TOXINS FROM VENOMS OF POISONOUS SNAKES         INDIGENOUS TO MALAYSIA: A REVIEW

Professor Tan, Nget Hong

Department of Molecular Medicine, Faculty of Medicine

University of Malaya, Kuala Lumpur, Malaysia

tanngethong@yahoo.com.sg    

                                                     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.

1. Introduction

Snakebite 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.

The 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 to Malaysia.

2. Poisonous Snakes of Malaysia

Poisonous 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 several genera.

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 (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.

Only 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

Other poisonous snakes indigenous to Malaysia that are potentially dangerous to human include Bungarus candidus (Malayan krait), Bungarus fasciatus  (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.

3. Composition of snake venoms

Dried 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.

Venoms 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. Several presynaptic phospholipase A₂ toxins have been isolated from krait and sea snake venoms.

Besides 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 1-2 mg/gm.

Enzymes 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).

Enzymes 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  

Venom 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

Abbreviations 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; PRO:  protease, unit/mg, AEH:  arginine ester hydrolase, mmole/min/mg; PME:  alkaline 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).  

4. 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 LD<sub>50</sub>'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).

4.1 Naja naja sputatrix venom     

The major biochemical constituents of the venom include high molecular weight proteins and enzymes, phospholipase A₂ enzymes, postsynaptic neurotoxins and polypeptide cardiotoxins.

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).

Phospholipase 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, 0.28 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.

The 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.

Postsynaptic 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).

Polypeptide 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)

Table 3: The amino acid sequences of some Malayan cobra toxins  

4.2 Naja naja kaouthia venom

The major biochemical constituents of the N.n.kaouthia venom also consist of high molecular weight proteins and enzymes, phospholipase A₂ enzymes, postsynaptic neurotoxins and cardiotoxins.

The 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).

Postsynaptic neurotoxins account for 23% of the venom dry weight. The major polypeptide neurotoxins are `long' neurotoxins with 71 amino acid residues (Karlsson and  Eaker, 1972). Neurotoxins are the major lethal toxins of the venom.

Cardiotoxins, 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.

4.3 Differences between N.n.sputatrix and N.n.kaouthia venoms.

It 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.

5. Venom of Calloselasma rhodostoma (Malayan pit viper)

Calloselasma 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 than 2%.

The 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).

Malayan 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 environmentally induced.

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)

There 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).

The 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 membrane.

Epitope 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).

Malayan 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 heterodimer. The platelet aggregation inducers presumably play a role in the thrombocytopenic syndrome caused by Malayan pit viper venom poisoning.

Several 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 a_IIbb₃ 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 in function.   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 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.

              Table 4:  Amino Acid Sequence of Some Malayan Pit Viper Venom Toxins

Malayan 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, 1986).

The 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.

6. Venom of Ophiophagus hannah (King cobra)

King 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).

King 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 phosphomonoesterases.

Venom 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).

The 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.

The 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.

7. Venoms of the Bungarus (kraits)

There 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.

7.1 Bungarus fasciatus (Banded krait) venom

Banded 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).

The 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.

The 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 0.04 mg/g (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 (Chang et al., 1983; Gong et al., 1989).

Banded 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.

7.2 Bungarus candidus (Malayan krait) venom

Malayan 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.

7.3 Bungarus flaviceps (Red-headed krait) venom

Red-headed 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).  Bungarus flaviceps venom 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 results).

8. Venoms of Trimeresurus (Asian lance-headed vipers)

The Trimeresurus 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 and T.purpureomaculatus have been reported (Reid et al., 1963a) but they rarely result in serious poisoning. T.sumatranus is considered to be a dangerous species as the snake is reported to be aggressive (Lim, 1982). Bites from other Malaysian Trimeresurus are rare.

8.1 Trimeresurus wagleri (Speckled pit viper) venom

Speckled 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).

8.2 Trimeresurus purpureomaculatus (shore pit viper) venom

Shore 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 cholinesterase.

The 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)

There are four phospholipase A₂ isoenzymes in the venom (Tan et al., 1989c).  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 edema-inducing activity.

The 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.

Unlike 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.

8.3 Trimeresurus popeorum (Pope's tree viper) venom

Pope's 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 al., 1989a).

8.4 Trimeresurus albolabris (White-lipped tree viper) venom

White-lipped 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 al., 1989a).

8.5 Trimeresurus sumatranus (Sumatran pit viper) venom

The 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).

9. Venom of Enhydrina schistosa (beaked sea snake)

There 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 klossi.

Beaked 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).

The 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 acetylcholinesterase.

Postsynaptic 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 toxins.

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).

Enhydrina schistosa 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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