TOXINS FROM VENOMS OF
INDIGENOUS TO MALAYSIA: A REVIEW
Tan, Nget Hong
of Molecular Medicine, Faculty of Medicine
of Malaya, Kuala Lumpur, Malaysia
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A B S T R A C T
In Malaysia and the coastal waters of the region, there are at least 18 different species of venomous front fanged land snakes and more than 22 different species of sea snakes. However, only a few of the Malaysian poisonous snakes can be regarded as of medical importance, and these include the elapids Naja naja, Bungarus candidus, B.fasciatus, Ophiophagus hannah and Enhydrina schistosa, and the crotalids Calloselasma rhodostoma, Trimeresurus purpureomaculatus, T.wagleri and T.sumatranus. Dried snake venom contains mainly proteins and polypeptides. The major toxic constituents of Malaysian elapid venoms include polypeptide neurotoxins, pharmacologically active phospholipase A2 enzymes and polypeptide cardiotoxins, while the major toxic constituents of Malaysian crotalid venoms include hemorrhagic and non-hemorrhagic proteases, thrombin-like enzymes and platelet aggregation inducers and inhibitors. Both Malaysian elapid and crotalid venoms also exhibit many other enzymatic activities that may also be involved in the toxic action of the venoms as well as in the digestion of the prey.
is a serious medical problem in Malaysia. During
the period 1958 to 1980, as many as 55000 cases
of snakebites were admitted to the hospitals in
Malaysia (Reid, et al., 1963a; Lim and
Ibrahim, 1970; Lim, 1982). While the mortality
rate of snakebite in Malaysia is only 0.3 per
100000 population (Reid, 1968), the local
necrotic effect of some venom can cause prolonged
morbidity or crippling deformity.
symptomology, pathology and treatment of
snakebites in Malaysia have been adequately
documented (Reid 1961a,b; Reid 1962; Reid et
al.,1963b,c; Reid, 1964; Reid, 1968; Reid,
1979; Warrell, 1986). The systematics,
zoogeography and identification of the Malaysian
poisonous snakes have also been described by
Tweedie (1983) and Lim (1982). The present review
will focus on the biochemistry of the venoms of
medically important poisonous snakes indigenous
Poisonous Snakes of Malaysia
snakes are usually classified into 3 families:
Colubridae, Viperidae and Elapidae (Underwood,
1979). The two families Viperidae and Elapidae
are front fanged poisonous snakes.
Viperidae is subdivided into three
subfamilies Azemiopinae, Viperinae (viper) and
Crotalinae (pit viper), whereas Elapidae is
subdivided into five subfamilies: Elapinae,
Laticaudinae, Hydrophiinae, Ephalophiini and
Hydrophiini. Each subfamily usually consists of
Malaysia and the coastal waters of the region,
there are at least 18 different species of
venomous front fanged land snakes and more than
22 different species of sea snakes (Tweedie,
1983). These poisonous snakes belong to the
following 5 subfamilies: 1) Crotalinae,
represented by the two genera Calloselasma and
Trimeresurus; 2) Elapinae, represented by the
five genera Naja, Bungarus, Ophiophagus, Maticora
and Calliophis; 3) Laticaudinae, represented by
the genus Laticauda; 4) Hydrophiini, represented
by the six genera Enhydrina, Kerilia, Hydrophis,
Thalassophis, Pelamis and Kolpophis; and 5)
Ephalophiini, represented by the only genus
Aipysurus. The Crotalinae and Elapinae
subfamilies are land snakes whereas the other
three subfamilies are sea snakes.
a few of the Malaysian poisonous snakes can be
regarded as of medical importance (Reid et al.,
1963a; Sawai et al., 1972). Reid et al
(1963a) reported that during 1958-1959, of the
824 snakebite cases in West Malaysia in which the
snake was reliably identified, the bites were
mainly due to four species of land snake, i.e.,
Calloselasma rhodostoma (Malayan pit
viper), Naja naja (Asian common cobra), Trimeresurus
purpureomaculatus (shore pit viper) and Trimeresurus
wagleri (Wagler's pit viper). The
epidemiological study by Sawai et al.
(1972) yielded similar findings (Table 1).
Table 1: Snakebites in West Malaysia:
Species of snake identified*
* Report from monthly statistics of 28 hospitals throughout Malaysia, 1965-1971 (Sawai et al., 1972). Note the large number of cases in which the snakes causing the bites were unidentified
poisonous snakes indigenous to Malaysia that are
potentially dangerous to human include Bungarus
candidus (Malayan krait), Bungarus
(banded krait), Ophiophagus hannah
(king cobra), Trimeresurus sumatranus
(Sumatran pit viper) and the sea snakes. In most
of the snakebite cases in East Malaysia, however,
the snakes were unidentified.
Composition of snake venoms
snake venom contains mainly proteins (70-90%) and
small amounts of metals, amino acids, peptides,
nucleotides, carbohydrates, lipids and biogenic
amines (Tu, 1977). The protein components include
enzymes and non-enzymatic proteins.
of many elapid snakes (cobra, krait and sea
snakes etc) produce flaccid paralysis and
respiratory failure in animals. These effects
have been attributed to the neurotoxins of the
venoms (Lee, 1972). Snake venom neurotoxins can
be classified into two groups according to their
mode of action (Karlsson, 1979). The first group
of neurotoxin produces neuromuscular block of the
antidepolarizing type and is termed postsynaptic
neurotoxin. Snake venom postsynaptic neurotoxins
are generally basic polypeptides containing 60-70
amino acid residues with molecular weights of
about 7000 and intravenous LD50's
(median lethal doses) of approximately 0.1mg/gm
mouse. Postsynaptic neurotoxins isolated from
cobra, krait and sea snake venoms are very
similar in their amino acid sequences. The second
group of neurotoxin produces neuromuscular block
by acting presynaptically on the motor nerve
endings and is known as the presynaptic
neurotoxin. The presynaptic neurotoxins generally
have molecular weights exceeding 11000 and
exhibit phospholipase A2 activity.
phospholipase A2 toxins have
been isolated from krait and sea snake venoms.
neurotoxins, elapid venoms also contain various
pharmacologically active basic polypeptides. In
cobra venom, the basic polypeptide cardiotoxins
may constitute 25-60% of the venom dry weight.
They are non-enzymatic polypeptides with
molecular weights of 6000-7000 (Karlsson, 1979).
Cardiotoxins isolated from various cobra venoms
are similar in their amino acid sequences. The
toxin affects both excitable and non-excitable
cells, causing irreversible depolarization of the
cell membrane and consequently impairing the
structure and function of various cells, thus
contributing to muscle paralysis and leading to
circulatory and respiratory failure and systolic
arrest (Lee, 1972). The intravenous LD50
of cardiotoxin in mice is usually in the range of
are also important constituents of snake venom.
Generally, elapid venoms contain fewer enzymes
than viperid (viper) and crotalid (pit viper)
venoms. Table 2 lists the enzymatic activities of
venoms from the common poisonous snakes of
Malaysia. It should be noted, however, that there
are marked individual variations in the enzymatic
compositions of snake venoms, and that venom
enzymatic composition may vary depending on the
season of collection, age of snakes, differences
in processing of venoms or individual variation
in snakes (Iwanaga and Suzuki, 1979).
are involved in many levels of venom action
besides functioning in the digestion of the prey
(Zeller, 1977; Iwanaga and Suzuki, 1979). For
example, certain venom phospholipase A2
enzymes exhibit potent toxic effects such as
neurotoxic, direct hemolytic, anticoagulant,
myonecrotic or hemorrhagic activities (Rosenberg,
1986). Some arginine ester hydrolases are
thrombin-like enzymes that cause defibrination
syndrome in vivo while other arginine
ester hydrolases exhibit bradykinin-releasing
properties. Hyaluronidase may serve as spreading
factor for venom toxins. Other venom enzymes that
may contribute to the toxic action of snake venom
include protease (some exhibiting hemorrhagic or
edema inducing activity), L-amino acid oxidase,
acetylcholinesterase, phosphodiesterase and
5'-nucleotidase (Zeller, 1977).
Table 2: Enzymatic
Activities and Lethalities of the Venoms of Some Malaysian Poisonous Snakes
yield (mg venom dry weight per snake):
N.n.sputatrix: 170-250 mg, O.hannah: 350-500 mg,
B.fasciatus: 25-50 mg; H.cyanocinctus: 5-0 mg;
E.schistosa: 10-15 mg; C.rhodostoma: 40-60 mg;
T.wagleri: 35-90 mg
and units: LAAO: L-amino acid oxidase, mmole/min/mg;
HYA: hyaluronidase, national formulary unit/mg;
ACE: acetyl cholinesterase, mmole/min/mg;
NUC: 5'-nucleotidase, nmole/min/mg; PDE:
phosphodiesterase, nmole/min/mg; PLA:
phospholipase A, mmole/min/mg;
arginine ester hydrolase, mmole/min/mg;
phosphomonoesterase, nmole/min/mg; CE: clotting
enzyme, NIH unit/mg, LD50:
Median Lethal Dose (μg/g). (For
methods and definitions of units see Tan and Tan
(1988a) and Tan et al. 1989a).
Venoms of Malaysian cobras
There are two subspecies of Naja naja (Asian cobra) in Peninsular Malaysia (Tweedie, 1983) The species found throughout most of Peninsular Malaysia is Naja naja sputatrix (Malayan cobra). N.n.sputatrix is quite black with some white marks on the throat. This species of cobra has the habit of spraying or `spitting' venom. In the northern states, the common species found is Naja naja kaouthia, the monocellate cobra. It is grey brown with throat almost white. On the back of the hood there is a centrally placed white circle. Both species are also very common in Thailand. Venoms of N.n.sputatrix and N.n.kaouthia are immunologically different: the monospecific antivenom raised against N.n.sputatrix venom was ineffective against N.n.kaouthia venom (Theakston and Reid, 1983), suggesting that there are important differences in the toxin composition of the two cobra venoms. The intravenous LD50's for N.n.sputatrix and N.n.kaouthia venoms are 0.8 mg/g and 0.4 mg/g, respectively. Local necrosis is the main feature of cobra venom poisoning while the main systemic effects are myoneural curare-like (`neurotoxic') effect and cardiovascular effect (Reid, 1964; Viravan et al., 1986).
Naja naja sputatrix venom
major biochemical constituents of the venom
include high molecular weight proteins and
enzymes, phospholipase A2 enzymes,
postsynaptic neurotoxins and polypeptide
The high molecular weight proteins and enzymes, with molecular weights exceeding 30000, account for approximately 5% of the venom dry weight. The enzymes present include phosphodiesterase, alkaline phosphomonoesterase, 5'-nucleotidase, L-amino acid oxidase, hyaluronidase, acetylcholinesterase and protease (Tan and Tan, 1987; 1988a). Several acetylcholinesterases have been partially purified from the venom; all have molecular weights of approximately 60000. Together, however, the acetylcholinesterases only account for less than 0.5% of the venom dry weight. The high molecular weight proteins and enzymes do not play an important role in the lethal action of Malayan cobra venom (Tan, 1983a).
A2 enzymes, with molecular weight of
approximately 14000, account for 15% of the venom
dry weight. Three acidic lethal phospholipase A2
enzymes, sputatrix PLA2-1, sputatrix
PLA2-2 and sputatrix PLA2-3,
have been isolated and purified from the venom
(Tan, 1982a, Tan and Arunmozhiarasi, 1989a,b).
The three enzymes have intravenous LD50's
of 0.27 mg/g,
and 0.86 mg/g,
respectively. Sputatrix PLA2-3
accounts for approximately 10% of the venom
protein. It exhibits weak anticoagulant and
edema-forming activity and induces contracture of
muscle in a chick biventer cervicis nerve-muscle
preparation (Geh and Tan, 1988). Sputatrix PLA2-1
and PLA2-2 together account for 3% of
the venom protein. These two enzymes also exhibit
potent anticoagulant activity in vitro (Tan and
Arunmozhiarasi, 1989b) and are the major
anticoagulants of the venom. The role of the
anticoagulant activity in the pathophysiology of
Malayan cobra bite has not been evaluated.
cDNA encoding the three phospholipases A2
have been cloned and characterized (Armugam et
al., 1997). The specific characteristics of
the isoforms were attributed to mutations at
nt139 and nt 328 from G to C and G to A,
respectively. The sequences are shown in Table 3.
neurotoxins account for approximately 4.5% of the
venom dry weight. Both the two major neurotoxins,
sputa-neurotoxin 1 (SN1) and sputa-neurotoxin 2
(SN2) isolated from the
venom are `short' neurotoxins with 62 and
61 amino acid residues respectively, and
molecular weights of approximately 7000 (Tan,
1983b). The intravenous LD50's of the
two toxins are 0.09 mg/g
and 0.07 mg/g
respectively and they possess amino acid
sequences similar to those of other cobra venom
neurotoxins (Table 3). The cDNAs encoding two
neurotoxins in the venom have been cloned and
characterized (Afifiyan et al., 1997).
cardiotoxins account for 60% of the venom dry
weight. Three polypeptide cardiotoxins, sputa-cardiotoxin
A, B and C have been isolated from the venom
(Tan, 1982b). They are basic polypeptides with
59-60 amino acid residues and molecular weights
of approximately 7000. The amino acid
compositions of the three cardiotoxins are also
very similar to those of other cobra venom
cardiotoxins. The intravenous LD50's
of the three toxins are 1.0, 1.05 and 1.1 mg/g,
respectively. All three cardiotoxins exhibit
direct lytic effect on human and guinea pig
erythrocytes and strong edema-inducing activity.
The local necrotic action of the venom may be due
to the synergistic action of the cardiotoxins and
phospholipase A2 enzymes. The
structural gene and cDNA encoding a cardiotoxin
in Naja naja sputatrix venom has been
cloned and characterized (Yeo et al.,
1993, see Table 3)
3: The amino acid sequences of some Malayan cobra
4.2 Naja naja kaouthia
major biochemical constituents of the N.n.kaouthia
venom also consist of high molecular weight
proteins and enzymes, phospholipase A2
enzymes, postsynaptic neurotoxins and
high molecular weight proteins (MWt > 30000)
and enzymes account for 8% of the venom dry
weight and are not important in the toxic action
of the venom. There are two forms of
phospholipase A2 enzymes (MWt 14000)
which together account for 7% of the venom dry
weight. The two phospholipase A2
enzymes are acidic proteins that exhibit weak
anticoagulant activity but not lethal toxicity (Karlsson
and Pongsawasidi, 1980).
neurotoxins account for 23% of the venom dry
weight. The major polypeptide neurotoxins are
`long' neurotoxins with 71 amino acid residues (Karlsson
1972). Neurotoxins are the major lethal toxins of
which account for 45% of the venom dry weight,
also contribute to the lethal toxicity of the
venom. The amino acid sequences of the three
major cardiotoxins are homologous to other cobra
venom cardiotoxins (Joubert and Taljaard, 1980).
The intravenous LD50's for the
cardiotoxins are 1.2 mg/g.
Differences between N.n.sputatrix and N.n.kaouthia
is apparent that N.n.sputatrix venom
differs from N.n.kaouthia venom in their
compositions: N.n.kaouthia venom has a
higher neurotoxin and a lower cardiotoxin
contents. The three acidic phospholipase A2
enzymes of N.n.sputatrix venom are lethal
to mouse, while phospholipase A2
enzymes from N.n.kaouthia venom are not.
Besides, the major neurotoxins from N.n.kaouthia
venom are `long' (71 amino acid residues) toxins
that are immunologically different from the
`short' toxins (62-63 amino acid residues) of N.n.sputatrix
venom. These differences may account for the
observation that monospecific antivenom raised
against N.n.kaouthia venom was ineffective
against N.n.sputatrix venom.
Venom of Calloselasma rhodostoma (Malayan
rhodostoma (Malayan pit viper) was previously known as Agkistrodon
rhodostoma or Ancistrodon rhodostoma.
In Malaysia, confirmed cases of Malayan pit viper
bites are confined only to the northern states of
Kedah, Perlis and Penang. The snake is reddish or
purplish brown with series of dark brown
cross-bands. Head is triangular with large scales
on the crown and the average length of the snake
is 23-32 inches. It is a bad tempered snake,
quick to strike if disturbed and is the commonest
cause of snakebite in Peninsular Malaysia (Table
1). Fortunately, only 10% of those bitten
developed severe poisoning and death rate is less
intravenous LD50 for Malayan pit viper
venom is 6.1 mg/g
mouse, which is considerably less lethal than
venoms of most other poisonous snakes (Table 2).
This partially explains the low mortality rate of
Malayan pit viper bites. Swelling always follows
Malayan pit viper envenomation. Local necrosis
occurred in 11 % of the patient bitten by the pit
viper. In 15% of the cases there was an overt
hemorrhagic syndrome. The principal
characteristics of systemic Malayan pit viper
venom poisoning was, however, systemic bleeding
characterized by defibrination and
thrombocytopenia (Reid et al. 1963b).
pit viper venom exhibits very strong
thrombin-like enzyme and moderate hemorrhage
activities. The biological constituents of the
venom that have been characterized include
thrombin-like enzymes, hemorrhagins, platelet
aggregation inducers, platelet aggregation
inhibitors, fibrinogenase, L-amino acid oxidase,
proteases, phospholipase A2 enzymes
and arginine esterases.
There is significant intraspecific
variation in the biological activities of the
venom, including phosphodiesterase,
alkalinephosphomonoesterase, L-amino acid oxidase,
arginine ester hydrolase, 5’-nucleotidase,
thrombin-like enzyme and hemorrhagic activity (Daltry
et al., 1996). Studies of captive-bred
snakes indicate that the intraspecific variation
in venom is genetically inherited rather than
The venom contains several thrombin-like enzymes. The major form, Ancrod (or arvin), accounts for approximately 7% of the venom dry weight and is a glycoprotein with a molecular weight of 55000 (Esnouf and Tunnah, 1967). The enzyme coagulates fibrinogen solution by catalyzing the release of fibrinopeptide A, AP and AY. Clots that formed with arvin are not cross-linked and are susceptible to rapid lysis by plasmin. When injected into human/animals, it causes continual microcoagulation of fibrinogen but the resulting fibrin is virtually simultaneously disposed off. Thus, although ancrod is a `coagulant' in vitro, the in vivo effect is non-clotting blood because of the very low fibrinogen level. Ancrod exhibits arginine ester hydrolase activity but is not lethal to mouse even at doses exceeding 10 mg/g.
Ancrod is a serine proteinase. The cDNA sequence reveals that Ancrod is synthesized as a pre-zymogen of 259 amino acids. The amino acid sequence (Table 4) exhibits a high degree of sequence similarity to those of mammalian serine proteinases as well as reptilian fibrinogenases, most notably the catalytic triad and many conserved cysteine positions (Burkhart et al., 1992). Ancrod is heavily glycosylated (36% by mass) with five N-linked glycosylation sites (Pfeiffer et al, 1993)
The use of Ancrod in anticoagulation therapy has been examined in many clinical trials. Ancrod provides optimal therapy for patients suspected of having heparin associated thrombocytopenia and thrombosis. Depletion of fibrinogen with Ancrod results in anticoagulation comparable to therapy with heparin (Cole et al., 1990)
are at least five different forms of hemorrhagin
in Malayan pit viper venom (Tan and Kanthimathi,
1989). Snake venom hemorrhagins are generally
metalloproteinases that probably act by
destruction of the collagenous basement membrane
and other connective tissue collagens with
consequent weakening of the blood vessel wall
causing hemorrhagic effect (Ohsaka, 1979).
major Malayan pit viper venom hemorrhagin, termed
rhodostoxin (or Kistomin) has been isolated and
its amino acid sequence is homologous to other
venom hemorrhagins (Ponnudurai et al.,
1993, Tan, 1998). It is a zinc-metalloproteinase
with 203 amino acid residues (Table 4), a
molecular weight of 30000 and carbohydrate
content of 15%. It exhibited potent hemorrhagic
and edema-inducing activities but was not lethal
to mice at a dose of 6 mg/g
(i.v.). It also exhibits anti-platelet activity
(Huang et al., 1993). It exhibits the extended consensus
active-site sequence of MXHEXGHNLGXXHD (residues
140 to 153), and the predicted secondary
structure in the zinc-binding region of the
hemorrhagin is similar to that of other snake
venom metalloproteinases and thermolysin (Ponnudurai,
et al.1993). Rhodostoxin, however, is
unique in that it contains four disulfide
bridges. The structure of the oligosaccharides
attached to the protein has also been elucidated
(Chung et al., 1996). Deglycosylation
causes changes in the interactions between
rhodostoxin and the relevant substrate in the
mapping of rhodostoxin using polyclonal
antibodies showed that the antigenic sites of
rhodostoxin include residues 45-76, 81-88,
111-120 and 165-177, and that the oligosaccharide
moieties of rhodostoxin were not part of the
antigenic determinants (Ponnudurai, 1995).
pit viper venom contains several platelet
aggregation inducers (Ouyang et al., 1986,
Shin and Morita, 1998). The platelet aggregation
inducer, aggretin (Rhodocytin) is a nonenzymatic
protein with two subunits, containing 136 and 123
amino acid residues, respectively. (Chung et
al., 1999; Wong et al (2001). Some authors suggested
that it is an endothelial integrin α2β1
agonist but recent report indicated the inducer
does not recognize that α2β1
I. It is a C-type lectin-like
Theplatelet aggregation inducers presumably play a role in the thrombocytopenic syndrome caused by Malayan pit viper venom poisoning.
platelet aggregation inhibitors (or disintegrins)
have been isolated from the Malayan pit viper
venom (Teng and Huang, 1991; Huang et al.,
1987; Ouyang et al., 1983). Rhodostomin,
the first disintegrin isolated from the venom, is
a 68 amino acid residue polypeptide with a
hairpin loop that presents the binding sequence
RGD (Arg-Gly-Asp). It inhibits platelet
aggregation by blocking the binding of fibrinogen
to the integrin aIIbb3
of platelet. The amino acid sequence (Table 4)
deduced from the cDNA sequence indicates that the
68-amino acid sequence of rhodostominis located
at the carboxyl terminus of the precursor
protein, which also contains the sequence of
rhodostoxin, the major hemorrhagin (Ponnudurai,
1995). It appears that rhodostomin and
rhodostoxin share a common gene sequence,
suggesting that these proteins may be synergistic
Another Malayan pit viper
venom disintegrin that has been characterized is
Rhodocytin. It is a bigger molecule (262 amino
acid residues). It is RGD-independent (Bergmeier et al., 2001). Platelet
aggregation inhibitors are probably involved in the hemorrhagic action of
Platelet aggregation inhibitors are probably involved in the hemorrhagic action of the venom.
Kistomin is a metalloproteinase purified from the venom. It dose and time-dependently prolonged the latent period of platelet aggregation and inhibited ATP secretion of human washed platelets stimulated by thrombin (Huang et al., 1993)
L-Amino acid oxidase is a major constituent of the venom, constituting 30% of the crude venom dried weight. The enzyme is an acidic glycoprotein (Geyer et al., 2001) with a molecular weight of 132000, and is composted of two subunits of equal molecular weight (Ponnudurai et al., 1994). The enzyme contains 2 mol of flavin mononucleotide per molecule of enzyme. The N-terminal sequence of the protein was ADDRNPLAEEFQENNYEEFL. Kinetic studies suggest the presence of an alkyl side-chain binding site in the enzyme and that the binding site comprises at least four hydrophobic subsites. The characteristics of the binding site differ slightly from those of cobra venom L-amino acid oxidases. Preliminary studies indicated that the enzyme was not lethal but exhibited strong edema-inducing activity in rat. The enzyme may also cause impairment of platelet aggregation and therefore involve in the hemorrhagic action of the venom.
Amino Acid Sequence of Some Malayan Pit
Viper Venom Toxins
pit viper venom also contains at least four
different basic proteases with molecular weights
of approximately 25000, all of which exhibit
moderate edema inducing activity (Tan,N.H.,
unpublished data); four acidic phospholipase A2
enzymes that exist in dimeric form (Tan and
Kanthimathi, 1989); and more than nine different
forms of arginine ester hydrolases, five of which
exhibit thrombin-like enzyme activity. Two minor
forms of arginine ester hydrolase also exhibit
arginine amidase activity (Tan, 1984). Both the
phospholipase A2 enzymes and arginine
ester hydrolases were not lethal to mouse
(Tan and Kanthimathi, 1989; Tan et al,
primary defect in systemic bleeding caused by
Malayan pit viper venom poisoning is apparently
hemostatic failure due to thrombocytopenia
aggravated by defibrination syndrome, while the
local swelling, hemorrhage and necrosis may be
due to the action of the hemorrhagins and
edema-inducing factors as well as
autopharmacological actions of the venom. Shock,
which may lead to death, is caused partly by
hypovolemia from loss of fluids into the bitten
limb and probably partly due to bradykinin
release caused by autopharmacological actions of
the venom. The nature of the autopharmacological
factor of the venom has not been investigated.
Venom of Ophiophagus hannah (King cobra)
cobra is one of the world's most dangerous
snakes. It is olive brown or greenish yellow and
can attain a length of 16-18 feet.
The intravenous LD50 for king
cobra venom is approximately 1.2 ug/g mouse. The
snake is not aggressive and king cobra bite in
man appears to be infrequent (Table 1).
cobra venom has much greater enzyme content than
venoms of common cobras. The major lethal toxins
are the polypeptide neurotoxins and the main
systemic effect of king cobra venom bite appears
to be neurotoxic poisoning (Ganthavorn, 1971).
The biologically active constituents of king
cobra venom that have been characterized include
polypeptide neurotoxins, hemorrhagic and
non-hemorrhagic proteases, phospholipase A2
enzymes, L-amino acid oxidase and alkaline
of king cobra in Malaysia or Thailand contains
two lethal postsynaptic neurotoxins with 73 amino
acid residues each (Joubert, 1973; Tan and
Saifuddin, 1989a). The two toxins are basic
polypeptides with intravenous LD50 of 0.2 mg/g
and account for 20% of the venom dry weight. The
amino acid sequences of these two toxins are
homologous to neurotoxins from common cobra and
krait venoms. Venom of king cobra in Southern
China contains more than four postsynaptic
neurotoxins (Sun et al., 1981).
venom also contains five proteases, of which
probably two re hemorrhagic proteases. The
molecular weights of these proteases are 70000
(Tan, N.H., unpublished data; Yamakawa and
Omori-Satoh, 1988). The proteases account for 7%
of the venom dry weight. All five proteases
exhibit strong edema inducing activity. The major
hemorrhagic protease has been purified and is a
zinc containing metalloprotease with an
isoelectric point of 5.3 (Tan and Saifuddin,
1990a; Weissenberg et al., 1987). It
accounts for 2% of the venom dry weight. The
hemorrhagic protease is not lethal to mouse but
kills rabbit at doses above 0.18 mg/g
when injected intravaenously.
There are at least five forms of alkaline phosphomonoesterase with molecular weights of 95100 (Qin and Qi, 1986) and more than four phospholipase A2 isoenzymes. The phospholipase A2 isoenzymes together account for 4% of the venom dry weight. The two major isoforms of phospholipase A2 enzymes have isoelectric points of 3.81 and 3.89, respectively (Tan and Saifuddin, 1990b) and are not lethal to mouse at a dose of 10 mg/g but exhibit moderate anticoagulant and edema inducing activities.
major L-amino acid oxidase from the venom has
been purified to homogeneity (Tan and Saifuddin,
1989b). It has a molecular weight of 68000, an
isoelectric point of 4.5 and an intravenous LD50
of 5 mg/g
in mice. Unlike other venom L-amino acid oxidases,
king cobra venom L-amino acid oxidase exhibits
exceptional thermal and alkaline stability and is
therefore a suitable source enzyme for the study
of mechanism of action of L-amino acid oxidase.
It accounts for 5% of the venom dry weight.
Venoms of the Bungarus (kraits)
are three species of krait in Malaysia: Bungarus
fasciatus (banded krait), Bungarus
candidus (Malayan krait) and Bungarus
flaviceps (Red-headed krait). The reported
incidence of krait bite is low (Table 1) but
mortality is high.
Bungarus fasciatus (Banded krait) venom
krait is a common krait throughout Southeast
Asia. It has a pattern of alternating light
(usually yellow) and dark bands encircling the
body. It is a surprising quiet, inoffensive snake
and is generally believed to be harmless. Bites
by banded krait are very rare but fatal cases of
neurotoxic envenoming by banded krait have been
reported (Viravan et al., 1986).
intravenous LD50 for banded krait
venom is 1.4 mg/g,
which is considerably less lethal than venoms of
other kraits. The biological constituents of
banded krait venom that have been characterized
include postsynaptic and presynaptic neurotoxins,
cardiotoxic proteins and acetylcholinesterase.
venom contains three postsynaptic neurotoxins
which bind with high affinity to nicotinic
cholinergic receptors (Lu and Lo, 981; Kruck and
Logan, 1982). The toxins are basic polypeptides
with molecular weight of 14200. The venom also
contains ceruleotoxin, a phospholipase A2
neurotoxin with an intravenous LD50 of
(Bon and Saliou, 1983). It irreversibly blocks
the postsynaptic response of Torpedo
electroplaques to cholinergic agonists without
preventing the binding of acetylcholine to its
receptor. In addition, there are four presynaptic
neurotoxins in the venom and the molecular
weights of the toxins range between 10800 to
19100 (Kruck and Logan, 1982). The cardiotoxic
proteins are phospholipase A2 enzymes
exhibiting cardiotoxin-like properties with
intravenous LD50's of between 0.2 mg/g to 4.8 mg/g
al., 1983; Gong et al., 1989).
krait venom acetylcholinesterase has also been
purified to homogeneity (Kumar and Elliott,
1973). This enzyme has a molecular weight of
126000 and accounts for less than 1% of the venom
dry weight. It does not exhibit lethal toxicity.
Bungarus candidus (Malayan krait) venom
krait is black above with about thirty white
cross-bands in body and tail. This snake is well
distributed and may cause severe neurotoxic
envenomation in man (Warrell et al.,
1983). The venom is highly lethal with an
intravenous LD50 of 0.1 mg/g. It contains two lethal, basic
phospholipase A toxins with intravenous LD50's
of 0.02 mg/g
and 0.18 mg/g
respectively and two polypeptide toxins with
intravenous LD50's of 0.17 mg/g
and 0.83 mg/g,
respectively (Tan et al., 1989b). Candidus
toxin, the highly lethal basic phospholipase A
toxin with intravenous LD50 of 0.02 mg/g,
accounts for 11% of the venom dry weight and is
the major lethal toxin of the venom.
Bungarus flaviceps (Red-headed krait)
krait is a very rare snake. It is blue black
above with the head, neck and tail bright red.
The venom contains kappa-flavitoxin, a
postsynaptic neurotoxin which is a potent
inhibitor of nicotinic transmission in autonomic
ganglia (Chiappinelli et al., 1987).
had an intravenous LD50 of 0.32 mg/g and exhibited enzymatic
activities similar to other Bungarus
venoms, with very low protease and high
acetylcholinesterase and phospholipase A
activities. It did not exhibit hemorrhagic,
procoagualtn or anticoagualtn activity. The
lethality of the venom could be neutralized
effectively by a commercial antisera prepared
against Bungarus .multicinctus and Naja
naja atra venoms (Tan, N.H. unpublished
Venoms of Trimeresurus
(Asian lance-headed vipers)
are pit vipers in which all the scales on the top
of the head are small and irregularly arranged.
There are at least seven known species of Trimeresurus
in Malaysia, including T.wagleri
(Wagler's pit viper or speckled
pit viper), T.popeorum
(Pope's tree viper), T.sumatranus
(Sumatran pit viper), T.puniceus
(flat-nosed pit viper), T.monticola
(mountain pit viper), T.purpureomaculatus
(shore pit viper) and T.hageri.
Snakebite cases by T.wagleri
have been reported (Reid et
al., 1963a) but they rarely result in serious
is considered to be a dangerous species as the
snake is reported to be aggressive (Lim, 1982).
Bites from other Malaysian Trimeresurus are rare.
wagleri (Speckled pit viper) venom
pit viper is abundant in lowland, primary forest
and secondary forest. This is the species that is
kept at the Snake Temple at Penang. According to
Brattstrom (1964), T.wagleri
has many morphological characteristics that
distinguish it from other species of Trimeresurus
and he put this species in the subgenus Tropidolaemus.
The intravenous LD50
for the venom is 0.9 mg/g. Unlike other pit viper venom, T.wagleri
venom does not exhibit hemorrhagic activity
(Minton, 1968). The venom also exhibits only
feeble thrombin-like activity. The major lethal
toxins of the venom are two basic polypeptide
toxins, wagleri toxin 1 and wagleri toxin 2,
which have intravenous LD50's of 0.17 and 0.19 mg/g,
respectively and molecular weights of 8900 (Tan
and Tan, 1989a).
purpureomaculatus (shore pit viper) venom
pit viper (also known as mangrove pit viper) is a
lowland snake and is very numerous in the
mangrove and swamp forests. The venom has an
intravenous LD50 of 0.9 mg/g and exhibits moderate thrombin-like enzyme
and strong hemorrhagic activities (Tan et
al., 1989a). The lethal action of the venom
appears to be due to synergistic action of
several venom constituents (Tan and Tan, 1988b).
Biological constituents of the venom that have
been characterized include thrombin-like enzymes,
hemorrhagin, arginine ester hydrolases,
phospholipase A2 enzymes and acetyl
venom contains several thrombin-like enzymes and
the major form has been purified. It was termed
purpurase. Purpurase has a molecular weight of
35000 and its N-terminal sequence is homologous
to many other venom serum proteinases (VVGGDECNINEHRSLVAIF).
The enzyme exhibited both arginine ester
hydrolase and arginine amidase activities. The
clotting activity by purpurase was in the
following decreasing order: cat dibrinogen>human
fibrinogen >dog fibrinogen>goat
fibrinogen>>rabbit fibrinogen. Analysis of
the products of its action on bovine fibrinogen
showed that only fibrinopeptide A was released
(Tan, 1985; Lu and Tan, 1998)
are four phospholipase A2
isoenzymes in the venom (Tan et
The molecular weights of the enzymes are
approximately 25000, indicating that they exist
in dimeric form. The isoelectric points of the
enzymes are 4.03, 4.5, 4.8 and 5.3, respectively.
They are not lethal to mouse but exhibit
major hemorrhagic of the venom has been purified
(Tan and Fung, 2002) and was termed maculatoxin.
It is a protease with a molecular weight of
136500 and a pI of 4.2. The N-terminal sequence
was TPEQQRFPPTYIDLGIFVDHGMYAT and is homologous
to other snake venom metalloproteinases.
Maculatoxin has a minimum hemorrhagic dose of
0.84 ug in mice but was not lethal to mice at a
dose of 1 mg/g
(i.v.). It was inactivated by EDTA and partially
inhibited by ATP and citrate. Both indirect and
double sandwich ELISA showed extensive
cross-reaction between venoms from the Crotalidae
family (in particular the Trimeresurus
genus) with rabbit anti-maculatoxin.
most other crotalid venoms, shore pit viper venom
exhibits acetylcholinesterase activity. The
enzyme is an acidic protein with kinetic
properties similar to acetylcholinesterases
isolated from cobra and krait venoms (Tan and
Tan, 1988c). It accounts for less than 0.1% of
the venom dry weight and is unlikely to play any
important role in the toxic action of the venom.
popeorum (Pope's tree viper) venom
tree viper is widely distributed but strictly
confined to hilly areas. The snake is greenish in
colour and its appearance is very similar to
several other green tree pit vipers such as T.macrops,
T.erythrurus and T.albolabris.
Pope's tree viper venom poisoning results in
hemorrhage and thrombocytopenia (Mitrakul and
Impun, 1973). The venom has an intravenous LD50
of 1.9 mg/g
and exhibits strong thrombin-like enzyme and
hemorrhagic activities (Tan et
albolabris (White-lipped tree viper) venom
tree viper occurs in East Malaysia. In severe
white-lipped tree viper venom poisoning,
hemorrhage, defibrination, thrombocytopenia and
increased fibrinolytic activity were observed (Mahasandana
et al., 1980). The venom has an intravenous
LD50 of 0.5 mg/g
and exhibits moderate thrombin-like enzyme and
hemorrhagic activities (Tan et
sumatranus (Sumatran pit viper) venom
Sumatran pit viper is green with dark cross-bands
and grows to rather over one meter. The snake is
found in forest in inland localities. T.sumatranus venom has an intravenous LD50
of 0.6 mg/g.
It exhibits very potent hemorrhagic and very weak
thrombin-like activities. The major lethal
component of the venom may be the hemorrhagic
principle (Tan and Tan, 1989b).
Venom of Enhydrina schistosa (beaked sea
are more than 18 species of sea snake in the
coastal waters of Malaysia. The commonest species
is Enhydrina schistosa (beaked sea snake),
which is also the most aggressive species (Smith,
1926; Reid and Lim, 1957; Reid, 1979). Other
common sea snakes include Hydrophis
cyanocinctus, Hydrophis spiralis and Hydrophis
sea snake is usually uniform grey above and
whitish beneath with a maximum length of 1.4
meter. The nostril in front gives a
characteristic beak-like appearance. In animals, E.schistosa
venom is neurotoxic, acting mainly at the
neuromuscular junction. In human, however, the
venom is primarily myotoxic (Reid, 1979). The
myonecrosis causes generalized muscle movement
pains, myoglobinuria and much of the paresis in
severe sea snakebite poisoning (Reid 1961 a,b).
venom contains mainly polypeptides and the
content of enzymes is very low. The biological
constituents of the venom that have been
characterized include postsynaptic neurotoxins,
phospholipase A2 enzymes and
neurotoxins account for 70% of the venom dry
weight. The two major neurotoxins which accounts
for 14% and 25% respectively of the venom
proteins are polypeptides with 60 amino acid
residues (Karlsson et al., 1972) and the sequences are
homologous to the cobra neurotoxins (Fryklund et
al., 1972). At a dose of 0.1 mg/g the toxins produce respiratory paralysis
and death in guinea pigs within 1-2 hr after i.p
injection (Geh and Toh, 1978). These toxins cause
neurotoxic poisoning in animals. Human, however,
is probably less susceptible to the action of the
venom contains more than seven highly lethal
basic phospholipase A toxins with intravenous LD50
of less than 0.1 mg/g,
and two non-lethal, acidic phospholipase A2 enzymes, which however are
able to depress muscle excitability due to direct
stimulation as well as the response of the muscle
to nerve stimulation and to exogenous
acetylcholine (Geh and Lin-Shiau, 1987). The
basic and acidic phospholipase A2 enzymes account for 13% and 4%, respectively,
of the venom dry weight (Tan, 1982c). The major
basic phospholipase A toxin, which accounts for
7% of the venom protein, also exhibits strong
myotoxic activity (Fohlman and Eaker, 1977). When
injected into mice the myotoxic phospholipase A
toxin causes breakdown of myofilament and
mitochondrial cristae with invasion of phagocytic
cells (Geh and Toh, 1978). The myonecrotic action
of E.schistosa venom is presumably due to the
action of these myotoxic phospholipase A toxins.
The amino acid sequence of the major myotoxic
phospholipase A toxin is closely homologous to
other elapid venom phospholipase A2
as well as the non-toxic bovine phospholipase A2 (Lind and Eaker, 1981).
venom acetylcholinesterase has been partially
purified (Kanthimathi, 1980). The enzyme, which
accounts for less than 0.1% of the venom dry
weight, has a molecular weight of 77000 and does
not exhibit significant lethal toxicity.
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