Purification of an Angiotensin-Converting Enzyme-Lik

16 Pages·2016·1.86 MB·English

toxins
Article
Insights into the Hypertensive Effects of
Tityus serrulatus Scorpion Venom: Purification of
an Angiotensin-Converting Enzyme-Like Peptidase
DanielaCajado-Carvalho1,AlexandreKazuoKuniyoshi1,BrunoDuzzi1,LeoKeiIwai2,
ÚrsulaCastrodeOliveira2,InáciodeLoiolaMeirellesJunqueiradeAzevedo2,
RobertoTadashiKodama1andFernandaVieiraPortaro1,*
1 ImmunochemistryLaboratory,ButantanInstitute,SãoPauloCEP05503-900,SP,Brazil;
[email protected](D.C.-C.);[email protected](A.K.K.);
[email protected](B.D.);[email protected](R.T.K.)
2 SpecialLaboratoryforAppliedToxinology,ButantanInstitute/CenterofToxins,Immune-ResponseandCell
Signaling(CeTICS),SãoPauloCEP05503-900,SP,Brazil;[email protected](L.K.I.);
[email protected](Ú.C.d.O.);[email protected](I.d.L.M.J.d.A.)
* Correspondence:[email protected];Tel.:[phone]
AcademicEditor:RonaldA.Jenner
Received:7October2016;Accepted:16November2016;Published:24November2016
Abstract: The number of cases of envenomation by scorpions has grown significantly in Brazil
since 2007, with the most severe cases being caused by the Tityus serrulatus scorpion. Although
envenomedpatientsmostlysufferneurotoxicmanifestations,othersymptoms,suchashypertension,
cannotbeexclusivelyattributedtoneurotoxins. Omicsanalyseshavedetectedplentifulamountsof
metalloproteasesinT.serrulatusvenom.However,therolesplayedbytheseenzymesinenvenomation
arestillunclear. Endeavoringtoinvestigatethefunctionsofscorpionvenomproteases,wedescribe
here for the first time an Angiotensin I-Converting Enzyme-like peptidase (ACE-like) purified
fromT.serrulatusvenom. Thecrudevenomcleavednaturalandfluorescentsubstratesandthese
activitieswereinhibitedbycaptopril. Regardingtheserumneutralization,thescorpionantivenom
was more effective at blocking the ACE-like activity than arachnid antivenom, although neither
completelyinhibitedthevenomcleavageaction,evenathigherdoses.ACE-likewaspurifiedfromthe
venomafterthreechromatographicstepsanditsidentitywasconfirmedbymassspectrometricand
transcriptomicanalyses. BioinformaticsanalysisshowedhomologybetweentheACE-liketranscript
sequencesfromTityusspp. andhumantestisACE.Thesefindingsadvanceourunderstandingof
T.serrulatusvenomcomponentsandmayimprovetreatmentofenvenomationvictims,asACE-like
maycontributetoenvenomationsymptoms,especiallytheresultinghypertension.
Keywords: ACE-like;Tityusserrulatusvenom;proteases;antivenom;hypertension
1. Introduction
AccordingtoBrazil’sMinistryofHealth, since2007scorpionstingshavebeenthemainform
ofenvenomationbyanimalsinthiscountry. AnepidemiologicalsurveyconductedbytheMinistry
of Health shows that, between 2010 and 2013, cases of scorpion envenomation represent 49% of
poisoningsbyvenomousanimalsinBrazil, surpassingthosebysnakes(17%)andspiders(18.5%).
This scenario is mainly attributed to the proliferation of Tityus serrulatus scorpions, synanthropic
animalsthatreproducebyparthenogenesis[1]andwhosepotentvenomcontributestotheoccurrence
ofcriticalclinicalenvenomation. Thus,T.serrulatusvenom(Tsv)isoneofthemoststudiedBrazilian
scorpionvenoms[2].
Toxins2016,8,348;doi:10.3390/toxins8120348 www.mdpi.com/journal/toxins
Toxins2016,8,348 2of16
NeurotoxinsinTsvcausemostoftheenvenomationsymptomsbypromotingneurotransmitter
release from the autonomic nervous system and the adrenal medulla onto several organs [3].
Theseeventsresultinclinicalmanifestationssuchasrestlessness,excessivesalivation,lacrimation,
hypertensionfollowedbyhypotension,heartfailure,andcardiogenicshock,amongothers. Notall
symptoms,however,areattributedtoadirectactionofspecificneurotoxinsonorgans[4],andrecent
studies suggest that other molecules may contribute to these effects [5–7]. Metallopeptidases,
hyaluronidase, biogenic amines, antimicrobial peptides (AMP), and other oligopeptides are also
presentinTsv[7–9];however,theroleofthesemoleculesinenvenomationisstillunclear.
Peptidases present in animal venoms often play an important role in the envenomation
syndrome, astheycancleaveproteinsandpeptidesthatarekeyfactorsforphysiologicalsystems.
Forinstance,insnakevenoms,serinepeptidasescanaffectthecoagulationcascadeandkallikrein–kinin
system,whilemetallopeptidasestargetcomponentsofbloodcoagulationandplateletaggregation,
bothresultinginanimbalanceofthehemostaticsystem[10,11]. Althoughsnakevenompeptidases
are the most studied, due to their abundance, proteolytic activity has recently been described in
jellyfish[12],conesnail[13],wasp[14],spider[15],andscorpion[16]venoms.
Thusfar,metallopeptidasesandhyaluronidasescomprisetheonlyclassesofenzymespurified
andwiththeirfunctionalactivitiesdeterminedinTityusspp. venoms[6,9],althoughtranscriptomic
studieshaveidentifiedotherclassesofenzymesaswell[8,17].Studiesfromourgrouphaveshownthat
Tsvproteasescleavemammalpeptidesinvitro,andthatthisactivityisinhibitedbyhighconcentrations
ofcommerciallyavailableantivenomsusedinthetreatmentofenvenomedpatientsinBrazil[16,18].
Moreover,endopeptidases,aminopeptidases,andcarboxypeptidasesthatcleaveendogenouspeptides
from the Tsv and inactivate human bioactive peptides have been detected [18,19]. One of these,
antarease,isametallopeptidaseubiquitousinthevenomofscorpionsoffamilyButhidae[20],towhose
actionthepancreatitisobservedinmiceaftervenominjectionisattributed[21].
The human somatic angiotensin-I converting enzyme (sACE, EC 3.4.15.1) is a dipeptidyl
carboxypeptidasethatplaysacrucialroleinbloodpressureregulationthroughtherenin–angiotensin
(RAS)andkalikrein–kinin(KKS)systems. sACEconvertsangiotensinIintoangiotensinIIinRAS,
andinactivatesbradykinininKKS,leadingtobloodpressureincrease. sACEhastwohighlysimilar,
independent catalytic domains, the N- and C-terminal domains. Although both have proteolytic
activity,regulationofbloodpressuremostlydependsontheC-terminaldomain,asitisresponsible
for producing angiotensin II [22]. A smaller isoform of sACEis also present in mammals: known
as testicular ACE (tACE), it contains only the C-terminal domain [23,24]. The same gene encodes
bothsomaticandtesticularACE[23,25]buttACEisonlyfoundafterpubertyinmalegerminalcells,
whereitparticipatesinspermmaturation,incontrastwiththeconstantandwideexpressionofsACE
thatactsalloverthebody[26].
ACE-likeenzymesarehighlyconserved,havingalsobeendetectedininsects,wheretheyplay
aroleinfeeding,reproduction,andtheproductionofpeptidehormonesandneurotransmitters[27].
Also, functional ACE-like activity was detected in animal venoms of the fish-hunting cone
snail [13], vampire snail [28], and solitary endoparasitic wasp [14]. In scorpions, ACE-like
peptidasesweredetectedbytranscriptomeanalysisinHottentottajudaicus[29],Tityusstigmurus[30],
andTityusbahiensis[17]venoms.
HerewedescribeandcharacterizeforthefirsttimeanAngiotensinI-ConvertingEnzyme-like
peptidaseactivityinTityusserrulatusvenomandtheevaluationofcommerciallyavailableantivenoms
toneutralizeit. WeusedproteolyticactivityassaystodetectanACE-likepeptidase,thenpurifiedand
confirmeditsidentitybytrypticdigestion/massspectrometricandtranscriptomicanalysis.
Thisreportmaycontributetoourunderstandingoftheroleofproteasesfromscorpionvenomsin
theenvenomationprocess,asanACE-likepeptidasemaycontributetothehypertensionobservedin
humanvictims.
Toxins2016,8,348 3of16
Toxins 2016, 8, 348   3 of 16 
22.. RReessuullttss 
22..11.. FFRREETT SSuubbssttrraatteess SSppeecciiffiicc ffoorr CCaarrbbooxxyy‐- aanndd EEnnddooppeeppttiiddaasseess oonn TTssvv 
WWee ffiirrsstt eevvaalluuaatteedd TTssvv aaccttiivviittyy wwiitthh ppeeppttiiddaassee iinnhhiibbiittoorrss,, uussiinngg ttwwoo fflluuoorreesscceenntt ssuubbssttrraatteess: :
AAbbzz‐-FFRRKK((DDnnpp))PP‐-OOHH, ,wwhhiicchh  wwaass  ddeessiiggnneedd  ffoorr  AACCEE  ssttuuddiieess  [[3311]],,  aanndd  AAbbzz‐-GGGGFFLLRRRRVV‐-EEDDDDnnpp, ,
hhoommoollooggoouuss ttoo tthhee ddyynnoorrpphhiinn 11––1133 sseeqquueennccee aanndd pprreevviioouussllyy ddeetteerrmminineedd aass ssuubbssttrraattee ffoorr TTssvv 
eennddooppeeppttiiddaasseess [[1188]].. RReessuullttss aarree pprreesseenntteedd iinn FFiigguurree 11.. 
Abz-FRK(Dnp)P-OH Abz-GGFLRRV-EDDnp
100
80
)
%
( 60
n
o
i
t
bi 40
hi
n
I
20
0
Captopril BPP 10c P M SF antroline EDTA
n
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Figure 1. Fluorimetric assays for carboxy- and endopeptidases activities on Tsv using peptidase
Figure 1. Fluorimetric assays for carboxy‐ and endopeptidases activities on Tsv using peptidase 
inhibitors. Captopril (100 nM), BPP 10c (16 µM), EDTA (50 mM), 1,10-phenantroline (2 mM),
inhibitors. Captopril (100 nM), BPP 10c (16 μM), EDTA (50 mM), 1,10‐phenantroline (2 mM), PMSF (2 
PMSF(2mM)weretestedonthewholeTityusserrulatusvenom(1µg)inafluorometricassaywith
mM) were tested on the whole Tityus serrulatus venom (1 μg) in a fluorometric assay with 
Abz-FRK(Dnp)P-OH(darkgrey)andAbz-GGFLRRV-EDDnp(lightgrey).Thereactionsoccurredin
A10b0z‐mFRMKT(Drisn,p5)0P‐mOMH N(daaCrkl, g10reµyM) aZndn CAlbzb‐uGfGfeFr,LpRHRV7.‐0EaDtD37np◦C (l.iEghxpt egrriemy)e.n Ttshew reeraecdtioonnes ionccduurprelidca itne .
2
1T0h0e mSDM oTfrkisi,n 5e0ti mcrMes uNltasCiln, 1e0a cμhMca ZsenCwla2 sbunfefveer,r pgHre a7t.e0r atth 3a7n °5C%. Eoxfpthereimvaelnutes owbetarein deodn.e in duplicate. 
The SD of kinetic results in each case was never greater than 5% of the value obtained. 
Although only metallopeptidases were detected in Tsv (due to inhibition of EDTA and
Although  only  metallopeptidases  were  detected  in  Tsv  (due  to  inhibition  of  EDTA  and 
1,10-phenantroline),weobservedthatdifferentproteasesactoneachsubstrate.ForAbz-FRK(Dnp)P-OH,
1,10‐phenantroline),  we  observed  that  different  proteases  act  on  each  substrate.  For 
thetwoclassicalACEinhibitors,captopril(100nM)andBPP10c(16µM),significantlyinhibitedTsv
Abz‐FRK(Dnp)P‐OH, the two classical ACE inhibitors, captopril (100 nM) and BPP 10c (16 μM), 
activity(98%and90%,respectively),whileAbz-GGFLRRV-EDDnphydrolysiswasnotaffectedby
significantly inhibited  Tsv  activity  (98%  and  90%,  respectively),  while  Abz‐GGFLRRV‐EDDnp 
theseinhibitorsatthesameconcentrations. However,PMSF(2mM)hadnoeffectontheactivityof
hydrolysis was not affected by these inhibitors at the same concentrations. However, PMSF (2 mM) 
thevenomonanyfluorescentsubstratestested. Thus,itseemsthatAbz-GGFLRRV-EDDnpiscleaved
had no effect on the activity of the venom on any fluorescent substrates tested. Thus, it seems that 
mainlybymetalloendopeptidasesandAbz-FRK(Dnp)P-OHbymetallodipeptidylcarboxypeptidases.
Abz‐GGFLRRV‐EDDnp is cleaved mainly by metalloendopeptidases and Abz‐FRK(Dnp)P‐OH by 
metallo dipeptidyl carboxypeptidases. 
2.2. InVitroSerumNeutralizationAssaysUsingAbz-FRK(Dnp)P-OHasSubstrate
2.2. InW Veittreos Steedrunmin Needuitfrfaelrizeanttiocno Ancsesanytsr aUtisoinngs Aofbza‐rFaRchKn(Didn(pA)PA‐OV)Ha ansd Ssucbosrtrpaitoen  (SAV)antivenomsto
determinetheirefficacyinneutralizingTsvhydrolysisofAbz-FRK(Dnp)P-OH(Figure2).
We tested nine different concentrations of arachnid (AAV) and scorpion (SAV) antivenoms to 
SAVpartiallyinhibitedtheactivityevenatlowvenom:antivenomratios,whereasAAVstarted
determine their efficacy in neutralizing Tsv hydrolysis of Abz‐FRK(Dnp)P‐OH (Figure 2). 
inhibiting Tsv at a higher dose, after 25 µg of antivenom (1:25), but also reached higher levels of
inhibitionthanSAVatconcentrationsof1:500µg. NeitherantivenomfullyneutralizedTsvactivityat
thehighestdosetested(1:1000),withinhibitionreaching94%forSAVand98%forAAV.
Toxins2016,8,348 4of16
Toxins 2016, 8, 348   4 of 16 
Toxins 2016, 8, 348   4 of 16 
SAV AAV
SAV AAV
100
100
80
80
)
%)
%
on (n ( 6600
io
nhibithibiti 4400
In
I
20
20
0
0
1 2 0 5 0 0 0 0 0
1:1:1 1:1:2 1:1:110 1:1:225 1:1:550 1:1:11000 1:1:22550 1:1:55000 1:1:1100000
g venom:g antivenom
g venom:g antivenom

FFFiigigguuurrreee  222.. . IInInn  vvviititrtrrooo  ssseeerrruuummm  nnneeeuuuttrtrraaallilizizzaaattitioioonnn  oooff f AAAbbbzzz‐-F‐FFRRRKKK((D(DDnnnppp))P)PP‐-O‐OOHHH  hhhyyydddrrrooollylyysssiisiss  bbbyyy  TTTsssvvv  uuusssiininnggg  ssscccooorrrpppiioioonnn  
((SSAAVV)) aanndd aarraacchhnniidd ((AAAAVV)) ccoommmmeerrcciiaall aannttiivveennoommss.. TThhee vveennoomm wwaass iinnccuubbaatteedd ffoorr 3300 mmiinn aatt rroooomm 
(SAV) and arachnid (AAV) commercial antivenoms. The venom was incubated for 30 min at room 
tteemmppeerraattuurree wwiitthh nninineec oconncecnentrtartaitoinonsso foafn atinvteivneonmom(w (ewigehitgrhatt iroatoifo voefn ovmen:aonmti:vanentiovmen)—om1):1—;11::21;; 11:1:20;; 
temperature with nine concentrations of antivenom (weight ratio of venom:antivenom)—1:1; 1:2; 
11::1205;; 11::5205;; 11::15000; ;11:1:20500; ;11::255000;, 1a:n5d001,: 1a0n0d0 —1:1a0n0d0—theanndth tehflenu otrhees cfelunotrseusbcsetnrta tseuwbsatsraatde dwedas. Tahdedereds.u Tlthies 
1:10; 1:25; 1:50; 1:100; 1:250; 1:500, and 1:1000—and then the fluorescent substrate was added. The 
reexspurlet sisse edxapsre%ssiendh aibs it%io innhofibpiteipotni doafs peeapcttiidvaitsye iancttihveitvye inno tmhe( %ve)n.oTmhe (r%es).u Tlthree prerseuseltn rtsepthreesmenetas nthoef 
result is expressed as % inhibition of peptidase activity in the venom (%). The result represents the 
mtweoanin odfe tpweon dinednetpexenpdereinmt eenxtps.erTihmeeSnDts.o Tfhkein SeDti corfe ksiunletstiicn reeascuhltsc ainse ewacahs cnaesvee wrgarse nateevretrh garnea5%tero tfhtahne 
mean of two independent experiments. The SD of kinetic results in each case was never greater than 
5v%al uoef tohbet avianleude. obtained. 
5% of the value obtained. 
2.3. ESSfAfAeVcVt po pfaaCrrthtiailaolrllyliyd ie ninIhohnibibCitioteenddc te thnheter aa atcictotinviviotityny eT evsvveenan na adtt sl olAowCw Ev veennoomm:a:annttiviveennoomm r raattioioss, ,w whheerreeaass A AAAVV s sttaarrtteedd  
inhibiting Tsv at a higher dose, after 25 μg of antivenom (1:25), but also reached higher levels of 
inhibiting Tsv at a higher dose, after 25 μg of antivenom (1:25), but also reached higher levels of 
AschlorideionsareknowntoaffectACEactivity[32],wecomparedTsvandACEhydrolysisof
inhibition than SAV at concentrations of 1:500 μg. Neither antivenom fully neutralized Tsv activity 
tihnehAibbitzio-FnR tKh(aDn nSpA)PV- OatH cosuncbesntrtartaetiionntsh oefp 1r:e5s0e0n μcego. fNdeiiftfheerer natnCtilv−encoonmc efnutlrlyat nioenustr(aFliigzuerde T3s)v. activity 
at the highest dose tested (1:1000), with inhibition reaching 94% for SAV and 98% for AAV. 
at the highest dose tested (1:1000), with inhibition reaching 94% for SAV and 98% for AAV. 
AsshowedinFigure3,bothenzymeswerealreadyactiveintheabsenceofNaCl,withhigher
proteolytic activities observed as NaCl concentrations increased. At 10 mM NaCl, Tsv and ACE
2.3. Effect of Chloride Ion Concentration on Tsv and sACE 
2.3. Effect of Chloride Ion Concentration on Tsv and sACE 
activities increased by 15% and 25%, respectively, and, at the highest NaCl concentration tested
(50 mAAMss c) chhhlyolodrrirdiodele yi oisonisnss oa afrreAe k bknzno-oFwwRnnK t (toDo a nafffpfee)cPctt -A OACHCEEb a aycctTtivisvvitityiyn [ [3c3r22e]],a ,ws weede c cbooymmmppaoarrreeeddt Th Tsasvnv a 3an5nd%d A ,ACaCnEEd h hayyrdodruroonlyldyssi4sis6 o %off  
tbthhyee AA ACbbEzz‐.‐FFRRKK((DDnnpp))PP‐‐OOHH s suubbssttrraattee i nin t thhee p prreesseennccee o off d dififffeerreenntt C Cl−l −c coonncceennttrraattioionnss ( (FFigiguurree 3 3)). . 

Figure 3. Effect of chloride ions concentration on (A) angiotensin-converting enzyme (ACE) and
Figure 3. Effect of chloride ions concentration on (A) angiotensin‐converting enzyme (ACE) and (B) 
Figure 3. Effect of chloride ions concentration on (A) angiotensin‐converting enzyme (ACE) and (B) 
(B)Tityusserrulatusvenom.Forboth,Abz-FRK(Dnp)P-OHhydrolysiswasdeterminedinTris100mM,
Tityus serrulatus venom. For both, Abz‐FRK(Dnp)P‐OH hydrolysis was determined in Tris 100 mM , 
ZTnitCyuls 1se0rrµuMlatubsu vffeenr,omw.i tFhofro buorthd,i fAfebrze‐nFtRcKo(nDcnepnt)rPa‐tOioHn shyodfrNolayCsils: w0,a1s 0demteMrm, 2in0edm iMn ,Tarnisd 10500 mmMM ., 
2
ZTZnhnCeClr2le 2s1 1u00l tμ μrMeMp b rbeuusfeffefnertr,s ,w twhietihtmh f eofoauunrr od dfifitffweferoreeninnt tdc ecoopnnecnceednnetrtnratatiteoixonpnses ro iofm fN eNnaaCtCsl.:l T:0 0h, ,e1 10S0 Dm mMoMf,k ,2 i2n00 em tmicMMr,e ,sa aunnldtds 5 i50n0 m emaMcMh. .cT Tahsheee  
result represents the mean of two independent experiments. The SD of kinetic results in each case 
wreassunlte vreeprrgerseeanttesr tthhea nm5e%ano fotfh tewvoa liunedeopbetanidneendt. experiments. The SD of kinetic results in each case 
was never greater than 5% of the value obtained. 
was never greater than 5% of the value obtained.
Toxins2016,8,348 5of16
2.4. HydrolysisofACENaturalSubstratesbyTsv
Angiotensin I and bradykinin were incubated with Tsv in the classical ACE working buffer,
100mMTris,50mMNaCl,10µMZnCl ,pH7.0. Afterverifying,withreservephasechromatography,
2
that both peptides were substrates for Tsv, we manually collected the products of hydrolysis and
analyzedthembymassspectrometry. Additionally,wedeterminedthespecificactivityofthevenom
oneachpeptide,includinghemopressin,anddeterminedthecleavagesites(Table1).
Table1.HydrolysisofbiologicallyactivepeptidesbyTityusserrulatusvenomandreleasedfragments.
Peptide FragmentSequence FragmentIdentification MW VenomSpecificActivity(µM/µg/min)
DRVY Ang(1–4) 551.2
IHPFHL Ang(5–10) 762.4
HPFHL Ang(6–10) 649.3
AngiotensinI DRVYIHP Ang(1–7) 898.4 0.050
DRVYIHPF AngII 1045.4
RVYIHPF AngIII 930.5
FHL Ang(8–10) 415.2
RPPGF BK(1–5) 572.3
Bradykinin RPPGFSP BK(1–7) 756.3 0.045
PGFSPFR BK(3–9) 806.4
PVNFKFL Hemo(1–7) 863.4
PVNFKF Hemo(1–6) 750.4
Hemopressin PVNFK Hemo(1–5) 603.3 0.400
KFLSH Hemo(5–9) 630.35
FLSH Hemo(6–9) 502.25
Assayswerecarriedoutin100mMTrisbuffercontaining50mMNaCl,10µMZnCl2,pH7.0,at37◦C,inafinal
volumeof100µL.Theresultsareshownasthemeanofthreeindependentexperiments.TheSDofkineticwas
nevergreaterthan5%ofthevalueobtained.
Hemopressin was the best substrate for the whole venom (0.40 µM/µg/min), followed by
angiotensinI(0.05µM/µg/min)andbradykinin(0.045µM/µg/min). Oneofthefragmentscollected
fromangiotensinIcorrespondedtoangiotensinII,duetotheremovalofHis9-Leu10. Otherfragments
werealsoformedfromangiotensinIhydrolysis,suchasAng ,Ang ,Ang ,andAng ,
(1–4) (5–10) (1–7) (8–10)
probablyduetoendopeptidaseactivity;andAngIIIandAng ,probablyduetoaminopeptidase
(6–10)
activity. Bradykinin hydrolysis by the venom formed the fragments BK , BK , the expected
(1–7) (1–5)
productsofACE-likeactivity,andBK .Hemopressincleavagesitesweredeterminedpreviously[18],
(3–7)
butthehydrolysisrateincreased5.6timeswhenthebuffercontaining10µMZnCl wasused.
2
2.5. InhibitionAssayonReversePhaseChromatography
As new substrates and hydrolysis rates were observed for Tsv when using the ACE buffer,
weevaluatedinhibitionbycaptopril,apotentandspecifichumanACEinhibitor,andbyEDTA(Table2).
Table2.InhibitionofTsvactivityonbioactivepeptidesbycaptoprilandEDTA.
InhibitoryActivity(%)
Peptide
EDTA Captopril100nM Captopril1µM
Angiotensin-I 100 8.2 48.6
Bradykinin 100 36.3 60.0
DynorphinA 100 0.0 1.8
Hemopressin 100 37.8 8.9
Experimentsweremadeusing100mMTris,50mMNaCl,10µMZnCl2buffer,pH7.0at37◦CwithTsv(1µg),
at37◦C,using30µMofeachbiologicallyactivepeptidesubstrate.Theincubationsvariedaccordingtothe
particularityofeachsubstrate,asdescribedinSection4.6.Theresultsshownarethemeanofthreeindependent
experiments.TheSDofkineticwasnevergreaterthan5%ofthevalueobtained.
Toxins 2016, 8, 348   6 of 16 
Table 2. Inhibition of Tsv activity on bioactive peptides by captopril and EDTA. 
Inhibitory Activity (%)
Peptide 
EDTA Captopril 100 nM  Captopril 1 μM 
Angiotensin‐I  100  8.2  48.6 
Bradykinin  100  36.3   60.0 
Dynorphin A  100  0.0  1.8 
Hemopressin  100  37.8  8.9 
Experiments were made using 100 mM Tris, 50 mM NaCl, 10 μM ZnCl2 buffer, pH 7.0 at 37 °C with 
Tsv (1 μg), at 37 °C, using 30 μM of each biologically active peptide substrate. The incubations varied 
according to the particularity of each substrate, as described in Section 4.6. The results shown are the 
Toxins2016,8,348 6of16
mean of three independent experiments. The SD of kinetic was never greater than 5% of the value 
obtained. 
EDTAfullyinhibitedtheactivityofTsvonallsubstrates.Captopril,ineitherofthetwoconcentrations
EDTA fully inhibited the activity of Tsv on all substrates. Captopril, in either of the two 
tested, failed to inhibit the cleavage of dynorphin 1–13 in any concentration, which was expected,
concentrations tested, failed to inhibit the cleavage of dynorphin 1–13 in any concentration, which 
asasimilarresultwasobservedwiththeanalogfluorescentsubstrate,Abz-GGFLRRV-EDDnp(Figure1).
was  expected,  as  a  similar  result  was  observed  with  the  analog  fluorescent  substrate, 
Ontheotherhand,captoprilinhibitedbradykininandangiotensinIhydrolysesinadose-dependent
Abz‐GGFLRRV‐EDDnp  (Figure  1).  On  the  other  hand,  captopril  inhibited  bradykinin  and 
manner(Table2). Hydrolysisofhemopressinwasalsostronglyinhibited(37.8%)by100nMcaptopril,
angiotensin I hydrolyses in a dose‐dependent manner (Table 2). Hydrolysis of hemopressin was also 
butlesssowithahigherdose(1µM)ofthisinhibitor. TheRP-HPLCprofileofhemopressinhydrolysis
strongly inhibited (37.8%) by 100 nM captopril, but less so with a higher dose (1 μM) of this 
by Tsv (Figure 4), shows that, when captopril was present (in both doses), one of the products
inhibitor. The RP‐HPLC profile of hemopressin hydrolysis by Tsv (Figure 4), shows that, when 
(PVNFKFL)wasnotformed,indicatingthatthedipeptidylcarboxypeptidaseactivityofthewhole
captopril was present (in both doses), one of the products (PVNFKFL) was not formed, indicating 
venom was abolished. Nevertheless, the other products (PVNFK and FLSH) were not affected by
that the dipeptidyl carboxypeptidase activity of the whole venom was abolished. Nevertheless, the 
thisinhibitor.
other products (PVNFK and FLSH) were not affected by this inhibitor. 

FFiigguurree 44.. HHememoporpersessisni nhyhdyrdolryoslyiss bisy bTyityTuisty suersruselartruusl avteunsovme noonm RPo‐nHPRLPC-H. (PAL)C H.e(mAo)pHreesmsionp, 3re0s μsiMn,, 
30 µM, without venom; (B) hemopressin after 2 h incubation with Tsv (1 µg), and its fragments;
without venom; (B) hemopressin after 2 h incubation with Tsv (1 μg), and its fragments; (C) and (D) 
(C,D) hemopressin hydrolyzed by Tsv in presence of 100 nM and 1 µM of captopril, respectively,
hemopressin hydrolyzed by Tsv in presence of 100 nM and 1 μM of captopril, respectively, showing 
sthhoawt tihneg pthroadttuhcet p(PrVodNuFcKt(FPLV) NisF nKoFt Lfo)rimsneodt. fEoxrpmeerdim.Eenxptse wrimereen dtsonwee irne dduonpelicinatde.u plicate.
22.6.6.. PPuurriifificcaattiioonn ooffA ACCEE-‐LLiikkeeP Peeppttididaasseef rfroommT T..s seerrrurulalatutussV Venenomom 
IInn oorrddeerrt toop puurriiffyy tthhee AACCEE-‐lliikkee ppeeppttiiddaassee pprreesseenntt iinn TTssvv,,w weep peerrfoforrmmeeddt hthrreeeec chhrroommaattooggrraapphhiicc 
sstteeppsst otoo botabitnaianh oam  hoogmenoegoeunseaonuds aacntidv eaecntzivyem ee.nFziyrmst,et.h eFiwrsht,o ltehTes vwwhaoslea pTpslive dwtoasa naHppPlLieCd- DtEo AaEn 
cHolPuLmCn‐,DaEnAdEth ceoAluCmEn-l,i kaendac tthivei tAyCwEa‐sliikdee nactitfiiveidty( 7w7aUs Fi/dµengt/ifmieidn )(i7n7 fUraFc/tμiogn/m1,inw)h iinc hfrdaicdtionnot 1i,n wtehraiccht 
wdiitdh nthoet cinoltuermacnt (wdaittha nthoet schooluwm).nT h(deant,af rnaoctti osnho1ww).a sThloeand, efdraocntitoona 1D wioal-s3 0lo0acdoeldu monnt,ofr oam Dwiohl‐i3c0h0 
fcroaclutimonns, wferroemc ollwechtiecdh (Ffirgaucrteio5n,sp anweelrAe )acnodllescctreede ne(dFibgyurAe bz5-F, RKpa(Dnenlp  )PA-)O  Hanhdy drsoclryeseins.edSi ncbey 
fArabczti‐oFnRFK1(-D2nsph)oPw‐OedHt hehybdesrtohlyysdisr.o lySsiniscrea tefrsa(c8t8ioUnF /Fµ1g‐2/ msihno),witedw asthfeu rtbheesrt frahcytdiornoelydswis ithraPteAs- C(M88 
iUonF/eμxgch/maning)e, ciht rwomasa tfougrrtahperh yf.raAcfttieorntehdis wlaistths tPeAp,‐CwMeo ibosne revxecdhoannegea ccthivreomfraactotigorna,pphryes. eAntfitnegr tahsiisn glalset 
bsatenpd, owfe7 0obksDerav(eFdig ounree a5c,tpivaen eflraBc)t,iowni,t hpraesspenectiinfigc aac stiinvgitlye obfa2n2d0 oUf F7/0µ kgD/am (iFnigounreth 5e, FpRanEeTl sBu)b, wstritahte a. 
Isnptehceiffiicn aalcctihvriotym aotfo g22ra0p UhyF/sμtegp/,mthine pounr itfhicea tFioRnEfTa cstuorbsotbrtaatien. eIdn wthase 2f2in.9awl cithhroamyiaetlodgorfap1%hy( Tsatbeple, St1h)e. 
puriTfihcaetiporno ftaecintorc oonbtteanintefdro wmast h22e.9S DwSit-hP Aa GyiEeldb aonf d1%w (aTsaebxletr Sa1c)t.e d and subjected to MS/MS for
peptidefingerprintanalysis. UsingadatabasethatcombinedsequencesrestrictedtoTityusgenus
from UNIPROT and the transcript sequences of ACE-like peptidase from Tityus scorpion venom

glands, PeaksDB was able to identify, with high confidence (FDR ≤ 1%), two unique ACE-like
peptidesfromTityusserrulatus(TserSP00939)(Figure6). Additionally,thepureenzymewasableto
convertangiotensinIintoangiotensinII,inadditiontoAbz-FRK(Dnp)P-OH,withspecificactivityof
0.01µM/µg/min(Figure5,panelC).
Toxins2016,8,348 7of16
Toxins 2016, 8, 348   7 of 16 

Figure5.PurificationofACE-likeenzymefromTityusserrulatusvenom.(A)Fraction1wasfragmented
Figure  5. Purification of ACE‐like enzyme from Tityus serrulatus venom. (A) Fraction 1 was 
ingelfiltrationDiol-300column,andF1-2wastheonlyfractionabletocleavetheFRETsubstrate.
fragmented in gel filtration Diol‐300 column, and F1‐2 was the only fraction able to cleave the FRET 
SDS-PAGEshowedaseparationoflowmolecularweightbands. (B)ProfileofthefractionF1-2on
substrate. SDS‐PAGE showed a separation of low molecular weight bands. (B) Profile of the fraction 
acationexchangecolumn(blackline),withF1-2.7beingthefractionwiththehighestpeptidaseactivity,
F1‐2 on a cation exchange column (black line), with F1‐2.7 being the fraction with the highest 
andresultinginasingleproteinbandin13%SDS-PAGE.Inordertomaintaintheactivity,aftereach
peptidase activity, and resulting in a single protein band in 13% SDS‐PAGE. In order to maintain the 
stepthebufferwasimmediatelychangedto100mMTris,50mMNaCl,10µMZnCl buffer,pH7.0,
2
activity, after each step the buffer was immediately changed to 100 mM Tris, 50 mM NaCl, 10 μM 
using a 10 kDa molecular weight cutoff membrane. The grey line represents the NaCl gradient.
ZnCl2 buffer, pH 7.0, using a 10 kDa molecular weight cutoff membrane. The grey line represents the 
(C)ConversionofangiotensinIintoangiotensinIIbythepurifiedACE-likeenzyme.Thedetailsofthe
experimentsaredescribedinSection4.8.
Toxins 2016, 8, 348   8 of 16 
NaCl gradient. (C) Conversion of angiotensin I into angiotensin II by the purified ACE‐like enzyme. 
The details of the experiments are described in Section 4.8. 
The protein content from the SDS‐PAGE band was extracted and subjected to MS/MS for 
peptide fingerprint analysis. Using a database that combined sequences restricted to Tityus genus 
from UNIPROT and the transcript sequences of ACE‐like peptidase from Tityus scorpion venom 
glands, PeaksDB was able to identify, with high confidence (FDR ≤ 1%), two unique ACE‐like 
peptides from Tityus serrulatus (TserSP00939) (Figure 6). Additionally, the pure enzyme was able to 
convert angiotensin I into angiotensin II, in addition to Abz‐FRK(Dnp)P‐OH, with specific activity of 
Toxins2016,8,348 8of16
0.01 μM/μg/min (Figure 5, panel C). 

FFiigguurree 66.. TTryrypptitci cppepeptitdidese sfrformom frfarcatciotino nF1F‐12-.72 .7thtaht amt amtcahtcehde dwiwthi tthhet hperepdriecdteicdt eAdCAEC‐liEk-eli kfreomfro Tm. 
sTe.rsreurlrautulast u(Gs(eGneBnaBnakn TksTesreSrPS0P00903993)9 o)botbatianiende dfrformom trtarnanscsrcirpiptotommici caannaalylysissi.s .TThhee ppeepptitdideess wweerree ffoouunndd 
uussiinngg PPeeaakkss DDBB wwiitthh FFDDRR ≤≤ 11%%. .
2.7. Sequence Analysis 
2.7. SequenceAnalysis
We aligned the amino acid sequences of ACE‐like peptidases obtained from transcriptomics 
WealignedtheaminoacidsequencesofACE-likepeptidasesobtainedfromtranscriptomicsdata
data analysis from Tityus serrulatus, T. obscurus and T. bahiensis with each other and with human 
analysisfromTityusserrulatus,T.obscurusandT.bahiensiswitheachotherandwithhumantesticular
testicular ACE (Table 3). 
ACE(Table3).
TTaabbllee 33.. IIddeennttiittyy ((wwhhiittee)) aanndds siimmilialarritiyty( g(grereyy))b betewtweeenenA ACCE-Eli‐kliekefr ofrmomth tehvee vneonmomof oBfr BazrialziailniaTni tTyuitsyusps .
sspc osrcpoiropniosnasn adnHdo Hmoomsoa psiaepnisentes stteicstuilcaurlaArC AEC(EA (AAAA6A0661016.111)..1).  
TesticularACE Homo  T. serrulatus  T. bahiensis  T. obscurus 
Proteins 
TesticularACEHomosapiens T.serrulatus T.bahiensis T.obscurus
Proteins sapiens (AAA60611.1)  (TserSP00939)  (JAG85170)  (Tobs01141) 
(AAA60611.1) (TserSP00939) (JAG85170) (Tobs01141)
testicularACE Homo sapiens 
testicularACEHomosapiens 39.18%  23.12%  39.18% 
(AAA60611.1)    39.18% 23.12% 39.18%
(AAA60611.1)
T. serrulatus (TserSP00939 )  46.39%  67.21%  90.34%
T.serrulatus(TserSP00939) 46.39% 67.21% 90.34%
T. bahiensis (JAG85170)  28.16%  67.34%  61.22%
T.bahiensis(JAG85170) 28.16% 67.34% 61.22%
T obscurus (Tobs01141)  46.53%  92.51%  62.31% 
Tobscurus(Tobs01141) 46.53% 92.51% 62.31%
The identity with human tACE varied from 23% to 39%; however, the metallopeptidase motif 
was Thhigehildye nctointysewrvitehd htuhmroaungthAoCutE  tvhaer iesdpefcroiems 2(F3i%gutroe 39S%1);. hTohwee vAeCr,Et‐hliekme esteaqlluoepnecpetsi doafs eTmityoutisf 
swerarsuhlaitguhsl y(TcosenrsSePrv00ed93t9h)r oaungdh oTuittythues sopbescciuersu(sF i(gTuorbesS011)1.4T1h) ehAaCdE  -9l2ik.5e1s%eq uoef nsciemsiolafrTiittyy,u swsherirleu latthues 
t(bTasheArSCPE00‐l9i3k9e) asenqduTeintycues oshbsacruerdu s6(2T.o3b1s%01 1a4n1d) h6a7d.3942%.5 1s%imoiflasirmityil awritiyth,w Th.i loebtshceurtbuash AanCdE -Tli.k esesrerquulaetnucse, 
rsehsapreecdti6v2e.l3y1.%  and67.34%similaritywithT.obscurusandT.serrulatus,respectively.
3. Discussion
3. Discussion 
Using enzymatic assays, protein purification, mass spectrometry analysis, and comparative
Using enzymatic assays, protein purification, mass spectrometry analysis, and comparative 
studiesofthetranscriptsequencesinthevenomglandsofTityusspp. scorpions,weshowforthefirst
studies of the transcript sequences in the venom glands of Tityus spp. scorpions, we show for the 
timethepresenceofanACE-likepeptidaseinthevenomofTityusserrulatus.
first time the presence of an ACE‐like peptidase in the venom of Tityus serrulatus. 
ThevenomcleavesphysiologicallyimportantACEsubstrates,suchasangiotensinI,bradykinin,
The  venom  cleaves  physiologically  important  ACE  substrates,  such  as  angiotensin  I, 
andhemopressin,andcaptoprilinhibitsthisactivity. Expectedproductsfromthesehydrolyseswere
bradykinin, and hemopressin, and captopril inhibits this activity. Expected products from these 
observed,suchashemopressin1–7,angiotensinII,BK ,andBK ,consistentwiththeACEspecificity
hydrolyses were observed, such as hemopressin 1–7,1 –a5ngiotensi1n–7 II, BK1–5, and BK1–7, consistent with 
forthesesubstrates[33,34]. However,thepresenceofotherfragmentsindicatestheparticipationof
the ACE specificity for these substrates [33,34]. However, the presence of other fragments indicates 
morevenomproteasesinthecleavageofthesesubstrates.Inaddition,Abz-FRK(Dnp)P-OH,asubstrate
the  participation  of  more  venom  proteases  in  the  cleavage  of  these  substrates.  In  addition, 
specificallydesignedforACE[31],wasanimportanttooltoidentifyandpurifytheACE-likepeptidase
Abz‐FRK(Dnp)P‐OH, a substrate specifically designed for ACE [31], was an important tool to 
onT.serrulatusvenom,alongwiththesuccessfulACEinhibitionbycaptoprilandBPP10c[31,35].

At the end of the third chromatography step, we observed a single protein band in the
silver-stainedgelwithmolecularmassaround70kDathatconvertsangiotensinIintoangiotensinII.
Thus, the ACE-like enzyme present in Tsv has a molecular mass similar to tACE. Besides the
precursorofACE-likepeptidasebeingsequencedbyRNAsequencing(GenBankTserSP00939)and
theidentificationoftheACE-likepeptidasefromT.serrulatusthroughmassspectrometricanalysis,
theisoformsequencesarealsopresentintranscriptsfromthevenomglandofT.bahiensis(GenBank
JAG85170.1), T. stigmurus [30], and T. obscurus (GenBank Tobs01141 and GenBank Tobs00978) [36].
Thus,thesesequenceshelpedtovalidatethepurificationofthisenzyme,achievingtwouniquepeptides
Toxins2016,8,348 9of16
fromT.serrulatus’sACE-likesequence. Moreover,thealignmentindicatedhighhomologybetweenthe
ACEsequencesfromBrazilianTityusspeciesandaconservedmetallopeptidasemotifHHEMGHV
withhumantACE.
The expressive number of ACE-like enzymes described in invertebrates indicates that this
metallopeptidase is conserved throughout evolution [27]. Also, in accordance with our results,
the ACE-like enzymes in invertebrates include only a C-domain, similar to the human testicular
formoftheenzyme. DespitethemanystudieswithACEininvertebrates,naturalsubstratesforthese
enzymesareyettobedescribed[27],and,therefore,theirphysiologicalrolestillneedsclarification.
Ingeneral,ACE-likepeptidasesshowbroadsubstratespecificity,cleavingalargenumberofpeptides
invitro[33]. Theexceptionsarepeptideswithprolineresiduesatthepenultimateposition,suchas
BPPs[37].
StudieshaveshownthatmalefruitflieswithmutationsintheACEgenearesterile[38]. Other
studiessuggestapossiblephysiologicalroleofACE-likepeptidasesinthemidgutofinvertebrates,
butsolelybasedontheirpresenceinthisregion[39,40]. Also,thepresenceofACE-likeenzymesinthe
neuropilareaofinsectbrains,andtheabilityoftheenzymetocleaveinsecttachykinin(LomTK-1)
invitro,mayindicatethatthisenzymeparticipatesintheprocessingofneuropeptides[41,42]. Inthis
scenario,wehypothesizethattheACE-likepeptidasepresentinT.serrulatusvenomcontributestothe
abilitytocaptureorkillprey.
Moreover, the presence of this enzyme in the venom may be related to forming endogenous
peptidesinTsv,sinceaproteomicstudyidentifiedpost-translationalmodificationsoftoxinsinthis
venomandshowedthatabout80%ofthevenommoleculesaredegradedbyendogenouspeptidases,
particularlyaminoandcarboxypeptidases[19]. Also, arecentreportfromourgroupshowedthat
metallocarboxypeptidasescontributetoinactivateaseriesofhumanbioactivepeptides[18].
Lastly, we cannot exclude the involvement of this carboxypeptidase in the envenomation.
AccordingtoSafavi-Hemamiandcollaborators(2013),whodescribedanangiotensinI-converting
enzymeinthevenomofconesnails,thisenzymemaypromotevasoconstrictionofbloodvesselsatthe
stingsitebyangiotensinII,alongwithcatecholamine[13]. Infact,animalsandpatientsenvenomed
byscorpionsfromtheButhidaefamilypresentelevationofplasmarenin–angiotensinactivityand,
consequently,anincreaseofangiotensinII[43,44]. Thisobservationmight,atleastinpart,beexplained
by the action of the venom ACE-like enzyme, as we showed here that it is capable of releasing
angiotensinII,andvictimsofTityusserrulatusstingpresenttransienthypertension[45,46]. Untilnow,
neurotoxinswereconsideredthemaincauseofhypertensioninstungpatients,duetocatecholamine
release[7]. However,asAngIIcanalsostimulatethereleaseofcatecholamines,thismaycontribute
synergisticallytothehypertensionobservedinhumanvictims[47,48].
BesidesAngIIformationbythewholevenomandthepurifiedpeptidase,wealsoobservedthat
TsvactsonAngI,releasingAngIII,ahypertensivepeptidewithactivitycomparabletoAngII[49].
Differently, Ang 1–7, another product of the action of Tsv, promotes vasodilation, which would
seemtobeacontradictoryactionofthevenom. However,envenomedpatientscommonlypresent
persistenthypotensionafterthetransienthypertension[7].Heretofore,hypotensioncouldbeexplained
by the presence of hypotensins, which are bradykinin-potentiating peptides that do not inhibit
human ACE activity [50]. Our results suggest yet another factor that can contribute to this effect.
Inaddition,andsinceanimalvenomsaimtodestabilizehomeostasisintheprey,thejointactionof
multiplemoleculesinthevenom(neurotoxins,proteases,amongothers)isimportanttocausethe
envenomationsyndrome.
Furthermore,takingintoaccountthepossibleinvolvementofACE-likeduringtheenvenomation,
especially on hypertension, the invitro serum neutralization of Tsv is an important assay.
TheantivenomserarepresenttheonlyspecifictherapyavailableandrecommendedbytheWorld
Health Organization (WHO) to treat incidents involving animal venoms. In case of scorpion
envenomation,theadministrationofeitherscorpion(SAV)orarachnid(AAV)antivenomsisprescribed;
inthemostseverecases,itisrecommendedtouse4–6ampoules. OurresultsshowthatSAVandAAV
Toxins2016,8,348 10of16
produceddifferentlevelsofneutralization: whileSAVcausedpartialinhibitionatlowerdoses,AAV
wasslightlymoreefficientatthehighestdoses. TheeffectofthelowerSAVconcentrationisprobably
duetoACE-likepeptidasebeingpresentinbothscorpionvenomsusedfortheSAVimmunizationpool
(T.serrulatus,50%andT.bahiensis,50%),asisshowninFigureS1,whilethevenomsusedtoobtain
arachnidantivenomincludeonlyonescorpionspecies(T.serrulatus,57%),togetherwithtwospider
species(PhoneutrianigriventerandLoxoscelesgaucho). Theinhibitionobservedusinghighdosesofboth
antivenomscouldbearesultofsomesortofinteractionimpairmentbetweentheACE-likeandthe
FRETsubstrateduetothehighnumberofnon-specificantibodies,andthatmightnotrepresentareal
neutralizationbytheantivenom. However,theuseofhigherdosesofantivenommaybeinjuriousto
patients,asitcancauseadversereactionsknownas“serumsickness”[51].
In summary, here we describe, for the first time, the purification and characterization of
ACE-like enzyme in Tityus serrulatus venom, as well as two fluorescent substrates specific for
endo-andcarboxypeptidasesinthisvenom. Also, sinceweobservedformationofAngIIinvitro,
wehypothesizethatthisenzymemayalsocontributetothehypertensionobservedintheenvenomation
syndrome,alongwithothermolecules. Consideringthatscorpionbitesareacriticalpublichealth
probleminBrazil,thisstudydemonstratestheimportanceofcharacterizingnewmoleculesrelated
toenvenomationprocessesaswellasevaluatingantivenomtherapies. Thehighconcentrationsof
commerciallyavailableantivenomsnecessarytopartiallyblocktheACE-likeactivityinTsvmaybe
harmfultovictims.
4. MaterialsandMethods
4.1. Reagents
Phenylmethanesulfonylfluoride (PMSF), 1,10-phenanthroline, dynorphin 1–13 (Dyn 1–13),
hemopressin (hemo), angiotensin I (Ang I), bradykinin (BK), captopril, and rabbit lung somatic
angiotensinI-convertingenzyme(ACE)werepurchasedfromSigma-Aldrich(St. Louis,MO,USA).
Acetonitrileandtrifluoroaceticacid(TFA)wereacquiredfromJ.T.Baker. Thefluorescentresonance
energytransfer(FRET)substratesAbz-GGFLRRV-EDDnpandAbz-FRK(Dnp)P-OH,synthesizedwith
automated solid-phase synthesis [52], were kindly provided by Dr. Adriana Carmona, from the
DepartmentofBiophysicsofUNIFESP-EPM,SãoPaulo,Brazil.
4.2. VenomsandAntivenoms
ThelyophilizedvenomofTityusserrulatus(BatchNo. 2146andbatchNo. 744)wereprovided
by the Venom Section of Instituto Butantan, SP, Brazil. The venoms were submitted to a 10 kDa
molecularweightcutoff(AmiconUltra-15CentrifugalFilterDevices)andstocksolutionwasprepared
in50mMsodiumphosphateand50mMNaCl,pH6.0. Thescorpionandarachnidantivenoms(SAV
andAAV,respectively)werefromtheHyperimmunePlasmasProcessingSection,InstitutoButantan,
SP,Brazil. TheSAV(batchNo. 0905104/A)andtheAAV(batchNo. 0706121)proteinconcentrations
were8.43g/dLand15.4g/dL,respectively. TheantivenomsfromInstitutoButantanareproduced
throughhyperimmunizationofhorses,withapoolofT.serrulatus(50%)andT.bahienses(50%)venoms
forSAV,andT.serrulatus(57%),Phoneutrianigriventer(21.5%)andLoxoscelesgaucho(21.5%)venoms
forAAV.
4.3. FluorescentSubstrate-SpecificforCarboxy-andEndopeptidases,andPeptidaseInhibitors
The substrate-specific assay was carried out using 1 µg Tityus serrulatus venom (Tsv) and the
fluorescentsubstratesAbz-GGFLRRV-EDDnp(5µM)[18]andAbz-FRK(Dnp)P-OH(4µM),in100mM
Tris,50mMNaCl,10µMZnCl buffer,pH7.0. Allreactionsweremonitoredbymeasuringhydrolyses
2
using a fluorimeter (Victor 3™, Perkin-Elmer, Waltham, MA, USA; λem 420 nm and λex 320 nm),
at a stable temperature of 37 ◦C. The measurements of peptidase activity were made for 15 min
continuously(onereadperminute). ThefluorometricassayswereanalyzedusingGrafit5.0from