J O U R NA L OF PR O TE O MI CS 73 ( 20 1 0 ) 1 7 5 8– 1 7 7 6

available at www.sciencedirect.com

www.elsevier.com/locate/jprot

Snake venomics and antivenomics of Crotalus durissus

subspecies from Brazil: Assessment of geographic variation
and its implication on snakebite management

Johara Boldrini-Françaa , Carlos Corrêa-Nettob , Marliete M.S. Silvac , Renata S. Rodriguesd ,

Pilar De La Torree , Alicia Péreze , Andreimar M. Soaresa , Russolina B. Zingalib ,
Romildo A. Nogueirac , Veridiana M. Rodriguesd , Libia Sanze , Juan J. Calvetee,⁎
a
Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, Brazil
b
Instituto de Bioquímica Médica, Instituto Nacional de Ciência e Tecnologia de Biologia Estrutural e Bioimagem-INBEB and Rede Proteomica do
Rio de Janeiro-Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil
c
Departamento de Morfologia e Fisiologia Animal, Universidade Federal Rural de Pernambuco, Recife, Brazil
d
Instituto de Genética e Bioquímica, Universidade Federal de Uberlândia, Uberlândia, Brazil
e
Instituto de Biomedicina de Valencia, Consejo Superior de Investigaciones Científicas (CSIC), Valencia, Spain

AR TIC LE I N FO ABS TR ACT

Article history: We report the comparative proteomic and antivenomic characterization of the venoms of
Received 10 May 2010 subspecies cascavella and collilineatus of the Brazilian tropical rattlesnake Crotalus durissus.
Accepted 3 June 2010 The venom proteomes of C. d. collilineatus and C. d. cascavella comprise proteins in the
range of 4–115 kDa belonging to 9 and 8 toxin families, respectively. Collilineatus and
cascavella venoms contain 20–25 main toxins belonging to the following protein families:
Keywords: disintegrin, PLA2, serine proteinase, cysteine-rich secretory protein (CRISP), vascular
Crotalus durissus subspecies endothelial growth factor-like (VEGF), L-amino acid oxidase, C-type lectin-like, and snake
Snake venom proteomics venom metalloproteinase (SVMP). As judged by reverse-phase HPLC and mass
Geographic venom variation spectrometry, cascavella and collilineatus share about 90% of their venom proteome.
Individual venom variability However, the relative occurrence of the toxin families departs among the two C. durissus
Crotamine subspecies venoms. The most notable difference is the presence of the myotoxin crotamine
Mass spectrometry in some C. d. collilineatus specimens (averaging 20.8% of the total proteins of pooled venom),
Antivenomics which is absent in the venom of C. d. cascavella. On the other hand, the neurotoxic PLA2
Anti-crotalic antivenom crotoxin represents the most abundant protein in both C. durissus venoms, comprising 67.4%
of the toxin proteome in C. d. collilineatus and 72.5% in C. d. cascavella. Myotoxic PLA2s are also
present in the two venoms albeit in different relative concentrations (18.1% in C. d. cascavella
vs. 4.6% in C. d. collilineatus). The venom composition accounts for the clinical manifestations
caused by C. durissus envenomations: systemic neurotoxicity and myalgic symptoms and
coagulation disturbances, frequently accompanied by myoglobinuria and acute renal
failure. The overall compositions of C. d. subspecies cascavella and collilineatus venoms
closely resemble that of C. d. terrificus, supporting the view that these taxa can be considered
geographical variations of the same species. Pooled venom from adult C.d. cascavella

⁎ Corresponding author. Instituto de Biomedicina de Valencia, CSIC, Jaume Roig 11, 46010 Valencia, Spain. Tel.: +34 96 339 1778; fax: +34 96
369 0800.
E-mail address: jcalvete@ibv.csic.es (J.J. Calvete).

1874-3919/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.jprot.2010.06.001
 J O U R NA L OF PR O TE O MI CS 7 3 ( 2 01 0 ) 1 7 5 8– 1 7 7 6 1759

and neonate C.d. terrificus lack crotamine, whereas this skeletal muscle cell membrane
depolarizing inducing myotoxin accounts for ∼20% of the total toxins of venom pooled
from C.d. collilineatus and C.d. terrificus from Southern Brazil. The possible relevance of
the observed venom variability among the tropical rattlesnake subspecies was assessed
by antivenomics using anti-crotalic antivenoms produced at Instituto Butantan and
Instituto Vital Brazil. The results revealed that both antivenoms exhibit impaired
immunoreactivity towards crotamine and display restricted (∼60%) recognition of PLA2
molecules (crotoxin and D49-myotoxins) from C. d. cascavella and C. d. terrificus venoms. This
poor reactivity of the antivenoms may be due to a combination of factors: on the one hand,
an inappropriate choice of the mixture of venoms for immunization and, on the other hand,
the documented low immunogenicity of PLA2 molecules. C. durissus causes most of the
lethal snakebite accidents in Brazil. The implication of the geographic variation of venom
composition for the treatment of bites by different C. durissus subspecies populations is
discussed.
© 2010 Elsevier B.V. All rights reserved.

1. Introduction heterodimeric PLA2 molecule exhibiting presynaptic β-neuro-

Rattlesnakes (genera Crotalus and Sistrurus) represent a
monophyletic clade of pitvipers which likely originated in
the pine–oak, woodlands of the Sierra Madre Occidental in the
north-central part of the Mexican Plateau several million years
after a single lineage of an Asian pitviper crossed to North
America via the Behring land bridge during the late Oligocene
or early Miocene, 22–24 Mya [1–6]. Rattlesnakes dispersed
northward into North America and southward into South
America [7,8] giving rise to the approximately 70 species and
subspecies (http://www.reptile-database.org), which are cur-
rently discontinuously found from southern Canada to
northern Argentina [9–11]. In Brazil, genus Crotalus is repre-
sented by a single species, the Neotropical rattlesnake Crotalus
durissus [12]. Biogeographical data suggest a relatively recent
South American dispersal of C. durissus across a central trans-
Amazonian corridor during the middle Pleistocene (1.1–
1.0 Mya) followed by vicariance and dispersal into northeast-
ern Brazil [12,13]. The assemblage of forms currently known as
the subspecies of the Neotropical rattlesnake constitute a set
of closely related parapatric forms, whose mutual relations
and mechanisms of speciation remain largely elusive. Seven
subspecies of C. durissus are recognized in Brazil (C. d. dryinas,
C. d. marajoensis, C. d. ruruima, C. d. trigonicus, C. d. terrificus, C. d.
cascavella, and C. d. collilineatus) [14] (Fig. 1). However, some
authors consider C. d. cascavella and C. d. collilineatus in the
synonymy of C. d. terrificus [12,15].
Species of the C. durissus complex pose a serious medical
problem in many parts of their range, being responsible for
most of the lethal snakebite accidents in Brazil [14,16], where C.
d. cascavella, C. d. collilineatus and C. d. terrificus display wide
ecological and geographical distribution (Fig. 1). Venoms of
these rattlesnakes are notorious for their ability to decouple
neuromuscular transmission and for provoking myalgic
symptoms causing progressive paralysis [17,18]. Systemic
neurotoxicity is frequently accompanied by rhabdomyolysis
causing acute tubular necrosis and renal failure, which
represents the primary cause of death [17,18]. Systemic
neuro- and myotoxicity upon C. durissus ssp. bites are mainly
attributable to the high concentration of crotoxin [19], a
toxicity [20–22]. Crotoxin typically account for 70–90% of C.
durissus venom toxins [23,24]. Crotalus durissus ssp. venoms
also contain variable amounts of crotamine, a myonecrotic
toxin acting on voltage-sensitive Na+ channels [25,26]. Com-
parative venomics of the Central American rattlesnake
C. simus and the South American C. durissus complex points
to neurotoxicity and lethal venom activities to rodents,
associated to an increased concentration of neurotoxins
crotoxin and crotamine, as an adaptive paedomorphic trend
along Crotalus dispersal in South America [24]. Identifying the
molecular basis of adaptations in natural populations is an
important yet largely unrealized goal in evolutionary biology
[27–31]. Given the central role that diet has played in the
adaptive radiation of snakes [32], venom represents a key
factor in the diversification of these animals. On the other
hand, adequate treatment of envenoming is critically depen-
dent on the ability of parenteral-administered antivenoms to
neutralize the lethal toxins reversing thereby the pathological
symptoms [33,34]. Hence, besides ecological implications,
knowledge of the natural history and toxin composition of
venoms is of fundamental importance for the treatment of bite
victims and in the selection of specimens for the preparation
of venom pools for antivenom production [35,36]. This is
particularly relevant for highly adaptable and widely distrib-
uted species, in which intraspecific (ontogenetic, individual,
and geographic) venom variability represents a well documen-
ted phenomenon [37–39] and a source of diversity of the
pathological effects of snakebites in different geographical
regions [40,41]. Here we sought to characterize the venom
phenotypes of adult specimens of subspecies cascavella and
collilineatus of Brazilian C. durissus and neonate C. d. terrificus.
The immunoreactivity of the anti-crotalic antivenoms pro-
duced at Instituto Vital Brazil (Niterói, Rio de Janeiro, Brazil)
and Instituto Butantan (São Paulo, Brazil) against bites of these
taxa was investigated using an antivenomics approach. Our
results reveal population-specific venom features and the lack
of immunoreactivity of the two antivenoms towards crota-
mine, highlighting the need to revisit current immunization
protocols for producing an improved anti-crotalic antivenom
displaying broad-ranging clinical use in Brazil.
1760 J O U R NA L OF PR O TE O MI CS 73 ( 20 1 0 ) 1 7 5 8– 1 7 7 6

Fig. 1 – Distribution and evolutionary trends among South American rattlesnake. (A) Geographic distribution of C. d. cascavella,
C. d. collilineatus and C. d. terrificus in Northern, Midwestern, and Southern Brazil, respectively. Symbols indicate the
approximate regions of origin of the snakes sampled in this work: ▲, C. d. cascavella; ■, C. d. collilineatus; ● and ♦, C. d. terrificus.
Pictures of C. d. collilineatus, C. d. cascavella and C. d. terrificus were generously provided by Renata S. Rodrigues, Breno
Handan (Nucleo Regional de Ofiologia e Animais Peçonhentos da Bahia (NOAP/UFBA)), and Rodolfo Capdevielle (http://www.
serpientes-snakes.com.ar), respectively. Intraperitoneal median lethal dose ranges (LD50) have been reported in the literature
[15,102] and are expressed in μg/kg. LNC (lethal neurotoxicity coefficient) is defined as the ratio between the average LD50 of the
venom and the neurotoxin (crotoxin + crotamine) concentration [24]. (B) The evolutionary trend toward increasing neurotoxicity
to mice among C. durissus ssp. venoms involves decrease of myotoxic D49-PLA2 concentration concomitantly to an enhanced
expression of crotoxin and crotamine along the dispersal axis of this taxa.

from neonates was obtained by aspiration from the fangs using

2. Experimental section
2.1. Venoms and antivenoms
Crude venoms of adult Crotalus durissus terrificus were pooled
from specimens collected in Juiz de Fora (Minas Gerais)
(Southeastern population) and in the state of Rio Grande do
Sul (Southern population), and from the captive-born 12-weeks
old offspring of specimens from the Southern population, all
kept in the serpentarium of the Instituto Vital Brazil, Niterói-RJ,
Brazil. Venom from C. d. collilineatus was pooled from adult
specimens collected in the state of Goiás, Brazil, and kept in
captivity in the serpentarium Bioagents of Batatais-SP, Brazil.
Venom of C. d. cascavella was pooled from adult specimens
collected in Bahia, Brazil and kept in the serpentarium of
Nucleo Regional de Ofiologia e Animais Peçonhentos da Bahia
(NOAP/UFBA), BA, Brazil. The venom from adult animals was
collected by snake biting on a parafilm-wrapped jar. Venom
an Eppendorf pipette. Crude venoms were centrifuged at low
speed to remove cells and debris, lyophilized, weighed on a
microbalance, and stored at − 20 °C until used. The anti-crotalic
antivenoms used for the antivenomic study were kindly
provided by Instituto Butantan (But, São Paulo, Brazil, http://
www.butantan.gov.br) and Instituto Vital Brazil (IVB, Niterói,
RJ, Brazil; http://www.ivb.rj.gov.br). These antivenoms were
produced by hyperimmunization of horses with a pool of
venoms. Instituto Butantan uses a mixture of equal amounts
of C. d. terrificus and C. d. collilineatus venoms collected in
Southeastern and Midwestern Brazil, in the states of São Paulo,
Mato Grosso and Minas Gerais (Marisa Maria Teixeira da Rocha,
Instituto Butantan, personal communication); the antivenom
manufactured at IVB was produced in horses using venom
pooled from C. d. terrificus specimens from South-Eastern Brazil
(Dr. Aníbal Melgarejo, Instituto Vital Brazil, personal commu-
nication). Both antivenoms comprise purified F(ab′)2 frag-
ments generated by pepsic digestion of ammonium sulphate-
 J O U R NA L OF PR O TE O MI CS 7 3 ( 2 01 0 ) 1 7 5 8– 1 7 7 6
precipitated IgGs. F(ab′)2 concentration was determined spec-
trophotometrically using an extinction coefficient (ε) of 1.4 for
a 1 mg/ml concentration at 280 nm using a 1 cm light path-
length cuvette [42].
2.2. Isolation and characterization of venom proteins
Venom proteins were separated by reverse-phase HPLC using
a Teknokroma Europa C18 (0.4 cm × 25 cm, 5 mm particle size,
300 Å pore size) column as described [24,35,36,43]. Protein
detection was at 215 nm and peaks were collected manually
and dried in a Speed-Vac (Savant). The relative abundances (%
of the total venom proteins) of the different protein families in
the venoms were estimated from the relation of the sum of the
areas of the reverse-phase chromatographic peaks containing
proteins from the same family to the total area of venom
protein peaks. In a strict sense, and according to the Lambert–
Beer law, the calculated relative amounts correspond to the “%
of total peptide bonds in the sample”, which is a good estimate
of the % by weight (g/100 g) of a particular venom component.
The relative contributions of different proteins eluting in the
same chromatographic fraction were estimated by densitom-
etry after SDS-PAGE separation.
HPLC fractions were analyzed by SDS-PAGE (on 12 or 15%
polyacrylamide gels) and N-terminal sequencing (using a
Procise instrument, Applied Biosystems, Foster City, CA,
USA). Amino acid sequence similarity searches were per-
formed against the available databanks using the BLAST
program [44] implemented in the WU-BLAST2 search engine
at http://www.bork.embl-heidelberg.de. Molecular mass de-
termination was performed by MALDI-TOF mass spectrometry
(using an Applied Biosystems Voyager-DE Pro™ instrument)
and electrospray ionization (ESI) mass spectrometry (using an
Applied Biosystems QTrap™ 2000 mass spectrometer). Protein
bands of interest were excised from Coomassie Brilliant Blue-
stained SDS-PAGE gels and subjected to automated reduction,
alkylation, and in-gel digestion with sequencing grade porcine
pancreatic trypsin (Promega). Doubly- or triply-charged ions of
peptides selected from the MALDI-TOF mass fingerprint
spectra of the tryptic digests were sequenced by CID-MS/MS
using an Applied Biosystem's QTrap 2000. CID spectra were
interpreted manually or using a licensed version of the
MASCOT program (http://www.matrixscience.com) against a
private database containing viperid protein sequences depos-
ited in the SwissProt/TrEMBL database plus the previously
assigned peptide ion sequences from snake venomics projects
carried out in our laboratory. MS/MS mass tolerance was set to
±0.6 Da. Carbamidomethyl cysteine and oxidation of methio-
nine were fixed and variable modifications, respectively.
2.3. Western blot analysis
For assessing the immunoreactivity of the IVB and But anti-
crotalic antivenoms towards C. durissus venom proteins by
Western blot analysis, the reverse-phase HPLC chromato-
graphic fractions were electrophoresed in SDS-PAGE (12 or
15%) polyacrylamide gels under non-reduced conditions
followed by electrotransfer to Hybond™-P membrane (GE-
Healthcare) [45] using a Bio-Rad Semi-Dry minitransfer cell
operated at 200 V during 120 min. Transfer efficiency was
1761
evaluated by staining the nitrocellulose membranes with
Ponceau-S Red. Membranes were then destained in water,
incubated in 1% bovine serum albumin in 0.12 M NaCl, 0.04 M
sodium phosphate, pH 7.2 (blocking buffer), for 30 min at room
temperature to block non-specific reactive sites, and incubat-
ed overnight at 4 °C with antivenom diluted 1:2000 (v/v) in
blocking buffer. After washing 4 times with blocking buffer
containing 0.05% Tween-20, the membranes were incubated
for 2 h at room temperature with alkaline phosphatase-
conjugated anti-horse IgG (Sigma) diluted 1:2000, washed 4
times as above, and developed using ECL PLUS Western
Blotting Detection Reagents (GE-Healthcare).
2.4. Antivenomics: immunodepletion of venom proteins by
polyvalent antivenoms
We have coined the term “antivenomics” for the proteomic
identification and quantitation of venom proteins escaping
antivenom recognition [35,36,39,46–50]. Briefly, 2 mg of crude
venom were dissolved in 70 μL of 20 mM phosphate buffer, pH
7.0, mixed with 4 mg of But or IVB antivenom, and incubated
with gentle stirring overnight at 37 °C. 6 mg (350 μL) of rabbit
anti-horse IgG antiserum (Sigma) were then added and the
mixture was allowed to react for 2 h at 37 °C. Immunocom-
plexes were precipitated by centrifugation at 13.000 ×g for
30 min in an Eppendorf centrifuge, and the supernatant was
analyzed by reverse-phase HPLC as above. Control venom
samples were subjected to the same procedure except that
non-immune IgG instead of antivenom was included in the
reaction mixture.
2.5. Generation of rabbit antibodies against C. d. terrificus
venom
To assess immunogenicity of crotamine, an experimental
antiserum was raised in rabbits by subcutaneous injections of
sublethal amounts of a mixture of crotalid venoms (to be
reported elsewhere), including crotamine-positive C. d. terrifi-
cus venom pooled from Southern Brazil specimens. First
injection comprised 100 μg venom in 100 μL of PBS (20 mM
phosphate, 135 mM NaCl, pH 7.3) emulsified with an equal
volume of Freund's complete adjuvant. Booster injections
comprising increasing amounts (200–500 μg) of immunogen
emulsified in Freund's incomplete adjuvant were adminis-
tered every 2 weeks for a period of 2–3 months (depending on
the titer of the antiserum determined by a standard ELISA
procedure). Terminal cardiac blood collection, done by intra-
cardiac puncture performed under general anesthesia, was
approved by the IBV's Ethics Commission. The IgG fraction
was purified by ammonium sulphate precipitation followed by
affinity chromatography on Sepharose-Protein A (Agarose
Bead Technologies, Tampa, FL, USA) following the manufac-
turer's instructions. For Western blot analysis, purified IgG
was used at a concentration of 10 μg/ml. For antivenomics,
1 mg of C. d. terrificus venom in 300 μlL of 0.2 M phosphate, pH
7.0, was incubated with 10 mg of rabbit IgG antibodies
overnight at room temperature and with gentle stirring. IgG–
antigen complexes were immunoprecipitated with Agarose-
Protein-A (ABT), and the supernatant was analyzed by
reverse-phase HPLC as above.
1762 J O U R NA L OF PR O TE O MI CS 73 ( 20 1 0 ) 1 7 5 8– 1 7 7 6

belonging to 8 protein families (Fig. 4, Tables 1 and 2). PLA2

3. Results and discussion molecules, particularly neurotoxic crotoxin isoforms, which
accounts for 70–80% of the total venom proteins, constitute
3.1. The venom proteomes of C. d. collilineatus and C. d. the major toxin of both taxa. Disintegrins, thrombin-like and
cascavella gyroxin-like serine proteinases, CRISP, svVEGF, L-amino acid
oxidase, C-type lectin-like convulxin, and PIII-SVMPs all
The proteome of venom pooled from adult C. d. collilineatus represent minor components, none of which is present in
(Fig. 2A) and C. d. cascavella (Fig. 3) specimens share toxins concentration higher than 2% of the total venom proteins

Fig. 2 – Characterization of the venom proteome of Crotalus durissus collilineatus. Panels A and B display, respectively,
reverse-phase HPLC separations of the proteins of pooled and individual venoms. Two milligrams of total venom proteins were
applied to a Europa Protein-300 C18 column, which was then developed with the following chromatographic conditions: isocratically
(5% B) for 10 min, followed by (5–15% B) for 20 min, (15–45% B) for 120 min, and (45–70% B) for 20 min. Fractions were collected
manually and submitted to N-terminal sequencing and molecular determination by SDS-PAGE, ESI and MALDI mass spectrometry.
The results are shown in Table 1. Inset panels: Coomassie blue-stained SDS-AGE showing the protein composition of the
reverse-phase HPLC-separated venom protein fractions displayed in the respective panel and run under non-reduced (upper panels)
and reduced (lower panels) conditions. Molecular mass markers (in kDa) are indicated at the side of each gel. Selected protein bands
were excised and characterized by mass fingerprinting and CID-MS/MS of selected doubly- or triply-charged peptide ions (Table 1).
 J O U R NA L OF PR O TE O MI CS 7 3 ( 2 01 0 ) 1 7 5 8– 1 7 7 6 1763

Fig. 3 – Characterization of the venom proteome of Crotalus durissus cascavella. The proteins contained in two milligrams of
whole dried pooled venom were separated by reverse-phase HPLC and analyzed as described in the legend of Fig. 2. The results
are shown in Table 1. Inset, Coomassie blue-stained SDS-PAGE of the reverse-phase HPLC protein fractions run under
non-reduced (upper panels) and reduced (lower panels) conditions. Molecular mass markers (in kDa) are indicated at the side of
each gel. Selected protein bands were excised and characterized by mass fingerprinting and CID-MS/MS of selected doubly- or
triply-charged peptide ions (Table 1).

(Fig. 4, Table 2). Shared venom components between C. d.
collilineatus and C. d. cascavella are apparently identical as
judged by reverse-phase HPLC elution profile, N-terminal
sequencing, SDS-PAGE, MALDI-TOF mass fingerprinting and
MS/MS analysis, and are identified in the respective HPLC
separations and in Table 1 with the same numbering.
However, the relative abundance of these proteins varied
among the venoms, each venom comprising some unique
components. The most notable departure between the venom
pools of C. d. collilineatus and C. d. cascavella is the large amount
of the myotoxin crotamine in the former, and the increased
concentration of acidic D49-PLA2 [C9E7C4] (fraction 14) in the
latter (Fig. 4, Tables 1 and 2). Notwithstanding these differ-
ences, C. d. collilineatus and C. d. cascavella share about 90% of
their venom proteomes, and closely resemble that of C. d.
terrificus [24, but see also Fig. 5], supporting the view that, from
a venomics perspective, these three taxa might be considered
geographic variations of the same species [12,15]. However, a

Fig. 4 – Overall protein composition of the venoms of C. d. collilineatus and C. d. cascavella. Pie charts of relative abundance of
different toxin families in the pooled (A) and individual (B) venom of C. d. collilineatus and the pooled venom of C. d. cascavella (C).
svVEGF, snake venom vascular endothelial growth factor; CRISP, Cysteine-rich secretory protein; LAO, L-amino acid oxidase;
CTL, C-type lectin-like protein; PIII-SVMP, snake venom metalloproteinase from class III; SerProt, serine proteinase; PLA2,
phospholipase A2. Details of the individual proteins are shown in Table 1 and the percentages of the different toxin families in
the venoms are listed in Table 2.
1764 J O U R NA L OF PR O TE O MI CS 73 ( 20 1 0 ) 1 7 5 8– 1 7 7 6

Table 1 – Assignment of the reverse-phase fractions from the venom pools of Crotalus durissus collilineatus and Crotalus
durissus cascavella, isolated as in Figs. 2A and 3A, to protein families by N-terminal Edman sequencing, mass spectrometry,
and collision-induced fragmentation by nESI-MS/MS of selected peptide ions from in-gel digested protein bands separated
by SDS-PAGE (insets in Figs. 2A and 3A). X, Ile or Leu; Z, pyrrolidone carboxylic acid. Cp, propionamide cysteine. Unless
other stated, for MS/MS analyses, cysteine residues were carbamidomethylated; Molecular masses of native proteins were
determined by electrospray ionization (±0.02%) or MALDI-TOF (±0.2%) mass spectrometry. Apparent molecular masses were
determined by SDS-PAGE of non-reduced (■) and reduced (▼) samples.
HPLC N-terminal Molecular Peptide MS/MS-derived Protein family
fraction mass ion sequence

m/z z

1–3 YKQCHKKGGHCFPKE 4884 Da 610.6 2 ICLPPSSDFGK Myotoxin Crotamine 1 [P01475]

4982 Da Myotoxin Crotamine isoform
4 (R)IECDCGSIENPCCYA 7305 Da 812.6 2 NDDTCpTGQSADCpPR Disintegrin [COL2T8] 408–474
5 (R)IECDCGSIENPCCYA 7519 Da 812.6 2 NDDTCpTGQSADCpPR Disintegrin [COL2T8] 408–476
7721 Da ∼ Disintegrin [COL2T8] 408–478
(R)IECDCGSIENPCCYA 7593 Da ∼ Disintegrin [COL2T8] 408–477
IECDCGSIENPCCYAT 7364 Da Disintegrin [COL2T8] 408–476
6–9 SSYGCYCGAGGQGWP 9■/▼ kDa Crotoxin acid chain [P08878]
A-chain (38–83)-SS-B-chain
(84–124)-SS-C-chain (125–138)
7 Blocked 16.5■/ 831.5 2 LTGCDPTTDVYTYR Crotoxin acid chain [P08878]
7▼ kDa
780.3 2 CCFEHDCCYAK
10 SLLQFNKMIKFETRK 14,197 Da 871.8 2 GTWCEEQICECDR ∼ Crotoxin basic chain
[P62022, I109]
633.7 2 YGYMIYPDSR
11 a HLLQFNKMIKFETRK 14,248 Da Crotoxin basic chain [P0CAS2]
b NLLQFNKMIKEETGK 14,143 Da ∼ Presynaptic neurotoxic PLA2
[AAB34413]
12 a HLLQFNKMIKFETRK 14,187 Da 649.6 2 YGYMFYPDSR Crotoxin basic chian 1 [P62022]
450.3 2 HLLQFNK
871.5 2 GTWCEEQICECDR
592.8 2 WDIYPYSLK
720.3 3 NAIPFYAFYGCYCGWGGR
b SLLQFNKMIKFETRK 14,284 Da ∼ Crotoxin basic chain [P62022]
c NLLQFNKMIKEETGK 14,143 Da ∼ Presynaptic neurotoxic PLA2
[AAB34413]
13 a Blocked 25,733, 472.2 2 PVCVTVXR svVEGF [∼ ACN22040]
25,835 Da
682.4 2 XEVMQFTEH(249.2)
750.3 3 (374.3)VSILDEYPSEVAHLFR
706.3 2 (342.2)PFMEVYER
b SVDFDSESPRKPEIQ 23 kDa▼ 569.7 2 SVDFDSESPR CRISP
c SLVEFETLMMKIAGR 14 kDa▼ 831.4 2 LTGCDPTTDVYTYR ∼ Crotoxin acid chain [P08878]
14, 15 SLLDFEMMIIKVAKK 13,836 Da 752.8 2 CCFVHDCCYGK ∼ acidic D49–PLA2 [C9R7C4]
511.2 2 QFPAENCR
670.6 2 SLLDFEMMIIK
836.8 2 QTCECDGVAAVCFR
13–18 SLLDFEMMIIKVAKK 14,245 Da 518.2 2 QFPAENCpR ∼ acidic D49–PLA2 [C9E7C4]
15 VIGGDECNINEHNFL 28 kDa▼ 728.4 2 THFLIYVGVHDR ∼ Serine porteinase Gyroxin-like
B1_4
661.4 2 HILIYVGVHDR [B0FXM2]
568.8 2 SVQFDKEQRR
16 VIGGDECNINEHNFL 28 kDa▼ 728.4 2 THLIYVGVHDR ∼ Serine proteinase Gyroxin-like
B1_4
874.3 2 ILCAGVLEGGIDTCNR [B0FXM2]
773.8 3 NNEHIAPLSLPSSPPSVGSVCR
568.8 2 SVQFDKEQRR
647.8 2 XNXXDYEVCR
17 GLHCPSDWYYYDQH 110■/ 534.3 2 WFVASCIGK C-type lectin convulxin-α
14–16▼ kDa [Ο93426]
GFCCPSHWSSYDRY 774.3 3 GLHCPSDWYYYDQHCYR
829.8 2 GFCCPSHWSSYDR C-type lectin convulxin-β
[Ο93427]
849.4 2 STFFWIGANNIWNK
809.9 3 TFDNQWLSAPCSDTYSFVCR
 J O U R NA L OF PR O TE O MI CS 7 3 ( 2 01 0 ) 1 7 5 8– 1 7 7 6 1765

Table 1 (continued)
HPLC N-terminal Molecular Peptide MS/MS-derived Protein family
fraction mass ion sequence

m/z z
■/▼
18 a Blocked 115 kDa 577.9 2 EGNHYGYCRKEQNTK PIII-metalloproteinase [C5H5D1]
b N.D. 58 kDa▼, ■ 485.6 2 VQVHFNAR L-amino acid oxidase [∼ P56742]
519.3 2 FWEDDGIR
532.3 2 NPLEECFR
583.3 2 IKFEPPLPPK
682.6 2 SAAQLYVESLRK
757.8 2 ETDYEEFLEIAR
619.2 2 SAAQLYVESLR
935.2 2 NNPGLLEYPVKPSEEGK
612.2 2 DWYANLGPMR
557.8 2 VIEIQQNDR
c N.D. 28 kDa▼, ■
728.4 2 THFLIYVGVHDR Serine porteinase Gyroxin-like B1_4
661.4 2 HILIYVGVHDR [B0FMX2]
15–18 N.D. 15 kDa▼, ■
649.6 2 YGYMFYPDSR Crotoxin basic chain 1[P62022]
871.5 2 GTWCEEQICECDR
753.3 2 CCFVHDCCYGK
19 VVGHPCNINEHRSL 28 kDa▼ Serine proteinase
20 VIGGHPCNINEHNFL 28 kDa▼ Serine proteinase

thorough proteomic investigation of the 2DE-separated
venom proteome of C. d. terrificus [51] identified eight 5′-
nucleotidase isoforms, 2 phosphodiesterase spots, and one
glutaminyl cyclase, which comprised respectively 7–8%, 1.9%,
and 1% of total venom proteins, but have been detected
neither in previous reverse-phase separation of C. d. terrificus
[24] nor in C. d. collilineatus and C. d. cascavella venoms
[this work]. Furthermore, Georgieva et al. [51] did not found
L-amino acid oxidase and the C-type lectin-like convulxin in
C. d. terrificus venom, whereas these proteins are clearly
present in the C. d. collilineatus and C. d. cascavella venoms
sampled in this work (Table 2), and have been reported to
comprise about 3.6% and 0.2% of the venom proteome of C. d.
terrificus in a previous work [24]. Our results also show the
presence of PIII-metalloproteinase(s) in the three C. durissus
subspecies [24, Table 2] although this toxin family was not
detected in the 2DE-separated venom proteome of C. d.
terrificus [51]. In addition, C. d. collilineatus and C. d. cascavella,
but not C. d. terrificus [24,51], contain CRISP (1.1–2.6% of total
reverse-phase-separated venom proteins) (Table 2). As a
whole, these evidences indicate the occurrence of individual
or geographic variability among C. durissus spp. venoms.
In line with the proteomic outcome reported here, a recent
analysis of toxin-encoding ESTs from a C. d. collilineatus cDNA
library [23] identified crotoxin as the most expressed tran-
script, comprising 88% of all toxin-coding sequences. Minor
components (accounting for less than 3% of the total venom
proteins) found in both, transcriptome and proteome include
svVEGF, serine proteinases (including gyroxin), the C-type
lectin-like convulxin, and SVMPs (Table 2). However, low
abundant transcripts encoding nerve growth factor, cardio-
toxin, angiotensin-converting enzyme inhibitor, C-natriuretic
peptide, ohanin, and PLA2 inhibitor, which comprised 2.5% of
toxin-coding ESTs, are not mirrored in the venom proteome
(Tables 1 and 2). In addition, crotamine, which was identified
in the transcriptome as a singleton [23], represents 21% of
the total toxins of C. d. collilineatus pooled venom (Table 2).
However, this toxin was not found in the individual venom
(Table 2), indicating the existence of crotamine-positive and
crotamine-negative C. d. collilineatus specimens. The transla-
tional rate of the crotamine mRNA has not been studied, and
thus it remains uncertain whether the specimen used for the
transcriptomic analysis belonged to a crotamine-positive or to
a crotamine-negative population. In the few instances in
which transcriptomics and proteomics databases have been
compared [52–54], a low degree of accordance has been
reported, indicating that the cDNA libraries lacked many
transcripts encoding venom-expressed proteins. On the other
hand, the occurrence of non-translated messengers point
out to transcripts exhibiting an individual or a temporal
expression pattern over the life time of the snake [37].
The cocktail of toxins present in C. durissus ssp. venoms
explains the clinical picture observed upon envenomations in
Brazil. The serious neurotoxic symptoms are attributable to the
high amounts of the β-neurotoxin crotoxin [19–22,55]. Crotoxin
has also been recognized as the main component responsible
for acute nephrotoxicity [56,57], although the myonecrotic
activity of crotamine [25,26,58] may also contribute to rhabdo-
myolysis and acute kidney failure. Other potentially life-
threatening clinical effects encountered throughout the
range of C. durissus such as hemostatic disturbances, myocar-
dial damage, hypotension and shock [18,19] may involve the
combined or synergistic effect of the less abundant toxins.
Hence, RGD disintegrin [COL2T8] [23] (HPLC fractions 4, 5)
blocks platelet aggregation; thrombin-like serine proteinases
(i.e. gyroxin [B0FXM2] found in fractions 15, 16, and 18–20) may
produce hypofibrinogenemia and incoagulable blood [59,60];
gyroxin, in addition, induces the neurological syndrome of
“barrel rotation”, characterized by opisthotonos and rotations
around the long axis of the animal [61]. The acidic D49-PLA2
[C9E7C4] [62] eluted in HPLC fractions 13–18 may induce local
tissue damage associated to inflammation [63]. Fraction 18a
1766 J O U R NA L OF PR O TE O MI CS 73 ( 20 1 0 ) 1 7 5 8– 1 7 7 6

corresponds to crotastatin-1 [C5H5D1], a vascular apoptosis- venom proteins in the Southern population but only 2% in the
inducing (VAP) dimeric PIII-SVMP [64]. The L-amino acid venom from the Southeastern pool. Venom pooled from
oxidase eluted in fraction 18b was previously isolated by neonates born from mothers from the Southeastern popula-
Toyama and co-workers [65] from C. d. cascavella venom tion were all crotamine-negative (Fig. 5C). Except for the
and exhibited strong platelet aggregation activity. Tetrameric absence of crotamine, neonate and adult snakes from the
C-type lectin-like convulxin [O93427] (HPLC fraction 17) has same geographical pool both express type II venoms exhibit-
been also characterized as a potent platelet aggregation agent ing highly similar toxin profiles, thus essentially supporting
[66–68]. the predicted trend. Nonetheless, serine proteinases 15 and 16
showed higher expression level in venom pooled from
3.2. Change in the PLA2 profile of C. d. cascavella and neonates (∼ 12%) (Fig. 5C) than in venom from adults (∼ 0.5–
C. d. collilineatus venoms: an adaptive specialization 4%) (Fig. 5B and A, respectively). This may explain the higher
to neurotoxicity coagulant and fibrinolytic activities reported in the venom of
newborn versus adult C. d. terrificus [71].
Venoms of Neotropical Crotalus subspecies belong to one of Fig. 1 shows the geographic distribution of C. d. cascavella,
two distinct phenotypes, which broadly correspond to type I C. d. collilineatus and C. d. terrificus along with the estimated
(high levels of SVMPs and low toxicity, LD50 > 1 μg/g mouse LNC for their venoms, calculated using LD50 values reported in
body weight) and type II (low metalloproteinase activity and the literature [15]. The data showing a clear tendency of C.
high toxicity, LD50 < 1 μg/g mouse body weight) venoms durissus ssp. venoms towards decreasing LNC values from
defined by Mackessy [69,70]. In a previous comparative Northeastern through Southern Brazil, is in agreement with
venomic study, we reported the occurrence of ontogenetic the hypothesis that neurotoxicity to mammals represents the
change from type II (neurotoxic) to type I (hemorrhagic) key axis along which overall venom toxicity has evolved
venom in Central American C. simus, and hypothesized that during the southward dispersion of C. durissus ssp. in South
neurotoxicity represented a paedomorphic trend and an America [24]. In line with this view, Powell and Lieb [72] have
adaptive trait along the dispersal of Crotalus durissus ssp. in predicted that the extremely high neurotoxicity exhibited by
South America [24]. This hypothesis predicted i) the absence of North American rattlesnakes represents a transitory popula-
ontogenetic change in venoms of Crotalus durissus ssp., and tional phenomenon associated with novel prey bases. The
ii) a gradual decreasing of the “lethal neurotoxicity coefficient” venoms of these species contain Mojave neurotoxin [acid
(LNC, defined as the ratio between the average LD50 of the chain, P18998; basic chain, P62023], the North American
venom and its relative amount of neurotoxins) along the homolog of South American crotoxin [73]. Niche colonization
dispersal route of Crotalus southward of the Amazon basin. by development of venom neurotoxicity has also been
The first prediction, lack of ontogenetic venom variation, was described in European Viperid snakes. The major toxic
tested through comparison of the toxin profiles of venoms component of the V. a. meridionalis venom is vipoxin
pooled from neonate and adult specimens of C. d. terrificus [P14420], a heterodimeric postsynaptic PLA2 neurotoxin
(Fig. 5). It was found that venoms pooled from adult snakes [74,75]. Since 1992, the Marseille Poison Center has regularly
from the Southeastern (Fig. 5A) and Southern (Fig. 5B) observed the occurrence of cases of human envenomation
populations strongly depart in their crotamine content. The after Vipera ammodytes ammodytes bites in south-east France
relative abundance of this toxin averaged 22% of the total involving unexpected neurological signs [76]. In France,
neurological symptoms had previously only been described
in cases of envenomation by Vipera aspis zinnikeri located
mainly in the south-west of France. Venom PLA2 profiling,
Table 2 – Overview of the relative occurrence of proteins genetic analyses, and epidemiological survey of snake bites in
(in percentage of the total HPLC-separated proteins) of the
various regions of France all indicate that the neurotoxic V. a.
different families in the venoms of C. durissus subspecies
collilineatus and cascavella. ammodytes population may have originated from a “crypto-
neurotoxic” V. a. zinnikeri population that may have colonized
Proteins family % of total venom proteins
northern areas from the ice refuges of the southwest of France,
C. d. collilineatus C. d. cascavella by moving along the rivers, which constitute thermal corridors
[77].
Pooled Individual Pooled
C. d. cascavella venom contains, in addition to neurotoxic
Crotamine 20.8 – – PLA2, a considerable amount (18% of the total venom proteins)
Disintegrin 0.5 0.4 0.2 of the acid D49-PLA2 [C9E7C4] (HPLC peak 14 in Fig. 3; Table 2).
PLA2 72 90.9 90.6
The amount of this myotoxin is much reduced in the pooled
f Crotoxin
Other PLA2s
67.4
4.6
79.6
11.3
72.5
18.1
venoms of C. d. collilineatus (4.6%) (Fig. 2; Table 2), and C. d.
Serine Proteinase 1.9 0.5 1.2 terrificus (2.1%). This observation, which is in line with
CRISP 1.8 1.6 0.9 previous biochemical evidences showing that C. durissus
svVEGF 2.1 2.6 1.1 cascavella venom presented the highest PLA2 activity, whereas
L-amino acid 0.5 2.6 < 0.1 C. durissus collilineatus the lowest [15]. We interpret the
oxidase decrease of non-neurotoxic PLA2 as part of the venom
C-type lectin-like <0.1 < 0.1 < 0.1
specialization trend of C. durissus ssp. toward neurotoxicity
PIII-Zn2+- 0.4 2.3 < 0.1
metalloproteinase
during the colonization of new ecological niches on their
southward dispersion. A prediction of this view is that the
 J O U R NA L OF PR O TE O MI CS 7 3 ( 2 01 0 ) 1 7 5 8– 1 7 7 6 1767

venom from Paraguayan and North Argentinian populations
of C. d. terrificus may follow the trend toward lower levels of
myotoxic D49-PLA2 in favor of an increase in the concentra-
tion of crotoxin.
3.3. The trend toward increasing concentration and
frequency of crotamine in C. d. durissus populations from
northeastern Brazil to northern Argentina suggests an
adaptive role for this myotoxin
The outcome of the present investigation suggests that in
South America C. durissus crotamine, a small basic myone-
crotic toxin with β-defensin scaffold (PDB accession code
1Z99) targeting voltage-sensitive Na+ channels, inducing
thereby skeletal muscle membrane depolarization [78], may
also represent an adaptive trait. Crotamine-positive and
crotamine-negative populations were described more than
50 years ago [79,80]. Now, we show an increasing expression
of crotamine among C. durissus ssp. coincident with the
direction of the dispersal of this taxa. Thus, the proteomic
analysis of pooled venom from C. d. cascavella (Fig. 3) corro-
borates the reported absence of crotamine in rattlesnakes
from northeastern Brazil (Fig. 1) [15], and the occurrence of
crotamine-positive and crotamine-negative populations of

Fig. 5 – Lack of ontogenetic venom variation in C. d. terrificus. The venom proteomes of adult C. d. terrificus from Southeast
(A) and Southern (B) Brazil, and from the pooled venom of captive-born 12-weeks old offspring of specimens from the Southern
population (C) were separated by reverse-phase HPLC and analyzed as described in the legend of Fig. 2. Peak numbering
corresponds to proteins listed in Table 1.
1768 J O U R NA L OF PR O TE O MI CS 73 ( 20 1 0 ) 1 7 5 8– 1 7 7 6
Figure 5 (continued).
C. d. collilineatus (Fig. 2) and C. d. terrificus (Fig. 5) in midwestern
and south Brazil, respectively (Fig. 1).
Schenberg [79] reported an increase in the number of
crotamine-positive rattlesnakes to the west of the states of
São Paulo and Paraná, until a region was reached where only
crotamine-secreting snakes were found [80]. Furthermore,
C. d. terrificus from Northern Argentina and Paraguay appear to
comprise genetically pure crotamine-secreting populations
[81]. This geographical distribution indicates the Mendelian
character of crotamine secretion [79]. Crotamine-like proteins
have evolved convergently in the venom of distant lineages,
such as lepidosaurs and monotremes [82], indicating that
coopting these β-defensin proteins seems to be a common
strategy for evolving venoms. In Crotalus, crotamine evolved
in the venom gland by recruitment, duplication, and acceler-
ated evolution of its paralogous pancreatic crotasin gene [83].
The gene coding for crotamine (Crt-p1) has been located to the
end of the long arm of chromosome 2 of crotamine-positive
specimens [84]. The crotamine gene exists from 1 to 32
copies per haploid genome, and the concentration of crota-
mine in the venom appears to be positively correlated with
the number of gene copies [85]. The variability in the copy
number of crotamine genes might be explained by its
subtelomeric location resulting in accelerated rate of dupli-
cation and rearrangement [85,86]. The increased frequency of
crotamine-positive populations from northeastern Brazil to
northern Argentina, suggests an adaptive value for crotamine.
However, the mean LD50 values of crotamine for mice, 6.9 mg/
kg (0.07–32.8 mg/kg, depending on the method used in
determining lethality, such as mouse strain, site of inocula-
tion, etc.) [86], is two orders of magnitude higher than the LD50
of crotoxin (60–110 μg/kg). Thus, fixation of crotamine in the
venom proteome concomitantly with C. durissus ssp. dispersal
in South America may reflect an adaptation to different
ecological niches and prey availability. Relevant to this
argument, the habitat of C. d. cascavella (dry thorn scrub, the
Caatinga region) is quite different from that of C. d. collilineatus
and C. d. terrificus (semideciduous forest, the Cerrado region).
In addition, the feeding habits of C. d. cascavella consists on
lizard, birds and small mammals, whereas C. d. collilineatus
and C. d. terrificus feed exclusively on rodents from birth
[87,88]. The adaptive value of crotamine may relay in a rapid
paralyzation of rodents, thus securing the meal and avoiding
the risk of injury to the snake. Supporting this view,
crotamine has been shown to induce a potent analgesic effect
when injected (s.c. and i.p.) or administered orally into mice
[89].
3.4. Assessing the immunodepleting capability of
Brazilian anti-crotalic antivenoms against the venoms of
Crotalus durissus subspecies using antivenomics
Given the wide distribution of C. durissus subspecies in Brazil,
and the occurrence of notable geographic venom variations,
we applied an antivenomics approach [35,36,39,46–50] to
investigate the in vitro immunoreactivity of the anti-crotalic
antivenoms produced at Instituto Vital Brazil and Instituto
Butantan. These two institutions, together with Centro de
Produção e Pesquisa de Imunobiológicos (Secretaria de Estado
da Saúde do Paraná), and Fundação Ezequiel Dias (FUNED,
Secretaria de Estado de Saúde de Minas Gerais), form part of
the network of institutions that manufacture antivenoms for
the Ministério da Saúde do Brasil. The Butantan antivenom
(But) efficiently immunodepleted serine proteinases, L-amino
acid oxidase, SVMPs and C-type lectin-like molecules from
the venom of adult (Fig. 6A) and juvenile (Fig. 6C) C. d.
terrificus. However, this antivenom lacks immunoreactivity
towards crotamine and disintegrin molecules (Fig. 6A) and
exhibits restricted (∼ 60%) immunodepletion ability of PLA2
molecules (crotoxin and D49-myotoxin) from C. d. terrificus
(Fig. 6A) and C. d. cascavella (Fig. 7A) venoms. The But
antivenom also showed negligible immunodepleting activity
 J O U R NA L OF PR O TE O MI CS 7 3 ( 2 01 0 ) 1 7 5 8– 1 7 7 6 1769

towards serine proteinase 19 from C. d. cascavella (Fig. 7A).
The immunodepleting ability of the IVB antivenom (Figs. 6B
and 7B) did not significantly depart from that of the But
antivenom. However, in Western blot analysis, the IVB
antivenom, but not the antivenom from Instituto Butantan,
showed very weak immunorecognition of crotamine (Fig. 8).
The distinct affinity of IVB and But antivenoms against
crotamine may be due to a combination of i) the different
mixtures of venoms used for hyperimmunizing horses and ii)
low immunogenicity of crotamine. The first possibility is
supported by the fact that Instituto Butantan uses a mixture
of C. d. terrificus and C. d. collilineatus venoms collected in
crotamine-negative regions of Southeastern and Midwestern
Brazil. On the other hand, the low amount of crotamine in the
venom pooled from C. d. terrificus specimens from Southeast-
ern Brazil (Fig. 5A) may explain the very weak immunor-
ecognition capability of the IVB antivenom toward crotamine.
On the other hand, the failure of snake antivenoms to

Fig. 6 – Immunodepletion of proteins from C. d. terrificus venoms by Brazilian anti-crotalic antivenoms. Panels A and B display,
respectively, reverse-phase separations of the proteins from pooled venom of adult C. d. terrificus recovered after antivenomic
treatment of the crude venom with the anti-crotalic antivenoms manufactured in Instituto Butantan (But) and Instituto Vital
Brazil (IVB). Panel C, reverse-phase HPLC of the non-immunodepleted proteins from the pooled venom of neonate C. d. terrificus
after incubation with antivenom from Instituto Butantan.
1770 J O U R NA L OF PR O TE O MI CS 73 ( 20 1 0 ) 1 7 5 8– 1 7 7 6
Figure 6 (continued).
recognize specific low molecular components of the venom
used for hyperimmunization, predominantly PLA2 proteins
and α-neurotoxins has been also reported [46–49,90–92]. The
possibility that lack of immunoreactivity against crotamine
was due to weak immunogenicity of the toxin was investi-
gated by immunizing rabbits against crotamine-positive C. d.
terrificus venom. The results, shown in Fig. 9, clearly
demonstrate that crotamine is able to induce a strong
immune response and that the purified rabbit anti-crotamine
IgG antibodies efficiently immunoprecipitate this toxin.
3.5. Concluding remarks and perspectives
This work supports a previous report pointing to neurotoxicity
as an adaptive trend along Crotalus dispersal in South
America [24]. In addition, results presented here indicate
that this venom specialization trend occurred concomitantly
with decrease of non-neurotoxic D49-PLA2 concentration and
with an enhanced expression of crotamine among C. durissus
ssp. coincident with the direction of the dispersal of this taxa.
The increased frequency of crotamine-positive populations
from northeastern Brazil to northern Argentina, suggests an
adaptive value for crotamine. Besides ecological and taxono-
mical implications, the identification of evolutionary trends,
as reported here, may have an impact in snakebite manage-
ment. Hence, adequate treatment of snakebite envenoming is
critically dependent on the parenteral administration of an
appropriate antidote that neutralizes the venom-induced
pathology, and the antivenom design requires a careful
consideration of the venom mixtures to be used in the
immunization protocol [34,36].
Accidental envenomations by subspecies of C. durissus are
usually serious and frequently fatal in the absence of adequate
treatment. In Brazil, 9% of the 26,688 snakebite cases reported
to have occurred between 2000 and 2007 were inflicted by
Crotalus durissus ssp. [93], and the case fatality rate ranged
from 0.8% to 2.53% [94], thus constituting a relevant public
health issue. The anti-crotalic antivenom produced at Insti-
tuto Butantan (But) (São Paulo) against C. d. terrificus venom
shows a very high effectiveness in the neutralization of the
lethal, myotoxin, and neurotoxic effects of venoms of C.
durissus subspecies [63]. The anti-crotalic antivenom manu-
factured at Instituto Vital Brazil (IVB) (Niterói, Rio de Janeiro)
has also been reported to neutralize the lethal activity of C. d.
terrificus venom [95]. This activity is mainly attributable to
crotoxin-induced neurotoxicity [16,17], and monospecific anti-
crotoxin antibodies neutralize the lethal activity of C. d.
terrificus [96] and C. d. cascavella [97] venoms. Our antivenomic
results indicate that both, the But and the IVB antivenoms
exhibit suboptimal crotoxin immunodepleting activity. This
does not seem to be due to low immunogenicity of crotoxin, as
evidenced by the fact that an experimental antiserum against
the venom of C. d. terrificus developed in rabbits in our
laboratory is capable of quantitatively immunoprecipitate
the neurotoxin (Fig. 9).
Envenoming by C. durissus is also frequently associated
with haemostatic disorders, likely due to the action of
thrombin-like enzymes [98]. Our results indicating an im-
paired capability of the two Brazilian anti-crotalic antivenoms
sampled to immunodeplete serine proteinase 19 from the
venom of C. d. cascavella (Fig. 7) should raise some concern
among antivenom producers to try to improve this activity.
However, the most unexpected and disturbing outcome of our
work is the absolute ineffectiveness of both, the But and IVB,
antivenoms to immunorecognize crotamine. Although from
its high LD50, crotamine seems not represent a life-threaten-
ing toxin, its role in human envenomation has not been
entirely clarified. Crotamine produces muscle contracture of
peripheral origin, as well as spastic paralysis of skeletal
muscle in mammals [99,100], and its reported myolytic
 J O U R NA L OF PR O TE O MI CS 7 3 ( 2 01 0 ) 1 7 5 8– 1 7 7 6 1771

Fig. 7 – Immunodepletion of proteins from C. d. cascvavella venom by Brazilian anti-crotalic antivenoms. Panels A and B display,
respectively, reverse-phase separations of the proteins from pooled venom of adult C. d. cascavella recovered after antivenomic
treatment of the crude venom with the anti-crotalic antivenoms manufactured in Instituto Butantan (But) and Instituto Vital
Brazil (IVB).

activity may be consequence of impairing voltage-gated
sodium channels of skeletal muscle sarcolemma, and of
lysosomal membrane permeabilization leading to activation
of calcium-dependent proteases [101]. Crotamine is able to
induce an immune response in rabbits, and therefore the
negligible titer of the But and IVB antivenoms against
crotamine may be due to an unfortunate choice of the venoms
(from crotamine-negative populations) used in the immuni-
zation mixture. The increasing frequency of crotamine in
southern populations of C. d. terrificus should be reason enough
to warn about the need to develop an antivenom capable of
neutralizing this toxin.
As a whole, our data suggest that the current immunization
protocols need to be revised in order to generate an improved
anti-crotalic antiserum. Optimization of the dose-efficacy of
the antivenom will result in the need for lower volumes to
effect treatment, with a consequent decrease in the risk of
serum-sickness and anaphylactic adverse effects. Venom
phenotyping by reverse-phase HPLC may be considered to
select the set of venoms containing all desired immunogens.
Our antivenomic approach is simple and easy to implement,
and may thus represent a useful proteomic tool for monitoring
and quantifying the onset of the hyperimmunized animal's
immune response during antivenom production.
1772 J O U R NA L OF PR O TE O MI CS 73 ( 20 1 0 ) 1 7 5 8– 1 7 7 6

Fig. 8 – Western blot analysis. Crotamine isolated by reverse-phase HPLC from pooled C. durissus collilineatus venom (lane 1),
and the proteins from pooled C. d collilineatus venom (lane 2); pooled C. durissus terrificus venom (lane 3); individual C. d.
collilineatus venom (lane 4), and pooled C. d. cascavella venom were separated by SDS-PAGE (left panel), electroblotted onto
nitrocellulose membrane and probed with the anti-crotalic antivenoms manufactured in Instituto Butantan (Panel A) and
Instituto Vital Brazil (Panel B). The two-headed arrow indicates the position of crotamine.

Program, Fundação Carlos Chagas Filho de Amparo à

Acknowledgements
This work has been financed by grants BFU2007-61563 and
BFU2010-17373 from the Ministerio de Ciencia e Innovación,
Madrid, Spain, 2008/11688-5 and 2009/09698-5 from Fundação
de Amparo a Pesquisa do Estado de São Paulo (FAPESP),
and grants from Conselho Nacional de Desenvolvimento
Científico e Tecnológico (CNPq), CNPq National Institute
Pesquisa do Estado do Rio de Janeiro (FAPERJ), Financiadora
de Estudos e Projetos (FINEP), and Fundação de Amparo a
Pesquisa do Estado de Minas Gerais (FAPEMIG). CCN and RSR
are recipients of fellowships from CNPq and FAPEMIG,
respectively. MMSS gratefully acknowledges Fundación Car-
olina (http://www.fundacioncarolina.es) for providing a Fel-
lowship of the Mobility Programme for Brazilian University
Professors.
 J O U R NA L OF PR O TE O MI CS 7 3 ( 2 01 0 ) 1 7 5 8– 1 7 7 6 1773

Fig. 9 – Immunodepletion of proteins from C. d. terrificus venom by an experimental antivenom generated in rabbits against
C. d. terrificus venom. Reverse-phase separation of the proteins from the crotamine-positive venom of adult C. d. terrificus
(Southern population) recovered after antivenomic treatment of the crude venom with an experimental antivenom raised in
rabbits against the same venom. Insets, left panel, specificity control: reverse-phase separation of the proteins from the
crotamine-positive venom of adult C. d. terrificus (Southern population) recovered after treatment of the crude venom with
non-immune IgG; Right panel: lane a, SDS-PAGE of the C. d. terrificus venom used as immunogen; lane b, molecular mass
markers; lane c, Ponceau red-stained nitrocellulose membrane showing electroblotted C. d. terrificus venom proteins;
lanes d and e, nitrocellulose membranes probed with purified non-immune and immune IgG, respectively. Ctx, crotoxin; Cro,
crotamine.

REFERENCES Sistrurus. Venomous animals and their venoms, vol. 2.

[1] Gloyd HK. The rattlesnakes, Genera Sistrurus and Crotalus: A
study in zoogeography and evolution. Chicago: Chicago
Academy of Sciences; 1940. p. 1–270. Special Publication
No. 4.
[2] Brattstrom BH. Evolution of the pitvipers. Transactions of the
San Diego Society of Natural History, vol. 13; 1964. p. 185–268.
[3] Knight A, Styler D, Pelikan S, Campbell JA, Densmore LD,
Mindrell DP. Choosing among hypotheses of rattlesnake
phylogeny: a best-fit rate test for DNA sequence data. Syst
Biol 1993;42:356–67.
[4] Klauber LM. Rattlesnakes: their habitats, life histories, and
influence on mankind. Second edition. Berkeley: University
of California Press; 1997.
[5] Place AJ, Abramson CI. A quantitative analysis of the
ancestral area of rattlesnakes. J Herpetol 2004;38:152–6.
[6] Castoe TA, Daza JM, Smith EN, Sasa M, Kuch U, Campbell JA,
et al. Comparative phylogeography of pitvipers suggests a
consensus of ancient Middle American highland biogeogra-
phy. J Biogeogr 2009;36:88–103.
[7] Greene H. Snakes: the evolution of mystery in nature.
Berkeley: Univ. of California Press; 1997.
[8] Parkinson CL, Campbell JA, Chippindale P. Multigene
phylogenetic analysis of pitvipers, with comments on their
biogeography. In: Höggren M, Schuett GW, Greene H, Douglas
ME, editors. Biology of the vipers. Eagle Mountain, UT: Eagle
Mountain Publishing; 2002. p. 93–110.
[9] Klauber LM. Classification, distribution and biology of the
venomous snakes of northern Mexico, the United States and
Canada. In: Bücherl W, Buckley EE, editors. Crotalus and
Venomous vertebratesNew York & London: Academic Press;
1971. p. 115–56.
[10] Campbell JA, Lamar WW. The venomous reptiles of the
Western Hemisphere. Ithaca (NY) and London: Comstock
Publishing Associates; 2004.
[11] Hoser RT. A reclassification of the rattlesnakes; species
formerly exclusively referred to the genera Crotalus and
Sistrurus. Aust J Herpetol 2009;6:1–21.
[12] Wüster W, Ferguson JE, Quijada-Mascareñas JA, Pook CE,
Salomão MG, Thorpe RS. Tracing an invasion: landbridges,
refugia and the phylogeography of the Neotropical
rattlesnake (Serpentes: Viperidae: Crotalus durissus). Mol Ecol
2005;14:1095–108.
[13] Quijada-Mascareñas JA, Ferguson JE, Pook CE, Salomão MG,
Thorpe RS, Wüster W. Phylogeographic patterns of
trans-Amazonian vicariants and Amazonian biogeography:
the Neotropical rattlesnake (Crotalus durissus complex) as an
example. J Biogeogr 2007;34:1296–312.
[14] Cardoso JLC. Animais peçonhentos no Brasil: biologia clínica
e terapêutica dos acidentes. São Paulo: Sarvier; 2003.
[15] Santoro ML, Sousa-e-Silva MCC, Gonçalves LRC,
Almeida-Santos SM, Cardoso DF, Laporta-Ferreiro LI, et al.
Comparison of the biological activities in venoms from three
subspecies of South-American rattlesnake Crotalus durissus
terrificus, C. durissus cascavella and C. durissus collilineatus.
Comp Biochem Physiol 1999;122C:61–73.
[16] Oshima-Franco Y, Hyslop S, Prado JF, Cruz-Hoffling MA,
Rodriguez LS. Neutralizing capacity of antisera raised in
horses and rabbits against Crotalus durissus terrificus
(South-American rattlesnake) venom and its main toxin,
crotoxin. Toxicon 1999;37:1341–57.
1774 J O U R NA L OF PR O TE O MI CS 73 ( 20 1 0 ) 1 7 5 8– 1 7 7 6
[17] Vital-Brazil O. Pharmacology of crystalline crotoxin. II.
Neuromuscular blocking action. Mem Inst Butantan 1966;33:
981–92.
[18] Azevedo-Marques MM, Hering SE, Cupo P. Acidente crotálico.
In: Cardoso JLC, Franca FOS, Wen FH, Málaque CMS, Haddad
V, editors. Animais Peçonhentos no Brasil. Biología, Clínica e
Terapeutica dos Acidentes, 2nd editionSao Paulo: Sarvier;
2009. p. 108–15.
[19] Warrell DA. Snakebites in Central and South America:
epidemiology, clinical features, and clinical management.
In: Campbell JA, Lamar WW, editors. The venomous reptiles
of the Western Hemisphere. Ithaca and London: Comstock
Publishing Associates; 2004. p. 709–61.
[20] Bon C, Bouchier C, Choumet V, Faure G, Jiang MS, Lambezat
MP, et al. Crotoxin, half-century of investigations on a
phospholipase A2 neurotoxin. Act Physiol Pharmacol
Latinoam 1989;39:439–48.
[21] Bon C. Multicomponent neurotoxic phospholipases A2. In:
Kini RM, editor. Venom phospholipase A2 enzymes:
structure, function and mechanism. Chichester: Wiley; 1997.
p. 269–85.
[22] Sampaio SC, Hyslop S, Fontes MR, Prado-Franceschi J,
Zambelli VO, Magro AJ, et al. Crotoxin: novel activities for a
classic beta-neurotoxin. Toxicon 2010;55:1045–60.
[23] Boldrini-França J, Rodrigues RS, Fonseca FP, Menaldo DL,
Ferreira FB, Henrique-Silva F, et al. Crotalus durissus
collilineatus venom gland transcriptome: analysis of gene
expression profile. Biochimie 2009;91:586–95.
[24] Calvete JJ, Sanz L, Cid P, de la Torre P, Flores-Díaz M, dos
Santos MC, et al. Snake venomics of the Central American
rattlesnake Crotalus simus and the South American Crotalus
durissus complex points to neurotoxicity as an adaptive
paedomorphic trend along Crotalus dispersal in South
America. J Proteome Res 2010;9:528–44.
[25] Chang CC, Tseng H. Effect of crotamine, a toxin of South
American rattlesnake venom, on the sodium channel of
murine skeletal muscle. Br J Pharmacol 1978;63:551–9.
[26] Oguiura N, Boni-Mitak M, Rádis-Baptista G. New view
on crotamine, a small basic polypeptide myotoxin from
South American rattlesnake venom. Toxicon 2005;46(18):
363–70.
[27] Lewontin RC. The genetic basis of evolutionary change.
New York: Columbia University Press; 1974.
[28] Orr HA, Coyne JA. The genetics of adaptation — a
reassessment. Am Nat 1992;140:725–42.
[29] Golding GB, Dean AM. The structural basis of molecular
adaptation. Mol Biol Evol 1998;15:355–69.
[30] Gibbs HL, Mackessy SP. Functional basis of a molecular
adaptation: prey-specific toxic effects of venom from
Sistrurus rattlesnakes. Toxicon 2009;53:672–9.
[31] Gibbs HL, Sanz L, Calvete JJ. Snake population venomics:
proteomics-based analyses of individual variation reveals
gene regulation effects on venom protein expression in
Sistrurus rattlesnakes. J Mol Evol 2009;68:113–25.
[32] Greene HW. Dietary correlates of the origin and radiation of
snakes. Am Zool 1983;23:431–41.
[33] Espino-Solis GP, Riaño-Umbarila L, Becerril B, Possani LD.
Antidotes against venomous animals: state of the art and
prospectives. J Proteomics 2009;72:183–99.
[34] Gutiérrez JM, León G. Snake antivenoms. In: De Lima ME,
Pimenta AMC, Martin-Euclaire MF, Zingali RB, editors.
Animal toxins: state of the art. Perspectives in health and
biotechnologyBelo Horizonte, Brazil: Editora UFMG; 2009.
p. 393–421.
[35] Calvete JJ, Sanz L, Angulo Y, Lomonte B, Gutiérrez JM.
Venoms, venomics, antivenomics. FEBS Lett 2009;583:
1736–43.
[36] Gutiérrez JM, Lomonte B, León G, Alape-Girón A, Flores-Díaz
M, Sanz L, et al. Snake venomics and antivenomics:
proteomic tools in the design and control of antivenoms for
the treatment of snakebite envenoming. J Proteomics
2009;72:165–82.
[37] Chippaux JP, Willians V, White J. Snake venom variability:
methods of study, results and interpretation. Toxicon
1991;29:1279–303.
[38] Alape-Girón A, Sanz L, Escolano J, Flores-Díaz M, Madrigal M,
Sasa M, et al. Snake venomics of the lancehead pitviper
Bothrops asper: geographic, individual, and ontogenetic
variations. J Proteome Res 2008;7:3556–71.
[39] Núñez V, Cid P, Sanz L, De La Torre P, Angulo Y, Lomonte B,
et al. Snake venomics and antivenomics of Bothrops atrox
venoms from Colombia and the Amazon regions of Brazil,
Perú and Ecuador suggest the occurrence of geographic
variation of venom phenotype by a trend towards
paedomorphism. J Proteomics 2009;73:57–78.
[40] Warrell DA. Geographical and intraspecies variation in the
clinical manifestations of envenoming by snakes. In: Thorpe
RS, Wüster W, Malhotra A, editors. Venomous snakes:
ecology, evolution and snakebite. Oxford: Clarendon Press;
1997. p. 189–203.
[41] Chippaux JP. Impact of the environment on envenomation
incidence and severity. Med Sci 2009;25:858–62.
[42] Fasman DG. Practical Handbook of Biochemistry and
Molecular Biology. Boston: CRC Press; 1992.
[43] Calvete JJ, Juárez P, Sanz L. Snake venomics. Strategy and
applications. J Mass Spectrom 2007;42:1405–14.
[44] Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z,
Miller W, et al. Gapped BLAST and PSI-BLAST: a new
generation of protein database search programs. Nucleic
Acids Res 1997;25:3389–402.
[45] Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of
proteins from polyacrylamide gels to nitrocellulose sheets:
procedure and some applications. Proc Natl Acad Sci USA
1979;76:4350–4.
[46] Lomonte B, Escolano J, Fernández J, Sanz L, Angulo Y,
Gutiérrez JM, et al. Snake venomics and antivenomics of the
arboreal neotopical pitvipers Bothriechis lateralis and
Bothriehis schlegelii. J Proteome Res 2008;7:2445–57.
[47] Gutiérrez JM, Sanz L, Escolano J, Fernández J, Lomonte B,
Angulo Y, et al. Snake venomic of the Lesser Antillean pit
vipers Bothrops caribbaeus and Bothrops lanceolatus:
correlation with toxicological activities and
immunoreactivity of a heterologous antivenom. J Proteome
Res 2008;7:4396–408.
[48] Calvete JJ, Borges A, Segura A, Flores-Díaz M, Alape-Girón A,
Gutiérrez JM, et al. Snake venomics and antivenomics of
Bothrops colombiensis, a medically important pitviper of the
Bothrops atrox-asper complex endemic to Venezuela:
contributing to its taxonomy and snakebite management.
J Proteomics 2009;72:227–40.
[49] Gutiérrez JM, Sanz L, Flores-Díaz M, Figueroa L, Madrigal M,
Herrera M, et al. Impact of regional variation in Bothrops
asper snake venom on the design of antivenoms: integrating
antivenomics and neutralization approaches. J Proteome Res
2010;9:564–77.
[50] Calvete JJ, Cid P, Sanz L, Segura A, Villalta M, León G, Harrison
R, Durfa N, Nasidi A, Theakston RG, Warrell D, Gutiérrez JM.
Antivenomic Assessment of the Immunological Reactivity of
EchiTAb-Plus-ICP®, an Antivenom for the Treatment of
Snakebite Envenoming in sub-Saharan Africa Am. J Trop
Med Hyg 2010;82:1194–201.
[51] Georgieva G, Öhler M, Seifert J, von Bergen M, Arni RK, Genov
N, Betzel C. Snake venomic of Crotalus durissus terrificuss:
correlation with pharmacological activities. J Proteome Res
2010;9:2302–16.
[52] Sanz L, Escolano J, Ferretti M, Biscoglio MJ, Rivera E, Crescenti
EJ, et al. Snake venomics of the South and Central American
Bushmasters. Comparison of the toxin composition of
 J O U R NA L OF PR O TE O MI CS 7 3 ( 2 01 0 ) 1 7 5 8– 1 7 7 6
Lachesis muta gathered from proteomic versus
transcriptomic analysis. J Proteomics 2008;71:46–60.
[53] Calvete JJ, Marcinkiewicz C, Sanz L. Snake venomics of Bitis
gabonica gabonica. Protein family composition, subunit
organization of venom toxins, and characterization of
dimeric disintegrins bitisgabonin-1 and bitisgabonin-2.
J Proteome Res 2007;6:326–36.
[54] Wagstaff SC, Sanz L, Juárez P, Harrison RA, Calvete JJ.
Combined snake venomics and venom gland transcriptomic
analysis of the ocellated carpet viper, Echis ocellatus.
J Proteomics 2009;71:609–23.
[55] Howgood BJ. Crotoxin, the phospholipase A2 neurotoxin
from the venom of Crotalus durissus terrificus. Mem Inst
Butantan 1990;52:21–2.
[56] Martins AMC, Toyama MH, Havt A, Novello JC, Marangoni S,
Fonteles MC, et al. Determination of Crotalus durissus
cascavella venom components that induce renal toxicity in
isolated rat kidneys. Toxicon 2002;40:1165–71.
[57] Amora DN, Souza TS, Martins AMC, Barbosa PSF, Magalhães
MR, Toyama MH, et al. Effects of Crotalus durissus collilineatus
venom in the isolated rat kidney. Toxicon 2006;47:260–4.
[58] Gutiérrez JM, Cerdas L. Mechanism of action of myotoxins
isolated from snake venoms. Rev Biol Trop 1984;32:213–22.
[59] De Sousa-e-Silva MC, Tomy SC, Tavares FL, Navajas L,
Larsson MH, Lucas SR, et al. Hematological, hemostatic and
clinical chemistry disturbances induced by Crotalus durissus
terrificus snake venom in dogs. Hum Exp Toxicol 2003;22:
491–500.
[60] de Oliveira DGL, Murakami MT, Cintra ACO, Franco JJ,
Sampaio SV, Arni RK. Functional and structural analysis of
two fibrinogen-activating enzymes isolated from the
venoms of Crotalus durissus terrificus and Crotalus durissus
collilineatus. Acta Biochim Biophys Sin 2009;41:21–9.
[61] Alexander G, Grothusen J, Zepeda H, Schwartzman RJ.
Gyroxin, a toxin from the venom of Crotalus durissus terrificus,
is a thrombin-like enzyme. Toxicon 1988;26:953–60.
[62] Guarnieri MCI, Melo ESLI, Melo KMSII, Albuquerque-Modesto
JCIII, Prieto-da-Silva ARBIV, Rádis-Baptista G. Cloning of a
novel acidic phospholipase A2 from the venom gland of
Crotalus durissus cascavella (Brazilian northeastern rattle-
snake). J Venom Anim Toxins Incl Trop Dis 2009;15:4. doi:
10.1590/S1678-91992009000400012.
[63] Saravia P, Rojas E, Arce V, Guevara C, López JC, Chaves E,
et al. Geographic and ontogenic variability in the venom of
the neotropical rattlesnake Crotalus durissus:
pathophysiological and therapeutic implications. Rev Biol
Trop 2002;50:337–46.
[64] Tavares NA, Correia JM, Guarneri MC, Lima-Filho JL,
Prieto-da-Silva AR, Radis-Baptista G. Expression of mRNAs
coding for VAP1/crotastatin-like metalloproteases in the
venom glands of three South American pit vipers assessed
by quantitative real-time PCR. Toxicon 2008;52:897–907.
[65] Toyama MH, Toyama Dde O, Passero LF, Laurenti MD,
Corbett CE, Tomokane TY, et al. Isolation of a new L-amino
acid oxidase from Crotalus durissus cascavella venom. Toxicon
2006;47:47–57.
[66] Prado-Franceschi J, Brazil OV. Convulxin, a new toxin from
the venom of the South American rattlesnake Crotalus
durissus terrificus. Toxicon 1981;19:875–87.
[67] Murakami MT, Zela SP, Gava LM, Michelan-Duarte S, Cintra
ACO, Arni RK. Crystal structure of the platelet activator
convulxin, a disulfide-linked 44 cyclic tetramer from the
venom of Crotalus durissus terrificus. Biochem Biophys Res
Commun 2003;310:478–82.
[68] Batuwangala T, Leduc M, Gibbins JM, Bon C, Jones EY.
Structure of the snake venom toxin convulxin. Acta
Crystallogr 2004;D60:46–53.
[69] Mackessy SP. Venom composition in rattlesnakes: trends
and biological significance. In: Hayes WK, Beaman KR,
1775
Cardwell MD, Bush SP, editors. The biology of rattlesnakes.
Loma Linda, California: Loma Linda University Press; 2008.
p. 495–510.
[70] Mackessy SP. Evolutionary trends in venom composition in
the Western Rattlesnakes (Crotalus viridis sensu lato):
Toxicity vs. tenderizers. Toxicon 2010;55:1463–74.
[71] Furtado MFD, Santos MC, Kamiguti AS. Age-related
biological activity of South American rattlesnake (Crotalus
durissus terrificus) venom. J Venom Anim Toxins Incl Trop Dis
2003;9:186–201.
[72] Powell RL, Lieb CS. Perspectives on venom evolution in
Crotalus. In: Hayes WK, Beaman KR, Cardwell MD, Bush SP,
editors. The biology of rattlesnakes. Loma Linda, California:
Loma Linda University Press; 2008. p. 551–6.
[73] John TR, Smith LA, Kaiser II. Genomic sequences encoding
the acidic and basic subunits of Mojave toxin: unusually high
sequence identity of non-coding regions. Gene 1994;139:
229–34.
[74] Tchorbanov B, Grishin E, Aleksiev B, Ovchinnikov Y. A
neurotoxic complex from the venom of the Bulgarian viper
(Vipera ammodytes meridionalis) and a partial amino acid
sequence of the toxic phospholipase A2. Toxicon 1978;16:
37–44.
[75] Banumathi S, Rajashankar KR, Nötzel C, Aleksiev B, Singh TP,
Genov N, et al. Structure of the neurotoxic complex vipoxin
at 1.4 Å resolution. Acta Crystrallogr 2001;D57:1552–9.
[76] de Haro L, Robbe-Vincent A, Saliou B, Valli M, Bon C,
Choumet V. Unusual neurotoxic envenomations by Vipera
aspis aspis snakes in France. Hum Exp Toxicol 2002;21:137–45.
[77] Ferquel E, de Haro L, Jan V, Guillemin I, Jourdain S, Teynié A,
d'Alayer J, Choumet V. Reappraisal of Vipera aspis venom
neurotoxicity. PLoS ONE 2007;2(11):e1194.
[78] Fadel V, Bettendorff P, Hermann T, de Azevedo Jr WF,
Oliveira EB, Yamana T, Wüthrich K. Automated NMR
structure determination and disulfide bond identification of
the myotoxin crotamine from Crotalus durissus terrificus.
Toxicon 2005;46:759–67.
[79] Schenberg S. Geographical pattern of crotamine distribution
in the same rattlesnake subspecies. Science 1959;129:1361–3.
[80] Gonçalves JM, Arantes EG. Estudos sobre venenos de
serpentes brasileiras. III — Determinação quantitativa de
crotamina no veneno de cascavel brasileira. An Acad Bras
Ciênc 1956;28:369–71.
[81] Barrio A, Vital Brazil O. Neuromuscular action of the Crotalus
terrificus terrificus (Laur.) poisons. Acta Physiol Latinoam
1951;1:291–308.
[82] Warren WC, Hillier LW, Marshall Graves JA, Birney E, Ponting
CP, Grützner F, et al. Genome analysis of the platypus reveals
unique signatures of evolution. Nature 2008;453:175–83.
[83] Rádis-Baptista G, Kubo T, Oguiura N, Da Silva ARBP, Hayashi
MAF, Oliveira EB, et al. Identification of crotasin, a
crotamine-related gene of Crotalus durissus terrificus. Toxicon
2004;43:751–9.
[84] Rádis-Baptista G, Kubo T, Oguiura N, Svartman M, Almeida
TMB, Batistic RF, et al. Structure and chromosomal
localization of the gene for crotamine, a toxin from the South
American rattlesnake, Crotalus durissus terrificus. Toxicon
2003;42:747–52.
[85] Oguiura N, Collares MA, Furtado MFD, Ferrarezzi H, Suzuki H.
Intraspecific variation of the crotamine and crotasin genes
in Crotalus durissus rattlesnakes. Gene 2009;446:35–40.
[86] Oguiura N, Boni-Mitake M, Rádis-Baptista G. New view on
crotamine, a small basic polypeptide myotoxin from South
American rattlesnake venom. Toxicon 2005;46:363–70.
[87] Vanzolini PE, Ramos-Costa AMM, Vitt LJ. Répteis das
Caatingas. Rio de Janeiro: Academia Brasileira de Ciências;
1980. p. 67–8.
[88] Salomão MG, Almeida-Santos SM, Puorto G. Activity pattern
of Crotalus durissus (Viperidae, Crotalinae): feeding,
1776 J O U R NA L OF PR O TE O MI CS 73 ( 20 1 0 ) 1 7 5 8– 1 7 7 6
reproduction and snake bite. Stud Neotrop Fauna Environ
1995;30:101–6.
[89] Mancin AC, Soares AM, Adrião-Escarso SH, Faça VM, Greene
LJ, Zuccolotto S, et al. The analgesic activity of crotamine, a
neurotoxin from Crotalus durissus terrificus (South American
rattlesnake) venom: a biochemical and pharmacological
study. Toxicon 1998;36:1927–37.
[90] Fry BG, Wickramaratna JC, Jones A, Alewood PF, Hodgson
WC. Species and regional variations in the effectiveness of
antivenom against the in vitro neurotoxicity of death adder
(Acanthophis) venoms. Toxicol Appl Pharmacol 2001;175:140–8.
[91] Judge RK, Henry PJ, Mirtschin P, Jelinek G, Wilce JA. Toxins
not neutralized by brown snake antivenom. Toxicol Appl
Pharmacol 2006;213:117–25.
[92] Correa-Netto C, Teixeira-Araujo R, Aguiar AS, Melgarejo AR,
De-Simone SG, Soares MR, et al. Immunome and venome of
Bothrops jararacussu: a proteomic approach to study the
molecular immunology of snake toxins. Toxicon 2010;55:
1222–35.
[93] De Oliveira RC, Wen FH, Sifuentes DN. Epidemiologia dos
accidentes poe animais peçonhentos. In: Cardoso JLC, França
FOS, Wen FH, Málaque CMS, Haddad V, editors. Animais
Peçonhentos no Brasil. Biologia, Clínica e Terapêutica dos
Acidentes, 2nd editionSao Paulo: Sarvier; 2009. p. 6–21.
[94] Jorge MT, Ribeiro LA. Epidemiology and clinical picture of
accidental bites by the South American rattlesnake (Crotalus
durissus). Rev Inst Med Trop São Paulo 1992;34:347–54.
[95] Brazilian Pharmacopeia. Brazilian Pharmacopeia
Convention. 4nd ed. São Paulo: Atheneu; 2002.
View publication stats
[96] Rodriguez JP, De Marzi M, Maruñak S, Malchiodi EL, Leiva LC,
Acosta O. Rabbit IgG antibodies against phospholipase A2
from Crotalus durissus terrificus neutralize the lethal activity
of the venom. Medicina (B Aires) 2006;66:512–6.
[97] Beghini DC, Hernandez-Oliveira S, Rodrigues-Simioni L,
Novello JC, Hyslop S, Marangoni S. Anti-sera raised in rabbits
against crotoxin and phospholipase A2 from Crotalus durissus
cascavella venom neutralize the neurotoxicity of the venom
and crotoxin. Toxicon 2004;22:141–8.
[98] Sano-Martins IS, Tomy SC, Campolina D, Dias MB, de Castro
SC, de Sousa-e-Silva MC, et al. Coagulopathy following lethal
and non-lethal envenoming of humans by the South
American rattlesnake (Crotalus durissus) in Brazil. QJM
2001;94:551–9.
[99] Bourillet F. Comparative neuromuscular action of crotamine
and veratrine. Ann Pharm Fr 1970;28:535–44.
[100] Cheymol J, Gonçalves JM, Bourillet F, Roch-Arveiller M. A
comparison of the neuromuscular action of crotamine and
the venom of Crotalus durissus terrificus var. crotaminicus.
Isolated preparations. Toxicon 1971;9:287–9.
[101] Hayashi MA, Nascimento FD, Kerkis A, Oliveira V, Oliveira
EB, Pereira A, et al. Cytotoxic effects of crotamine are
mediated through lysosomal membrane permeabilization.
Toxicon 2008;52:508–17.
[102] Gutiérrez JM, Dos Santos MC, Furtado MF, Rojas G.
Biochemical and pharmacological similarities between the
venoms of newborn Crotalus durissus durissus and adult
Crotalus durissus terrificus rattlesnakes. Toxicon 1991;29:
1273–7.