Toxicon 54 (2009) 949–957

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Toxicon
journal homepage: www.elsevier.com/locate/toxicon

Biochemistry and toxicology of toxins purified from the venom of

the snake Bothrops asper
Yamileth Angulo a, b, Bruno Lomonte a, *
a
Instituto Clodomiro Picado, Facultad de Microbiologı́a, Universidad de Costa Rica, San José, Costa Rica
b
Departamento de Bioquı́mica, Escuela de Medicina, Universidad de Costa Rica, San José, Costa Rica

a r t i c l e i n f o a b s t r a c t

Article history: The isolation and study of individual snake venom components paves the way for a deeper
Received 11 November 2008 understanding of the pathophysiology of envenomings – thus potentially contributing to
Accepted 9 December 2008 improved therapeutic modalities in the clinical setting – and also opens possibilities for
Available online 16 December 2008
the discovery of novel toxins that might be useful as tools for dissecting cellular and
molecular processes of biomedical importance. This review provides a summary of the
Keywords:
different toxins that have been isolated and characterized from the venom of Bothrops
Bothrops asper
asper, the snake species causing the majority of human envenomings in Central America.
Snake
Venom This venom contains proteins belonging to at least eight families: metalloproteinase, serine
Toxin proteinase, C-type lectin-like, L-amino acid oxidase, disintegrin, DC-fragment, cystein-rich
secretory protein, and phospholipase A2. Some 25 venom proteins within these families
have been isolated and characterized. Their main biochemical properties and toxic actions
are described, including, in some cases, their possible relationships to the pathologic
effects induced by B. asper venom.
Ó 2008 Published by Elsevier Ltd.

1. Introduction (Jiménez-Porras, 1970; Tu, 1977). This heterogeneous

The harmful and even fatal consequences of snakebites
have been noted by mankind since the times of ancient
civilizations. Venomous snakes can inspire both fear and
fascination, and have been linked to mystical and religious
concepts in many cultures throughout history. It is not
surprising that with the advent of modern science, snake
venoms became the subject of intense studies aimed at
understanding their biochemical composition, and the
modes by which they cause harmful effects.
Snake venoms are toxic secretions produced by a pair of
specialized exocrine glands connected to the fangs by ducts
(Kochva et al., 1980; Mackessy and Baxter, 2006). Such
secretions are complex mixtures of molecules of different
biochemical nature, with a predominance of proteins,
many of which are endowed with enzymatic activities
* Corresponding author.
E-mail address: blomonte@cariari.ucr.ac.cr (B. Lomonte).
nature of venom composition was evidenced since the
earliest analytical studies, and hence associated with the
wide variety of bioactivities, both in vitro and in vivo, that
were observed clinically or experimentally. Thus, it became
widely established that specific activities of a snake venom
could be attributed to particular components or toxins.
While this general principle has been very useful and
important in the study of venoms, it is not always valid, as
there may be effects that are caused by two or more toxins
acting in combination, i.e. synergistically. Moreover, a given
toxin may have more than one specific activity, and
therefore, it may play multiple roles in the overall effects of
envenoming. Notwithstanding these considerations, the
isolation and characterization of individual venom
components constitutes the mainstay of toxinology, as
a key strategy to dissect and to analyze the complex series
of events involved in envenomings. Thus, steered by the
development and refinement of chromatographic tech-
niques, early studies of snake venoms using whole,
unfractionated secretions rapidly evolved into detailed

0041-0101/$ – see front matter Ó 2008 Published by Elsevier Ltd.

doi:10.1016/j.toxicon.2008.12.014
950 Y. Angulo, B. Lomonte / Toxicon 54 (2009) 949–957

analyses of their individual components. The purpose of
this review is to provide an updated summary of the
different toxins that have been isolated and characterized
from the venom of Bothrops asper, the snake species
causing the majority of human envenomings in Central
America (Gutiérrez, 1995).
2. Toxins isolated from B. asper venom
The first biochemical analyses of B. asper venom
attempting to dissect its constituents and to establish their
correlation with different enzymatic activities are probably
those of Jiménez-Porras (1964), who utilized starch gel
electrophoresis to separate at least 14 fractions from the
venom of this species (at the time classified as Bothrops
atrox). Differences in the venom composition of B. asper
from the Pacific and the Caribbean (Atlantic) regions of
Costa Rica were noticed since early studies on its
biochemical and toxicological properties (Jiménez-Porras,
1964; Gutiérrez et al., 1980; Aragón and Gubenšek, 1981).
Variations in the frequency of occurrence of some electro-
phoretic bands, as well as in the activity levels of several
enzymes and pharmacological effects were described. For
example, B. asper specimens inhabiting the Caribbean
versant of Costa Rica were shown to produce venom that is
more hemorrhagic and myotoxic than that of specimens
from the Pacific versant, which display higher proteolytic
activity (Gutiérrez et al., 1980).
Proteomic analyses have now revealed the presence of
proteins belonging to at least eight families in B. asper
venom: metalloproteinase (41–44%), phospholipase A2
(29–45%), serine proteinase (4–18%), L-amino acid oxidase
(5–9%), disintegrin (1–2%), C-type lectin-like (0.5%), cys-
tein-rich secretory protein (CRISP) (0.1%), and DC-frag-
ment (<0.1%) (Alape-Girón et al., 2008). Representative
toxins of some of these protein families have been iso-
lated, as listed in Table 1. A few of them have been
characterized biochemically and structurally, and have
been the subject of a number of studies aimed at
understanding their mechanisms of action. Other toxins
have only been described with partial biochemical and
functional characterizations. The following sections
summarize the main properties and actions of the toxins
listed in Table 1, and in cases where such information has
been obtained, their relationships to the pathologic
effects induced by B. asper venom.

Table 1
Proteins isolated from the venom of the snake Bothrops asper.

Protein family Toxin name Properties Sequence, PDBa Reference

Phospholipase A2
D49 PLA I acidic, 32 kDa – Ferlan and Gubenšek (1978)
PLA II acidic, 16 kDa – Ferlan and Gubenšek (1978)
PLA2 1 acidic, 11 kDa – Alagón et al. (1980)
PLA2 2 acidic, 11 kDa – Alagón et al. (1980)
PLA2 3 acidic, 29 kDa – Alagón et al. (1980)
myotoxin I basic, 15 kDa – Gutiérrez et al. (1984)
myotoxic PLA2 basic, 15 kDa – Mebs and Samejima (1986)
myotoxin III basic, pI 8.7, 15 kDa P20474 Kaiser et al. (1990)

K49 myotoxin II basic, pI 9.1, 15 kDa P24605, 1CLP Lomonte and Gutiérrez (1989)
myotoxin IV basic, 15 kDa – Dı́az et al. (1995)

Metalloproteinase
P-I proteinase G neutral, pI 7.1, 18 kDa – Aragón and Gubenšek (1987)
BaP1 basic, pI 8.2, 23 kDa P83512, 1ND1 Gutiérrez et al. (1995a)

P-III BaH1 acidic, pI 4.5, 64 kDa – Borkow et al. (1993)

BH2 acidic, pI 5.2, 26 kDa – Borkow et al. (1993)
BH3 acidic, pI 5.0, 55 kDa – Borkow et al. (1993)
BaH4 acidic, pI 5.3, 69 kDa – Franceschi et al. (2000)
basparin A acidic, 70 kDa – Lorı́a et al. (2003)

Serine proteinase
asperase acidic, 30 kDa – Aragón-Ortiz and Gubenšek (1978)
ficozyme acidic, 25 kDa – Fořtová et al. (1990)
thrombin-like acidic, 27 kDa Q072L6 Pérez et al. (2008)

L-amino acid oxidase

Laao 1 acidic, 125 kDa – Umaña (1982a)
Laao 2a acidic, 125 kDa – Umaña (1982a)
Laao 2b acidic, 125 kDa – Umaña (1982a)

C-type lectin-like
aspercetin acidic, 30 kDa – Rucavado et al. (2001)

Disintegrin
bothrasperin acidic, 8 kDa – Pinto et al. (2003)
a
Amino acid sequence entry code in UniProtKB/TrEMBL, and Protein Data Bank (PDB) entry code.
 Y. Angulo, B. Lomonte / Toxicon 54 (2009) 949–957
2.1. Myotoxic phospholipases A2 (PLA2) and PLA2 homologues
In similarity with other species of viperid snakes
(Serrano et al., 2005; Guércio et al., 2006; Calvete et al.,
2007; Sanz et al., 2008; Angulo et al., 2008; Lomonte et al.,
2008; Gutiérrez et al., 2008b), the venom of B. asper
contains a significant proportion of PLA2 enzymes (Alape-
Girón et al., 2008), both acidic and basic.
Ferlan and Gubenšek (1978) isolated two PLA2 enzymes
of this venom, named PLA I and PLA II, with approximate
molecular masses of 32 kDa and 16 kDa, respectively. On
the basis of their amino acid compositions, these should be
acidic. Their intravenous lethal activities for mice were
estimated at 2 and 10 mg/g body weight, respectively. These
lethal potencies are markedly lower than those corre-
sponding to the highly neurotoxic PLA2s present in the
venoms of many elapid snake species (Rosenberg, 1990),
and such finding would be in agreement with the lack of
neurotoxic symptoms in patients bitten by B. asper
(Gutiérrez, 1995), since no PLA2s with high lethal/neuro-
toxic potency have been found in this venom. Alagón et al.
(1980) also reported the isolation and biochemical char-
acterization of acidic PLA2s from B. asper venom, named
PLA2 1, PLA2 2, and PLA2 3 (Table 1). However, their possible
toxic activities were not determined. On the basis of reports
on acidic PLA2s isolated from the venoms of other Bothrops
species (Serrano et al., 1999; Andrião-Escarso et al., 2002;
Roberto et al., 2004; Modesto et al., 2006), it is likely that
acidic enzymes of B. asper may display effects upon platelet
aggregation, but other activities such as neuromuscular
blockade, myotoxicity, and hypotensive effect have also
been reported for these enzymes (Andrião-Escarso et al.,
2002; Cogo et al., 2006; Rodrigues et al., 2007). The
possible role(s) of the acidic PLA2 isozymes in the patho-
physiology of envenomings by B. asper needs to be evalu-
ated in future studies.
On the other hand, basic PLA2s are more abundant than
their acidic counterparts in B. asper venom (Alape-Girón
et al., 2008) and several isoforms have been isolated (Table 1).
All of them have been shown to damage skeletal muscle
tissue within minutes of their intramuscular injection in
mice, reproducing the drastic myonecrotic picture induced
by the whole venom (Gutiérrez and Lomonte, 1989, 1995,
1997, 2003). Thus, this group of proteins has been commonly
referred to as PLA2 myotoxins. Selective neutralization of
these basic PLA2s by the use of specific anti-myotoxin
antibodies nearly abrogates the muscle damage induced by
the whole venom of B. asper (Lomonte et al., 1987, 1990,
1992), therefore indicating that these toxins are the main
components responsible for myonecrosis in envenomings by
this species.
If administered systemically, i.e. by intravenous route,
the PLA2 myotoxins do not increase the plasma levels of
creatine kinase, an enzyme marker of skeletal muscle
damage, indicating that they behave as locally acting
myotoxins (Gutiérrez and Ownby, 2003; Gutiérrez et al.,
2008a). Biochemical characterization and amino acid
sequence determination have shown that these proteins,
all classified within the group IIA of secreted PLA2s, belong
to two distinct subgroups. The Asp49 subgroup includes
isoforms that are catalytically active, whereas the subgroup
951
of Lys49 PLA2 homologues does not display such enzymatic
activity (Lomonte et al., 2003; Chioato and Ward, 2003).
B. asper myotoxins I, III, as well as the PLA2 reported by
Mebs and Samejima (1986) are Asp49 isoforms, whereas
myotoxins II and IV are members of the Lys49 PLA2
homologues subgroup (Table 1). Noteworthy, B. asper
presents an ontogenetic regulation in the expression of all
these basic PLA2 myotoxins in its venom: they are absent in
newborns, and appear as snakes become juveniles, reach-
ing high proportions in the venom composition of adults
(Gutiérrez et al., 1980; Lomonte and Carmona, 1992; Saravia
et al., 2001; Alape-Girón et al., 2008).
The basic PLA2s of B. asper are made of 121–122 amino
acid residues, containing 14 Cys residues that engage in
seven disulfide bridges, and have pI values in the range of
8.5–9.5 (Kaiser et al., 1990; Francis et al., 1991). These
proteins are not glycosylated, and form stable, non-
covalently associated homodimers in their native state
(Lomonte and Gutiérrez, 1989; Francis et al., 1991). In the
case of myotoxin II, it was shown that under acidic pH
conditions (<5.0) the dimers dissociate into monomers with
a concomitant decrease in toxic potency, both in vitro and in
vivo (Angulo et al., 2005). Thus, it is possible that the dimeric
structure of these PLA2s may lead to a higher avidity for their
target(s) by means of a divalent interaction, in comparison
to the monovalent binding of the dissociated monomers.
A similar hypothesis has been presented by Montecucco and
Rossetto (2008) to explain the oligomeric structure of the
highly potent neurotoxic PLA2s from snake venoms.
Among the basic PLA2s of B. asper, myotoxin II (Lomonte
and Gutiérrez, 1989) is the most thoroughly studied toxin,
structurally and functionally. The three-dimensional
structure of this Lys49 protein was determined by Arni et al.
(1995) and Murakami et al. (2005) at resolutions of 2.8 Å
and 1.7 Å, respectively, allowing a deeper understanding of
its structure–function relationships. The mechanism of
action of myotoxin II involves the interaction of its highly
cationic C-terminal region with anionic moieties on target
membranes, which subsequently lose permeability control
and undergo a series of degenerative events related to Ca2þ
influx, ultimately leading to necrotic cell death (Lomonte
et al., 1994b, 2003; Gutiérrez and Ownby, 2003). Although
the main region responsible for toxicity on this protein has
been identified (Fig. 1A), knowledge on the nature and
identity of its acceptor molecule(s) on the target cells has
been elusive. In contrast to their Lys49 counterparts, the
Asp49 PLA2 myotoxins utilize their catalytic activity upon
membrane phospholipids as part of the muscle-damaging
mechanism, but the detailed events occurring at the
molecular and cellular level are largely unknown. The
mechanisms of action of both types of PLA2 myotoxins, i.e.
Asp49 and Lys49 proteins, have been recently reviewed
(Gutiérrez and Ownby, 2003; Lomonte et al., 2003; Soares
et al., 2004; Montecucco et al., 2008). Besides their prom-
inent myotoxic effect in vivo, other activities described for
the group of basic PLA2s of B. asper, include the induction of
edema (Gutiérrez et al., 1986; Lomonte and Gutiérrez, 1989;
Chaves et al., 1998), in vitro anticoagulant action (Gutiérrez
et al., 1986), induction of cytokines (Lomonte et al., 1993;
Rucavado et al., 2002; Chacur et al., 2004), cytotoxic action
(Lomonte et al., 1994a,b, 1999; Angulo and Lomonte, 2005),
952 Y. Angulo, B. Lomonte / Toxicon 54 (2009) 949–957

Fig. 1. Ribbon representations of the three-dimensional structures of (A) Bothrops asper myotoxin II (1CLP; Arni et al., 1995), a Lys49 PLA2 homologue, and
(B) B. asper BaP1 (1ND1; Watanabe et al., 2003), a P-I metalloproteinase with weak hemorrhagic activity. In (A), a myotoxin II homodimer is represented
highlighting its C-terminus region 115–129 (black), in the approximate orientation that approaches membrane bilayers (bottom), drastically altering their
permeability. The toxin interacts with anionic sites (AS) of unknown identity in skeletal muscle, which may be phospholipids or proteins. N- and C-termini are
labelled as N and C, respectively. In (B), the metalloproteinase BaP1 is represented with its Zn2þ atom (sphere) in the catalytic site. By hydrolyzing protein
components of the extracellular matrix (ECM) and of the basal membrane of capillaries, this enzyme alters dermo-epidermal junctions and disrupts the integrity
of microvessels and their endothelial cells (End), ultimately causing local dermonecrosis and hemorrhage. Images are not drawn to scale.

bactericidal effect (Páramo et al., 1998; Santamarı́a et al.,
2005), induction of hyperalgesia (Chacur et al., 2003, 2004),
activation of macrophages (Zuliani et al., 2005), apoptotic
effects (Mora et al., 2005, 2006), KDR/VEGF receptor-
binding (Fujisawa et al., 2008), and lympahtic vessel
contraction (Mora et al., 2008).
2.2. Metalloproteinases
Zn2þ-dependent metalloproteinases are abundantly
found in snake venoms, and classified into four structural
groups, from P-I to P-IV, on the basis of their domain
composition (Bjarnason and Fox, 1994; Fox and Serrano,
2005, 2008; Ramos and Selistre-de-Araujo, 2006). In
B. asper venom, the presence of metalloproteinases of the
first three groups has been demonstrated by proteomic
techniques (Alape-Girón et al., 2008), although only repre-
sentatives of the P-I and P-III groups have been isolated.
The first protein of this family isolated from B. asper was
metalloproteinase G (Aragón and Gubenšek, 1987), a gly-
cosylated enzyme of 18 kDa, which probably belongs to the
P-I group. This enzyme hydrolyzes a number of protein
substrates in vitro such as casein, hemoglobin, gelatin and
fibrinogen (particularly its alpha-chain). Despite this
activity, the enzyme was reported to lack hemorrhagic and
coagulant actions.
Borkow et al. (1993) purified three toxins, named BaH1,
BH2 and BH3, showing strong hemorrhagic activity. Their
inhibition by ortho-phenanthroline and by the chelating
agent EDTA identified these proteins as metalloproteinases.
On the basis of their molecular masses (Table 1), BaH1 and
BH3 should correspond to group P-III enzymes, whereas
BH2 is likely a P-I metalloproteinase. Interestingly, a clear
synergistic effect was observed in the hemorrhagic action
of these toxins in mice: one-quarter MHD (minimal
hemorrhagic dose) of each isolated toxin caused only
a vague hemorrhagic spot, whereas a mixture containing
one-eight MHD of each toxin resulted in hemorrhagic spot
of one MHD (Borkow et al., 1993). BaH1 was the most
potent of the three enzymes in inducing this effect, with
a minimum hemorrhagic dose of 0.18 mg.
The hemorrhagic action of BaH1 has been studied in
vivo at the ultrastructural level and using endothelial cell
cultures. Pathological alterations induced by this toxin in
vivo occur in endothelial cells and in their basal membrane,
which appears to be proteolytically degraded (Moreira
et al., 1994). However, BaH1 is not cytotoxic to cultured
endothelial cells, which detach from their substrate but still
retain viability (Lomonte et al., 1994a,b; Borkow et al.,
1995). A mild myonecrotic effect of slow onset has been
described for BaH1, probably secondary to ischemic
conditions caused by hemorrhage and blood flow impair-
ment (Gutiérrez et al., 1995b). The pathogenesis of
hemorrhage by BaH1 and other hemorrhagic toxins has
been recently reviewed (Gutiérrez and Rucavado, 2000;
Gutiérrez et al., 2005).
Gutiérrez et al. (1995a) isolated BaP1, an abundant P-I
metalloproteinase of 23 kDa in the venom of B. asper from
the Pacific region of Costa Rica. This protein is highly active
in the proteolysis of general substrates, but it is only weakly
hemorrhagic, having a minimum hemorrhagic dose of
20 mg. It lacks coagulant, defibrinating, and platelet aggre-
gation-inhibitory effects, but is capable of inducing a mild
myonecrosis (Gutiérrez et al., 1995a; Escalante et al., 2004).
 Y. Angulo, B. Lomonte / Toxicon 54 (2009) 949–957
Other activities displayed by this toxin include dermonec-
rosis and blistering (Rucavado et al., 1998; Jiménez et al.,
2008), complement activation (Farsky et al., 2000), and
induction of an inflammatory response including leukocyte
recruitment, hypernociception, and synthesis of matrix
metalloproteinases and cytokines (Rucavado et al., 2002;
Fernandes et al., 2006, 2007). In similarity with BaH1, BaP1
is not cytotoxic to endothelial cells in culture (Rucavado
et al., 1995a). However, BaP1 induces anoikis in these cells,
a particular form of apoptosis related to their detachment
from substrate (Dı́az et al., 2005). This in vitro effect was
also reported for the P-III hemorrhagic metalloproteinase
jararhagin (Tanjoni et al., 2005). Nevertheless, apoptosis of
endothelial cells does not seem to be detected in vivo
(Jiménez et al., 2008). The degenerative changes observed
after BaP1 injection include damage to capillary basement
membrane (Escalante et al., 2006) and endothelial cells, the
latter effect requiring an intact blood flow (Gutiérrez et al.,
2006). The hemorrhagic action of this toxin is exerted
locally, but not systemically, probably due to its inactivation
by the plasmatic protease inhibitor a2-macroglobulin
(Escalante et al., 2004). BaP1 is antigenically distinct from
BaH1, since no cross-reactivities were recorded by using
rabbit antibodies raised against each one (Rucavado et al.,
1995b). Watanabe et al. (2003) determined the primary
and three-dimensional structures of BaP1, which consists of
202 amino acid residues, including three disulfide bridges,
and contains the characteristic signature of the zinc-
binding region of the metzincin superfamily of metal-
loproteinases (Gomis-Rüth, 2003). The crystal structure of
BaP1 (Fig. 1B) will be most valuable to analyze the subtle
relationships between structure and function among met-
alloproteinases with markedly different hemorrhagic
activities.
BaH4 is another P-III metalloproteinase of B. asper
venom, with hemorrhagic activity (Franceschi et al., 2000).
This toxin is antigenically related to the previously
described BaH1 (Borkow et al., 1993), generating a pattern
of partial identity with the latter by double immunodiffu-
sion analysis. BaH4 differs from BaH1 by having a slightly
higher isoelectric point (pI 5.3), and a lower hemorrhagic
potency, with a minimum hemorrhagic dose of 2 mg
(Franceschi et al., 2000). Its median lethal dose (LD50) by
intravenous injection in mice was estimated at 0.37 mg/g
body weight, and this route of administration resulted in
prominent pulmonary hemorrhage. These functional
characteristics of BaH4 suggest that it may play a relevant
role in the systemic toxicity induced by B. asper venom.
Basparin A (Lorı́a et al., 2003) is a single-chain, glyco-
sylated, P-III metalloproteinase of 70 kDa and pI of 5.4. In
addition to its metalloproteinase domain, it presents dis-
integrin-like and high-cystein domains. This toxin displays
potent prothrombin-activating and platelet aggregation-
inhibitory activities in vitro, causing defibrin(ogen)ation
and thrombosis in mice. In contrast to other venom met-
alloproteinases, basparin A does not degrade the typical
components of the extracellular matrix. Its ability to inhibit
collagen-induced platelet aggregation is not dependent on
its proteolytic activity. The plasma proteinase inhibitors
a2-macroglobulin and murinoglobulin do not prevent its
ability to clot human plasma (Lorı́a et al., 2003). The
953
defibrination syndrome observed after the intravenous or
intramuscular administration of this enzyme, together with
its ability to induce formation of pulmonary thrombi after
intravenous injection, suggest that basparin A is likely to
play a relevant role in the coagulopathy observed during
envenomings by B. asper.
2.3. Serine proteinases
In B. asper venom, serine proteinases are abundant
constituents that account for 5–18% of the proteins,
depending on age and geographic region variations (Alape-
Girón et al., 2008). Of these enzymes, those with thrombin-
like clotting activity have been isolated and studied.
Asperase, the first serine proteinase with thrombin-like
activity isolated from B. asper venom, was described by
Ortiz and Gubenšek (1976) and Aragón-Ortiz and Gubenšek
(1978). This enzyme is glycosylated, with an estimated
molecular mass of 30 kDa. By intravenous route, asperase
had an LD50 of 0.18 mg/g body weight in mice. It is likely that
such enzyme is the same as ficozyme (Fořtová et al., 1990),
and the one recently characterized in more detail by Pérez
et al. (2008), a 27 kDa serine proteinase with in vitro
thrombin-like activity, which promotes defibrin(ogen)a-
tion in mice after intravenous injection. In addition to its
ability to induce coagulopathy, this toxic enzyme also
causes behavioral alterations such as loss of the righting
reflex, opisthotonus, and intermittent rotations over the
long axis of the body (Pérez et al., 2008). This feature
resembles that of gyroxin, also a thrombin-like enzyme,
from the venom of Crotalus durissus terrificus (Alexander
et al., 1988). Its N-terminal amino acid sequence (VIG-
GDECNIN EHRSLVVLFX SSGFLCAGTL VQDEWVLTAA
NCDSKNFQ) is identical to a serine proteinase cDNA cloned
from B. asper venom gland (UniProtKB/TrEMBL entry
Q072L6). The proportion of this thrombin-like enzyme in
the venom is very low, nearly 0.13%. Considering its in vitro
clotting potency for plasma (minimum coagulant
dose ¼ 4.1 mg) and for fibrinogen (4.2 mg), and its in vivo
defibrin(ogen)ating effect (minimum defibrin(ogen)ating
dose ¼ 1.0 mg) in mice, Pérez et al. (2008) concluded that its
contribution to the coagulopathy induced by B. asper
venom is probably of lower significance than that provided
by prothrombin-activating metalloproteinases such as
basparin A (Lorı́a et al., 2003).
2.4. L-Amino acid oxidase
Umaña (1982a,b) isolated three isoforms of the enzyme
L-amino acid oxidase from B. asper venom, with a similar
molecular mass of 125 kDa, but slight variations in their
electrophoretic mobility. Under reducing and denaturing
conditions, subunits of 60 kDa and 75 kDa were obtained
from these purified enzymes. However, no data for possible
bioactivities or toxicity profiles of these venom compo-
nents were described.
2.5. C-type lectin-like toxins
A disulfide-linked heterodimeric protein, with a molec-
ular mass of 29,759 and a pI of 4.5, was isolated from
954 Y. Angulo, B. Lomonte / Toxicon 54 (2009) 949–957
B. asper venom and named aspercetin (Rucavado et al.,
2001). Its N-terminal amino acid sequence identified it as
a member of the C-type lectin-like family of proteins, with
a high homology to botrocetin from the venom of Bothrops
jararaca (Andrews et al., 1989). Aspercetin is a potent
platelet-aggregating agent only in the presence of plasma
or von Willebrand factor, since its activity results from the
interaction of this factor with platelet receptor GPIb
(Rucavado et al., 2001). In contrast to other representatives
of the C-type lectin family of snake venoms, aspercetin
lacked anticoagulant and hemagglutinating activities. In
mice, this protein induced a rapid drop in circulating
platelet numbers, prolonged the bleeding time, and
enhanced the hemorrhagic activity of a purified metal-
loproteinase. All these observations strongly suggest that
aspercetin contributes significantly to the prominent
hemorrhagic effects manifested in envenomings by B. asper.
2.6. Disintegrins
Disintegrins are non-enzymatic polypeptides broadly
distributed in the venoms of viperid snakes, which antag-
onize the adhesive functions of several types of integrin
receptors (Calvete, 2005; Calvete et al., 2005). A medium-
sized disintegrin, named bothrasperin, was isolated from
B. asper venom (Pinto et al., 2003) and partially characterized.
This acidic protein showed an estimated molecular mass of
8 kDa, and its N-terminal amino acid sequence (EAGEEX
DXGTE) evidenced homology with disintegrins isolated from
the venoms of B. jararaca and other pitvipers (Scarborough
et al., 1993). In vitro, bothrasperin inhibited ADP-induced
human platelet aggregation, with an estimated IC50 of 50 nM.
Recent proteomic analyses of B. asper venom disclosed the
presence of several isoforms of this disintegrin, with slight
differences in mass (Alape-Girón et al., 2008). Further
biochemical and functional characterization of bothrasperin
and its variants is currently underway.
3. Concluding remarks
The isolation and study of individual snake venom
components paves the way for a deeper understanding of
the pathophysiology of envenomings – thus potentially
contributing to improved therapeutic modalities in the
clinical setting – and also opens possibilities for the
discovery of novel toxins that might be useful as tools for
dissecting cellular and molecular processes of biomedical
importance. The venom of B. asper has provided a source of
varied and interesting components, some of which have
been well characterized, most notably the Lys49 PLA2
homologue ‘‘myotoxin II’’ and the P-I metalloproteinase
‘‘BaP1’’ (Fig. 1). The intensive study of these two toxins has
allowed to gain significant insights into the pathological
phenomena of myotoxicity and hemorrhage, respectively.
However, many more components of this venom await to be
isolated, or to be characterized in greater detail. With the
recently established proteomic database on the composi-
tion of B. asper venom, and the ongoing work to define its
transcriptome, this task should be greatly facilitated,
opening a new era in the study of this venom. Within this
framework, venom components that are scarce or difficult
to isolate could be cloned and expressed in recombinant
form. As pointed out before, however, it should be kept in
mind that although the study of isolated toxins constitutes
an invaluable approach to dissect and analyze venom
actions in great detail, the effects induced by whole,
unfractionated venom should always be considered as an
important reference frame to envisage the relative contri-
butions of the isolated components within its context.
A number of clinically relevant toxins of B. asper remain
to be isolated and studied. One of the important conse-
quences of envenomings by this species is kidney damage,
and too little attention has been paid to this effect as well as
to the toxins responsible for it. Likewise, the acidic PLA2s
need to be studied further to assess their possible contri-
butions to the toxic actions of this venom. A number of
abundant serine proteinases and metalloproteinases,
which are likely to have significant roles in the patho-
physiology of envenomings, await to be isolated and char-
acterized. Components such as members of the CRISP
family of proteins are virtually unknown, and the group of
medium-sized disintegrins should be also focused in more
depth to define their role(s) in envenomings. It is hoped
that in addition to the fascinating basic knowledge to be
gained from the study of this venom and its toxins, strat-
egies to improve the clinical treatment of snakebite victims
may also emerge.
Acknowledgements
We thank all colleagues and collaborators that have
contributed to the study of B. asper venom at our Institute
as well as abroad, the Vicerrectorı́a de Investigación,
University of Costa Rica for support, and Dr José Marı́a
Gutiérrez for critical reading of the manuscript. We would
like to dedicate this review to the memory of Drs Róger
Bolaños and Luis Cerdas, former Directors of the Instituto
Clodomiro Picado, for their vision and their encouraging
support to new generations of researchers in the field of
toxinology.
Conflicts of interest
The authors declare that there are no conflicts of
interest.
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