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Research paper
Isolation of an acidic phospholipase A2 from the venom of the snake Bothrops
asper of Costa Rica: Biochemical and toxicological characterizationq
Julián Fernández a, José María Gutiérrez a, Yamileth Angulo a, Libia Sanz b,
Paula Juárez b, Juan J. Calvete b, Bruno Lomonte a,*
a Instituto Clodomiro Picado, Facultad de Microbiología, Universidad de Costa Rica, San José, Costa Rica
b Instituto de Biomedicina de Valencia, Consejo Superior de Investigaciones Científicas (CSIC), Jaume Roig 11, 46010 Valencia, Spain
a r t i c l e i n f o
Article history:
Received 19 October 2009
Accepted 8 December 2009
Available online 22 December 2009
Keywords:
Bothrops asper
Phospholipase A2
BaspPLA2-II
Snake venom
a b s t r a c t
Phospholipases A2 (PLA2) are major components of snake venoms, exerting a variety of relevant toxic
actions such as neurotoxicity and myotoxicity, among others. Since the majority of toxic PLA2s are basic
proteins, acidic isoforms and their possible roles in venoms are less understood. In this study, an acidic
enzyme (BaspPLA2-II) was isolated from the venom of Bothrops asper (Pacific region of Costa Rica) and
characterized. BaspPLA2-II is monomeric, with a mass of 14,212 
 6 Da and a pI of 4.9. Its complete
sequence of 124 amino acids was deduced through cDNA and protein sequencing, showing that it
belongs to the Asp49 group of catalytically active enzymes. In vivo and in vitro assays demonstrated that
BaspPLA2-II, in contrast to the basic Asp49 counterparts present in the same venom, lacks myotoxic,
cytotoxic, and anticoagulant activities. BaspPLA2-II also differed from other acidic PLA2s described in
Bothrops spp. venoms, as it did not show hypotensive and anti-platelet aggregation activities. Further-
more, this enzyme was not lethal to mice at intravenous doses up to 100 mg (5.9 mg/g), indicating its lack
of neurotoxic activity. The only toxic effect recorded in vivo was a moderate induction of local edema.
Therefore, the toxicological characteristics of BaspPLA2-II suggest that it does not play a key role in the
pathophysiology of envenomings by B. asper, and that its purpose might be restricted to digestive
functions. Immunochemical analyses using antibodies raised against BaspPLA2-II revealed that acidic and
basic PLA2s form two different antigenic groups in B. asper venom.

 2009 Elsevier Masson SAS. All rights reserved.
1. Introduction
The snake Bothrops asper is responsible for most cases of enve-
nomings in the Central American region [1,2]. Its venom contains
proteins that belong to at least eight families: serine proteinases,
disintegrins, metalloproteinases, L-amino acid oxidases, cysteine-
rich secretory proteins, DC fragments, C-type lectin-like proteins,
and phospholipases A2 (PLA2s) [3]. PLA2s are ubiquitous enzymes
that catalyze the hydrolysis of the C2 ester bond of 3-sn-phospho-
glycerides, producing lysophospholipids and free fatty acids in
a calcium-dependent reaction [4]. In snake venoms, PLA2s have
acquired during evolution the ability to exert different toxic activi-
ties in vivo, most notably neurotoxicity and myotoxicity [5e7]. The
PLA2 superfamily includes five types of enzymes (secreted PLA2s,
cytosolic PLA2s, calcium-independent PLA2s, lysosomal PLA2s, and
platelet-activating factor acetylhydrolases), classified within fifteen
groups [8]. Snake venom PLA2s are among the secreted PLA2s, and
those fromB. asper, in similarity to PLA2s of all viperids, belong to the
subgroup IIA. Proteins of this subgroup can be further subdivided
into two types: Asp49 PLA2s, which are catalytically active, and PLA2
homologues, which possessmost commonly a Lys49 residue and do
not have catalytic activity [9,10].
Both acidic and basic PLA2s can be found in snake venoms, in
variable proportions depending on the species. Nevertheless, the
basic isoforms appear to have acquired the highest toxicity, espe-
cially in the case of neurotoxic and myotoxic enzymes [11,12]. To
date, all acidic PLA2s purified from viperid venoms present an Asp
residue at position 49. These acidic isoforms usually have a higher
catalytic activity than basic PLA2s upon conventional substrates in
vitro [11,13,14]. In spite of this, many acidic PLA2s are not lethal or
show a weak lethal potency in mice [15e17].
Toxic effects induced by acidic PLA2s from Bothrops species were
demonstrated in early studies by Nisenbom et al. [18], who isolated
an enzyme from Bothrops alternatus causing severe tissue damage
in the liver, kidneys, lungs and heart of mice. More recent studies
have shown that acidic PLA2s from Bothrops spp. venoms may
express other toxic actions in vivo, such as myotoxicity and
q UniProt Knowledgebase accession number of BaspPLA2-II is P86389.
* Corresponding author. Tel.: þ506 22290344.
E-mail address: bruno.lomonte@ucr.ac.cr (B. Lomonte).
Contents lists available at ScienceDirect
Biochimie
journal homepage: www.elsevier .com/locate/biochi
0300-9084/$ e see front matter 
 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.biochi.2009.12.006
Biochimie 92 (2010) 273e283
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hypotensive activity, as well in vitro, such as neuromuscular
blockade and inhibition of platelet aggregation [14,17,19e23]. In the
case of B. asper, Ferlan and Gubensek [24] purified an acidic enzyme
from the venom of specimens from Costa Rica, PLA2 I, which
showed a lethal intravenous potency of 2 mg/g in mice. Alagón et al.
[25] characterized three acidic isoforms from the venom of B. asper
from Mexico, named PLA2 1, PLA2 2 and PLA2 3. This multiplicity of
acidic PLA2 isoforms in the venoms of B. asper from the Pacific and
Caribbean regions of Costa Rica has also been evidenced by iso-
electrofocusing techniques [26], andmore recently confirmed using
a proteomic approach [3].
The potential toxic activities of acidic PLA2s of B. asper venom
have not yet been identified, and therefore their possible roles in
the pathophysiology of envenoming are still unknown. In the
present work, an acidic PLA2 from B. asper venom (BaspPLA2-II) was
isolated and thoroughly characterized, to gain insights into its
possible biological roles and relevance in the pathophysiology of
envenomings by B. asper.
2. Materials and methods
2.1. Isolation of BaspPLA2-II
Crude venom was obtained from more than twenty specimens
of B. asper from the Pacific region of Costa Rica, kept at the ser-
pentarium of Instituto Clodomiro Picado. The venom was pooled,
centrifuged to remove debris, lyophilized, and stored at 20 C.
Batches of 500 mg of venom were dissolved in 6 ml of 0.1 M
ammonium acetate buffer, pH 7.0, and applied to a CM-Sephadex
C25 column (20  2 cm) equilibrated with the same buffer. Protein
elution was monitored at 280 nm using an Econo-system chro-
matograph (Bio-Rad), at 0.4 ml/min. The unbound fraction was
collected and applied to a DEAE-Sepharose column (23  3 cm),
which was eluted at 0.5 ml/min with a linear gradient of ammo-
nium acetate, from 0.1 to 1.0 M, at pH 7.0. Fractions were assayed
for PLA2 activity as described below, and freeze-dried. The fraction
with highest activity was subjected to reverse-phase high-perfor-
mance liquid chromatography (RP-HPLC) on a semi-preparative C8
column (Vydac, 250  10 mm, 5 mm particle size), monitored at
280 nm on an Agilent 1100 chromatograph. The protein was dis-
solved in 1 ml of buffer A (0.1% trifluoroacetic acid [TFA], 5%
acetonitrile, 95% water), injected, and eluted at 1 ml/min with
a linear gradient from 0 to 70% buffer B (0.1% TFA, 95% acetonitrile,
5% water) in 55 min. The main peak was collected, dried by vacuum
centrifugation, dissolved in 1 ml of 0.1 M ammonium acetate
buffer, pH 5.0, and finally applied to a CM-Sephadex column
(1  5 cm) equilibrated with the same buffer, to remove traces of
a contaminant. The unbound fraction was collected, freeze-dried,
and stored at 20 C. Homogeneity of the final preparation was
evaluated by sodium dodecylsulphate-polyacrylamide (15%) gel
electrophoresis (SDS-PAGE) under reducing and non-reducing
conditions, followed by Coomassie blue R-250 staining. In some
experiments, two basic PLA2s from B. asper venom were included
for comparative purposes: myotoxin I is a catalytically active Asp49
enzyme [27], whereas myotoxin II is a catalytically inactive Lys49
homologue [28].
2.2. Isoelectric point and molecular mass determinations
Two-dimensional polyacrylamide gel electrophoresis of Basp-
PLA2-II was performed on a Multiphor II (Amersham Bioscience)
apparatus. For the first dimension, 5 mg of enzyme were loaded
onto a 7 cm IPG Immobiline Dry Strip of pH range 3e10, and
focused at 200 V for 1 min, followed by 3500 V for 120 min. Second
dimension was run on 12% SDS-PAGE and stained by Coomassie.
The experimentally observed pI was compared with the theoreti-
cally predicted value based on the amino acid sequence, using the
Compute pI/MW tool at the ExPASy Proteomics Server (www.
expasy.ch/tools). The molecular mass of BaspPLA2-II was deter-
mined by electrospray ionization (ESI-MS) on a QTrap 2000
instrument (Applied Biosystems).
2.3. Amino acid sequence
The N-terminal sequence of BaspPLA2-II was obtained directly
by automated Edman sequencing on a Procise Instruments Seque-
nator (Applied Biosystems). Then, protein fragments were gener-
ated with cyanogen bromide and separated by RP-HPLC using an
Ettan LC system (Amersham)with a C18 column (250 4mm, 5 mm
particle size) eluted at a flow rate of 1 ml/minwith a linear gradient
of 0.1% TFA in water (buffer A) or in acetonitrile (buffer B): 5% B for
10 min, followed by 5e15% B over 20 min, 15e45% B for 120 min,
and 45e70% B over 20 min. Detection of peptides was monitored at
215 nm, and the main fragments recovered were subjected to
Edman sequencing. Additional internal peptides of BaspPLA2-II
were sequenced by tandem MS. Protein bands were excised from
Coomassie-stained, reduced 15% gels (SDS-PAGE) and subjected to
automated reduction with dithiothreitol, alkylation with iodoace-
tamide, and digestion with sequencing grade bovine pancreatic
trypsin (Roche) using a Progest Digestion Station (Genomic Solu-
tions), following manufacturer's instructions. A total of 0.65 ml of
the tryptic peptide mixtures (total volume of 20 ml) was spotted
onto a MALDI-TOF sample holder, mixed with an equal volume of
a saturated solution of a-cyano-4-hydroxycinnamic acid in 50%
acetonitrile containing 0.1% TFA, dried, and analyzed with
a Voyager-DE Pro MALDI-TOF mass spectrometer (Applied Bio-
systems), operated in delayed extraction and reflector modes. For
peptide sequencing, the protein digest mixture was loaded in
a nanospray capillary column and subjected to ESI-MS analysis on
a QTrap 2000 instrument equipped with a nanospray source (Pro-
tana). Doubly- or triply-charged ions of selected peptides from the
MALDI-TOF mass fingerprint spectra were analyzed in Enhanced
Resolution MS mode, and the monoisotopic ions were fragmented
using the Enhanced Product Ion tool with Q0 trapping. Enhanced
Resolution was performed at 250 amu/s across the entire mass
range. Settings for MS/MS experiments were as follows: Q1, unit
resolution; Q1-to-Q2 collision energy, 30e40 eV; Q3 entry barrier,
8 V; LIT (linear ion trap) Q3 fill time, 250 ms; and Q3 scan rate,
1000 amu/s. CID spectra were interpreted manually or using
a licensed version of MASCOT (www.matrixscience.com) against
a private database containing 927 viperid protein sequences
deposited in the Swiss-Prot/TrEMBL database, plus the previously
assigned peptide ion sequences from snake venomics projects
carried out in the laboratory of J.J. Calvete. MS/MS mass tolerance
was set to 
 0.6 Da. Carbamidomethylcysteine and oxidation of
methionine were fixed and variable modifications, respectively.
2.4. cDNA cloning and nucleotide sequencing
The complete sequence of BaspPLA2-II was deduced from the
cloning and nucleotide sequencing of its cDNA. Total RNA was
extracted from the venom glands of B. asper (Pacific Region) using
specifications of the RNEasy Protect Mini kit (Qiagen). BaspPLA2-II
specific mRNA underwent reverse transcription to obtain cDNA
with a FirstChoice RLM-RACE Kit (Ambion) using a rapid ampli-
fication of 30 cDNA ends polymerase chain reaction (30 RACE-PCR).
According to the kit specifications, the entire mRNA was first
transformed into cDNAusing an OligodTwith the following adapter
sequence: 50-GCGAGCACAGAATTAATACGACTCACTATAGGT12VN-30.
From the cDNA obtained, BaspPLA2-II sequencewas amplified using
J. Fernández et al. / Biochimie 92 (2010) 273e283274
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a gene specific primer for the enzyme. This primer was designed
on the basis of the N-terminal sequence data. The primer 50-
TGGCAATTCGGGCAAATGATG-30 corresponds to the N-terminal
portion WQFGQMM of the protein. The 30 RACE Outer Primer had
the sequence 50-GCGAGCACAGAATTAATACGACT-30, and the 30 RACE
Inner Primer had the sequence 50-CGCGGATCCGAATTAATACGA
CTCACTATAGG-30. The 30 RACE-PCR was conducted using the
M-MLV reverse transcriptase (Promega). The reaction occurred
under the following conditions: 5 min at 65 C and then 1 h at
42 C. For the second part of the reaction a touchdown PCR was
carried out from 60 to 50 C. The touchdown 60/50 PCR protocol
included an initial denaturation step at 95 C for 10min followed by
4 cycles of denaturation (30 s at 94 C), annealing (30 s at 60 C),
and extension (30 s at 72 C); 21 cycles starting with the above
conditions and, in subsequent cycles, decreasing the annealing
temperature by 0.5 C (reaching 50 C in cycle 21); 10 cycles of
denaturation (30 s at 94 C), annealing (30 s at 50 C), and extension
(30 s at 72 C); and a final extension for 10 min at 72 C. Products
were identified on 2% agarose gel electrophoresis, searching for
bands of approximately 0.4 kb. The cDNA obtainedwas treatedwith
the ExoSAP-IT kit (Affymetrix) for 15 min at 37 C and 15 min at
80 C to remove all contaminants. Then, dA tails were added to the
cDNA for 30 min at 72 C. Subsequently, the cDNA was cloned into
the pGEM-T vector (Promega) overnight at 4 C. Once the cDNA
was ligated to the vector, Escherichia coli strain DH5a (Novagen)
were transformed by electroporation. A PCR was used to detect the
presence of the vector with BaspPLA2-II sequence in the colonies.
Transformed bacteria were incubated overnight at 37 C, and then
the vector was extracted with the Wizard plus Minipreps DNA
purification system (Promega) DNA extraction kit. Final nucleotide
sequencing was performed with an Applied Biosystems model 377
instrument, using primers T7 and SP6.
2.5. Molecular modeling
Homology modeling using the Swiss-Model server (http://
swissmodel.expasy.org) was utilized to predict the three-dimen-
sional structure of BaspPLA2-II using the acidic PLA2 from Bothrops
jararacussu (PDB code 1ZL7) as a template, which has a sequence
identity of 81%, and has been crystallized and resolved at 1.6 Å [29].
Superposition of model and template structures, and r.m.s.d.
calculations were performed with Swiss-PdbViewer [30] and DS
ViewerPro (Accelrys).
2.6. Phospholipase A2 activity
Enzymatic activity of BaspPLA2-II was determined by the
colorimetric method of de Araujo and Radvanyi [31], with phenol
red as a pH indicator, upon micelles of 0.4% v/v Triton X-100
and 0.25% w/v sn-3-phosphatidylcholine as substrate. Twenty
microliters of a solution of enzyme, containing 500, 250, 125, or
62.5 ng, in water, were added to 1 ml of substrate in a thermo-
regulated cuvette at 30 C. After a stabilization period of 20 s, the
decrease in absorbance at 558 nmwas monitored continuously for
1 min. One unit of PLA2 activity was defined as the change of 0.001
in absorbance per min. Results obtained with this method were
additionally confirmed by means of the titrimetric assay of Dole
[32] using egg yolk phospholipids, as described [33], and expressed
as mEq/mg/min of enzyme activity.
2.7. Anticoagulant activity
Citrated (3.8% v/v) human plasma was obtained from the blood
of healthy volunteers. Aliquots of 0.2 ml were dispensed into glass
tubes and incubated in awater bath for 5min at 37 C. Then, 50 ml of
a BaspPLA2-II solution in phosphate-buffered saline (PBS; 0.12 M
NaCl, 0.04 M sodium phosphate, pH 7.2) containing 40 mg of
enzyme were added, and further incubated for 10 min at 37 C.
Control tubes contained plasma incubated with PBS only. Finally,
50 ml of 0.25 M CaCl2 was added to all tubes and the clotting time
was determined, in duplicate assays.
2.8. Anti-platelet aggregating activity
Fresh platelet-rich human plasma was prepared by centrifuga-
tion of citrated blood from healthy volunteers, at 135 g for 15 min.
Aliquots of 450 ml of this preparation were incubated with Basp-
PLA2-II at final concentrations up to 20 mg/ml plasma, for 5 min at
37 C. Then, platelet aggregation was initiated by adding 5 ml of
0.1 mM ADP and monitored through the increase in the light
transmittance using a model 530-VS aggregometer (Chrono-Log
Corporation). Platelet-poor plasma (450 ml), obtained after centri-
fugation at 1500 g for 15 min, was used as a blank. Platelet-rich
plasma incubated with 50 ml of ADP alone served as a positive
control for aggregation. Assays were performed in duplicate.
2.9. Cytotoxic activity
The cytotoxic activity of BaspPLA2-II on C2C12 skeletal muscle
cell cultures was determined as described [34]. Doses up to 40 mg of
the enzyme were diluted in assay medium (Dulbecco's modified
Eagle Medium supplemented with 1% fetal bovine serum) and
added to cells growing in 96-well plates, in a volume of 100 ml/well.
Control wells consisted of medium alone (0% toxicity), or 0.1%
Triton X-100 in medium (100% toxicity). After 3 h at 37 C, 40 ml of
the supernatant were taken to determine the activity of lactic
dehydrogenase released by damaged cells, using a kinetic assay
(LDH-P Mono, Biocon Diagnostik). Assays were performed in
duplicate.
2.10. Lethal activity
To evaluate the lethal activity of BaspPLA2-II, four CD-1 mice
(16e18 g body weight) received an intravenous injection of 100 mg
of enzyme, dissolved in 100 ml of PBS. As a control, two mice were
injected similarly with 100 ml of PBS alone. Animals were observed
up to 24 h after injection to record deaths. All animal experiments
were approved by the Institutional Committee for the Care and Use
of Laboratory Animals of the University of Costa Rica (CICUA).
2.11. Myotoxic activity
A group of five mice (18e20 g) received an intramuscular
injection of 50 mg of BaspPLA2-II, dissolved in 50 ml of PBS, in their
right gastrocnemius. A control group received an identical injection
of PBS alone. After 3 h, a tail blood sample was collected into
heparinized capillaries, centrifuged, and a plasma aliquot of 4 ml
was utilized to determine the activity of creatine kinase (CK; E.C.
2.7.3.2) using a kinetic assay (CK-Nac, Biocon Diagnostik). Enzyme
activity was expressed in U/L. Myotoxicity was also assessed by
histological evaluation. Twenty-four hour after BaspPLA2-II injec-
tion mice were sacrificed by inhalation of carbon dioxide, and
samples of their right gastrocnemius were obtained, fixed in 3.7%
formalin, and processed for hematoxylineeosin staining of
paraffin-embedded sections.
2.12. Histological evaluation of systemic toxicity
For histological assessment of the systemic toxicity of BaspPLA2-
II, twomice (16e18 g) received an intravenous injection of 100 mg of
J. Fernández et al. / Biochimie 92 (2010) 273e283 275
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the enzyme, dissolved in 100 ml of PBS. As a control, two mice were
injected identically with PBS alone. Animals were euthanized by
carbon dioxide inhalation 24 h after injection, and samples of liver,
lungs, heart, and kidneys were obtained. Tissues were fixed and
processed as described above.
2.13. Edema-forming activity
A group of four mice (18e20 g) received an injection of 10 mg of
BaspPLA2-II, dissolved in 50 ml of PBS, in the footpad. As a control,
another group received an identical injection of PBS alone. Footpad
thickness was measured with a low-pressure spring caliper (Odit-
est) before and at various intervals after injection (30, 60, 120, 180,
240, 300, and 360 min). Edema was expressed as the percentage
increase in thickness relative to readings obtained before injection.
2.14. Hypotensive activity
A non-invasive blood pressure monitoring system (CODA, Kent
Scientific Corporation) was utilized to evaluate the hypotensive
activity of BaspPLA2-II in a group of five mice (18e20 g). Blood
pressure was determined before, and at 5 and 30 min after the
intravenous injection of 10 mg of BaspPLA2-II, dissolved in 100 ml
PBS. As a positive control, another group of mice received 4 mg of
crude B. asper venom i.v., in 100 ml of PBS. A negative control group
received an i.v. injection of 100 ml of PBS alone.
2.15. Preparation of rabbit antibodies against BaspPLA2-II
Antibodies to BaspPLA2-II were prepared by immunization of
two rabbits with the purified enzyme, either intramuscularly or
subcutaneously. An initial dose of 100 mg, emulsified in complete
Freund's adjuvant, was followed by booster doses of 50 mg in
incomplete adjuvant, at weeks 5 and 10. Rabbits were bled at week
12 and their sera were separated, aliquoted, and stored at 20 C.
2.16. Immunochemical analyses
Rabbit antibodies raised against BaspPLA2-II, together with
previouslyobtained rabbit antibodies toB. aspermyotoxin I [35], and
the equine polyvalent (Crotalinae) antivenom produced at Instituto
Clodomiro Picado [36] were utilized to analyze the immunochem-
ical relationships between the acidic and basic PLA2s of B. asper.
Antibody characterization was performed by double immunodiffu-
sion in gel, enzyme-immunoassay (EIA), and immunoblotting.
Immunodiffusion was carried out in 1% agarose-PBS gels, loading
30 ml/well of undiluted sera, enzymes (0.2 mg/ml) or crude venom
(2 mg/ml), and read after 24 h. For the EIA, 0.2 mg/well of enzymes
(BaspPLA2-II or myotoxin I) were adsorbed onto microplates as
described [37]. Afterwashing andblockingexcess free siteswith PBS
containing 1% bovine serum albumin (BSA), varying dilutions of
antisera were added to triplicate wells and incubated for 1 h. After
five washings with FALC buffer (Tris 0.05 M, NaCl 0.15 M, ZnCl2
20 mM, MgCl2 1 mM, pH 7.4), bound antibodies were detected with
either anti-horse IgG or anti-rabbit IgGealkaline phosphatase
conjugates (1:5000) and p-nitrophenylphosphate as substrate.
Absorbances were recorded on a Multiskan RC microplate reader
(Labsystems) at 405 nm. Normal sera of the corresponding animal
species were utilized as negative controls. For immunoblotting,
30 mg of crude B. asper venom were separated by SDS-PAGE (15%)
under reducing conditions, followed by electrotransfer to nitrocel-
lulose in a Bio-Rad cell at 150 mA during 90 min. To assess transfer
efficiency, membranes were previsualized by reversible Ponceau-S
Red staining. Then, membranes were blocked in 1% BSAePBS for
30 min, and incubated for 90 minwith 1:1000 dilutions of antisera,
or the corresponding normal sera for each species. After washing
four times with PBS containing 0.1% BSA and 0.05% Tween-20, the
membraneswere incubatedwith the appropriate anti-IgGealkaline
phosphatase conjugates (1:2000) during 90 min. Membranes were
finally washed four times, and color development was performed
with the BCIP/NBT substrate (Chemicon).
2.17. Neutralization of BaspPLA2-II enzymatic activity
by rabbit and equine antibodies
BaspPLA2-II was preincubated for 30 min at 37 C with rabbit
antiserum or equine antivenom, at ratios of 0.5, 1, 2, and 4 ml
serum/mg enzyme. Then, aliquots containing 0.25 mg or 15 mg of
enzyme were assayed for PLA2 activity, as described above, using
the colorimetric or the titrimetric assays, respectively. Controls
included identical enzyme aliquots incubated with PBS alone, or
with normal sera from the corresponding species. Assays were
performed in duplicate.
2.18. Statistical analysis
Results are expressed as mean 
 S.D. The significance of
differences between the means of two experimental groups was
analyzed by Student's t-test, where a p value <0.05 was considered
significant.
3. Results
3.1. Isolation and biochemical properties of BaspPLA2-II
To ensure the removal of basic PLA2s and PLA2 homologues of B.
asper venom, the first chromatographic step was performed in CM-
Sephadex at pH 7.0 (Fig. 1A), where such components were
retained. The unbound material was subsequently resolved into
several peaks by the DEAE-Sepharose step, where the highest PLA2
activity eluted in fraction D1 (Fig. 1B). The subsequent RP-HPLC
separation of this fraction (Fig. 1C) eliminated most contaminants,
but traces of a procoagulant venom component still remained, only
detectable by its clotting activity upon human plasma (data not
shown). This minor contaminant, most likely a thrombin-like
serine proteinase [38], was successfully removed from BaspPLA2-II
by a final fractionation step on CM-Sephadex at pH 5.0, where it
was retained by the chromatographic support.
Electrophoretic analyses of BaspPLA2-II by SDS-PAGE showed
that this enzyme migrates as a monomer of approximately
15e16 kDa, both under reducing and non-reducing conditions
(Fig. 1D), consistent with the molecular mass of 14,212 
 6 Da
determined by ESI-MS. Experimental assessment of the pI of this
enzyme by 2D electrophoresis resulted in an estimated value of 4.9,
close to the theoretical pI value of 5.05 predicted on the basis of its
complete sequence.
The amino acid sequence of BaspPLA2-II was obtained by
a combination of Edman degradation, tandem mass spectrometry,
and nucleotide sequencing of its cloned cDNA. It is composed of 124
amino acid residues, containing the conserved Asp49 of catalyti-
cally active enzymes (Fig. 2). The calculated isotope-averaged
molecular mass for the amino acid sequence shown in Fig. 2
(14,179.97) is about 32 
 6 Da lower than the experimentally
determined mass, suggesting that the protein may contain modi-
fied residues. In line with this assumption, the C-terminal peptide
was sequenced by MS/MS analysis of the doubly-charged peptidic
ion atm/z 576.6 as NCQE(129)SEPC. The sequences of the b5 and y5
daughter ions were interpreted as NCQE(D-oMe) and (D-oMe)SEPC,
respectively, indicating that Asp120 was o-methylated. The
remaining 16 Da difference between experimental and calculated
J. Fernández et al. / Biochimie 92 (2010) 273e283276
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masses may correspond to oxidation of one of the 5 methionine
residues of the protein.
The PLA2 activity of BaspPLA2-II was confirmed, as shown in
Fig. 3. In comparison to myotoxin I, a basic Asp49 isoform from
B. asper venom, this acidic enzyme was slightly more active in
hydrolyzing phosphatidylcholine micelles.
Comparison of BaspPLA2-II with similar proteins in the Swis-
sProt database showed that its primary structure is closely related
to several group IIA acidic PLA2s of crotaline species, mostly to the
enzymes isolated from Bothrops jararaca, Bothrops insularis, and
B. jararacussu (Fig. 4). Multiple sequence alignment evidenced that
only BaspPLA2-II and the B. jararaca P81243 enzyme present 124
amino acids within this group of proteins, all others having 122, or
123 in the case of Gloydius ussurensis (Q7LZU4). This difference is
caused by the insertion of two residues, Thr67 and Tyr68, in both
BaspPLA2-II and the B. jararaca PLA2 (Fig. 4). A phylogenetic tree
Fig. 1. Isolation of BaspPLA2-II. (A) Fractionation of crude B. asper venom on CM-Sephadex at pH 7.0, eluted with ammonium acetate (0.1e1.0 M), as described in Materials and
Methods. The unbound fraction (star) was subjected to separation on DEAE-Sepharose (B) using an identical gradient as in (A). Fraction D1 (thick horizontal line) was further
purified by RP-HPLC on a semi-preparative C8 column (C), eluted with a 5e70% acetonitrile gradient over 55 min. (D) SDS-PAGE (15%) analysis of BaspPLA2-II under reduced (R) and
non-reduced (NR) conditions. LMW: low molecular weight markers, as indicated at the left, in kDa.
Fig. 2. Amino acid sequence of BaspPLA2-II. The first 44 amino acid residues
were determined by direct Edman degradation sequencing from the N-terminus.
Overlapping peptides were generated by protein cleavage with CNBr. Other internal
fragments, obtained after trypsin digestion, were sequenced de novo by ESI-MS/MS.
Molecular mass values of the fragments are indicated.
0
100
200
300
400
0 125 250 375 500
)ni
m
·gµ/U(
 ytivitcA
Phospholipase (ng) 
BaspPLA -II 
Mt-I
2
Fig. 3. Phospholipase A2 activity of BaspPLA2-II and myotoxin I (Mt-I) from B. asper
venom upon phosphatidylcholine micelles, determined by the phenol red assay, as
described in Materials and Methods. (C) BaspPLA2-II; (B) myotoxin I. Each point
represents mean 
 SD of duplicates.
J. Fernández et al. / Biochimie 92 (2010) 273e283 277
Author's personal copy
constructed with 15 acidic PLA2s confirmed the close evolutionary
relationship of BaspPLA2-II with the enzymes of B. jararaca, B.
insularis, and B. jararacussu from South America (Fig. 5), whereas
the acidic PLA2s from other South American Bothrops, such as B.
erythromelas and B. pictus, were more distant from the clade of
BaspPLA2-II. On the other hand, the basic PLA2 myotoxin I (P20474)
from B. asper venom was markedly distant from BaspPLA2-II in the
cladogram, serving as an outgroup (Fig. 5), and confirming the
divergent evolutionary pathways of acidic and basic PLA2s even
within the venom of a single viperid species.
A three-dimensional model of BaspPLA2-II was built using as
template the crystal structure of B. jararacussu acidic PLA2. Both
structures were superimposed, as shown in Fig. 6, resulting in
average r.m.s.d. value for a-carbon backbones of 1.27 Å. The main
structural deviation between the BaspPLA2-II model and its
template protein was predicted to occur immediately before the
Fig. 4. Multiple sequence alignment of BaspPLA2-II with related proteins in crotaline snake venoms. Protein codes correspond to the UniProtKB database at the ExPASy Proteomics
Server. Alignments and percent identity calculations were performed with ClustalW [57]. Identical positions are shaded in gray, and cysteine residues are shown in boldface. A black
background highlights the insertion of two amino acids at positions 69 and 70, only present in BaspPLA2-II and the PLA2 (P81243) of B. jararaca.
Q6H3C9 Trimeresurus stejnegeri
P81479 Trimeresurus gramineus
Q2HZ28 Bothrops erythromelas
Q9I8F8 Bothrops pictus
P20249 Gloydius blomhoffii
O42192 Gloydius halys
Q2TU95 Sistrurus catenatus
Q7SID6 Deinagkistrodon acutus
Q7LZQ4 Gloydius ussurensis
A8E2V8 Trimeresurus gracilis
P81243 Bothrops jararaca
BaspPLA2-II Bothrops asper
Q8QG87 Bothrops insularis
Q8AXY1 Bothrops jararacussu
C3W4R6 Vipera lebetina
Q91506 Protobothrops mucrosquamatus
P20474 Bothrops asper
Fig. 5. Phylogenetic relationships of BaspPLA2-II with other phospholipases A2 from
snake venoms. Protein codes are as described in Fig. 4. The cladogramwas constructed
using the maximum likelihood method implemented in the PhyML program at www.
phylogeny.fr [58], and graphically represented with TreeDyn. Support values for
branches are indicated as percentages. In addition to all proteins aligned in Fig. 4,
a basic PLA2 from Bothrops asper (P20474; myotoxin I), and an acidic PLA2 from a non-
crotaline viperid (Vipera lebetina; C3W4R6) were included as outgroups.
Fig. 6. Three-dimensional model of BaspPLA2-II (dark gray), superimposed on the
crystal structure of B. jararacussu acidic PLA2 (PDB code 1ZL7; light gray), in ribbon
representation. N- and C-termini are labeled. Amino acid side chains of T67 and Y68 of
BaspPLA2-II are shown in dark gray. The dashed circle highlights the large deviation
in segment 62e65, immediately before the b-wing region, which bulges out in
comparison to the structure of the template protein.
J. Fernández et al. / Biochimie 92 (2010) 273e283278
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“b-wing” region, where residues 62e65 bulge out, probably due to
the insertion of the additional Thr67 and Tyr68 (see alignments of
Fig. 4) within the constraints of the relatively rigid scaffold of PLA2s.
3.2. Biological activities of BaspPLA2-II in vitro and in vivo
BaspPLA2-II did not exert anticoagulant effect upon human
plasma in vitro, up to a concentration of 100 mg/ml. The mean time
for clot formation in plasma incubated with this enzyme was
230 
 34 s, while plasma incubated with PBS clotted after
217 
 21 s (p > 0.05). Under the same conditions, myotoxin I pro-
longed the clotting time of plasma to 2400 
 50 s (p < 0.05).
BaspPLA2-II also lacked anti-aggregating activity for ADP-stimu-
lated human platelets, up to a concentration of 10 mg/ml of enzyme.
Similarly, this enzyme did not lyse skeletal muscle C2C12myoblasts
in culture, in contrast to the basic Lys49 myotoxin II used as
a control (Fig. 7A). Exposure of these cells to BaspPLA2-II, up to
40 mg/well (400 mg/ml) for 3 h, did not induce morphological
alterations nor LDH release to the supernatants.
In vivo, the i.m. injection of BaspPLA2-II (50 mg) did not increase
plasma CK levels after 3 h (Fig. 7B), indicating its lack of myotoxic
activity. This was also confirmed by histological evaluation of the
injected gastrocnemius muscle, obtained after 24 h, which showed
a normal tissue morphology (Fig. 7C and D). Similarly to observa-
tions made on skeletal muscle, the histological evaluation of other
tissues, including liver, lungs, heart, and kidneys, after the i.v.
injection of 100 mg of BaspPLA2-II, indicated in all cases a normal
morphology, similar to the corresponding tissues of control mice
receiving a PBS injection (not shown). In addition, BaspPLA2-II
was not lethal to mice by the i.v. route, up to a dose of 100 mg
(5.6e6.2 mg/g). No changes in the blood pressure of mice were
recorded after the i.v. injection of this enzyme (10 mg), whereas
injection of the crude venom (4 mg) under identical conditions
caused a rapid and transient drop in this parameter (Fig. 8) One
mouse was also injected with 25 mg of BaspPLA2-II, and there were
no changes in blood pressure (data not shown). The only toxic effect
induced by BaspPLA2-II was a transient induction of local edema in
the mouse footpad assay (Fig. 9).
3.3. Immunochemical analyses of BaspPLA2-II
Rabbits immunized with BaspPLA2-II by i.m. or by s.c. routes,
respectively, produced an antibody response to the enzyme, as
shown by the ability of their sera to form a precipitin line against
both the purified BaspPLA2-II or crude B. asper venom by gel
immunodiffusion (Fig. 10A). The sera of these two rabbits had
similar titers by EIA (data not shown). These rabbit antibodies
recognized a single band of 15e16 kDa in crude B. asper venom
subjected to immunoblotting analysis (Fig. 10B), corresponding to
the expected migration of the enzyme, and further supporting the
homogeneity of the immunizing preparation as well as the
monospecificity of the antiserum. By EIA, rabbit antibodies to
BaspPLA2-II recognized the homologous antigen, but not the basic
PLA2 myotoxin I, resulting in a signal close to that of non-immune
sera (Fig. 10C). Reciprocally, rabbit antibodies to myotoxin I readily
recognized this basic protein in the EIA, but did not cross-react with
the acidic BaspPLA2-II (Fig. 10C). On the other hand, the equine
Fig. 7. Lack of muscle damaging activity of BaspPLA2-II. (A) Cytotoxicity was evaluated upon cultured C2C12 skeletal muscle cells, exposed to BaspPLA2-II or to B. aspermyotoxin II as
a control. Lactic dehydrogenase (LDH) release was determined after 3 h. Each point represents mean 
 SD of duplicate assays. (B) Myotoxic activity was evaluated by determining
plasma creatine kinase (CK) activity 3 h after i.m. injection of BaspPLA2-II (50 mg/ml) or B. asper myotoxin I (50 mg/ml) or PBS (50 ml) as controls. (C) Histologic evaluation of
hematoxylineeosin stained sections of gastrocnemius muscle 24 h after the i.m. injection of BaspPLA2-II (50 mg/50 ml) or (D) PBS (50 ml).
Fig. 8. Lack of hypotensive effect of BaspPLA2-II. Mean blood pressure of mice was
recorded before (0 min) and after (5 and 30 min) the i.v. injection of this enzyme
(10 mg), crude B. asper venom (4 mg), or PBS alone, under identical conditions. Points
represent mean 
 SD of five animals per group. The asterisk indicates a statistically
significant (p < 0.05) difference.
J. Fernández et al. / Biochimie 92 (2010) 273e283 279
Author's personal copy
polyvalent antivenom produced at Instituto Clodomiro Picado
clearly recognized BaspPLA2-II by EIA, resulting in a titration curve
comparable to that corresponding to antibodies against myotoxin I
(Fig. 11A). However, as shown in Fig. 11B and C, when the ability of
equine and rabbit antibodies to neutralize the enzymatic activity of
BaspPLA2-II was tested in preincubation assays, neutralization was
only partial in the case of the polyvalent antivenom, whereas
inhibition by the rabbit serumwas null, even at a very high serum/
enzyme ratio (4 ml/mg).
4. Discussion
The first complete biochemical and toxicological characteriza-
tion of an acidic PLA2 from the venom of B. asper, here named
BaspPLA2-II, is reported. This enzyme is monomeric, with a pI of 4.9
and a molecular mass of 14,212 
 6 Da. According to its structural
characteristics, this protein corresponds to the fraction described as
peak 12 in the venom proteome of B. asper (Pacific region of Costa
Rica), which matches its molecular mass, N-terminal and internal
peptide sequences, and pI on 2-D gel electrophoresis [3]. On this
basis, and considering the quantitative data generated by the
Fig. 10. (A) Gel immunodiffusion of rabbit antibodies to BaspPLA2-II. Immune sera from two rabbits (S1, S2) precipitated BaspPLA2-II, both in purified form (PLA2) or in the crude
B. asper venom (V), as identified by the fused precipitin lines. (B) Immunoblotting analysis of the rabbit serum to BaspPLA2-II. Crude B. asper venom (30 mg, reduced) was subjected
to SDS-PAGE (15%), transferred to nitrocellulose, and probed with the rabbit antibodies as described in Materials and Methods. (C) Antigenic comparison of BaspPLA2-II and B. asper
myotoxin I (Mt-I) by enzyme-immunoassay. Both antigens were adsorbed to microplates and probed with rabbit antibodies against BaspPLA2-II or Mt-I, respectively. The dashed
line represents the absorbance value of non-immune rabbit sera.
Fig. 9. Edema-forming activity of BaspPLA2-II. Footpad thickness was determined
before and after injection of the enzyme (10 mg dissolved in 50 ml PBS), and edema was
expressed as the percentage increase relative to readings obtained before injection.
(B) BaspPLA2-II; (C) PBS control. Each point represents mean 
 SD of four animals.
J. Fernández et al. / Biochimie 92 (2010) 273e283280
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proteomic analysis of this venom, it is estimated that BaspPLA2-II
represents 6.3% of its proteins [3,39].
BaspPLA2-II is composed of 124 amino acids, presenting Asp49
and the characteristic pattern of half-Cys residues of group IIA
PLA2s [8]. These findings place BaspPLA2-II within the catalytically
active enzymes, consistent with all acidic PLA2s purified from
viperid snake venoms, where Asp49 appears so far to be an
absolutely conserved position. The phospholipolytic activity of
BaspPLA2-II was confirmed using phosphatidylcholine micelles as
substrate, and shown to be slightly higher than the activity of
myotoxin I, a basic PLA2 of the same venom. In general, snake
venom acidic PLA2s tend to be more active in catalysis than basic
isoforms, in spite of the stronger toxicity of the latter [11,13,14].
The primary structure of BaspPLA2-II presents high identity
values in comparison to other acidic enzymes within the genus
Bothrops, particularly P81243 from B. jararaca [19], Q8QG87 from
B. insularis [40], and Q8AXY1 from B. jararacussu [17]. Of these, only
the PLA2 from B. jararaca shares with BaspPLA2-II the feature of
having 124 amino acids (as opposed to the pattern of 122 residues
of most of these enzymes), caused by two insertions at positions 67
and 68. Interestingly, the three proteins with highest similarity to
BaspPLA2-II express some toxic activities, such as inhibition of
platelet aggregation (P81243 and Q8AXY1), myotoxicity (Q8QG87),
and hypotensive effect (Q8AXY1), whereas the toxicological char-
acterization of BaspPLA2-II, here presented, evidenced none of
these actions. Negative results were obtained for anticoagulant,
anti-platelet aggregation, cytotoxic, myotoxic, hypotensive, and
lethal effects of BaspPLA2-II. The growing structural information on
acidic PLA2s that differ in their toxic activities, or even lack toxicity,
may become of value to address the complex structureefunction
relationships that govern this highly diverse group of snake venom
proteins. The elucidation of an increasing number of venom pro-
teomes, or venomes, has revealed that PLA2s constitute percentages
as large as 30e60% in some species [39,41], strongly arguing for
their relevance in such secretions. In the case of BaspPLA2-II, with
the exception of a transient, moderate edema-inducing effect, the
observed lack of toxic activities implies that its contribution to the
overall physiopathology of envenomings by B. asper is probably of
marginal relevance. Rather, the present results suggest that this
enzyme could play mainly a digestive function in this venom, by
contributing to the hydrolysis of phospholipids of the prey,
a hypothesis that would need to be addressed. Alternatively, this
“non-toxic” enzyme could have yet unknown toxic actions upon
the physiology of prey other than rodents. Since it is known that
neonate and juvenile specimens of B. asper feed on ectothermic
prey, i.e. frogs and lizards [42], it would be relevant to assess the
toxic profile of BaspPLA2-II in these prey.
The induction of edema by BaspPLA2-II is consistent with
reports of this activity being expressed by a number of acidic PLA2s
from snake venoms [14,16,17,20e22,40,43,44]. Mechanisms that
underlie this effect have been attributed to phospholipid hydro-
lysis, resulting in the release of precursors of eicosanoids and
platelet-activating factor, or to the degranulation of mast cells, with
subsequent release of vasoactive amines [14,45,46].
BaspPLA2-II was devoid of myotoxic activity in vivo, as deter-
mined by the lack of plasma CK increase and by histological
observation. This result was in agreement with the absence of
cytolytic action upon C2C12 cells, known to represent a good
correlate for myotoxicity in the case of group IIA PLA2s [34]. These
findings are consistent with observations in most of the acidic
PLA2s purified from snake venoms, which generally lack myotox-
icity. However, some recently isolated acidic PLA2s display
myotoxicity in vivo [14,20,21,40,47]. Although it is clear that the
catalytic activity of PLA2s is not a sufficient requirement to generate
myotoxicity per se, the structural determinants of such differences
in myotoxicity among acidic enzymes are unknown, and their
identification poses a challenging question.
The fact that BaspPLA2-II was not lethal up to a dose of 5.9 mg/g,
and the lack of systemic toxicity to major organs, is also in line with
literature reports for several acidic PLA2s which display a very low
or no lethal activity. It also indicates that BaspPLA2-II does not
correspond to the acidic PLA2 isolated from B. asper venom by
Ferlan and Gubensek [24], which had a lethal intravenous activity
of 2 mg/g. A comparison of partial amino acid sequences between
BaspPLA2-II and another acidic PLA2 isolated from the venom of
B. asper from Panamá (to be named BaspPLA2-I, personal commu-
nication of A.M. Soares, University of São Paulo, Brasil) revealed
several structural differences. Therefore, the different acidic PLA2
isoforms that are present in B. asper venom may vary in the
expression of toxic effects.
BaspPLA2-II did not present anticoagulant activity, an effect
which has been reported for a number of acidic PLA2s from viperid
0 20 40 60 80 100
Activity (%)
BaspPLA2-II 
BaspPLA2-II +  PAV
BaspPLA2-II  +  RAbs
0 20 40 60 80 100
Activity (%)
BaspPLA 2-II
BaspPLA2-II + PAV
BaspPLA2-II + RAbs
0,0
0,2
0,4
0,6
0,8
1,0
1,2
1,4
1,6
1/300 1/900 1/2700 1/8100 1/24300 1/72900
m
n
504ta
ec
nabr
osbA
Serum dilution
PAV + Mt-I
NHS+ Mt- I
PAV + BaspPLA2-II
NHS+ BaspPLA2-II
A
C
B
Fig. 11. Titration of antibodies to BaspPLA2-II or to B. asper myotoxin I (Mt-I) in the
equine polyvalent (Crotalinae) antivenom from Instituto Clodomiro Picado (PAV),
determined by EIA (A). NHS: normal horse serum. Points represent mean 
 SD of
triplicate wells. Neutralization of PLA2 activity of BaspPLA2-II by polyvalent antivenom
or rabbit immune serum (RAbs), determined by the phenol red (B) or titrimetric
(C) assays. Bars represent mean 
 SD of duplicates.
J. Fernández et al. / Biochimie 92 (2010) 273e283 281
Author's personal copy
snake venoms [20,22]. Moreover, anti-platelet aggregating activity
and hypotensive effect were also absent in BaspPLA2-II, again at
variance with other acidic PLA2s [14,17,19e23,48e50]. It will be
important to determine in future studies if the hypotensive effect of
whole B. asper venom is induced by other acidic PLA2 isoforms or by
proteins/peptides from other families. Interestingly, the proteomic
analyses of B. asper venoms from both versants of Costa Rica
(Caribbean and Pacific) indicate the absence of bradykinin-poten-
tiating peptides [3], known as important mediators of hypotension
in other Bothrops species [51].
Immunochemical analyses with antibodies raised against
BaspPLA2-II revealed that this acidic enzyme differs antigenically
from the basic PLA2s in B. asper venom. This conclusion was also
supported by the reciprocal analyses using antibodies to myotoxin I,
which did not recognize BaspPLA2-II. The antigenic divergence
between snake venom PLA2s from elapids and viperids has been
demonstrated in previous studies [52,53]. However, the present
results offer a first evidence of antigenic divergence among acidic
and basic PLA2s within viperids, in this case, within the venom of
a single species. Thus, despite a sequence identity of 52% between B.
asper myotoxin I and BaspPLA2-II, antibodies against each of the
two were not cross-reactive.
Interestingly, despite the clear presence of antibodies to Basp-
PLA2-II in both its homologous rabbit serum and in the equine
polyvalent antivenom, their ability to neutralize its catalytic activity
was poor. This suggests the possibility that BaspPLA2-II may induce
antibodies towards immunodominant non-neutralizing epitopes,
in sharp contrast with the case of basic PLA2s of this venom, which
are well neutralized by antibodies fromvarious sources [35,54e56].
Nevertheless, the lack of major toxic activities of BaspPLA2-II, as
shown in the present study, predicts that its poor neutralization by
antivenoms should not be of concern from a therapeutic point of
view.
In conclusion, the newly characterized protein BaspPLA2-II is
a monomeric, acidic PLA2 from B. asper (Pacific versant of Costa
Rica) venom which, due to its lack of major toxic actions reported
for this type of proteins, probably has a marginal role in enve-
nomings by this snake species. This enzyme might play a digestive
function. The elucidation of its complete primary structure, here
reported, will be of value in future comparative studies attempting
to identify the molecular determinants of toxic activities by some
acidic PLA2s in crotaline venoms.
Acknowledgements
Financial support to this study by grants from the Ministerio de
Educación y Ciencia (Madrid, Spain), the CRUSA-CSIC Program,
CONARE, and Vicerrectoría de Investigación (Universidad de Costa
Rica, project 741-A9-506) is gratefully acknowledged. We also
thank Dr Teresa Escalante, Dr Mahmood Sasa, and the staff of
Centro de Investigación en Hematología y Trastornos Afines
(CIHATA-UCR) for their valuable help in various aspects of this
study. This work was performed in partial fulfillment of the
requirements for the M.Sc. degree of J. Fernández at the University
of Costa Rica.
References
[1] J.M. Gutiérrez, Clinical toxicology of snakebite in Central America. in: J. Meier,
J. White (Eds.), Handbook of Clinical Toxicology of Animal Venoms and
Poisons. CRC Press, Boca Raton, 1995, pp. 645e665.
[2] M. Sasa, S. Vázquez, Snakebite envenomation in Costa Rica: a revision of
incidence in the decade 1990e2000. Toxicon 41 (2003) 19e22.
[3] A. Alape-Girón, L. Sanz, J. Escolano, M. Flores-Díaz, M. Madrigal, M. Sasa,
J.J. Calvete, Snake venomics of the lancehead pitviper Bothrops asper:
geographic, individual, and ontogenetic variations. J. Proteome Res. 7 (2008)
3556e3571.
[4] D.L. Scott, S.P. White, Z. Otwinowski, W. Yuan, M.H. Gelb, P.B. Sigler, Inter-
facial catalysis: the mechanism of phospholipase A2. Science 250 (1990)
1541e1546.
[5] R.M. Kini, Excitement ahead: structure, function and mechanism of snake
venom phospholipase A2 enzymes. Toxicon 42 (2003) 827e840.
[6] J.M. Gutiérrez, B. Lomonte, Phospholipase A2 myotoxins from Bothrops snake
venoms. in: R.M. Kini (Ed.), Venom Phospholipase A2 Enzymes: Structure,
Function and Mechanism. Wiley, Chichester, 1997, pp. 321e352.
[7] C. Montecucco, J.M. Gutiérrez, B. Lomonte, Cellular pathology induced by
snake venom phospholipase A2 myotoxins and neurotoxins: common
aspects of their mechanisms of action. Cell. Mol. Life Sci. 65 (2008)
2897e2912.
[8] R.H. Schaloske, E.A. Dennis, The phospholipase A2 superfamily and its group
numbering system. Biochim. Biophys. Acta 1761 (2006) 1246e1259.
[9] B. Lomonte, Y. Angulo, L. Calderón, An overview of lysine-49 phospholipase
A2 myotoxins from crotalid snake venoms and their structural determinants
of myotoxic action. Toxicon 42 (2003) 885e901.
[10] B. Lomonte, Y. Angulo, M. Sasa, J.M. Gutiérrez, The phospholipase A2
homologues of snake venoms: biological activities and their possible adap-
tive roles. Protein Pept. Lett. 16 (2009) 860e876.
[11] P. Rosenberg, The relationship between enzymatic activity and pharmaco-
logical properties of phospholipases in natural poisons. in: J.B. Harris (Ed.),
Natural Toxins Animal, Plant and Microbial. Oxford Science Publications,
Oxford, 1986, pp. 129e174.
[12] I. Krizaj, J. Siigur, M. Samel, V. Cotic, F. Gubensek, Isolation, partial charac-
terization, and complete amino acid sequence of the toxic phospholipase A2
from the venom of the common viper, Vipera berus berus. Biochim. Biophys.
Acta 1157 (1993) 81e85.
[13] P. Rosenberg, Phospholipases. in: W.T. Shier, D. Mebs (Eds.), Handbook of
Toxinology. Marcel Dekker, New York, 1990, pp. 67e277.
[14] N.A. Santos-Filho, L.B. Silveira, C.Z. Oliveira, C.P. Bernardes, D.L. Menaldo,
A.L. Fuly, E.C. Arantes, S.V. Sampaio, C.C. Mamede, M.E. Beletti, F. de Oliveira,
A.M. Soares, A new acidic myotoxic, anti-platelet and prostaglandin I2
inductor phospholipase A2 isolated from Bothrops moojeni snake venom.
Toxicon 52 (2008) 908e917.
[15] A.L. de Araújo, F. Radvanyi, C. Bon, Purification of an acidic phospholipase A2
from Bothrops lanceolatus (fer de lance) venom: molecular and enzymatic
properties. Toxicon 32 (1994) 1069e1081.
[16] J.J. Daniele, I.D. Bianco, G.D. Fidelio, Kinetic and pharmacologic character-
ization of phospholipases A2 from Bothrops neuwiedii venom. Arch. Biochem.
Biophys. 318 (1995) 65e70.
[17] S.H. Andrião-Escarso, A.M. Soares, M.R. Fontes, A.L. Fuly, F.M. Corrêa, J.C. Rosa,
L.J. Greene, J.R. Giglio, Structural and functional characterization of an acidic
platelet aggregation inhibitor and hypotensive phospholipase A2 from
Bothrops jararacussu snake venom. Biochem. Pharmacol. 64 (2002) 723e732.
[18] H.E. Nisenbom, J.C. Perazzo, A.J. Monserrat, J.C. Vidal, Contribution of phos-
pholipase A2 to the lethal potency of Bothrops alternatus (víbora de la cruz)
venom. Toxicon 24 (1986) 807e817.
[19] S.M. Serrano, A.P. Reichl, R. Mentele, E.A. Auerswald, M.L. Santoro,
C.A. Sampaio, A.C. Camargo, M.T. Assakura, A novel phospholipase A2, BJ-
PLA2, from the venom of the snake Bothrops jararaca: purification, primary
structure analysis, and its characterization as a platelet-aggregation-inhib-
iting factor. Arch. Biochem. Biophys. 367 (1999) 26e32.
[20] D.F. Ketelhut, M.H. de Mello, E.L. Veronese, L.E. Esmeraldino, M.T. Murakami,
R.K. Arni, J.R. Giglio, A.C. Cintra, S.V. Sampaio, Isolation, characterization and
biological activity of acidic phospholipase A2 isoforms from Bothrops jarar-
acussu snake venom. Biochimie 10 (2003) 983e991.
[21] R.S. Rodrigues, L.F. Izidoro, S.S. Teixeira, L.B. Silveira, A. Hamaguchi,
M.I. Homsi-Brandeburgo, H.S. Selistre-de-Araújo, J.R. Giglio, A.L. Fuly,
A.M. Soares, V.M. Rodrigues, Isolation and functional characterization of
a new myotoxic acidic phospholipase A2 from Bothrops pauloensis snake
venom. Toxicon 50 (2007) 153e165.
[22] P.G. Roberto, S. Kashima, S. Marcussi, J.O. Pereira, S. Astolfi-Filho, A. Nomizo,
J.R. Giglio, M.R. Fontes, A.M. Soares, S.C. França, Cloning and identification of
a complete cDNA coding for a bactericidal and antitumoral acidic phos-
pholipase A2 from Bothrops jararacussu venom. Protein J. 23 (2004) 273e285.
[23] J.C. de Albuquerque Modesto, P.J. Spencer, M. Fritzen, R.C. Valença, M.L. Oliva,
M.B. da Silva, A.M. Chudzinski-Tavassi,M.C. Guarnieri, BE-I-PLA2, a novel acidic
phospholipase A2 from Bothrops erythromelas venom: isolation, cloning and
characterization as potent anti-platelet and inductor of prostaglandin I2
release by endothelial cells. Biochem. Pharmacol. 72 (2006) 377e384.
[24] I. Ferlan, F. Gubensek, Phospholipases of Bothrops asper venom. Period. Biol.
80 (1978) 31e36.
[25] A.C. Alagón, R.R. Molinar, L.D. Possani, P.L. Fletcher, J.E. Cronan, J.Z. Julia,
Venom from the snake Bothrops asper Garman. Purification and character-
ization of three phospholipases A2. Biochem. J. 185 (1980) 695e704.
[26] E. Moreno, A. Alape, M. Sánchez, J.M. Gutiérrez, A new method for the
detection of phospholipase A2 variants: identification of isozymes in the
venoms of newborn and adult Bothrops asper (terciopelo) snakes. Toxicon 26
(1988) 363e371.
[27] J.M. Gutiérrez, C.L. Ownby, G.V. Odell, Isolation of a myotoxin from Bothrops
asper venom: partial characterization and action on skeletal muscle. Toxicon
22 (1984) 115e128.
[28] B. Lomonte, J.M.Gutiérrez, Anewmuscle damaging toxin,myotoxin II, from the
venom of the snake Bothrops asper (terciopelo). Toxicon 27 (1989) 725e733.
J. Fernández et al. / Biochimie 92 (2010) 273e283282
Author's personal copy
[29] M.T. Murakami, A. Gabdoulkhakov, N. Genov, A.C.O. Cintra, C. Betzel,
R.K. Arni, Insights into metal ion binding in phospholipases A2: ultra high-
resolution crystal structures of an acidic phospholipase A2 in the Ca2þ free
and bound states. Biochimie 88 (2006) 543e549.
[30] K. Arnold, L. Bordoli, J. Kopp, T. Schwede, The SWISS-MODEL Workspace:
a web-based environment for protein structure homology modelling. Bio-
informatics 22 (2006) 195e201.
[31] A.L. de Araújo, F. Radvanyi, Determination of phospholipase A2 activity by
a colorimetric assay using a pH indicator. Toxicon 25 (1987) 1181e1188.
[32] V.P. Dole, A relation between non-esterified fatty acids in plasma and the
metabolism of glucose. J. Clin. Invest. 35 (1956) 150e154.
[33] J.M. Gutiérrez, B. Lomonte, F. Chaves, E. Moreno, L. Cerdas, Pharmacological
activities of a toxic phospholipase a isolated from the venom of the snake
Bothrops asper. Comp. Biochem. Physiol. 84C (1986) 159e164.
[34] B. Lomonte, Y. Angulo, S. Rufini, W. Cho, J.R. Giglio, M. Ohno, J.J. Daniele,
P. Geoghegan, J.M. Gutiérrez, Comparative study of the cytolytic activity of
myotoxic phospholipases A2 on mouse endothelial (tEnd) and skeletal
muscle (C2C12) cells in vitro. Toxicon 37 (1999) 145e158.
[35] B. Lomonte, J.M. Gutiérrez, E. Moreno, L. Cerdas, Antibody neutralization of
a myotoxin from the venom of Bothrops asper (terciopelo). Toxicon 25 (1987)
443e449.
[36] Y. Angulo, R. Estrada, J.M. Gutiérrez, Clinical and laboratory alterations in
horses during immunization with snake venoms for the production of
polyvalent (Crotalinae) antivenom. Toxicon 35 (1997) 81e90.
[37] B. Lomonte, J.M. Gutiérrez, G. Rojas, L. Calderón, Quantitation by enzyme-
immunoassay of antibodies against Bothrops myotoxins in four commer-
cially-available antivenoms. Toxicon 29 (1991) 695e702.
[38] A.V. Pérez, A. Rucavado, L. Sanz, J.J. Calvete, J.M. Gutiérrez, Isolation and
characterization of a serine proteinase with thrombin-like activity from the
venom of the snake Bothrops asper. Braz. J. Med. Biol. Res. 41 (2008) 12e17.
[39] J.J. Calvete, P. Juárez, L. Sanz, Snake venomics. Strategy and applications.
J. Mass Spectrom. 42 (2007) 1405e1414.
[40] J.C. Cogo, S. Lilla, G.H. Souza, S. Hyslop, G. de Nucci, Purification, sequencing
and structural analysis of two acidic phospholipases A2 from the venom of
Bothrops insularis (jararaca ilhoa). Biochimie 12 (2006) 1947e1959.
[41] J.J. Calvete, L. Sanz, Y. Angulo, B. Lomonte, J.M. Gutiérrez, Venoms, venomics,
antivenomics. FEBS Lett. 583 (2009) 1736e1743.
[42] M. Sasa, D.K. Wasko, W.W. Lamar, Natural history of the terciopelo Bothrops
asper (Serpentes: Viperidae) in Costa Rica. Toxicon 54 (2009) 904e922.
[43] A.L. Fuly, A.L. de Miranda, R.B. Zingali, J.A. Guimarães, Purification and
characterization of a phospholipase A2 isoenzyme isolated from Lachesis
muta snake venom. Biochem. Pharmacol. 63 (2002) 1589e1597.
[44] I.H. Tsai, Y.M. Wang, Y.H. Chen, A.T. Tu, Geographic variations, cloning, and
functional analyses of the venom acidic phospholipases A2 of Crotalus viridis
viridis. Arch. Biochem. Biophys. 411 (2003) 289e296.
[45] E.C. Landucci, R.C. de Castro, M. Toyama, J.R. Giglio, S. Marangoni, G. De Nucci,
E. Antunes, Inflammatory oedema induced by the Lys49 phospholipase A2
homologue piratoxin-I in the rat and rabbit. Effect of polyanions and
p-bromophenacyl bromide. Biochem. Pharmacol. 59 (2000) 1289e1294.
[46] C. Teixeira, Y. Cury, V. Moreira, G. Picolo, F. Chaves, Inflammation induced by
Bothrops asper venom. Toxicon 54 (2009) 988e997.
[47] A.L. Fuly, S. Calil-Elias, R.B. Zingali, J.A. Guimarães, P.A. Melo, Myotoxic
activity of an acidic phospholipase A2 isolated from Lachesis muta (bush-
master) snake venom. Toxicon 38 (2000) 961e972.
[48] X. Liu, H. Pan, G. Yang, X. Wu, G. Zhou, Cloning, expression and biochemical
characterization of a basiceacidic hybrid phospholipase A2-II from Agkis-
trodon halys pallas. Biochim. Biophys. Acta 1431 (1999) 157e165.
[49] A.J. Magro, M.T. Murakami, S. Marcussi, A.M. Soares, R.K. Arni, M.R. Fontes,
Crystal structure of an acidic platelet aggregation inhibitor and hypotensive
phospholipase A2 in the monomeric and dimeric states: insights into its
oligomeric state. Biochem. Biophys. Res. Commun. 323 (2004) 24e31.
[50] Y. Wang, G. Cui, M. Zhao, J. Yang, C. Wang, R.W. Giese, S. Peng, Bioassay-
directed purification of an acidic phospholipase A2 from Agkistrodon halys
pallas venom. Toxicon 51 (2008) 1131e1139.
[51] M.A. Hayashi, A.C. Camargo, The bradykinin-potentiating peptides from
venom gland and brain of Bothrops jararaca contain highly site specific
inhibitors of the somatic angiotensin-converting enzyme. Toxicon 45 (2005)
1163e1170.
[52] A. Ménez, Molecular immunology of snake toxins. Pharmacol. Ther. 30
(1985) 91e113.
[53] J.L. Middlebrook, I.I. Kaiser, Immunological relationships of phospholipase A2
neurotoxins from snake venoms. Toxicon 27 (1989) 965e977.
[54] B. Lomonte, J.M. Gutiérrez, E. Mata, Isolation from a polyvalent antivenom of
antibodies to a myotoxin in Bothrops asper snake venom. Toxicon 23 (1985)
807e813.
[55] B. Lomonte, J.M. Gutiérrez, E. Carmona, M.E. Rovira, Equine antibodies to
Bothrops asper myotoxin II: isolation from polyvalent antivenom and
neutralizing ability. Toxicon 28 (1990) 379e384.
[56] B. Lomonte, J.M. Gutiérrez, M. Ramírez, C. Díaz, Neutralization of myotoxic
phospholipases A2 from the venom of the snake Bothrops asper by mono-
clonal antibodies. Toxicon 30 (1992) 239e245.
[57] D. Higgins, J. Thompson, T. Gibson, J.D. Thompson, D.G. Higgins,
T.J. Gibson, CLUSTAL W: improving the sensitivity of progressive multiple
sequence alignment through sequence weighting, position-specific gap
penalties and weight matrix choice. Nucleic Acids Res. 22 (1994)
4673e4680.
[58] A. Dereeper, V. Guignon, G. Blanc, S. Audic, S. Buffet, F. Chevenet,
J.F. Dufayard, S. Guindon, V. Lefort, M. Lescot, J.M. Claverie, O. Gascuel,
Phylogeny.fr: robust phylogenetic analysis for the non-specialist. Nucleic
Acids Res. 36 (web server issue) (2008) W465eW469.
J. Fernández et al. / Biochimie 92 (2010) 273e283 283