Revista Electrónica:
Depósito Legal: ppi 201502ZU4665 / / ISSN electrónico: 2477-944X
Revista Impresa:
Depósito Legal: pp 199102ZU46 / ISSN 0798-2259
MARACAIBO, ESTADO ZULIA, VENEZUELA
Vol. XXX (3) 2020
UNIVERSIDAD DEL ZULIA
REVISTA CIENTÍFICA
FACULTAD DE CIENCIAS VETERINARIAS
DIVISIÓN DE INVESTIGACIÓN
142
Description of hutmannin-1 a new piii-metalloprotease from the venom / Pineda, M. y col.
ABSTRACT
The objective was to characterise hutmannin-1 (hut-1), a new ~
62 kDa P-III-class metalloprotease from Porthidium lansbergii
hutmanni (P.l.h) (Margarita Island, Venezuela). To characterise
this protein, the crude venom of P.l.h was fractionated by size
exclusion chromatography, anion exchange chromatography and
High Performance Liquid Chromatography (HPLC). Hutmannin-1
was identied by MALDI-TOF/TOF mass spectrometry, and the
venom was analysed by SDS-PAGE. The lethality, minimum
haemorrhagic dose (MHD), eect of temperature on the activity,
procoagulant activity on human plasma, and anticoagulant,
debrinating, gelatinolytic brinolytic, and brinogenolytic and
platelet aggregation activities of hut-1 were determined. Antigenic
recognition assays were performed on P.l.h crude venom and
hut-1 by a venezuelan polyvalent anti-ophidic serum (PAOS)
Hut-1 had strong brinogenolytic and moderate brinolytic
activity. These activities and the haemorrhagic activity of hut-
1 were completely inhibited by EDTA. P.l.h crude venom had
potent anticoagulant activity on recalcied plasma and inhibited
the platelet aggregation induced by thrombin, ADP, collagen and
ristocetin. In contrast, the anticoagulant, coagulant and platelet
aggregation inhibition of hut-1 were not observed with any of the
agonists. This result suggests that other proteins in the crude
venom, markedly impact platelet functions and/or coagulation
factors. Commercial venezuelan antivenin showed limited ability
to neutralise the haemorrhagic activity of hut-1.
Key words: A n t i c o a g u l a n t ; a n t i v e n i n ; c o a g u l a t i o n ; h a e m o s t a s i s ;
haemorrhage; snake venom
RESUMEN
El objetivo de este trabajo fue caracterizar hutmannin-1 (hut-
1), una nueva metaloproteasa de clase P-III de ~ 62 kDa de la
serpiente Porthidium lansbergii hutmanni (P.l.h) (Isla Margarita,
Venezuela). Para caracterizar esta proteína, el veneno crudo de
P.l.h se fraccionó mediante cromatografía de exclusión molecular,
cromatografía de intercambio aniónico y cromatografía líquida
del alto rendimiento (HPLC). Hutmannin-1 se identicó por
espectrometría de masas MALDI-TOF / TOF, y el veneno se
analizó por SDS-PAGE. Se determinó la letalidad, la dosis
hemorrágica mínima (MHD), el efecto de la temperatura sobre
la actividad, la actividad procoagulante en el plasma humano
y las actividades anticoagulantes, desbrinantes, brinolíticos
gelatinolíticos y brinogenolíticos y de agregación plaquetaria de
hut-1. Se realizaron ensayos de reconocimiento antigénico en
P.l.h veneno crudo y hut-1 mediante suero anti-ofídico polivalente
(PAOS) venezolano. Hut-1 tuvo una fuerte actividad brinolítica
y brinolítica moderada. Estas actividades y la actividad
hemorrágica de hut-1 fueron completamente inhibidas por
ethylenediaminetetraacetic acid (EDTA). El veneno crudo de P.l.h
tuvo una potente actividad anticoagulante en plasma recalcicado
e inhibió la agregación plaquetaria inducida por trombina, ADP,
colágeno y ristocetina. En contraste, la inhibición de la agregación
plaquetaria, actividad anticoagulante y coagulante de hut-1 no
se observó con ninguno de los agonistas. Este resultado sugiere
que otras proteínas en el veneno crudo, afectan notablemente
las funciones plaquetarias y / o los factores de coagulación. El
antiveneno comercial venezolano mostró una capacidad limitada
para neutralizar la actividad hemorrágica de la hut-1.
Palabras clave: A n t i c o a g u l a n t e ; a n t i t o x i n a ; c o a g u l a c i ó n ;
hemostasia; hemorragia; veneno de serpiente
Recibido: 24/01/2020 Aceptado: 12/08/2020
DESCRIPTION OF HUTMANNIN-1, A NEW PIII-METALLOPROTEASE
FROM THE VENOM OF THE NEOTROPICAL LANSBERG’S HOGNOSE
VIPER (Porthidium lansbergii hutmanni) WITH FIBRINO(GENO)LYTIC
AND HAEMORRHAGIC ACTIVITIES
DESCRIPCIÓN DE HUTMANNIN-1, UNA NUEVA METALOPROTEASA PIII DEL VENENO
DE LA SERPIENTE NEOTROPICAL MAPANARE DE LANSBERG (Porthidium lansbergii
hutmanni) CON ACTIVIDADES FIBRINO (GENO) LÍTICAS Y HEMORRÁGICAS
María Eugenia Pineda
1
, Jonás Perales
2*
, Elda Eliza Sánchez
3
, Tomas Hermoso
4
, María Narvaez
4
,
Alexander Chapeaurouge
2
, Alba Marlene Vargas
1
and Alexis Rodríguez-Acosta
5*
1
Biotechnology Unit of the Pharmacy Faculty, Universidad Central de Venezuela, Caracas, Venezuela.
2
Laboratory of Toxinology, Oswaldo Cruz Institute, Fiocruz, Rio de Janeiro, Brazil.
3
National Natural Toxins Research Center, Department of Chemistry, Texas A&M University-Kingsville, Kingsville, Texas, USA.
⁴Parasite Biochemistry Section, Tropical Medicine Institute of the Universidad Central de Venezuela, Caracas, Venezuela.
5
Immunochemistry and Ultrastructural Laboratory, Anatomical Institute, Universidad Central de Venezuela, Caracas, Venezuela.
E-Mail: rodriguezacosta1946@yahoo.es jonasperales@gmail.com
143
Revista Cientíca, FVC-LUZ / Vol. XXX, N° 3, 142 - 156, 2020
INTRODUCTION
Snake bite is a signicant work-related and countryside
menace in the tropical and sub-tropical Countries. Precise
statistics of the occurrence of snakebite and its morbidity and
mortality throughout dierent geographical areas does not exist;
nevertheless, it is sure that it is higher than what is ocially
reported. Clinical and toxinologically, description of snake
envenomations are considered into haemotoxic, neurotoxic, and
myotoxic pathological conditions.
Haemorrhagic signs are the characteristic symptoms associated
with Viperidae snake bites. This activity has been attributed to
haemorrhagic enzymes, usually metalloproteases. Several
studies have investigated the haemostatic eects of Viperidae
snake venoms and their isolated protein components [10, 16,
37, 40, 41]. Proteolytic enzymes and myotoxins are the central
components in the venom of members of the Viperidae family,
among which snake venom metalloproteases (SVMPs) induce
symptoms such as haemorrhages [36, 45]. These proteases
interact with dierent targets to regulate haemostasis or with
important tissues associated with vital physiological functions
in prey and predators, causing the most palpable eect:
haemorrhages [3]. The dierent actions of these proteases
involve dierent targets, such as the activation of coagulation
factors [43], brinogen [14], and the endothelial extracellular
matrix of capillary vessels [21].
The taxonomic classication of species in the Porthidium genus
has been controversial over the past several years. Species of
this genus were previously included in the literature as members
of the Bothrops genus [29] however, based on taxonomic
criteria and molecular studies, phylogenetic relationships
have been established among Viperidae family members in
the Porthidium, Atropoides and Cerrophidion genera in the so-
called “Central American Lineage», which is widely distributed in
Central America [2, 6, 23]. In this lineage, the Porthidium genus is
the only genus found in Venezuela [4, 11, 44]; this lineage forms
a paraphyletic group of «South American Lineage» genera and
comprises Bothrocophias, Bothrops and Bothriopsis [4, 11, 31].
The main objective of this work was the purication and the
biological-biochemical characterisation of a haemorrhagic
component found in the venom of the snake Porthidium lansbergii
hutmanni (P.l.h).
MATERIALS AND METHODS
Reagents
The next materials were used for electrophoresis: reagents
(Bio-Rad, USA) and immobilized pH gradient (IPG) strips, pH
3-10, 11 centimetres (cm) (Bio-Rad, USA). The following materials
were used for haemostasis: human brinogen (Sigma, MO, USA)
and bovine thrombin (Sigma, MO, USA). These materials were
used for immunoblotting: equine peroxidase-coupled-equine IgG
antibody (Santa Cruz Biotechnology, CA, USA); nitrocellulose
membrane (Bio-Rad, USA); and SuperSignal West Pico®
chemiluminescence development kit (ThermoScientic, USA).
These materials were used for MALDI-TOF/TOF: α-cyano-4-
hydroxycinnamic acid matrix (α-CHCA) (Sigma, MO, USA); ACN;
triuoroacetic acid (TFA); and diethyl ether (Sigma, MO, USA). The
next materials were used for LC-MS/MS: OFFGEL RoomTemp
HighRes® Kit (Agilent Technologies, USA); IPG strips, pH 3-10,
24 cm strips (GE Healthcare, USA); swine trypsin (PROMEGA);
electro spray calibrant solution 63606; and Calibration Tune Mix
ESI (Sigma-Fluka, USA). Working solutions were composed
of reagents of high purity (≥98%, Merck and Riedel de Haen,
Germany). Polyvalent antiophidic serum (PAOS) was obtained
from Biotecfar C.A., Caracas, Venezuela.
Software
Prism® (GraphPad, Software) [51] was used for statistical
analyses. For one dimension gels analysis and two-dimensional
gels electrophoresis analysis QuantityOne® (Bio-Rad, USA)(54)
and PDQuest® (Bio-Rad) [48], respectively, were used. For the
MALDI/TOF experiment, the Compass 1.2 SR1 for Flex Analysis
(BrukerDaltonics)[30} software was employed.
The liquid chromatography (LC)-MS/MS analysis was done
with the Compass 1.2 SR1 program for Microtof/Maxis® (Bruker-
Daltonics).
Experimental animals
The animals were purchased from the National Institute of
Hygiene “Rafael Rangel” (Caracas, Venezuela) animal facility. The
mice were kept in cages at room temperature with 12 hours (h) of
natural light and ad libitum water and food until experimentation.
Male mice (Mus musculus) of the National Institute of Health
(NIH) strain weighing 20 to 22 grams (g) and 25 to 27 g were used
to determine lethality and haemorrhagic activity, respectively.
Venom
The pooled P.l.h venom was obtained by manually milking
11 adult specimens of both sexes that were captured at 3
metres above sea level (m.a.s.l) in the at regions of Margarita
Island, Nueva Esparta State (Venezuela),The animals were
captured in Macanao peninsula, geographically located at
Longitude: “O64°16’59.99” and Latitude: “N11°1’0.01”). The
area these specimens originate from has a climate favourable
to xerophytes; the climate is inuenced by northeast trade
winds (“vientos aliseos”), the tropical oor as an average
annual temperature of 28°C and an annual rainfall less than
800 millimetres (mm), and the vegetation is very similar to the
Venezuelan coastal inland vegetation. The majority of this area
comprises at terrain, and common vegetation is the arborescent
cacti group known as “Cardonal”, which is mainly characterised
by columnar cacti (“cardones”) and spiny Mimosaceae with a
squat appearance (“cujíes”). The prevailing vegetation near
144
Description of hutmannin-1 a new piii-metalloprotease from the venom / Pineda, M. y col.
the seashore is these “Cardonales”; however, spine bushes or
“Espinares” (“cujíes”) are common inland [38].
On the other hand, the Bothrops colombiensis venom originated
from the pooled venom of 12 adult specimens of both sexes,
which were captured in dierent Venezuelan regions. All snakes
were maintained in captivity in the Serpentarium of the Pharmacy
School Faculty, Universidad Central de Venezuela (Caracas,
Venezuela). Once obtained, the venom was crystallized under
vacuum in a desiccator (Pyrex
®
, 2.4L Small Knob Top Desiccator
Corning, USA) containing CaCl
2
as a desiccant and maintained
at 4°C (Frigidaire FGVU21F8QF Vertical Freezer, USA) until use.
Fractionation of P.l.hcrudevenombygelltration
The fractionation of the P.l.h crude venom was initiated with
a Sephadex
®
G-100 molecular sieve chromatography column
(90 x 2.5 centimetres (cm) following the method by Grillo and
Scannone [18]. Venom samples were dissolved in 5 millilitres (mL)
of mobile phase, composed of 0.2 Molar (M) ammonium acetate
buer at pH 6.8 (four runs were performed, for a total of 1000
milligrams (mg of venom). Protein elution was achieved by mobile
phase at a ow rate of 7 mL/h. The eluates were monitored at
280 nanometres (nm). Fractions were lyophilised, weighed, and
stored at -20°C (Frigidaire FGVU21F8QF Vertical Freezer, USA)
until used to evaluate haemorrhagic action. To determine which of
the fractions (FI to FIV) obtained from P.l.h venom by molecular
exclusion had the highest haemorrhagic activity, a single dose
of 1 microgram (μg) in 0.1 mL of the FI and FII fractions diluted
in 0.85% saline solution was inoculated in experimental animals,
and four animals were used per group. The haemorrhagic
area was established as described for the determination of the
Minimum Haemorrhagic Dose (MHD). FI had the highest activity
and was selected for further purication with anionic exchange
chromatography.
Anion exchange chromatography
The fractionation was carried out in an automated work station
(Bio-rad, USA) by means of an anion exchange column [1] in
several stages. Briey, FI was dissolved in a 20 millimolar (mM)
acetic acid/sodium acetate buer at pH 5.4 (“A” solution). Then,
a gradient between solution “A” and an elution buer solution “B”
(20 mM acetic acid/sodium acetate, pH 3.0) was established, with
the percentage of the “B” solution increasing by 10% at each step.
Finally, the proteins that failed to be eluted under these conditions
were subjected to a linear gradient of NaCl at a nal concentration
of 2 M.
Throughout this process, the ow rate of the mobile phase
was 1 mL/minute (min), and protein detection was carried out at
280 nm. A total of 20 runs (100 mg) were performed to obtain a
batch of each fraction. Each individual batch was concentrated
and desalted by centrifugation at 2,500 x G), using concentrator
tubes, with a 3 kilodaltons (kDa) cut-o, until 90% of the volume
was reduced. Then, the samples were resuspended in deionised
water. This process was repeated three times for each fraction
until an aqueous solution with a neutral pH was obtained at an
appropriate protein concentration. The fractions obtained at the
end of this process were stored at -20 °C (Frigidaire FGVU21F8QF
Vertical Freezer, USA) until use.
Selection of the anion exchange fraction of interest
A single dose of 1 µg of protein from each ion exchange
fraction was intradermically injected into experimental animals,
and four animals were used per group. The fraction with the
highest haemorrhagic activity, protein content, and purity as
evidenced by one-dimensional electrophoresis was selected for
toxicological characterization and proteomic identication.
Protein determination
The protein content in the fractions obtained by anion exchange
chromatography was determined [32] using bovine serum albumin
as a standard for the calibration curve.
High-performance liquid chromatography of hutmannin-1
The puried fraction (100 μg) was dissolved in 200 μL of 1%
TFA in deionised water and subjected to a reverse phase C-18
column on an High Performance Liquid Chromatography (HPLC)
(Waters Alliance,) instrument.
A linear gradient from 0 to 100% acetonitrile (ACN) in 0.1%
TFA was established over one h at a ow rate of 1 mL/min. The
eluates were detected at 280 nm. The appearance of a single,
acute and symmetric peak was considered the purity criterion of
the component named hut-1.
Identicationofhutmannin-1withMALDI-TOF/TOFmass
spectrometry
The identication of hut-1 was carried out at the Toxicology
Laboratory, Department of Physiology and Pharmacodynamics,
Oswaldo Cruz Institute, Rio de Janeiro, Brazil. For this method,
band fragments from SDS-PAGE were treated with 65 mM DL-
Dithiothreitol (DTT) (Sigma-Aldrich, USA) for 30 min at 56°C to
reduce the protein disulphide bonds, and then the samples were
subjected to alkylation with 100 μL of 200 mM iodoacetamide
for 30 min. Later, the gel fragments were decolourised with 50%
ACN in 25 mM ammonium bicarbonate at pH 8.0, dehydrated
with 200 μL of ACN and trypsinised with 15 μL of a (20 ng/μL)
trypsin solution, prepared in 40 mM ammonium bicarbonate.
The obtained peptides were analysed by MALDI-TOF/TOF mass
spectrometry [14, 39]. The mass spectrum was obtained, and de
novo sequencing of the analysed peptides was carried out. The
obtained sequence was compared with the protein sequences
deposited in the NCBI (National Center for Biotechnology
Information, USA) with the BLAST program.
Assessment of FI and hutmannin-1 haemorrhagic potency
145
Revista Cientíca, FVC-LUZ / Vol. XXX, N° 3, 142 - 156, 2020
ateachpuricationstep
The performance of FI and hut-1 was dened as the per-cent
increase in haemorrhagic potency at each purication step.
SDS-PAGE analysis of venom
SDS-PAGE (12% gel) was carried out [28]. Briey,
samples were dissolved at a concentration of 5 μg/μL in a
protease inhibitor cocktail composed of 4-(2-aminoethyl)-
benzene-sulphonyl uoride (AEBSF), E-64, bestatin, leupeptin,
aprotinin and disodium
EthylendiamineTetraacetic acid (EDTA), and then the samples
were diluted to the optimum concentration for visualisation in 0.5
M Tris-HCl buer at pH 6.8, 10% SDS, 1% glycerol and 0.02%
bromophenol blue. For hut-1, a sample under reduced conditions
was also prepared. Once loaded in their respective gel wells, the
samples were run at 100 V for approximately 120 min. Afterward,
the appropriate gels were selected for Blue Silver staining [5],
which has a sensitivity of 1 ng per band. Next, the gels were
washed with deionised water to remove excess dye and digitised.
Each experiment was performed in triplicate.
Hutmannin-1 lethality
Venom lethality (deaths and signs of toxicity) was determined
in mice intravenously injected with 50 μg or 25 μg of hut-1
samples, corresponding to doses of 2.5 mg/kg and 1.25 mg/kg,
respectively. These doses were selected on basis of the lethality
of the crude venom and FI. The animals were autopsied, and the
macroscopic observations of the haemorrhages were performed.
Determination of the minimum haemorrhagic dose (MHD)
To determine the MHD of the P.l.h hut-1, a modied
method [27] was used. Serial doses of hut-1 in the range of 0.0088
μg to 0.044 μg were intradermally injected into the depilated
backs of mice (Mus musculus). The mice were sacriced, and the
skin was removed after 2 h. The diameter of the haemorrhage on
the skin was measured, and the MHD was dened as the amount
of venom protein required to induce a 10 mm haemorrhage.
With the experimental data, a dose response graph was
constructed. Linear regression analysis was performed to estimate
the MHD from the equation line with Prism® (GraphPad). This
procedure was repeated in triplicate, and the mean and standard
deviation of the MHD were calculated.
Eectoftemperatureonhutmannin-1activity
Hutmanin-1 was prepared in 0.1 mL of 0.85% saline solution
such that the amount of protein corresponded to 10x MHD. From
this solution, 2 mL aliquots were incubated for 30 min at dierent
temperatures: 40, 50, 60, 70, 80, and 90°C. After the incubation
period, 0.1 mL of each sample was injected into groups of four
mice. The diameter of the produced haemorrhagic area was
calculated as indicated for the determination of the MHD. The per-
cent haemorrhagic activity was calculated relative to the activity
of the control, which was a sample incubated for 30 min at 30°C.
Determination of procoagulant activity on human plasma
The ability of P.l.h crude venom and hut-1 to induce blood
coagulation was determined through the physical observation
of clot formation [47] Briey, dierent venom or fraction dilutions
were prepared in a coagulation solution composed of 0.02
M phosphate-saline buered solution (PBS) at pH 7.4. Aliquots
of 50 μL of crude venom and hut-1 dilutions were added to 200
μL of citrated human plasma from healthy laboratory donors. In
the case of crude venom, concentrations ranging from 0.1 μg to
100 μg per 50 μL were used. For hut-1, the concentration ranged
from 5 to 40 μg. The coagulation time was recorded, and the
samples that induced plasma coagulation in less than 30 min
were considered procoagulants.
Four replicates were carried out for each dilution. Additionally,
four tubes of the coagulation control solution without P.l.h crude
venom were prepared. A sample 5 μg of B. colombiensis venom
and 200 μL of plasma as a positive control was also carried out, in
which the coagulation time should not exceed 60 seconds (sec).
Anticoagulant activity determination
In addition to the previous experiment, whether the P.l.h crude
venom or hut-1 inhibited or promoted plasma coagulation when
recalcied was determined [13]. The procedure consisted of
making dilutions of venom or hut-1 preparation in coagulation
solution to contain the required dose in 50 μL of solution. With
crude venom, doses ranging from 0.1 μg to 100 μg were used.
For hut-1 doses from 5 μg to 40 μg, 50 μL of each dilution was
added to 200 μL of citrated plasma and incubated at 37°C for 10
min. During this interval, it was observed if plasma coagulation
occurred. If not, 100 μL of 1 M CaCl
2
was supplemented to the tube
and again incubated, recording the coagulation time for another
30 min. Four replicates were made for each trial. The experimental
control consisted of 50 μL of 0.85% saline solution and incubated
with plasma in the absence of venom or hut-1.
Determinationofdebrinatingactivity
The ability of venom or fractions to degrade brinogen in
vivo was assessed [13]. Briey, groups of ve (20-22 g) mice were
intravenously injected with dierent dilutions of P.l.h crude venom
(7.5 μg to 120 μg) or a single dose of 25 μg of hut-1, prepared in
0.2 mL 0.85% saline solution. One hour after the injection, blood
was drawn from the axillary plexus of each experimental animal
under anaesthesia. Samples were stored in glass tubes for two
h at room temperature, and clot formation was observed. The
minimal debrinating dose (MDD) was dened as the minimum
amount of venom that induced incoagulability in all inoculated
mice. In addition, a control group was inoculated with 0.85%
saline solution and developed a rm clot one h after collection.
146
Description of hutmannin-1 a new piii-metalloprotease from the venom / Pineda, M. y col.
Determination of the brinogenolytic activity of P.l.h crude
venom and hutmannin-1
To evaluate the proteolytic activity of P.l.h crude venom and hut-
1 on the α, β, and γ chains of brinogen, we proceeded according
to a modied protocol [34]. In brief, a stock solution of puried
human brinogen was prepared at a concentration of 2 mg/mL in
a 0.1 M Tris-HCl buer solution at pH 7.4. Dierent concentrations
of P.l.h crude venom (0.03 μg to 2 μg) and hut-1 (0.5 μg to 32 μg)
were incubated at 37°C for 30 min with pre-prepared aliquots of
brinogen solution (50 μL/100 μg). After incubation, each sample
was diluted 1:1 with reducing solution containing 0.5 M Tris at pH
6.8, 10% SDS, 1% glycerol, 0.02% bromophenol blue, and 3%
2-β-mercaptoethanol. Then, the samples were placed in a water
bath (Whip Mix, WPM-05350, USA) at 100°C for 5 min. An aliquot
of 15 μL of each sample was electrophoresed as indicated.
The degradation of dierent brinogen chains was observed.
A brinogen sample was run under the same conditions in the
absence of P.l.h crude venom or hut-1.
Determinationofbrinogenolyticactivityasafunctionof
time
After establishing the lowest amount of crude venom (0.25
μg) and hut-1 (1 μg) capable of completely degrading the α chain
of human brinogen under the conditions described above, the
appropriate concentrations of venom and hut-1 were incubated
with 100 μg of brinogen at 37°C for the following incubation
periods: 30 seconds, 1 min, 5 min, 15 min, 30 min, 60 min, 3 h
and 24 h. Subsequently, to determine the brinogenolytic activity,
these samples were electrophoresed (SDS-PAGE) as indicated
and compared to the electrophoretic pattern of a control sample
consisting of the corresponding dose of crude venom or fraction,
incubated with 100 μg of brinogen and immediately subjected to
the reducing action of the reducing solution (time 0).
Eectofproteaseinhibitorsonbrinogenolyticactivity
Constant amounts of P.l.h crude venom (1 μg) and hut-1 (2 μg)
were incubated at 37°C for 30 min with 100 μg of human brinogen
in a 0.02 M Tris-HCl buer solution at pH 7.5. To evaluate the
brinogenolytic serine protease activity, 2 mM benzamidine
was added to the incubation mixture, whereas to evaluate the
metalloprotease activity, EDTA was added to the samples. As
controls, samples without protease inhibitors were used. After the
incubation period, the samples were evaluated by SDS-PAGE as
indicated to determine brinogenolytic activity.
EectofthepHonbrinogenolyticactivity
A constant dose of hut-1 (50 µL/3 μg) was prepared in the
following buer solutions at dierent pH values: citric acid/0.1
MNa2HPO4 (pH 3, pH 4, pH 5 and pH 6) and 0.1 M Tris-HCl (pH
7, pH 8, pH 9 and pH 10). The mixtures were incubated at 37°C for
30 min with (50 µL/100 μg) human brinogen, and then SDS-
PAGE was performed as indicated to determine brinogenolytic
activity.
Determinationofbrinolyticactivity
The ability of P.l.h crude venom and hut-1 to degrade brin
was determined [34]. Briey, 1.5 mL of 0.1% brinogen solution
in imidazole-buered 0.85% saline solution at pH 7.4 was added
to Petri dishes (3 cm). Then, 75 μL of 10 U/mL bovine thrombin
containing 0.025 M CaCl
2
was added to form a uniform brin
layer. Afterward, 10 μL (1 μg/μL) of crude venom or 10 μL of (1
μg/μL) hut-1 in 0.85% saline solution was placed in the centre of
the brin layer and incubated for 24 h at 37°C. After the incubation
period, the diameter of the lysis area on the brin surface was
determined. Fibrinolytic activity was expressed as the diameter
(mm
2
) of the lysis area per microgram of venom or fraction.
Determination of proteolytic activity on gelatine
The modied methodology proposed by Terra et al. [46] was
followed for this step. Discontinuous 12.5% polyacrylamide gels
copolymerised with 1% gelatine were run. Then, 3 mL of each
sample of P.l.h crude venom and hut-1 at a concentration of 2 μg/
μL in 0.5 M Tris buer at pH 6.8, 10% SDS, 1% glycerol and 0.02%
bromophenol blue, was added to the gel. In addition, as a positive
control, 1 μL (2 μg/μL) of B. colombiensis venom was added to
the gel. After electrophoresis, the gel was equilibrated in a 2.5%
Triton X-100 solution, stirring for 1 h at room temperature, washed
with double distilled water (two washes of 10 min each) and
incubated at 37°C for 18 h in a buer solution of 20 mM Tris-HCl
at pH 7.4, 150 mM NaCl, and 5 mM CaCl2. The proteolytic activity
on gelatine was evidenced by the zones of degradation in the
gel, which were observed as translucent areas after Coomassie
R-250 blue staining. [5].
DeterminationoftheeectsofP.l.h crude venom and
hutmannin-1 on platelet aggregation
The eects of venom and hut-1 on platelet aggregation were
assessed by the turbidimetry method [7]. Briey, blood was
obtained from healthy laboratory donors and centrifuged at 190 x
G and 20°C for 15 min to obtain platelet-rich plasma (PRP). After
counting platelets, an aliquot was subjected to a second
centrifugation at 1700 x G for 15 min to obtain the platelet-poor
plasma (PPP). The plasma used during the trials consisted of the
PRP at a concentration of 1x10
5
platelets/mL adjusted with the
PPP.
Each measurement was obtained in an aggregometer (Crono-
Log
®
560, USA), and aliquot suspensions of 500 μL were
placed under agitation at 37°C in a silicon cuvette during the
determination. A total of 10 μL of dierent dilutions of crude venom
(0.6 μg to 16 μg), prepared in normal saline solution, were added to
each sample. For hut-1, an amount corresponding to ve times the
IC
50
of crude venom value was used. After 4 min, the aggregation
agonists ADP (10 μM), ristocetin (1.25 mg/mL), collagen (8 μg/
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mL) and thrombin (1 U/mL) were added. The aggregation curve
was recorded over 8 min for all assays. As a reaction control, the
agonists were placed on the platelet suspension without venom
and instead adding 10 μL of 0.85% saline solution. In the case
of crude venom, a dose response curve was prepared with the
obtained results, and IC
50
of each agonist was determined. The
IC
50
was dened as the amount of venom capable of reducing
platelet aggregation by 50% with respect to the control.
Neutralisation and antigenic recognition assays of P.l.h crude
venom and hutmannin-1 by the polyvalent anti-ophidic serum
Neutralisation of haemorrhagic activity
The antivenom used in the neutralisation experiments was
PAOS, produced by the Biotechnology Centre of the Faculty of
Pharmacy of the Universidad Central de Venezuela (Biotecfar C.
A), lot L-162. PAOS consists of F(ab)
2
fragments of hyperimmune
immunoglobulins obtained from horses. (Equus ferus caballus)
The species used for the immunisation were Crotalus durissus
cumanensis, Crotalus vegrandis, Crotalus pifanorum,
Crotalus ruruima, Bothrops atrox, B. colombiensis, Bothrops
venezuelensis, and P.l.h.
The capacity of PAOS neutralising the haemorrhages induced
by P.l.h crude venom, FI and hut-1 was determined. Briey,
crude venom, F1 or hut-1 was combined with dierent antivenom
dilutions, using as a reference value of the titre declared by the
PAOS manufacturer for Bothrops genus (1 mL of PAOS must
neutralise the activity of 2 mg of Bothrops venom).
The neutralising test was prepared to obtain 10 MHD in 0.1
mL of the mixture and dierent venom/antivenom proportions of
crude venom, F1 or hut-1. The mixtures were incubated for 30
min at 37°C and centrifuged at 2,500 x G for 10 min to eliminate
the antigen-antibody complexes that formed.
The experimental animals were assigned to groups of ve mice
each. Each mouse in the groups was injected with 0.1 mL of the
appropriate venom/antivenom mixture.
Additionally, there was a venom control group challenged with the
crude venom or fraction and a serum control group that received
the highest PAOS dose used in the experiments. Two h after the
injection, the haemorrhagic lesion as described for the MHD was
evaluated. The per-cent reduction in the haemorrhagic lesion
diameter induced by each dose was calculated with respect to the
control. The ED
50
was dened as the amount of PAOS capable of
reducing the diameter of the haemorrhagic lesion by 50%.
Immunoblotting assays
The PAOS reactivity against the epitopes present in
the P.l.h crude venom and hut-1 were evaluated using western
immunoblotting. In this determination, the selected gel was
incubated for 10 min in transfer solution (50 mM Tris-HCl at pH
8.0, containing 380 mM glycine, 0.1% SDS and 20% methanol).
Then, the gel was placed in a transfer chamber, allowing the
proteins to pass from the polyacrylamide matrix to a nitrocellulose
membrane. This process was carried out at 180 milliampers (mA)
for 2 h.
After the transfer, the nitrocellulose membrane was blocked for
2 h at room temperature with a 0.2 M PBS solution at pH 7.0, with
5% (w/v) skimmed milk and 0.1% (w/v) Tween 20. Later, three
washes were performed for 5 min, each with a solution of 0.05%
(w/v) Tween 20 and 0.2 M PBS at pH 7.0. The membrane was
incubated again at room temperature for another 90 min, with
PAOS diluted to 1:3000 in blocking solution. After the incubation
period, the membrane was washed three times with the washing
solution for ve min each wash. Immediately after the washes, the
secondary antibody anti-equine IgG (coupled to horseradish
peroxidase) diluted 1:7000 in blocking solution was added. Then,
the membrane was incubated at room temperature for another 90
min and washed, as indicated. The electrophoretic bands
recognised by PAOS were visualised using a chemiluminescence
development kit, and the image was analysed.
Eectofproteaseinhibitorsonthehaemorrhagicactivityof
hutmannin-1
The eect of protease inhibitors on the haemorrhagic activity of
hut-1 was tested using EDTA, a metalloprotease activity inhibitor,
and benzamidine, a trypsin or trypsin-like inhibitor. In each case,
the proteases were preincubated with the corresponding inhibitor
at 37°C for 30 min.
Six experimental groups, consisting of four mice each, were
used. Each mouse was inoculated with dose of hut-1 that
corresponded to 5 times the MHD. The rst group received hut-1
preincubated with 2 mM EDTA, the second group was inoculated
with hut-1 preincubated with benzamidine, and the third group
received a dose of hut-1 preincubated in 0.85% saline solution,
representing the haemorrhage control group. The remaining
groups were inoculated with the following vehicles: 0.85% saline
NaCl, 2mM EDTA and 2 mM benzamidine. Each animal was
injected and the haemorrhagic area was determined as indicated
to determine MHD. The arithmetic mean was calculated for the
results of each group. The per-cent reduction induced by the
protease inhibitors was calculated by considering the diameter
of the haemorrhagic area for the crude venom control group as
100%.
Statistical analysis
The MHD and neutralisation were analysed by linear regression.
The data were expressed as the mean ± standard deviation.
To determine dierences between experimental groups (three
replicates of each condition), one-way ANOVA with Dunnett’s post
hoc test was used to compare the experimental conditions to the
control conditions. Results with an error probability <0.01 were
148
Description of hutmannin-1 a new piii-metalloprotease from the venom / Pineda, M. y col.
considered signicant. Analysis was completed using SPSS
version 2.0 [35]
RESULTS AND DISCUSSION
The majority of snake venoms exert their actions on almost
all tissues, and their pharmacological activities are determined
by several biologically active fractions [21]. The most signicant
components of these bioactive fractions are SVMPs, the major
components of the venom produced by the Porthidium genus [23,
29]. SVMPs are enzymes considered to have the highest
haemorrhagic potential among the components of Viperidae
snake venoms. These proteins are capable of degrading
extracellular matrix proteins such as laminin, nidogen,
bronectin, type IV collagen (constituents of vessel walls) and
proteoglycans in the endothelial basement membrane, which
promotes the diusion of venom through the membranes and
weakens the capillary structure. Together with the hydrostatic
pressure generated inside the blood vessel, SVMPs can produce
blood extravasation [9]. Other toxic activities attributed to these
enzymes include brinogenolytic activity, prothrombin and Factor
X activation, apoptosis induction, platelet aggregation inhibition,
proinammatory activity, and inactivation of serine protease
blood inhibitors [26, 33]. Most Viperidae snake venoms alter
blood coagulation, but there are a few venoms, such as those
produced by Bothriechis lateralis and Porthidium nasutum, that
do not alter blood coagulation; however, these venoms do induce
haemorrhages due to other protease activities [19] According to
the current analysis of P.l.h crude venom, only high concentrations
of F1 showed procoagulant activity, indicating that the proportion
of proteins with procoagulant activity is low in this venom.
Other researchers [25, 37] have reported that mammalian
experimental models with disrupted platelet aggregation did not
present abnormalities in coagulation tests, despite showing signs
of systemic haemorrhaging.
In the size exclusion chromatography, four protein peaks
from the P.l.h venom were obtained (data not shown). Two
predominant fractions were isolated (FI and FII) and used for
further purication. These fractions presented the largest areas in
the chromatogram. FI (1 μg) produced the largest haemorrhagic
area (22.29 ± 2.79 mm) on mouse skin and was selected for the
next purication stage using anion exchange chromatography. FII
at this dose did not produce any haemorrhagic lesions, and it was
discarded.
After passing FI through an anion exchange chromatographic
column, seven well-dened peaks were obtained [FIG.1]. A total
of 20 runs were carried out, and the fractions (named according
to the percentage of buer “B” composing the mobile phase) were
grouped and homogenised as follows: rst, a fraction called F0%
(tubes 5 and 6), whose protein content did not interact with the
negatively charged resin. Second, a series of fractions were bound
to the resin with dierent anities, according to their isoelectric
point and that required a decreasing pH mobile phase for elution:
F20% eluted at pH ~ 5.0 (tubes 28 and 29); F30% eluted at pH ~
4.5 (tubes 37 and 38); F40% eluted at pH ~ 4.0 (tubes 46 and 47);
F60% eluted at pH ~ 3.5 (tubes 56 and 57); and F100% (tubes
66 and 67) eluted at pH ~ 3.0. The peak obtained with the salt
gradient was also collected and called FNaCl (tubes 81-82).
FIGURE 1. PURIFICATION OF HUT-1 BY ANION EXCHANGE
CHROMATOGRAPHY. Fraction I obtained from the crude venom
of P. lansbergii hutmanni was applied to a Q1 column (BioRad,
USA). Elution was performed by establishing a pH step gradient
with buer pH 3 in ve steps (20, 30, 40, 60 and 100%) during
rst 73 min of the run (showed on X axis), following with a NaCl
2M gradient, evidenced by an increasing of conductivity value
expressed in mS/cm. The fraction with the highest haemorrhagic
activity was called hut-1 and was selected to check its homogeneity
by means of HPLC.
Mice were intradermally injected with 1 μg of the protein
from the anion exchange fractions, and the haemorrhagic
lesions that resulted from these injections are shown in TABLE
I. The fractions F40%, F60%, F100% and FNaCl had a strong
haemorrhagic action. The fraction F100% was selected for further
biochemical and toxicological characterisation because it has the
highest haemorrhagic activity.
TABLE I
HAEMORRHAGIC LESIONS PRODUCED BY ANION
EXCHANGE FRACTIONS
Anion exchange
fraction
Haemorrhagic lesion (10 mm
diameter)
(μg protein/fraction)
F0% NH
F20% NH
F30% NH
F40% 20.7 ± 0.5µg
F60% 20.9 ± 0.6µg
F100% 22.5 ± 0.3µg
FNaCl 17.8 ± 0.8µg
NH: No haemorrhages were observed.
It was possible to purify a 62 kDa protein called hut-1 using a
combination of size exclusion chromatography and anion exchange
chromatography. Hut-1 showed a single protein peak that eluted
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Revista Cientíca, FVC-LUZ / Vol. XXX, N° 3, 142 - 156, 2020
during the acetonitrile (ACN) gradient under high-performance
liquid chromatography (HPLC). As shown in FIG.2, this peak was
symmetric, and no major peaks corresponding to protein sample
contaminants were observed. This protein was composed of a
single polypeptide chain, as evidenced by treatment with the
reducing agent B-mercaptoethanol. Hut-1 was identied as a
member of the SVMP family, with homology to class P-III of the
zinc-dependent metalloprotease domain family based on the
molecular mass determined by tandem mass spectrometry (MS/
MS). The toxic action of hut-1 demonstrated that, similar to other
SVMPs [45], its main toxicological targets were haemostasis
components.
symmetric, and no major peaks corresponding to protein
sample contaminants were observed. This protein was composed
of a single polypeptide chain, as evidenced by treatment with
the reducing agent B-mercaptoethanol. Hut-1 was identied as
a member of the SVMP family, with homology to class P-III of
the zinc-dependent metalloprotease domain family based on the
molecular mass determined by tandem mass spectrometry (MS/
MS). The toxic action of hut-1 demonstrated that, similar to other
SVMPs [45], its main toxicological targets were haemostasis
components.
FIGURE 2. HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
(HPLC) OF HUT-1. Hutmannin-1 (100 μg) was dissolved in 0.1%
triuoroacetic acid (TFA) and run in a C-18 reverse phase column
(HPLC) with a linear gradient from 0 to 100% acetonitrile (ACN),
in one h at a ow rate of 1mL/min.
Exploring the lethality of P.l.h FI and hutmannin-1 was obtained
that the LD
50
of FI (1.45 ± 0.16 mg/kg) was lower than that of
the crude venom of P.l.h (2.51 ± 0.16 mg/kg), showing that this
fraction possessed the majority of the toxic components in the
venom. Hut-1 showed high toxicity. It was not feasible to determine
the LD
50
of hut-1 because it was not possible to obtain enough
sample for this test; however, when doses as low as 2.63 mg/kg
and 1.32 mg/kg were tested, all the animals died within 24 h after
the injection, which indicated that the hut-1 LD
50
was below 1.32
mg/kg. Before the animals injected with both doses of hut-1 died,
they all presented motor incoordination, accid paralysis in the
anterior and posterior limbs, cyanosis, respiratory insuciency,
tachycardia and haematemesis. Autopsies of mice revealed atrial
thrombosis and massive pulmonary and hepatic haemorrhages,
indicating the high toxicity of hut-1 (data not shown).
The acute toxicity of hut-1 was found to be more potent than
those of FI and crude venom. This toxicity is notable when
comparing hut-1 with a PI class metalloprotease (Porthidin-1),
previously isolated from P.l.h venom [15], which was found to be
non-lethal in experimental mice intravenously injected with a dose
of 6 mg/kg.
The results obtained in each purication step are shown
in TABLE II. Here, the performance and eciency of the
purication process of hutmannin-1 (hut-1) were determined,
and the minimum haemorrhagic dose (MHD, µg) was obtained at
each purication step.
The MHD of hut-1 was 83 times higher than that of crude venom,
showing that hut-1 is one of the most powerful haemorrhagic toxins
described to date; this conclusion becomes evident when hut-1 is
compared with similar toxins from the venoms of various Viperidae
snakes [49]. Moreover, the autopsies of mice treated with hut-
1 showed extensive systemic haemorrhages. The macroscopic
study of the lung tissue of treated mice revealed profuse
haemorrhages in the lung, and these lesions rapidly appeared
15 min after injection. Observations of the structural changes that
occur following exposure to P.l.h crude venom [49] revealed the
appearance of erythrocytes and glomerular kidney oedema,
as well as the detachment from the basement membrane and
plasma membrane rupture of endothelial cell. This result could
demonstrate the possible systemic haemorrhagic activity of hut-1,
the haemorrhagic fraction from this venom. Previous studies that
investigated other metalloproteases from Bothrops snakes, such
as jararagin, have shown that similar pulmonary haemorrhages
occur in mice [8, 36]. The MHD value is shown in TABLE II. At
nanogram (0.0088 to 0.044 μg) doses, hut-1 presented a
high haemorrhagic capacity, resulting in bleeding skin lesions in
the experimental animals (data not shown).
TABLE II
PERFORMANCE AND EFFICIENCY OF THE
PURIFICATION PROCESS OF HUT-1. THE MHD
ACTIVITY (µG) RESULTS OBTAINED AT EACH
PURIFICATION STEP
Sample Amount
obtained
MHD
activity
(µg)
Purication
factor
Crude
venom
1000 mg 1.475 1
Fraction I 697.2 mg 0.102 14.5
Hutmannin-1 39 mg 0.021 69.9
Hut-1 maintained its haemorrhagic activity when
incubated for 30 min at 40°C, but haemorrhagic activity of hut-
1 decreased by approximately 20% after incubation at 50°C for
the same period of time. After exposure to temperatures equal
to or higher than 60°C, hut-1 completely lost its haemorrhagic
capacity [FIG. 3A]. This temperature restriction is similar to the
temperature restriction of other viperid metalloproteases, such
as uracoin-1 [1], and elapid metalloproteases, such
as EpyHTI and EcoHTI, which presented a 50% reduction
in haemorrhagic activity at temperatures close to 50°C and
completely lost activity at 70°C [49].
150
Description of hutmannin-1 a new piii-metalloprotease from the venom / Pineda, M. y col.
The haemorrhagic activity of hut-1 was completely abolished
by preincubation with 2 mM EDTA, a metal chelator, whereas
preincubation with 2 mM benzamidine, a trypsin or trypsin-like
inhibitor, did not induce signicant dierences from untreated hut-
1 (n=4, FIG. 3B), demonstrating the dependence of the activity of
hut-1 on divalent ions, as previously reported for a large variety of
metalloproteases [7,15,16,19, 36, 46].
FIGURE 3. EFFECT OF TEMPERATURE AND PROTEASE
INHIBITORS ON THE HAEMORRHAGIC ACTIVITY OF HUT-1.
(A) 1: Control; 2: 40
0
C; 3: 50
0
C; 4: 60
0
C; 5: 70
0
C; 6: 80
0
C. The
bars represent the standard error. Dunett test one-way ANOVA
analysis = * α <0.05 ** α <0.01. (B) 1) control; 2) Hut-1 + 2 mM
EDTA; 3) Hut-1 + 2mM benzamidine. The bars represent the
standard error. Dunett test one-way ANOVA analysis = ** α <0.01
The haemorrhagic potential of high class
P-III metalloproteases has been attributed to two main facts: (1)
the inability of α2-macroglobulin to inhibit these toxins and (2)
the presence of domains with disintegrin-like activity that are rich
in cysteine residues and specically degrade extracellular matrix
components, especially type IV collagen [22].
The identication hutmannin-1 by tandem mass spectrometry
showed that the fragmentations with matrix assisted
laser desorption/ionization (MALDI) time-of-ight mass
spectrometry (TOF) resulted in two main signals: m/z 3182.95
and 2197.84. The defragmentation of these peptides and
their subsequent de novo sequencing led to the identication of
two peptide sequences, NLLVAVTMAHELGHNL (m/z: 3182.95)
and VECETGECC (m/z: 2197.84), which are sequences of zinc-
metalloprotease domains found in SVMPs.
FIGURE 4. NCBI/BLAST search comparison of arrangements de novo peptide NLLVAVTMAHELGHNL and VECETGECC sequenced
from MS/MS spectrometry, of Hutmannin-1 to partial amino acid sequences of two snake venom metalloproteases, atrolysin-A 17 and
metalloprotease-8 [18]. The sequence similarities are shown in the gure
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Revista Cientíca, FVC-LUZ / Vol. XXX, N° 3, 142 - 156, 2020
After analysing these sequences with the BLASTp program,
hut-1 was identied as an SVMP, showing similarity with
adamalisin-1 (Crotalus adamanteus), atrolysin-A (Crotalus
atrox) (E-value 9e-07) [23], metalloprotease-8 (C. adamanteus)
(E-value 5e-07) [41], and an additional 43 SVMPs [24], all with
E-value estimates that are considered signicant (≤1e -04). The
similarity of the sequences obtained with the sequences of these
proteins is shown (FIG. 4).
The electrophoretic analysis of FI [FIG.5] revealed that the
chromatographic process was ecient; primarily high molecular
weight proteins were detected in this fraction (8 bands between
36 kDa and 170 kDa). Moreover, the predominance of the 62 kDa
band was observed. The concentration of the 170 kDa and 36
kDa bands was increased in FI compared with the total venom,
whereas the concentration of the 54 kDa band was decreased
in FI. No bands of proteins less than 36 kDa in mass were
detected. Next, the electrophoretic analysis of hut-1 [FIG.5]
showed a single 62 kDa band that was observed both in the
crude venom and in FI.
FIGURE 5. Porthidium l. hutmanni SDS-PAGE. A) molecular
weight markers; B) crude venom; C) FI; D) hutmannin-1 under
native conditions; E) hutmannin-1 under reduction conditions. All
samples were run with 25µg of protein.
After hut-1 was reacted with the reducing agent 2-β-mercaptoethanol
and subsequently underwent electrophoresis, a single band with a
slightly higher molecular weight than the unreduced protein (~ 69
kDa) was observed. This result suggested that hut-1 is composed
of a single polypeptide chain.
In the procoagulant activity of FI and hutmannin-1 was
demonstrated that FI of the P.l.h venom dose-dependently induced
the coagulation of human plasma, as measured by physical clot
formation; all the tested FI doses were markedly more eective
than 5 μg of B. colombiensis venom, in which the plasma
coagulation time was 40 sec (data not shown). Hut-1 did not show
plasma procoagulant activity during 30 min of incubation (data
not shown). On the other hand, Hut-1 did not show anticoagulant
activity on human plasma. Compared to the control, hut-1 did
not signicantly aect the recalcied plasma coagulation times
at the highest doses tested 200 μg (data not shown). The
experimental results indicated that mice treated with FI or hut-
1 maintained their coagulant capacity 2 h after the intravenous
injection of each fraction. This result shows that FI and hut-1 lack
debrinating activity.
In the brinogenolytic activity assay, P.l.hutmanni crude venom
degraded the brinogen Aα chain at the concentration of 0.5
μg venom/100 μg brinogen (data not shown). Moreover, hut-1
proved to have potent proteolytic action on the brinogen α chain
at the same hut-1/brinogen ratio of 0.5 μg/100 μg. However, it
was not easy to determine whether the degradation of the A chain
was complete at this ratio; at the 0.5 μg/100 μg ratio, Coomassie
blue staining revealed that the hut-1 band was located at the
FIGURE 6. (I) FIBRINOGENOLYTIC ACTIVITY OF HUT-1/FIBRINOGEN (µG/µG). A) molecular weights; B) 0 μg/100μg; C) 0.5μg/100μg;
D) 1.0μg/100μg; E) 2.0μg/100μg; F) 4.0μg/100μg; G) 8.0μg/100μg; H) 16.0μg/100μg; I) 32.0μg/100μg; J) hut-1 (32μg) in the absence
of brinogen. (II) FIBRINOGENOLYTIC HUT-1 EFFECT AS A TIME FUNCTION. A) molecular weight markers; B) Control (time 0); C)
incubation for 30 sec; D) incubation for 1 min; E) incubation for 5 min; F) incubation for 15 min; G) incubation for 30 min; H) incubation
for 1 h; I) incubation for 3 h; J) incubation for 24 h. (III) EFFECT OF PH FOR THE ACTIVITY OF HUT-1 ON FIBRINOGEN AT 37°C
INCUBATED FOR 30 MIN. A) molecular weight markers; B) pH 3; C) pH 4; D) pH 5; E) pH 6; F) pH 7; G) pH8; H) pH 9; I) pH 10
152
Description of hutmannin-1 a new piii-metalloprotease from the venom / Pineda, M. y col.
same position as the Aα chain gel due to the similar molecular
weights of these proteins. The degradation of the B or y chain
was not observed at any of the tested doses [FIG. 6I]. The eect
of hut-1 on the brinogen α chain was incubation time-dependent.
After incubating 1 μg of hut-1 with 100 μg of brinogen for
dierent time intervals, the degradation of the brinogen α chain
was observed. After 30 min of incubation, the brinogen Aα chain
began to degrade, and it was undetectable after 3 h of incubation.
After 24 h, the Bβ chain was completely degraded [FIG. 6II].
Hut-1 lacks the procoagulant activity of other P-III class
metalloproteases, such as the Factor V activator RVV-X and
the prothrombin activators ecarin and carinactivase-1 [26],
and the debrinating activity associated with the exacerbated
activation of some Porthidium venom coagulation factors, such
as Porthidin-1 [14]. Additionally, the fraction from which hut-1 was
isolated FI showed a procoagulant eect at high concentrations,
suggesting the presence of other low potency procoagulant toxins.
The eect of hut-1 on human brinogen was similar to that of crude
venom on human brinogen, indicating the strong degradation
power of hut-1 on the α subunit of brinogen. In addition, the
β chain was completely degraded after 24 h of incubation. The
optimum pH of this activity was between 7 and 9, similar to that
of colombienases 1 and 2, as previously reported [17] [FIG.6III].
min; G) incubation for 30 min; H) incubation for 1 h; I) incubation
for 3 h; J) incubation for 24 h. (III) EFFECT OF PH FOR THE
ACTIVITY OF HUT-1 ON FIBRINOGEN AT 37°C INCUBATED
FOR 30 MIN. A) molecular weight markers; B) pH 3; C) pH 4; D)
pH 5; E) pH 6; F) pH 7; G) pH8; H) pH 9; I) pH 10.
Hut-1 was completely inhibited by EDTA, conrming that
the metalloprotease action of hut-1 depends on brinogen
degradation [FIG.7].
FIGURE 7. EFFECT OF PROTEASE INHIBITORS ON THE
FIBRINOGENOLYTIC ACTIVITY OF HUT-1. A) Fibrinogen (Fb)
(100µg); B) Fb + hut-1 (100 µg+ 32 µg) ; C) Fb + hut-1 (100 µg+
32 µg) + 2mM benzamidine ; D) Fb + hut- 1 (100 µg+ 32 µg) +
EDTA 2mM.
Alternatively, the degradation of brin mesh induced by hut-1
proved to be less eective than that induced by the zymogen. The
lysis area obtained in the brin plates was 3.5 times
smaller than that obtained with crude venom, wh
ich suggests that toxins other than hut-1 were involved in
the brinolytic activity of P.l.h venom [FIG.8]. Hut-1 showed
more activity against the brinogen molecule than against the
polymerised brin mesh.
FIGURE 8. FIBRINOLYTIC ACTIVITY OF HUT-1. A) Negative
control PBS; B) Positive control B. colombiensis crude venom;
C) P.l.hutmanni crude venom; D) Hut-1.
No proteolytic activity of hut-1 on the hydrolysed collagen
that forms gelatine was observed [FIG.9].
Similarly, several authors have correlated gelatinolytic activity
with the haemorrhagic action of SVMPs [8,39,41,42]; however,
some P-III metalloproteases, such as alsophinase [50] and
VLH2 [20], have high brinogenolytic and haemorrhagic activity
but have not been shown to have gelatinolytic activity [12]
[FIG.9]. In the case of hut-1, it was necessary to evaluate this
activity by using dierent forms of colparticularly type IV collagen,
as collagen is one of the main toxicological targets for these
enzymes. However, when the hut-1 precursor FI was tested,
two lysis areas corresponding to ~ 37 and 27 kDa were clearly
evident, but no activity was observed in the region corresponding
to the molecular weight of hut-1.
FIGURE 9. GELATINOLYTIC ACTIVITY OF FRACTIONS OF
P.L.H . A) molecular weight markers; B) 6 μg of P.l.h crude venom;
C) 6 μg of P.l.h FI; D) 6 μg of hut-1; E) 2 μg of B. colombiensis
crude venom.
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In the platelet aggregation assay, hut-1 did not inhibit platelet
aggregation in response to the tested agonists (collagen, ADP,
ristocetin). This result contrasts with the potent inhibitory action
of P.l.h crude venom, suggesting that other proteins in this venom
induce platelet aggregation [52]
Compared to the titre, which is the volume of serum necessary
to neutralize the concentration of bothropic venom [47], as
determined by the manufacturer, PAOS had low ecacy at
neutralising the haemorrhages induced by the P.l.h crude venom,
nearly four times the amount recommended by the manufacturer
was required. PAOS was even less eective against hut.1 than
the crude venom; a concentration 11 times higher than the
recommended by the manufacturer was required to neutralise the
activity of hut-1 (1 mL of PAOS per 2 mg of venom).
To correlate these results with the antigenic recognition
evaluation by Western blot analysis, the antivenom exhibited
limited antigenic recognition of some protein bands,
specically, 54, 45 and 30 kDa [FIG.10]. These molecular weights
are within the range reported for PII and PI metalloproteases,
such as porthidin-1, a 23 kDa haemorrhagic metalloprotease that
was previously
isolated from this venom [14]. These observations could indicate
that additional metalloproteases exist in the P.l.h venom that are
not antigenically recognised by the immunoglobulins present in
the antivenom and, therefore, are not neutralised. These results
markedly contrast the results obtained for hut-1, which was
antigenically recognised by PAOS [FIG.10]; however, PAOS did
not eectively neutralise the haemorrhagic activity of hut-1, which
could be a consequence of the antigen binding at a site that does
not corresponding to the activity of hut-1. The ecological area of
the specimens whose venom were used corresponds, as detailed
in materials and methods, to the Macanao peninsula. It is known
that the manufacturer of antivenoms (Biotecfar CA) uses a pool of
venoms from specimens that are randomly collected throughout
the island, it is also known that venoms have intra-species
variability [42] and that geographic variability surely inuences the
synergistic strategies of predominant toxins components of snake
venoms [53].
FIGURE 10. WESTERN BLOT PA INTERACTION AGAINST
P.L.H FI AND HUT-1. A) molecular weight markers; B) SDS-
PAGE P.l.h FI (10 µg); C) Western blot PA against P.l.h FI; D)
SDS-PAGE hut-1 (10 µg); E) Western blot PA against hut-1.
CONCLUSIONS
P. lansbergii hutmanni is an epidemiologically important
venomous snake species located on Margarita Island (Venezuela).
Here, it has been shown that the P.l.h venom lacked the marked in
vitro procoagulant activity characteristic of bothropic venoms,
which could have implications in the diagnosis of envenomations
considered to be from bothropic snakes. The P.l.h crude venom
showed very high haemorrhagic and anticoagulant activities, and
hut-1, an ~ 62 kDa enzyme classied as a P-III metalloprotease,
was identied in this venom. Additionally, hut-1 had a strong
brinogenolytic and moderate brinolytic action and did not
exhibit anticoagulant activity. The antivenom PAOS was not
able to eectively neutralise the haemorrhagic activity of crude
venom, but PAOS did neutralise hut-1; therefore, treatment with
this antivenom could have reduced ecacy in the treatment of
envenomation by P.l.h.
Funding and Acknowledgments
The authors gratefully acknowledge the funding for the research
grant (PG: 09-8760-2013), from the Consejo de Desarrollo
Cientíco y Humanístico de la Universidad Central de Venezuela,
Bolivarian Republic of Venezuela and Conselho Nacional de
Desenvolvimento Cientíco e Tecnológico (CNPQ) y la Fundação
Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de
Janeiro (FAPERJ-Dr. J. Perales).
154
Description of hutmannin-1 a new piii-metalloprotease from the venom / Pineda, M. y col.
Ethical statement
Qualied sta arranged all the experimental methods relating
to the use of live animals. These methods were approved by the
Institute of Anatomy Ethical Committee of the Universidad Central
de Venezuela on 7 March 2018 under assurance number 07-
03-18 and followed the norms obtained from the Guidelines for
the Care and Use of Laboratory Animals, published by the US
National Institute of Health (1985). The research questions asked,
the technical methods chosen, and the conclusions reached are
exclusively responsibility of the authors.
ConictsofInterest
The authors declare no conict of interest.
BIBLIOGRAPHIC REFERENCES
[1] AGUILAR, I.; GIRÓN, M.E.; RODRÍGUEZ-ACOSTA, A. Puri-
cation and characterisation of a haemorrhagic fraction from
the venom of the Uracoan rattlesnake Crotalus vegrandis.
Biochim. Biophys. Acta. 548: 57-65. 2011.
[2] ANGULO, Y.; ESCOLANO, J.; LOMONTE, B.; GUTIERREZ,
J.M.; SANZ, L.; CALVETE, J. Snake venomics of Central
American Pittvipers: Clues for Rationalizing the Distinct En-
venomation Proles of Atropoides nummifer and Atropoides
picadoi. J. Proteom. Res. 7: 708-19. 2008.
[3] BERLING, I.; ISBISTER, G.K. Hematologic eects and
complications of snake envenoming. Transfus. Med. Rev.
29: 82-89. 2015.
[4] CAMPBELL, JA.; LAMAR, W. Porthidium genus. Venom-
ous reptiles of Latin America, Cornell. Univ. Press, Cornell
(USA). Pp. 313-329. 1989.
[5] CANDIANO, G.; BRUSCHI, M.; MUSANTE, L.; SANTUC-
CI, L.; GHIGGERI, G.M.; CARNEMOLLA, B.; ORECCHIA,
P.; ZARDI, L.; RIGHETTI, P. Blue silver: A very sensitive
colloidal Coomassie G-250 staining for proteome analysis.
Electrophor. 25: 1327-1333. 2004.
[6] CASTOE, A.; SASA, M.; PARKINSON, C. Modeling nucleo-
tide evolution at the mesoscale: The phylogeny of the Neo-
tropicalpitvipers of the Porthidium group (Viperidae: Crotali-
nae). Mol. Phylogenet. Evol. 37: 881–898. 2005.
[7] DA SILVA, M.; LUCENA, S.; AGUILAR, I.; RODRÍ-
GUEZ-ACOSTA, A.; SALAZAR, A.M.; SÁNCHEZ, E.E.;
GIRÓN, M.E.; CARVAJAL, Z.; AROCHA-PIÑANGO, C.;
GUERRERO, B. Anti-platelet eect of cumanastatin 1 a dis-
integrin isolated from venom of South American Crotalus rat-
tlesnake. Thromb. Res. 123: 731-739. 2009.
[8] ESCALANTE, T.; NÚÑEZ, J.; MOURA DA SILVA, A.M.; RU-
CAVADO, A.; THEAKSTON, R.D.; GUTIÉRREZ, J.M. Pul-
monary hemorrhage induced by jararhagin a metalloprotein-
ase from Bothrops jararaca snake venom. Toxicol. Appl.
Pharmacol. 193: 17–28. 2003.
[9] ESCALANTE, T.; RUCAVADO, A.; FOX, J.; GUTIÉRREZ,
J.M. Key events in microvascular damage induced by snake
venom hemorrhagic metalloproteinases. J. Proteomics. 74:
1781-1794 2011.
[10] FREITAS DE SOUSA, L.A.; COLOMBINI, M.; LOPES-FER-
REIRA, M.; SERRANO, S.M.T.; MOURA DA-SILVA, A.M. In-
sights into the mechanisms involved in strong hemorrhage
and dermonecrosis induced by Atroxlysin-Ia a PI-Class
snake venom metalloproteinase. Toxins. 9: 239. 2017.
[11] FUENTES, O.; RODRIGUEZ-ACOSTA, A. Sobre los géner-
os Bothriechis Bothriopsis Bothrops y Porthidium (Ser-
pentes: Crotalidae) existentes en Venezuela. Claves para
su identicación de interés biomédico. Acta. Biol. Venez.
17: 31-38. 1997.
[12] GAY, C.; LEIVA, L.; TEIBLER, P.; ACOSTA DE P, O. Proteo-
lytic edematogenic and miotoxic activities of a hemorrhagic
metalloproteinase isolated from Bothrops alternatus venom.
Toxicon. 46: 546-554. 2005;
[13] GENE, J.A.; ROY, A.; ROJAS, G.; GUTIÉRREZ, J.M.; CER-
DAS, L. Comparative study on the coagulant desbrinating
brinolytic and brinogenolytic activities of Costa Rican Cro-
talinae snake venoms and their neutralization by polyvalent
antivenom. Toxicon. 27: 841-848. 1989.
[14] GIDDEN J, DENSON J, LIYANAGE R, IVEY DM, LAY JO.
Lipid Compositions in Escherichia coli and Bacillus subtilis
During Growth as Determined by MALDI-TOF and TOF/TOF
Mass Spectrometry. Int. J. Mass. Spectrom. 2009;283:178-
184.
[15] GIRÓN, M.E.; ESTRELLA, A.; SÁNCHEZ, E.E.; GALAN,
J.; TAO, A.; GUERRERO, B.; SALAZAR, A.M.; RODRÍ-
GUEZ-ACOSTA, A. Purication and characterization of a
metalloproteinase Porthidin-1 from the venom of Lansberg’s
hog-nosed pitvipers (Porthidium lansbergii hutmanni). Toxi-
con. 57: 608-618. 2011.
[16] GIRÓN, M.E.; RODRÍGUEZ-ACOSTA, A.; SALAZAR, A.M.;
SÁNCHEZ, E.E.; GALAN, J.; IBARRA, C.; GUERRERO, B.
Isolation and characterization of two new non-hemorrhag-
ic metalloproteinases with brinogenolytic activity from the
mapanare (Bothrops colombiensis) Venom. Arch. Toxicol.
87: 197-208. 2013.
[17] GIRÓN, M.E.; SALAZAR, A.M.; AGUILAR, I.; PÉREZ,
J.; SÁNCHEZ, E.E.; AROCHA-PIÑANGO, C.; RODRÍ-
GUEZ-ACOSTA, A.; GUERRERO, B. Hemorrhagic coag-
ulant and brino(geno)lytic activities of crude venom and
fractions from mapanare (Bothrops colombiensis) snakes
155
Revista Cientíca, FVC-LUZ / Vol. XXX, N° 3, 142 - 156, 2020
Comp. Biochem. Physiol. Toxicol. Pharmacol. 147: 113-
121. 2008.
[18] GRILLO, O.; SCANNONE, H. Fractionation of Crotalus
durissus cumanensis venom by gel ltration. Toxicon. 14:
400-403. 1976.
[19] GUTIÉRREZ, J.M.; LEÓN, G.; LOMONTE, B.; ANGULO, Y.
Antivenoms for snakebite envenomings. Inamm.Allergy.
Drug. Targets. 10: 369-380. 2011.
[20] HAMZA, L.; GARGIOLI, C.; CASTELLI, S.; RUFINI, S.; LAR-
ABA-DJEBARI, F. Purication and characterization of a -
brinogenolytic and hemorrhagic metalloproteinase isolated
from Vipera lebetina venom. Biochimie. 92: 797- 805. 2010.
[21] HERNÁNDEZ, M.; SCANNONE, H.; FINOL, H.J.; PINEDA,
M.E.; FERNÁNDEZ, I.; GIRÓN, M.E.; AGUILAR, I.; RODRÍ-
GUEZ-ACOSTA, A. Alterations in the ultrastructure of car-
diac autonomic nervous system triggered by crotoxin from
rattlesnake (Crotalus durissus cumanensis) Venom. Exp.
Toxicol. Pathol. 59:129-37. 2007.
[22] HERRERA, C.; ESCALANTE, T.; VOISIN, M.; RUCAVA-
DO, A.; MORAZÁN, D.; KELE, J.; CALVETE, J.J.; SANZ,
L.; NOURSHARGH, S.; GUTIÉRREZ, J.M.; FOX, J.W Tis-
sue localization and extracellular matrix degradation by PI
PII and PIII snake venom metalloproteinases: Clues on the
mechanisms of venom-induced haemorrhage. PLoS. Negl.
Trop. Dis. 29: 1-20. 2015.
[23] JIA, L.G.; WANG, X.M.; SHANNON, J.D.; BJARNASON,
J.B.; FOX, J.W.; Function of disintegrin-like/cysteine-rich
domains of atrolysin A Inhibition of platelet aggregation by
recombinant protein and peptide antagonists. J.Biol. Chem.
272: 13094-13102. 1997.
[24] JIMÉNEZ-CHARRIS, E.; MONTEALEGRE-SANCHEZ, L.;
SOLANO-REDONDO, L.; MORA-OBANDO, D.; CAMACHO,
E.; CASTRO-HERRERA, F.; FIERRO-PÉREZ, L.; LOMON-
TE, B. Proteomic and functional analyses of the venom of
Porthidium lansbergii lansbergii (Lansberg’s hognose viper)
from the Atlantic Department of Colombia. J. Proteom. 114:
287-299. 2015;
[25] KAMIGUTI, A. Platelets as targets of snake venom metallo-
proteinases. Toxicon. 45: 1041-1049. 2005;
[26] KINI, M.; KOH, C. Metalloproteases Aecting blood coagu-
lation brinolysis and platelet aggregation from snake ven-
oms: denition and nomenclature of interaction sites. Tox-
ins. 8: 330-338. 2016.
[27] KONDO, H.; KONDO, S.; IKEZAWA, Y.; MURATA, R.;
OHSAKA, A. Studies on the quantitative method for the de-
termination of hemorrhagic activity of Habu snake venom.
Japan J. Med. Sci. Biol. 13: 43-51. 1960.
[28] LAEMMLI, U.K. Cleavage of structural proteins during the
assembly of the head of bacteriophage T4. Nature. 227:
680-85. 1970.
[29] LANCINI, A. Familia Viperidae. Genero Bothrops. Serpi-
entes de Venezuela. 2nd Ed. Ediciones Ernesto Armitano,
Venezuela. Pp. 203-204. 1986.
[30] LEE J.H.; CHOI, H.S.; NASR, K.A.; HA, M.; KIM, Y.; FR-
ANGIONI, J.V. High-throughput small molecule identication
using MALDI-TOF and a nanolayered substrate. Analytical
Chemistry. 83 (13):5283-5289. 2011.
[31] LOMONTE, B.; REY-SUÁREZ, P.; TSAIA, W.; ANGULO, Y.;
SASA, M.; GUTIÉRREZ, J.M.; CALVETE, J. Snake venom-
ics of the pit vipers Porthidium nasutum Porthidium ophryo-
megas and Cerrophidion godmani from Costa Rica: Tox-
icological and taxonomical insights. J. Proteom. 75:1675-
1689. 2012.
[32] LOWRY, O.; ROSEBROUGH, N.; FARR, A.; RANDALL, R.
Protein measurement with the Folin phenol reagent. J. Biol.
Chem. 93: 265-275. 1951.
[33] MARKLAND, F.; SWENSON, S. Snake venom metallopro-
teinases. Toxicon. 62: 3–18. 2013.
[34] MARSH, N.A.; AROCHA-PIÑANGO, C.L. Evaluation of the
brin plate method for estimating plasminogen activators.
Thromb. Diath. Haemorrh. 28: 75–88. 1972.
[35] MOURA-DA-SILVA, A.M.; BALDO, C. Jararhagin a hemor-
rhagic snake venom metalloproteinase from Bothrops jara-
raca. Toxicon. 60: 280-289. 2012;
[36] OYAMA, E.; TAKAHASHI, H. Structures and functions
of snake venom metalloproteinases (SVMP) from Protobo-
throps venom collected in Japan. Molecules. 22: E1305/1-
11. 2017.
[37] PIFANO, F. Investigación y docencia en Medicina Tropical.
Arch. Ven. Med. Trop. Parasitol. Med. 4: 1-203. 1961.
[38] REY-SUÁREZ, P.; NÚÑEZ, V.; GUTIÉRREZ, J.M.; LOMON-
TE, B. Proteomic and biological characterization of the ven-
om of the red-tail coral snake Micrurus mipartitus (Elapidae)
from Colombia and Costa Rica. J. Proteom. 75: 655-667.
2011.
[39] RODRÍGUEZ-ACOSTA, A.; SÁNCHEZ, E.E.; MÁRQUEZ,
A.; CARVAJAL, Z.; SALAZAR, A.M.; GIRÓN, M.E.; ES-
TRELLA, A.; GIL, A.; GUERRERO, B. Hemostatic properties
of Venezuelan Bothrops snake venoms with special refer-
ence to Bothrops isabelae Venom. Toxicon. 56: 926-935.
156
Description of hutmannin-1 a new piii-metalloprotease from the venom / Pineda, M. y col.
2010.
[40] ROKYTA, D.R.; WRAY, K.P.; LEMMON, A.R.; LEMMON,
E.M.; CAUDLE, S.B. High-throughput venom-gland tran-
scriptome for the Eastern Diamondback Rattlesnake (Cro-
talus adamanteus) and evidence for pervasive positive se-
lection across toxin classes. Toxicon. 57: 657-671. 2011.
[41] SALAZAR, A.M.; RODRÍGUEZ-ACOSTA, A.; GIRÓN, M.E.;
AGUILAR, I.; GUERRERO, B. A comparative analysis of the
clotting and brinolytic activities of the snake venom (Both-
rops atrox) from dierent geographical areas in Venezuela.
Thromb. Res. 120: 95-104. 2007.
[42] SÁNCHEZ, E.E.; GIRÓN, M.E.; UZCÁTEGUI, N.L.;
GUERRERO, B.; SAUCEDO, M.; CUEVAS, E.; RODRÍ-
GUEZ-ACOSTA, A. Biochemical and biological charac-
terisation of lancehead (Bothrops venezuelensis Sandner
1952) snake venom from the Venezuelan Central Coastal
range. Bol. Malariol. San. Ambient. 54:138-149. 2014.
[43] SANDNER-MONTILLA, F. Una nueva especie de Bothrops
lansbergii de la familia Crotalidae.Mem.Cient.Odiol. 9:1-
16. 1989.
[44] TAKEDA, S. Adam and Adamts family proteins and snake
venom metalloproteinases: A structural overview. Toxins.
8:155. 2016.
[45] TERRA, R.; PINTO, A.; GUIMARAES, J.; FOX, J. Proteomic
proling of snake venom metalloproteinases (SVMPs): In-
sights into venom induced pathology. Toxicon. 54: 836-844.
2009.
[46] THEAKSTON, R.D.G.; REID, H.A. Development of simple
standard assay procedures for the characterization of snake
venoms. Bull. WHO. 61: 949-956. 1983.
[47] VAN TILBURG, M.F.; SOUSA, S.D.; FERREIRA DE MELO,
R.B.; MORENO, F.B.; MONTEIRO-MOREIRA, A.C.; Proteo-
me of the rete testis uid from tropically-adapted Morada
Nova rams. Anim Reprod Sci. 2017n;176:20-31.
[48] VARGAS, A.M.; FINOL, H.J.; SCANNONE, H.; FERNAN-
DEZ, I.; RODRIGUEZ-ACOSTA, A. Eects of lansberg’s
hognose pit vipers (Porthidium lansbergii hutmanni) venom
on renal ultrastructure in experimental mice. Acta. Scien.
Vet. 39: 1-6. 2011.
[49] WAHBY, A.F.; ABDEL-ATY, A.; EL-KADY, E.M. Purication of
hemorrhagic SVMPs from venoms of three vipers of Egypt.
Toxicon. 59: 329-337. 2012;
[50] WANG, T.; ZHANG, S.J.; CAO, S.L.; GUO, W.Z.; YAN, B.;
FANG, H.B.. Protective eects of salubrinal on liver injury in
rat models of brain death. Chin. Med. J. (Engl.) 128(11):1523-
8. 2015.
[51] WELDON, C.L.; MACKESSY, S.P. Alsophinase a new P-III
metalloproteinase with α-brinogenolytic and hemorrhag-
ic activity from the venom of the rear-fanged Puerto Rican
Racer Alsophis portoricensis (Serpentes: Dipsadidae). Bio-
chimie. 94: 1189-1198. 2012.
[52] XIONG, S; HUANG,C. Synergistic strategies of predominant
toxins in snake venoms. Toxicol. Lett. 2018; 287: 142-154.
Vol, XXX, N
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