofinjectionson
as they can lead to death or serious sequelae. For example, animalsusedtoproduceantivenom,withoutimpairingtheir
inBrazil,theViperidaefamilyhasfourgeneraofvenomous immuneresponse.
snake,theBothropsgenusbeingresponsiblefor87.5%ofinci-
dentsreported,followedbyCrotalus(9.2%),Lachesis(2.7%), 2.MaterialandMethods
andsnakesthatbelongtotheElapidaefamily(0.6%)[1].
2.1.VenomsandChemicals. TheB.jararacussusnakevenom
Bothrops venom is rich in phospholipases A2 (PLA2), a pool, collected in Sa˜o Paulo state (𝑛 = 6), was provided
class of enzymes with catalytic activity on cell membranes
by L. H. A. Pedrosa (FMRP-USP). The proteins BthTX-I
ofspecifictissues.ThisobservationwouldsuggestthatPLA2
plays a crucial role in venom toxicity [2, 3]. Furthermore, (K49-PLA2-like)andBthTX-II(D49-PLA2)wereisolatedas
previously described [27, 28]. The venom (BjussuCV) was
snake venom PLA2 induces several other effects such as
dried over NaOH pellets in a vacuum desiccator at room
pre-orpostsynapticneurotoxicity[3–5],cardiotoxicity[6–9],
∘
temperatureimmediatelyaftercollectionandstoredat4 C.
myotoxicity[10,11],plateletaggregationinhibitionorinduc-
Chemicals such as acrylamide, bisacrylamide, TEMED,
tion [12, 13], edema [14–17] and hypotension [18–20]. Thus,
CoomassieBrilliantBlueG-250,p-bromophenacylbromide,
medicalandscientificinterestinPLA2sliesintheactionof
sinapinicacid(Sigma),bromophenolBlue(Merck),mercap-
these enzymes in various pathophysiological processes. In
toethanol(FlukaAG),CM-Sepharose(Pharmacia),pH5.0–
thisregard,severalresearchlinesaredevotedtosearchingfor
8.5buffalyte(Pierce),andFreundadjuvantwereofanalytical
drugsortoolstoinhibitorneutralizetheactionofPLA2.
grade.
Thedevelopmentofnaturalorsyntheticinhibitorswith
the capacity to neutralize the toxic effects of PLA2s has
2.2. Animals. Male Swiss mice (18–22g) and rabbits (2.0–
advanced our medical knowledge of the mechanisms of
2.5kg)wereobtainedfromtheCentralBioteriumofS.Paulo
actionandstructure-functionrelationshipsoftheseenzymes.
University,Ribeira˜oPreto,SP.Animalcarewasinaccordance
In this respect, our research group focuses on the develop-
withtheguidelinesoftheBrazilianCollegeforAnimalExper-
mentofbiotechnologicaltoolsthatinhibitthePLA2sandits
imentation(COBEA),andthepresentproposalwasapproved
toxiceffects[21,22].
by the Animal Ethics Committee of S. Paulo University
Snake venom PLA2s belong to classes I or II of this
(CEUA-USP) and The Brazilian Institute of Environment
enzyme family. In this regard, the PLA2 from venom of
(IBAMA).
Bothrops jararacussu belongs to the latter [2–4, 22]. The
enzymatic activity of the PLA2s is characterized by the 2.3. Alkylation of BjussuCV, BthTX-I, and BthTX-II. The
integrityoftheaminoacidresidueHis48intheactivesite.
BjussuCV, BthTX-I, and BthTX-II samples (12mg) were
TheliteraturedescribesthatPLA2losesitscatalyticactiv- dissolvedin1mLof100mMammoniumbicarbonatebuffer,
ity when His48 undergoes alkylation by p-bromophenacyl pH 7.8, containing 0.7mM EDTA. Then, 500𝜇L of BPB
bromide (BPB). In addition to this effect on the activity of
(1.5mg/mLethanol)wasadded,andthesampleswereincu-
PLA2s, the alkylation also leads to a reduction in the toxic bated at 25∘C for 24h. Excess reagent was removed by
andpharmacologicalactivityofthisenzyme[23].
ultrafiltrationthroughanAmiconYM3000membrane,and
The clinical treatment for Bothrops snake bites basically
the remaining protein solution was lyophilized [27, 28].
consists of the early administration of appropriate doses of
NativeproteinsarenamedBjussuCV,BthTX-I,BthTX-II,the
anti-Bothropic serum [24]. The production of antivenom,
proteins alkylated by BPB alk-BjussuCV, alk-BthTX-I, and
usually conducted with horses, involves the subcutaneous
alk-BthTX-II.
injectionofincreasingamountsofthecorrespondingvenoms
[25, 26]. Although these injections do not cause relevant
2.4. Immunization. 1.6mg of native or alkylated BjussuCV
systemic alterations in the animals, they induce significant
samples, as well as 0.5mg of native or alkylated BthTX-I
tissue damage at the site of injection (edema, hemorrhage,
and BthTX-II, was dissolved in 1mL of phosphate-buffered
and necrosis). Reduction of local tissue reaction at the site
saline (PBS), to which 1mL of Freund’s complete adjuvant
of inoculation, without impairing the immune response of
wasadded.Rabbitsweretheninjectedwith0.4mLofthese
the animals against venom components, is a major goal of
mixturesatfoursitesonthebackandthighs(s.c.).After12
laboratoriesproducingantivenoms.
days,asecondimmunizationatthesamesiteswasperformed
Here, wereportonthedevelopmentofabiotechnology usingFreund’sincompleteadjuvant.Finally,after45days,a
toolbasedonnativeBjussuCV(B.jararacussucrudevenom), firstbleedwascarriedoutbyintracardiacpuncture.Normal
BthTX-I (K49-PLA2-like) and BthTX-II (D49-PLA2), and andimmuneserawereprecipitatedwithasaturatedsolution
also all proteins alkylated by BPB. The enzymatic and bio- of ammonium sulphate, pH 6.5, left to stand for 30min at
logicalactivitiesofBjussuCV,BthTX-I,andBthTX-IIintheir 4∘C,andcentrifugedat3800×g.Theprecipitateobtainedwas
native and alkylated forms were evaluated and compared. washed with 40% ammonium sulfate, centrifuged, dialyzed
Rabbit polyclonal antibodies against native or chemically against20mMsodiumphosphatebuffer,pH7.5,andconcen-
modified BjussuCV, BthTX-I, and -II were produced, and trated through an Amicon YM10,000 membrane. Antibody
assays were performed to assess their capacity to neutralize purification was achieved by ion exchange chromatography
the phospholipasic, myotoxic, edema-inducing, and lethal on a 2 × 20cm DEAE-Sephacel column, using 0.02M