General biochemical and immunological characteristics of the venom

from Peruvian scorpion Hadruroides lunatus

F. Costal-Oliveira
a
, C.G. Duarte
a
, R.A. Machado de Avila
a
, M.M. Melo
c
, K.C.F. Bordon
d
,
E.C. Arantes
d
, N.C. Paredes
e
, B. Tintaya
e
, C. Bonilla
e
, R.E. Bonilla
a
, W.S. Suarez
e
, A. Yarleque
f
,
J.M. Fernandez
f
, E. Kalapothakis
b
, Carlos Chvez-Olrtegui
a,
*
a
Departamentos de Bioqumica e Imunologia, Universidade Federal de Minas Gerais, Belo Horizonte, 31270-901 Minas Gerais, Brazil
b
Biologia Geral, Instituto de Cincias Biolgicas, Universidade Federal de Minas Gerais, Belo Horizonte, 31270-901 Minas Gerais, Brazil
c
Escola de Veterinaria, Universidade Federal de Minas Gerais, Belo Horizonte, 31270-901 Minas Gerais, Brazil
d
Departamento de Fsica e Qumica, Faculdade de Cincias Farmacuticas de Ribeiro Preto-USP, Ribeiro Preto-SP, Brazil
e
Instituto Nacional de Salud, Lima, Peru
f
Universidad Nacional Mayor de San Marcos, Lima, Peru
a r t i c l e i n f o
Article history:
Received 3 May 2012
Received in revised form 1 June 2012
Accepted 20 June 2012
Available online 29 June 2012
Keywords:
Hadruroides lunatus scorpion venom
Phospholipasic activity
LD
50
Anti-H. lunatus sera
a b s t r a c t
This communication describes the general biochemical properties and some immuno-
logical characteristics of the venom from the Peruvian scorpion Hadruroides lunatus, which
is the most medically relevant species in Peru. The soluble venom of this scorpion is toxic
to mice, the LD
50
determined was 0.1 mg/kg and 21.55 mg/kg when the venom was
injected intracranial or intraperitoneally, respectively. The soluble venom displayed
proteolytic, hyaluronidasic, phospholipasic and cardiotoxic activities. High performance
liquid chromatography of the soluble venom resulted in the separation of 20 fractions. Two
peptides with phospholipasic activity were isolated to homogeneity and their molecular
masses determined by mass spectrometry (MALDI TOF). Anti-H. lunatus venom sera were
produced in rabbits. Western blotting analysis showed that most of the protein content of
this venom is immunogenic. H. lunatus anti-venom displayed consistent cross-reactivity
with venom antigens from the new World-scorpions Tityus serrulatus and Centruroides
sculpturatus venoms; however, a weaker reactivity was observed against the venom
antigens from the old World-scorpion Androctonus australis Hector.
2012 Elsevier Ltd. All rights reserved.
1. Introduction
Peru is a country in western South America. It is
bordered on the north by Ecuador and Colombia, on the
east by Brazil, on the southeast by Bolivia, on the south by
Chile, and on the west by the Pacic Ocean. This nation has
a rich and diverse herpetic and arachnid fauna, with wide
geographical distribution. This biodiversity has not,
however, been properly studied.
Hadruroides (Pocock, 1893) is a scorpion genus included
in the family Iuridae, subfamily Charaboctoninae. This
genus comprises sixteen species and there members
appear brown in color with darker stains and have median
size of 80 mm (Ochoa and Prendini, 2010; Maury, 1975).
Hadruroides scorpions have been reported in Bolivia, Chile,
Colombia, Ecuador, Peru, and Venezuela (Mello-Leito,
1945; Esquivel de Verde, 1968; Kinzelbach, 1973; Maury,
1975; Cekalovic, 1983; Sissom and Fet, 2000), but are
actually restricted to Ecuador, Peru, northern Chile, and
several offshore islands, including the Galpagos
(Cekalovic, 1966; Maury, 1975; Francke and Soleglad, 1981).
* Corresponding author. Departamento de Bioqumica e Imunologia,
Instituto de Cincias Biolgicas, Universidade Federal de Minas Gerais,
Belo Horizonte, Brazil. CP: 486. CEP: 31270-901. Tel.: 55 31 3409 2625;
fax: 55 313 441 5963.
E-mail address: olortegi@icb.ufmg.br (C. Chvez-Olrtegui).
Contents lists available at SciVerse ScienceDirect
Toxicon
j ournal homepage: www. el sevi er. com/ l ocat e/ t oxi con
0041-0101/$ see front matter 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.toxicon.2012.06.013
Toxicon 60 (2012) 934942
Species of Hadruroides inhabit inter-Andean valleys, Pacic
desert, and dry forest habitats (Ochoa and Prendini, 2010).
Hadruroides lunatus (escorpion de los pedregales) is
the most medically relevant species in Peru. According to
the Health Ministry of Peru (Ministerio de Salud del Per,
2004), the number of human envenomation cases repor-
ted has increased during recent years, with most incidents
occurring in the Central Coast of the country, which
corresponds with the main area of geographical distribu-
tion of H. lunatus scorpions (Zavaleta et al., 1981). Severe
toxic effects by H. lunatus stings have not been noted in
humans; however, intense pain, edema and ulceration are
frequently described as symptoms (Zavaleta et al., 1981).
Different approaches are adopted for the treatment of
scorpion envenomations such as local care, analgesics and
antihistaminics (Ministrio de Sald, Peru, 2004). Never-
theless, there are no scientic data to support these treat-
ments. The Instituto Nacional de Salud (INS) in Lima, Peru
does not produce specic scorpion anti-venon (Ministrio
de Sald, Peru, 2004). Consequently, the treatment of
scorpion envenomations with specic anti-venom for
Peruvian species does not exist.
Very little is known about the structural and functional
characteristics of Peruvian scorpion venoms. The rst
toxicological information was obtained from research on
the H. lunatus species (Delgado and Pesce, 1967; Aguilar,
1968; Aguilar and Meneses, 1970 and Zavaleta et al.,
1981). The pharmacological effects described by Zavaleta
et al. (1981) showed that H. lunatus crude venom has
a low lethality in mice (LD
50
, 68 mg/kg i.p.) and, in dogs,
induces a fall in blood pressure. Neurotoxic activity in
insects, crustaceans and mice and antibacterial peptides
from the Hadruroides sp. crude venoms were showed by
Escobar et al. (2002) and Escobar and Flores, (2008).
However, proteolytic and phospholipasic activities of this
scorpion venoms are not shown in these studies. Despite
these published works, the studies with Peruvian scorpions
are still very preliminary.
In view of the lack of information on the general
characteristics of Hadruroides scorpion venoms, the main
goal of this work was to report additional biochemical
and toxic characterization of H. lunatus scorpion venom.
In this paper, the hyaluronidase, proteolytic, phospholi-
pase, cardiotoxic and lethal activities of H. lunatus crude
venom were investigated. This communication also
describes the separation of the soluble venom compo-
nents by SDS-PAGE and by high performance liquid
chromatography (HPLC). Furthermore, the last part of this
study shows, some immunological characteristics of
soluble whole venom using specic polyclonal rabbit anti-
H. lunatus antibodies.
2. Materials and methods
2.1. Animals and venoms
H. lunatus scorpions were collected in the region of
Atocongo (Lima, Peru) and maintained in the herpetarium
of the Centro Nacional de Produccin de Biologicos of
Instituto Nacional de Salud (INS), in Lima, Peru. Scorpions
were maintained in plastic boxes with water ad libitumand
were fed weekly with cockroaches. The venom from
mature scorpions was obtained by electrical stimulation
(12 V) of the telsons. The venom collected in micropipettes
was diluted in ultrapure water, pooled and stored at 20

C
until use. The protein concentration was determined by the
method of Lowry et al. (1951).
Tityus serrulatus mature scorpions were collected in the
region of Belo Horizonte and maintained at the Seo de
Animais Peonhentos of Ezequiel Dias Foundation
(FUNED) of Belo Horizonte, Brazil. The crude venoms were
obtained by electrical stimulation of the telsons, lyophi-
lized and stored at 20

C in the dark until use. The venoms

from the scorpions Androctonus australis hector and Cen-
truroides sculpturatus were obtained from the Laboratoire
de Biochimie, Facult de Mdecine, Marseille, France and
fromSigma Chemical Company, St. Louis, USA, respectively.
Male and female Swiss and C57BL/6 mice (weighing 18
22 g) and male Wistar rats (weighing 110150 g) were
maintained at the Centro de Bioterismo of the Instituto de
Cincias Biolgicas of the Universidade Federal de Minas
Gerais (UFMG), Belo Horizonte, MG, Brazil. All animals
received water and food under controlled environmental
conditions. The experimental protocols were approved by
the Ethics Committee on the Use of Laboratory Animals of
UFMG (CETEA-UFMG). Eight- to nine-week-old New Zea-
land rabbits were used to produce anti-H. lunatus and anti-
T. serrulatus sera. Animals were maintained and handled as
described previously.
2.2. Toxic activities of H. lunatus venom
2.2.1. Determination of median lethal dose (LD
50
)
The lethality was assessed by intraperitoneal (i.p.) and
intracranial (i.c.) routes. For the intraperitoneal route,
groups of four mice were injected with different doses of
venom (from 11.53 mg to 32.95 mg per kg of body weight)
dissolved in 0.5 mL of PBSBSA 0.1%. For the intra-
cerebroventricular route, groups of six mice were injected
with various doses of venom (from 0.075 mg to 0.8 mg per
kg) dissolved in 5 mL of PBSBSA 0.1%. Twenty four hours
later deaths were recorded and the LD
50
was then calcu-
lated by Probit analysis (Finney, 1971).
2.2.2. Determination of proteolytic activity (PA)
Proteolytic activity was measured with dimethylcasein
(Sigma) as described by Lin et al. (1969) with modications
described in Sanchez et al. (2000). Dilutions corresponding
to 5, 10, 20 and 40 mg of venom were used and absorbance
values were determined at 340 nm. One unit was dened as
DA 340 nm/min. Activity was expressed relative to protein
concentration (mg).
2.2.3. Determination of phospholipase A
2
(PLA
2
) activity
PLA
2
activity was measured using an indirect hemolytic
assay (Gutierrez et al., 1988). Increasing concentrations of H.
lunatus crude venom(0.0625, 0.125, 0.25, 0.5 and 1 mg) were
prepared ina nal volume of 15 mL inPBS andadded to 2 mm
wells in agarose gels (0.8% in phosphate buffered saline, pH
8.1) containing 1.2% sheep erythrocytes, 1.2% egg yolk as
a source of lecithin, and 100 mM CaCl
2
. The main fractions
obtained in the purication of the venomby HPLC were also
F. Costal-Oliveira et al. / Toxicon 60 (2012) 934942 935
tested. Plates were incubated at 37

C for 18 h and the
diameters of the hemolytic halos were measured. As
a control, 15 mL of PBS was tested. One unit (Minimum
Phospholipasic Dose MPD) corresponds to a minor
concentration of venom which produced a hemolytic halo
of 1 cmdiameter. Experiments were conducted in duplicate.
2.2.4. Determination of hyaluronidase activity (HA)
Hyaluronidase activity of the venom and its molecular
mass determination was analyzed by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis and performed
according to Cevallos et al. (1992). Briey, SDS-PAGE gels
were prepared with hyaluronan, which was incorporated
into the gels as a hyaluronidase substrate in the 10%
resolving gel at a nal concentration of 0.5 mg/mL.
Venom samples (10 and 5 mg), dispersed in Laemmli
buffer under non-reducing conditions and room temper-
ature, were electrophoresed at 90 V at room temperature
until the indicator reached the end of the gel. After
electrophoresis, the gel was washed twice in 5% Triton X-
100 in sodium phosphate buffer 0.1 M, pH 5.8, with
0.15 M NaCl for 1 h, once in 0.05% Triton X-100 in buffer
pH 5.8 for an hour and nally in buffer pH 5.8 without
Triton X-100 for 10 min. All these steps were performed at
room temperature. Subsequently, the gel was placed in
buffer without Triton X-100 and incubated at 37

C for the
desired amount of time. After incubation, the gels were
washed twice for 15 min in 0.015 M TrisHCl, pH 7.95 and
then stained under gentle rotation for at least 5 h in the
dark. A stock solution of 0.1% Stains-all (1-ethyl-2-[3-(1-
ethylnaphthol [1,2-d] thiazolin-2-ylidene)-2-
methylpropenyl] Naphthol [1,2-d] thiazolium bromide;
Cat. No. 2718 fromEastman Kodak Company, Rochester,
NY, USA) in pure formamide was stored in a dark
container. The dye solution was freshly prepared by
combining 5 mL dye stock with 5 mL of formamide, 20 mL
of isopropanol, 1.5 mL of 1 M TrisHCl, pH 7.95, and
deionized water to a volume of 100 mL (Green et al.,
1973). The gel was destained in 5% formamide and 20%
isopropanol in 0.015 M TrisHCl, pH 7.95, until bands of
activity become clear. The protein molar mass standards
were always separated at the extreme end of the gel plate
and following electrophoresis, the line was carefully
sectioned and stained with Coomassie brilliant blue
R-250.
2.2.5. Determination of cardiotoxic activity
CK and CKMB levels in the serum of envenomed rats
were determined as a measured of the cardiotoxicity of H.
lunatus venom. Groups of six Wistar rats were injected
intraperitoneally (i.p.) with 750 mg of soluble venom or
ultra-pure water (control). The animals were kept under
inhalation anesthesia with morphine (2.5 mg/kg) and
diazepam (2.5 mg/kg), injected via the intramuscular route
(Flecknell et al., 1996). After 30 min of envenoming, blood
was collected by cardiac puncture. Blood was then centri-
fuged (3000 rpm for 5 min) and serum used for biochem-
ical analysis. The levels of creatine kinase isoenzyme MB
(CKMB) and total creatine kinase (CK) were measured
using commercial kits from Bioclin (Quibasa, Brazil) and
a Thermo Plate Analyzer Basic instrument.
2.3. Chromatography and mass spectrometry assays
Chromatographic fractionation of H. lunatus venom was
performed using high performance liquid chromatography
(HPLC). Briey, 1 mg of crude venom was applied to
a reverse phase column. The column used in this assay was
a Shimadzu-Pack CLC-ODS C18 (6 150 mm) eluted at
1 mL/min with a linear gradient of 0.1% TFA in water and
acetonitrile, solutions A and B, respectively. After column
equilibration the venom fractions were separated with
a linear gradient from solution A to 60% solution B, running
for 60 min.
Fractions were then subjected to MALDI-TOF-TOF anal-
yses. MS analysis was performed using a MALDI-TOF-TOF
AutoFlex III (Bruker Daltonics) instrument in positive/
reector mode controlled by the FlexControl software.
Instrument calibration was achieved by using Peptide
Calibration Standard IV (Bruker Daltonics) as a reference
and using sinapinic acid as a matrix. The peak was spotted
to MTP AnchorChip 400/384 (Bruker Daltonics) targets
using standard protocols for the dried droplet method.
2.4. Immunological studies
2.4.1. Immunization protocols
Adult New Zealand female rabbits were used for the
production of anti-H. lunatus and anti-T. serrulatus venom
antibodies. After collection of pre-immune sera, the
animals received an initial subcutaneous injection of 100 mg
of whole venom in complete Freunds adjuvant (day 1).
Three booster injections were made subcutaneously 14, 21
and 28 days later with a lower dose (50 mg) in incomplete
Freunds adjuvant. The animals were bled one week after
the last injection.
2.4.2. Indirect ELISA and immunoblotting assays
Maxisorp microtitration plates (Nalge Nunc, USA) were
coated overnight at 5

C with 100 mL of a 10 mg/mL solution
of H. lunatus, T. serrulatus, A. australis or C. sculpturatus
whole venomin carbonate buffer pH9.6. After blocking (3%
powdered milk in PBS) and washing (0.05% Tween-saline),
sera from pre-immune and immune rabbit were added in
different dilutions and incubated for 1 h at 37

C. Plates
were washed and incubated with anti-rabbit IgG conju-
gated with peroxidase diluted 1:4000, for 1 h at 37

C. After
washing, 100 mL of o-phenylenediamine (0.33 mg/mL in
citrate buffer, pH 5.2, in the presence of 0.04% hydrogen
peroxide) was added to the wells. The reaction was stopped
after 20 min by the addition of 20 mL of a 1:20 sulfuric acid
solution. Absorbance values were determined at 490 nm
using an ELISA plate reader (BIO-RAD, 680 models).
Duplicate readings were taken for all samples and the
means were calculated.
For the immunoblotting, an SDS-PAGE gel using H.
lunatus venom was run according to the method of
Laemmli (1970) using 12.5% gels and transferred onto
nitrocellulose membrane (Towbin et al., 1979). The
membrane was blocked with PBS-Tween 0.3% for 1 h. After
washing three times for 5 min with PBS-Tween 0.05%, the
membrane was incubated with anti-H. lunatus rabbit serum
(1:1500) for 1 h and 30 min. The membrane was washed
F. Costal-Oliveira et al. / Toxicon 60 (2012) 934942 936
(PBS-Tween 0.05%) more three times and immunoreactive
proteins were detected using anti-rabbit IgG conjugated to
peroxidase (1:8000) for 1 h at room temperature. After
washing three times for 5 min with PBS-Tween 0.05%, blots
were developed using DAB/chloronaphthol according to
manufacturers instructions.
3. Results and discussion
3.1. General venom characterization
The lethality of the H. lunatus soluble venom to
mammals was examined using mice. After intracranial (i.c.)
and intraperitoneal injection (i.p.) toxic and lethal effects
were observed. The LD
50
value was determined as 0.1 mg/
kg and 21.55 mg/kg (respectively). Injected mice displayed
typical symptoms of intoxication such as excitability,
agitation, salivation, eye secretions, sweating, convulsions
and paralysis of legs. The symptoms lasted for 30120 min
before death. The observed symptoms closely resemble
those produced by the venom of Buthidae scorpions of the
genera Centruroides or Tityus (Possani et al., 1977). The
venoms from some species of Brazilian scorpions were
analyzed regarding their lethality in mice by Nishikawa and
co-workers, in 1994 via intraperitoneal injection, the same
used by us. In this study, the venoms were grouped as
highly toxic {Tityus stigmurus (LD
50
0.773 mg/kg), Tityus
bahiensis (LD
50
1.062 mg/kg) and T. serrulatus
(LD
50
1.160 mg/kg)}; moderately toxic {Tityus cambridgei
(LD
50
12.136 mg/kg)} and practically non-toxic {Rhopa-
lurus agamemnon (LD
50
36.363 mg/kg) and Brotheas
amazonicus (LD
50
90.909 mg/kg)}. In view of the results
observed in our experiments and compared to the previous
data, H. lunatus venom can be classied as moderately
toxic. The value found for LD
50
of the venom of H. lunatus
(i.p. route) was more than three times lower than that
found by Zavaleta et al. (1981). This divergence can be
explained by the fact that in our experiments the venom
collected was immediately diluted in ultrapure water (milli
Q), pooled and stored at 20

C until use and never
lyophilized.
The proteolytic activity (caseinase) of H. lunatus venom
was detected, for the rst time, by the dimethylcasein
method (Lin et al., 1969; Sanchez et al., 2000). The
proteolytic activity of the venomwas determined as 0.46 U/
mg min and showed a dose dependent pattern (Fig. 1A).
This activity has already been investigated for the H. lunatus
venom, by Escobar and co-workers in 2002. However, in
this study, no proteolytic activity was found. This difference
may be due to the method used in the previous work that
may not have a comparable sensitivity to the method
chosen in this study. Enzymes with gelatinolytic activity
were detected in T. bahiensis and T. serrulatus venom
(Almeida et al., 2002). For these species Magalhes (1946)
suggested that proteolytic enzymes were present based
on observations of necrosis, hemolysis and gangrene
following experimental injection in different animals.
Necrosis is reported as one of the symptoms in humans
stung by H. lunatus scorpions (Anales de la Facultad de
Medicina, 1967). The role for proteolytic enzymes in scor-
pion venoms is not very clear, but they may be related to
tissue permeability, acting as spreading factors for the
venom. These enzymes may also have a direct toxic effect in
the development of pancreatitis presented by some enve-
nomed patients (Almeida et al., 2002; Renner et al., 1983).
The H. lunatus venom also displays phospholipase and
hyaluronidase activities (Figs. 1B and 2). The phospholipase
activity was examined through an indirect hemolytic assay,
as described by Gutierrez in 1988 and is dose dependent.
The minimum phospholipasic dose (MPD), which is the
dose capable of producing a 1 cm halo, was dened as
0.125 mg of soluble venom. Phospholipase activity has been
described for some scorpion venoms (Schwartz et al.,
2008), and phaiodactylipin was the rst molecule from
the venom of a scorpion belonging to the Iuridae family to
be described (Valdez-Cruz et al., 2007). This scorpion
phospholipase belongs to the A2 family, class III. It is
a heterodimeric protein with neurotoxic activity that acts
Fig. 1. Proteolytic and PLA
2
activities of Hadruroides lunatus venom. The proteolytic activity (Fig. 1A) was determined using dimethylcasein as a substrate. The
absorbance value (at 340 nm) showed the proteolytic activity of 5, 10, 20 and 40 mg of crude venom. One unit was dened as DA 340 nm/min, and the activity was
expressed relative to protein concentration (mg). PLA
2
activity (Fig. 1B) was determined via the indirect hemolytic assay in agarose gel containing rabbit
erythrocytes, egg yolk and CaCl
2
. The samples (15 mL) containing different concentrations of H. lunatus venomwere added to 2 mmwells on agarose, incubated at
37

C for 18 h and the diameters of the hemolytic halos measured.
F. Costal-Oliveira et al. / Toxicon 60 (2012) 934942 937
by inhibiting the ryanodine receptor (Zamudio et al., 1997).
Besides their importance in digestion, venom phospholi-
pases may also be related to other toxic symptoms such as
inammation, platelet aggregation, mionecrosis, hemo-
lysis, neurotoxicity and cardiotoxicity (Lambeau and
Lazdunski, 1999). The hyaluronidase activity appears
ubiquitous in all venom samples obtained from scorpion
venoms (Pessini et al., 2001; Seyedian et al., 2010).
Using the zymogram technique (Cevallos et al., 1992), we
have identied a component with a molecular mass
corresponding to 40 kDa as being responsible for the
hyaluronidase activity of H. lunatus venom. The presence of
hyaluronidase in scorpion venoms is probably related to
the spread and absorption of venoms toxic components
and may affect the stability of blood vessel walls (Veiga
et al., 2001; Seyedian et al., 2010). In addition, it was
recently shown that hyaluronidase combined with phos-
pholipase and citolytic peptides from insect venoms can
promote an IgE or IgG allergen antibody response in mice
or in susceptible humans (King and Wittkowski, 2011).
Total serum creatine kinase (CK) and its isoenzyme MB
(CKMB) are well established and widely accepted markers
for the diagnosis andfollow-upof heart injuryor myocardial
infarction (Bachmaier et al., 1995). Biochemical analyses
showed an increase in CK and CK-MB levels (Fig. 3A and B,
respectively). IncreasedCKconcentrations wereobservedin
envenomed animals (4850 UI/mL) compared to the control
group(injectionof PBS) (1293UI/mL). Levels of CKMBwere
also higher in the envenomed group (1980 UI/mL) than in
the control group (413 UI/mL). We have demonstrated
previously, in dogs envenomed with Tityus fasciolatus
scorpion venom (Pinto et al., 2010), that the occurrence of
myocardiumdamage is correlated with high serumlevels of
CK and CKMB. As we also observed higher levels of these
two markers of heart injury of the envenomed rats, we
suggest in this study that H. lunatus venom has cardiotoxic
effects, possibly through the action of neurotoxins acting on
voltage gated ion channels present in the heart (Chen and
Heinemann, 2001; Korolkova et al., 2004).
3.2. Venom fractionation
The soluble venom of H. lunatus was fractionated by
HPLC and showed more than 20 components (Fig. 4A). As
with other chromatographic proles of scorpion venoms
(Batista et al., 2004), the separation in C18 reverse column
is completed at approximately 60 min of the gradient, at
a ow rate of 1 mL/min. According to the authors
mentioned above, the fractionated components during the
rst 2040 min of the gradient would be the minor
peptides corresponding to K

- and Na

- channel neuro-
toxins. It is known that most scorpion toxic peptides have
Fig. 2. Determination of hyaluronidase activity of Hadruroides lunatus
venom using zymogram. Hyaluronidase activity was analyzed by SDSPAGE
on a 5% (w/v) concentration and 10% separation acrylamide gel co-
polymerized with hyaluronic acid as described in the Material and
methods. Lanes 2 and 3 contain 10 and 5 mg of crude H. lunatus venom.
The molecular weight marker of 45 kDa is show in lane 1.
Fig. 3. Serum levels of total creatine kinase (CK) and creatine kinase isoenzyme MB (CKMB) in rats envenomed with Hadruroides lunatus venom. Animals were
injected with 750 mg of soluble venom. Control rats were injected with PBS alone. (A) CK serum levels in rats of the control group and of the venom group. (B) CK
MB serum levels of the control animals and of the envenomed group (*, p < 0.05 vs. control, values shown represent mean plus standard errors).
F. Costal-Oliveira et al. / Toxicon 60 (2012) 934942 938
molecular masses lower than 10 kDa. These basic peptides
are neurotoxins of low molecular mass that bind to ion
channels with high afnity, exerting their noxious effect
(Catterall et al., 2007). These small proteins may be
responsible for the typical symptoms of neurotoxic enve-
noming observed in inoculated mice. Several components
puried after RT (retention time) 30 min showed PLA
2
activities (peaks 10 to 19, except the fraction 13). Fraction
15 (from 35 to 40 min RT), which showed highest PLA
2
activity, was further analyzed by MALDITOF and the
individual components clearly identied. The molar masses
(Fig. 4B) found were 11,914.5 Da and 13,650.6 Da.
3.3. Immunological studies
In the nal part of our study and as a preliminary step in
the production of an anti-H. lunatus anti-venom with
therapeutic properties, we have attempted to study by
ELISA and immunoblotting the antigenic/immunogenic
potential and cross-reactivity of rabbit anti-H. lunatus
serum. Immune sera anti-H. lunatus and anti-T. serrulatus
(for comparative purposes), were raised in rabbits and their
reactivities against H. lunatus, T. serrulatus, C. sculpturatus
and Androctonus australis hector venoms evaluated. Fig. 5
shows the ELISA (absorbance at 490 nm) at different
serum dilutions (1:100 to 1:12,800). Each specic serum
reacted strongly against their own venom antigens.
Notably, sera against Peruvian venom (Fig. 5A) reacted
moderately with their Brazilian and North American
counterparts and poorly with the venom from A. australis
(North African). The serum produced against T. serrulatus
(Fig. 5B) recognized strongly the Peruvian and North
American venoms coated on the ELISA plates and reacted
moderately with the North African scorpion venom. The
above observations suggest the presence of antigenic
identities or common epitopes across the four scorpion
genera studied. However, the recognition of anti-H. lunatus
and anti-T. serrulatus venomantibodies against the venoms
Fig. 4. General analysis of Hadruroides lunatus venom fractions. For HPLC separation 1 mg of crude venomwas applied to a reverse phase column (Shimadzu-Pack
CLC-ODS C18) (Fig. 4A). After column equilibration the venom fractions were separated with a linear gradient from solution A to 60% solution B (dashed line),
running for 60 min. The chromatographic prole is shown by the black line. Phospholipase activity (PLA
2
) of some fractions was performed using an indirect
hemolytic assay as previously described. The fractions 10, 11, 12, 14, 15, 16, 17, 18 and 19 presented phospholipase activity. The size of the halo produced by each
fraction is shown by the gray line located above the respective fraction. Fig. 4B shows the spectrophotometric analysis of the major phospholipasic fraction from
H. lunatus venom (fraction15). Two molecular masses were identied by MALDI-TOF-TOF analysis, one of 11914.5 and another of 13650.6 Da.
F. Costal-Oliveira et al. / Toxicon 60 (2012) 934942 939
of the American regions was signicantly higher than
against African scorpion venom. The antigenantibody
reactivity was also examined using Western blotting with
H. lunatus venomand rabbit polyclonal anti-H. lunatus anti-
venom. From the study (Fig. 6), at least six antigenic
components were identied. Bands were observed around
7, 15, 45 and 66 kDa. Molar masses higher than 66 kDa were
also recognized by anti-H. lunatus antibodies. The presence
of 45 kDa bands in H. lunatus venom observed by Western
blotting is due to the presence of the enzyme hyaluroni-
dase. Hyaluronidase, is present in all venom samples
obtained from scorpions (Pessini et al., 2001; ; Seyedian
et al., 2010). Antibodies against hyaluronidase can be
responsible for the cross-reactivity, observed in the ELISA
among the four scorpion venoms analyzed in this study.
Although the efciency of immunotherapy in treating
patients stung by scorpion remains controversial
(Theakston et al., 2003), certainly a complete analysis of the
neutralizing potency of rabbit anti-H. lunatus anti-venom
can provide convincing support that the anti-venom could
be an effective means of neutralizing the toxic effects of
H. lunatus scorpion toxins. Previous studies in our group in
Brazil (de Resende et al., 1995), strongly support the use of
anti-venom therapy in scorpionism, but the success of this
therapy remains dependent of the quality of the anti-
venoms and the spread at which the treatment is provided.
Fig. 5. ELISA cross reactivity of anti-H. lunatus and anti-T. serrulatus rabbit sera antibodies against different scorpion venoms. The reactivity of sera (dilutions
1:100 to 1:12800) from rabbits immunized with H. lunatus (Fig. 5A) and T. serrulatus (Fig. 5B) toward venoms (10 mg/mL) from the H. lunatus, T. serrulatus,
C. sculpturatus and A. a. hector was tested. Values are means SEM of triplicates. The absorbance of pre-immune serum used as negative control was subtracted.
F. Costal-Oliveira et al. / Toxicon 60 (2012) 934942 940
Acknowledgments
This research was supported by Coordenao de Aper-
feioamento de Pessoal de Nvel Superior, Brazil CAPES
(Toxinologia No 23038000825/2011-63), Fundao de
Amparo a Pesquisa do Estado de Minas Gerais, Brazil
(FAPEMIG) andbyfunds of theINCTTOXProgramof Conselho
Nacional de DesenvolvimentoCientcoe Tecnolgico, Brazil
(CNPq). The authors gratefully acknowledge the support and
assistance of the Instituto Nacional de Salud, Peru.
Conict of interest
None declared.
References
Aguilar, P., 1968. Nota sobre los escorpiones de Lima. Anales Cientcos
Universidad Nacional Agraria La Molina VI (3), 165172.
Aguilar, P., Meneses, O., 1970. Escorpiones y Escorpionismo en el Peru 1.
Nota preliminar sobre los Scorpionida peruanos. An. Cien. Univ. Nac.
Agraria, Lima 8 (12), 15.
Almeida, F.M., Pimenta, A.M., De Figueiredo, S.G., Santoro, M.M., Martin-
Eauclaire, M.F., Diniz, C.R., De Lima, M.E., 2002. Enzymes with
gelatinolytic activity can be found in Tityus bahiensis and Tityus ser-
rulatus venoms. Toxicon 40, 10411045.
Anales de la Facultad de Medicina, Universidad Nacional Mayor de San
Marcos, 1967. ISSN1025-5583.Vol. 50, p. 1.
Bachmaier, K., Mair, J., Offner, F., Pummerer, C., Neu, N., 1995. Serum
cardiac troponin T and creatine kinase-MB elevations in murine
autoimmune myocarditis. Circulation 92 (7), 19271932.
Batista, C.V., del Pozo, L., Zamudio, F.Z., Contreras, S., Becerril, B., Wanke, E.,
Possani, L.D., 2004. Proteomics of the venom from the Amazonian
scorpion Tityus cambridgei and the role of prolines on mass spec-
trometry analysis of toxins. J. Chromatogr. B Analyt Technol. Biomed.
Life Sci. 803 (1), 5566.
Catterall, W.A., Cestele, S., Yarov-Yarovoy, V., Yu, F.H., Konoki, K.,
Scheuer, T., 2007. Voltage gated ion channels and gating modier
toxins. Toxicon 49 (2), 124141.
Cekalovic, K.T., 1966. Contribucin al conocimiento de los escorpiones
chilenos. Museo Nacional De Historia Natural Noticiario Mensual
(Santiago) 10 (118), 18.
Cekalovic, K.T., 1983. Catlogo de los escorpiones de Chile (Chelicerata,
Scorpiones). Boletn De La Sociedad Biolgica De Concepcin 54, 43
70.
Cevallos, M.A., Navarro-Duque, C., Varela-Julia, M., Alagon, A.C., 1992.
Molecular mass determination and assay of venom hyaluronidases by
sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Toxicon
30 (8), 925930.
Chen, H., Heinemann, S.H., 2001. Interaction of scorpion alpha-toxins
with cardiac sodium channels: binding properties and enhance-
ment of slow inactivation. T.rnal of General Physiology 117 (6), 505
518.
Delgado, G.A., Pesce, H., 1967. La fauna ponzoosa de Valle de Rmac. Ann.
Fac. Med, Lima. 50,105.
De Rezende, N.A., Dias, M.B., Campolina, D., Chavez-Olortegui, C., Diniz, C.
R., Amaral, C.F., 1995. Efcacy of antivenom therapy for neutralizing
circulating venom antigens in patients stung by Tityus serrulatus
scorpions. Am. J. Trop. Med. Hyg. 52 (3), 277280.
Escobar, E., Rivera, C., Tincopa, L., Rivera, D., 2002. Puricacin parcial de
las toxinas Hl1, Hl2 y Hl3 del veneno del escorpin Hadruroides
lunatus KOCH, 1867(SCORPIONIDA, VEJOVIDAE). Revi. Peru. Biol 9 (1).
Escobar, E., Flores, L., 2008. Peptidos antibacterianos de los venenos de
Hadruroides mauryi y Centruroides margaritatus. Rev. Peru. Biol. 15 (1),
139142.
Esquivel de Verde, M.A., 1968. Notas sobre Scorpionidae de Venezuela,
Nuevos registros y comentarios sobre la distribucin de algunos
grupos en Venezuela. Acta Biologica Venezuelica 6 (2), 6670.
Finney, D.J., 1971. Probit Analysis, third ed. Cambridge University Press,
Cambridge, UK.
Flecknell, P.A., Cruz, I.J., Liles, J.H., Whelan, G., 1996. Induction of anaes-
thesia with halothane and isourane in the rabbit, a comparison of
the use of a face-mask or an anaesthetic chamber. Lab. Anim. Jan 30
(1), 6774.
Francke, O.F., Soleglad, M.E., 1981. The family Iuridae Thorell (Arachnida,
Scorpiones). J. Arachnology 9, 233258.
Green, M.R., Pastewka, J.V., Peacock, A.C., 1973. Differential staining of
phosphoproteins on polyacrylamide gels with a cationic carbocyanine
dye. Anal. Biochem.Nov 56 (1), 4351.
Gutirrez, J.M., Avila, C., Rojas, E., Cerdas, L., 1988. An alternative in vitro
method for testing the potency of the polyvalent antivenom
produced in Costa Rica. Toxicon 26, 411413.
King, T.P., Wittkowski, K.M., 2011. Hyaluronidase and hyaluronan in insect
venom allergy. Int. Arch. Allergy Immunol. 156 (2), 205211.
Kinzelbach, R., 1973. Scorpions from the Galapagos islands. In: Galapagos,
Studi e Ricerche. Spedizione L. Mares G.R.S.T.S. Museo Zoologico
dellUniversita` di Firenze, Firenze, pp. 112.
Korolkova, Y.V., Tseng, G.-N., Grishin, E.V., 2004. Unique interaction of
scorpion toxins with the hERG channel. J. of Molecular Recognition:
JMR 17 (3), 209217.
Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly
of the head of bacteriophage T4. Nature 227, 680685.
Lambeau, G., Lazdunski, M., 1999. Receptors for a growing family of
secreted phospholipases A2. Trends Pharmacol. Sci. 20 (4), 162170.
Lin, Y., Means, G.E., Feeney, R.E., 1969. The action of proteolytic enzymes
on N,N-dimethyl proteins. J. Biol. Chem. 244, 789793.
Lowry, O.H., Rosenbrough, N.J., Lewis Farr, A., Randal, R.J., 1951. Protein
measurement with the folin phenol reagent. J. Biol. Chem. 193 (1),
265275.
Magalhes, O., 1946. Escorpionismo IV. Mem. Inst. Oswaldo Cruz 3, 220.
Maury, E.A., 1975. Escorpiones y escorpionismo en el Per IV, Revisin del
gnero Hadruroides Pocock, 1893 (Scorpiones, Vejovidae). Revista
Peruana de Entomologa 17 (1), 921.
Fig. 6. Western blot analysis of Hadruroides lunatus scorpion venom after
12.5% SDS/PAGE. H. lunatus venom (20 and 10 mg) was analyzed by SDS-PAGE
under non-reducing conditions. Molecular size markers (MWM) are indi-
cated in the left (in kDa). Rabbit anti-H. lunatus serum and anti-rabbit IgG
conjugated to peroxidase were diluted 1:1500 and diluted 1:8000, respec-
tively. Experimental details are described in Material and methods.
F. Costal-Oliveira et al. / Toxicon 60 (2012) 934942 941
Mello-Leito, C., 1945. Escorpies sul-americanos. Arquivos do Museu
Nacional Rio de Janeiro 40, 7468.
Ministrio de Sald, Peru, 2004. Norma Tcnica sobre Prevencion y Tra-
tamiento de Acccidentes por Animales Ponzoosos. 16 March 2012.
http://www.minsa.gob.pe/dgsp/documentos/deais/Norma%20Final%
20Ponzo%C3%B1osos-2004.pdf.
Nishikawa, A.K., Caricati, C.P., Lima, M.L., Dos Santos, M.C., Kipnis, T.L.,
Eickstedt, V.R., Knysak, I., Da Silva, M.H., Higashi, H.G., Da Silva, W.D.,
1994. Antigenic cross-reactivity among the venoms from several
species of Brazilian scorpions. Toxicon 32 (8), 989998.
Ochoa, J.A., Prendini, L., 2010. The genus Hadruroides Pocock, 1893
(Scorpiones, Iuridae), in Peru, new records and descriptions of six
new species. Am. Museum Novitates 3687, 156.
Pessini, C., Takao, T.T., Cavalheiro, E.C., Vichnewski, W., Sampaio, S.V.,
Giglio, J.R., Arantes, E.C., 2001. A hyaluronidase from Tityus serrulatus
scorpion venom: isolation, characterization and inhibition by avo-
noids. Toxicon 39 (10), 14951504.
Pinto, M.C., Borboleta, L.R., Melo, M.B., Labarrre, C.R., Melo, M.M., 2010.
Tityus fasciolatus envenomation induced cardio-respiratory alter-
ations in rats. Toxicon 55 (6), 11321137.
Possani, L.D., Alagn, A.C., Fletcher Jr., P.L., Erickson, B.W., 1977. Purica-
tion and properties of mammalian toxins from the venom of Brazilian
scorpion Tityus serrulatus Lutz and Mello. Arch. Biochem. Biophys. 180
(2), 394403. Apr 30.
Renner, I.G., Pantoja, J.L., Abramson, S.B., Douglas, A.P., Russell, F.E.,
Koch, M.K., 1983. Effects of sorpion and rattlesnake venoms on the
canine pancreas following pancreaticoduodenal arterial injections.
Toxicon 21 (3), 405420.
Sanchez, E.F., Santos, C.I., Magalhes, A., Diniz, C.R., Figueiredo, S., Gilroy, J.
, Richardson, M., 2000. Isolation of a proteinase with plasminogen-
activating activity from Lachesis muta muta (bushmaster) snake
venom. Arch. Biochem. Biophys. 378, 131141.
Schwartz, E.F., Camargos, T.S., Zamudio, F.Z., Silva, L.P., Bloch Jr., C.,
Caixeta, F., Schwartz, C.A., Possani, L.D., 2008. Mass spectrometry
analysis, amino acid sequence and biological activity of venom
components from the Brazilian scorpion Opisthacanthus cayaporum.
Toxicon 51 (8), 14991508.
Seyedian, R., Pipelzadeh, M.H., Jalali, A., Kim, E., Lee, H., Kang, C., Cha, M.,
Sohn, E.T., Jung, E.S., Rahmani, A.H., Mirakabady, A.Z., 2010. Enzymatic
analysis of Hemiscorpius lepturus scorpion venom using zymography
and venom-specic antivenin. Toxicon 56 (4), 521525.
Sissom, W.D., Fet, V., 2000. FamilyIuridaeThorell, 1876. In: Fet, V., Sissom, W.
D., Lowe, G., Braunwalder, M.E. (Eds.), Catalog of the Scorpions of the
World (17581998). New York Entomological Society, pp. 409420.
Theakston, R.D., Warrell, D.A., Grifths, E., 2003. Report of a WHO
workshop on the standardization and control of antivenoms. Toxicon
41 (5), 541557.
Towbin, H., Staehelint, T., Gordon, J., 1979. Electrophoretic transfer of
proteins from polyacrylamide gels to nitrocellulose sheets, procedure
and some applications. Proc. Natl. Acad. Sci. U. S. A. 76 (9), 43504354.
Valdez-Cruz, N.A., Segovia, L., Corona, M., Possani, L.D., 2007. Sequence
analysis and phylogenetic relationship of genes encoding hetero-
dimeric phospholipases A2 from the venom of the scorpion Anu-
roctonus phaiodactylus. Gene 396 (1), 149158.
Veiga, S.S., Zanetti, V.C., Braz, A., Mangili, O.C., Gremski, W., 2001. Extra-
cellular matrix molecules as targets for brown spider venom toxins.
Braz. J. Med. Biol. Res. 34 (7), 843850.
Zamudio, F.Z., Conde, R., Arvalo, C., Becerril, B., Martin, B.M., Valdivia, H.
H., Possani, L.D., 1997. The mechanism of inhibition of ryanodine
receptor channels by imperatoxin I, a heterodimeric protein from the
scorpion Pandinus imperator. J. Biol. Chem. 272 (18), 1188611894.
Zavaleta, A., Navarro, J., Castro De La Mata, R., 1981. Pharmacological
effects of a Peruvian scorpion (Hadruroides lunatus) venom. Toxicon
19 (6), 906909.
F. Costal-Oliveira et al. / Toxicon 60 (2012) 934942 942