King Cobra Venom Research Articles

Hannalgesin from king cobra venom releases nitric oxide (NO) and activates nitric oxide synthase (NOS)

Hannalgesin from king cobra venom releases nitric oxide (NO) and activates nitric oxide synthase (NOS)

Chemical modification of cationic residues in toxin a from king cobra (Ophiophagus hannah) venom

The cationic groups of arginine and lysine residues in alpha-neurotoxin, Toxin a, isolated from king cobra (Ophiophagus hannah) venom were subjected to modification with trinitrobenzene sulfonate (TNBS) and p-hydroxyphenylglyoxal (HPG), respectively. The trinitrophenylated (TNP) derivatives of Toxin a at Lys-10, 56, or 71 showed approximately 25% residual lethality, and modifications on Lys-10 and 56 or Lys-10 and 50 resulted in a decrease of lethality by 84% and 86%, respectively. Modifications on Arg-34, 37, and 70 and Arg-34, 37, and 72 in Toxin a caused a decrease in lethality by 92% and 93%, respectively, and it almost completely lost its lethality and binding activity to nicotinic acetylcholine receptor (nAChR) when all four arginine residues were modified. These results indicate that in addition to the cationic residues on loop II (Arg-34, 37), loop III (Lys-50, 56), and the C-terminal tail (Arg-70, 72; Lys-71), Lys-10 on loop I is also related to the neurotoxicity of Toxin a.

A novel analgesic toxin (hannalgesin) from the venom of king cobra ( Ophiophagus hannah)

The pharmacological effects of a purified neurotoxin from king cobra ( Ophiophagus hannah) venom were studied. Using the hot-plate test, it is shown that this neurotoxin increased latency time dose-dependently when administered i.p. Similar analgesic action was observed when it was administered p.o. or i.c.v. The rota-rod performance, which is a good index for neurological deficits including sedation, muscle relaxant and impairment of motor activity and coordination, was not significantly affected in the dose range of 16–32 ng/g that caused analgesia. The toxin did not increase the convulsion threshold in the dose range of 8–64 ng/g in the maximal electroshock seizure tests. These results demonstrated that this neurotoxin produced analgesia in the dose range of 16–32 ng/g (i.p.) without causing any neurological or muscular deficits. It is further shown that such analgesic action was blocked by naloxone and l-N G- nitro-arginine methyl ester, suggesting the possible involvement of the opioid and nitric oxide systems, respectively. In view of the source of this neurotoxin ( O. hannah) and its potent analgesic action, it is proposed that this toxin be named hannalgesin.

Chemical modification of cationic groups of a novel alpha-neurotoxin (Oh-4) from king cobra (Ophiophagus hannah) venom.

The cationic groups of arginine and lysine residues in Oh-4, a novel alpha-neurotoxin from king cobra (Ophiophagus hannah) venom were subjected to modification with p-hydroxyphenylglyoxal (HPG) and trinitrobenzene sulfonate (TNBS), respectively. Monoderivatization of Arg-35, resulted in a drastic loss in neurotoxicity to 25% of the native toxin. The activity was decreased to a greater extent with the derivative extensively modified on Arg-35, -9, and -37. The Arg-35-modified derivative retained about a half of the antigenicity of the native toxin, and extensive modification on Arg-9 and Arg-37 caused a further decrease in the antigenicity of the toxin molecule. Selective trinitrophenylation (TNP-) of Lys-51 caused losses of neurotoxicity and antigenicity by 77 and 83%, respectively. These results indicate that Arg-35 and Lys-51 in Oh-4 have important roles in the neurotoxicity. In contrast to the Arg residues at 9, 35, and 37, Lys-51 plays a more critical role in the antigenicity.

Chemical modification of tryptophan residues in alpha-neurotoxins from Ophiophagus hannah (king cobra) venom.

Two alpha-neurotoxins, Oh-4 and Oh-7, from the king cobra (Ophiophagus hannah) venom were subjected to Trp modification with 2-nitrophenylsulfenyl chloride (NPS-Cl). One major NPS derivative was isolated from the modified mixtures of Oh-4 and two from Oh-7 by HPLC. Amino acid analysis and sequence determination revealed that Trp-27 in Oh-4, and Trp-30 and Trp-26 and 30 in the two Oh-7 derivatives, were modified, respectively. Sulfenylation of Trp-27 in Oh-4 caused about 70% drop in lethal toxicity and nicotinic acetylcholine receptor-binding activity. Modification of Trp-30 in Oh-7 resulted in the decrease of lethal toxicity by 36% and binding activity by 61%. The activities were further lost when the conserved Trp-26 in Oh-7 was modified. Sulfenylation of the Trp residues did not significantly affect the secondary structure of the toxins as revealed by the CD spectra. These results indicate that the Trp residues in these two long alpha-neurotoxins may be involved in the receptor binding.

Purification and characterization of l-amino acid oxidase from king cobra ( Ophiophagus hannah) venom and its effects on human platelet aggregation

Z.-Y. Li, T.-F. Yu and E. C.-Y. Lian. Purification and characterization of l-amino acid oxidase from king cobra ( Ophiophagus hannah) venom and its effects on human platelet aggregation. Toxicon 32, 1349–1358, 1994.—Venoms of several snake species contain large amounts of l-amino acid oxidase but its effects on human plasma coagulation and platelet aggregation have not been explored. We have purified l-amino acid oxidase from king cobra venom through CM-Sephadex C-25, Sephadex G-100 and DEAE Sephadex A-50 chromatographies. The purified enzyme has a mol. wt of 135,000 as determined by gel filtration and 65,000 by SDS-PAGE under non-reducing and reducing conditions. Incubation of plasma with l-amino acid oxidase at 200 μg/ml did not affect prothrombin time, activated partial thromboplastin time, or thrombin time. Upon addition of l-amino acid oxidase, platelets in platelet-rich plasma were aggregated. The enzyme-induced aggregation was abolished by catalase. The aggregation was also inhibited by indomethacin, aspirin, ethylenediaminetetraacetate, sodium nitroprusside, prostaglandin E 1, mepacrine and verapamil, but not by heparin, hirudin, creatine phosphate/creatine phosphokinase or antimycin/2-deoxy- d-glucose. These results suggest that l-amino acid oxidase induces human platelet aggregation through the formation of H 2O 2, and subsequent thromboxane A 2 synthesis requiring Ca 2+ but independent of ADP release. The platelet aggregation caused by l-amino acid oxidase is likely to contribute to toxicity inflicted by cobra venom.

Characterization of OhS1, an arginine/lysine amidase from the venom of king cobra ( Ophiophagus hannah)

Y. Zhang, W-h. Lee, Y-l. Xiong, W-y. Wang and S-w. Zu. Characterization of OhS1, and arginine/lysine amidase from the venom of king cobra ( Ophiophagus hannah). Toxicon 32, 615–623, 1994.—In this paper, we present the results of purification and characterization of an arginine/lysine amidase from the venom of Ophiophagus hannah (OhS1). It was purified by Sephadex G-75 gel filtration and ion-exchange chromatography on DEAE-Sepharose CL-6B. It is a protein of about 43,000, consisting of a single polypeptide chain. It is a minor component in the venom. The purified enzyme was capable of hydrolysing several tripeptidyl- p-nitroanilide substrates having either arginine or lysine as the C-terminal residue. We studied the kinetic parameters of OhS1 on six these chromogenic substrates. OhS1 did not clot fibrinogen. Electrophoresis of fibrinogen degraded with OhS1 revealed the disappearance of the α- and β-chains and the appearance of lower mol. wt fragments. OhS1 had no hemorrhagic activity. It did not hydrolyse casein, nor did it act on blood coagulation factor X, prothrombin and plasminogen. The activity of OhS1 was completely inhibited by NPGB, PMSF, DFP, benzamidine and soybean trypsin inhibitor, suggesting it is a serine protease. Metal chelator (EDTA) had no effect on it.

Kinetics of experimental envenomation by Ophiophagus hannah (king cobra) venom: N.-H. Tan, K.-K. Lim and M. I. Nik Jaafar (Department of Biochemistry, Faculty of Medicine, University of Malaya, 59100 Kuala Lumpur, Malaysia)

Kinetics of experimental envenomation by Ophiophagus hannah (king cobra) venom: N.-H. Tan, K.-K. Lim and M. I. Nik Jaafar (Department of Biochemistry, Faculty of Medicine, University of Malaya, 59100 Kuala Lumpur, Malaysia)

The antibody responses in rabbits immunized with king cobra (Ophiophagus hannah) venom or venom components: N.-H. Tan, M. I. Nik Jaafar and K.-K. Lim (Department of Biochemistry, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia)

The antibody responses in rabbits immunized with king cobra (Ophiophagus hannah) venom or venom components: N.-H. Tan, M. I. Nik Jaafar and K.-K. Lim (Department of Biochemistry, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia)

The edema inducing activity of Ophiophagus hannah (king cobra) venom L-amino acid oxidase: N.-H. Tan and S.-K. Choy (Department of Biochemistry, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia)

The edema inducing activity of Ophiophagus hannah (king cobra) venom L-amino acid oxidase: N.-H. Tan and S.-K. Choy (Department of Biochemistry, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia)

An investigation into the antigenic cross-reactivity of Ophiophagus hannah (king cobra) venom neurotoxin, phospholipase A 2, hemorrhagin and l-amino acid oxidase using enzyme-linked immunosorbent assay

The antigenic cross-reactivity of four Ophiophagus hannah (king cobra) venom components, the neurotoxin (OH-NTX), phospholipase A 2 (OH-PLA 2), hemorrhagin (OH-HMG) and l-amino acid oxidase (OH-LAAO) were examined by indirect and double sandwich ELISAs. The indirect ELISAs for OH-NTX, OH-PLA 2 and OH-HMG were very specific when assayed against the various heterologous snake venoms and O. hannah venom components, at 25 ng/ml antigen level. At higher antigen concentrations (100–400 ng/ml), there were moderate to strong indirect ELISA cross-reactions between anti- O. hannah neurotoxin and venoms from various species of cobra as well as two short neurotoxins. However, anti- O. hannah hemorrhagin did not cross-react with any of the venoms tested, even at these high antigen concentrations, indicating that O. hannah hemorrhagin is antigenically very different from other venom hemorrhagins. Examination of the indirect ELISA cross-reactions between anti- O. hannah PLA 2 and several elapid PLA 2 enzymes suggests that the elapid PLA 2 antigenic class has more than two subgroups. The antibodies to O. hannah l-amino acid oxidase, however, yielded indirect ELISA cross-reactions with many venoms as well as with OH-NTX, OH-PLA 2 and OH-HMG, indicating that OH-LAAO shares common epitopes even with unrelated proteins. The double sandwich ELISAs for the four anti- O. hannah venom components, on the other hand, generally exhibited a higher degree of selectivity than the indirect ELISA procedure.

Effects of an acidic phospholipase A 2 purified from Ophiophagus hannah (king cobra) venom on rat heart

M.-Z. Huang, Q.-C. Wang and G.-F. Liu. Effects of an acidic phospholipase A 2 purified from Ophiophagus hannah (king cobra) venom on rat heart. Toxicon 31, 627–635, 1993.—An acidic phospholipase A 2 (OHV A-PLA 2) purified from Ophiophagus hannah venom had a cardiotoxic action on rat heart. In rats OHV A-PLA 2 (2–4mg/kg) caused ECG abnormalities including decreased heart rate, prolonged P-R interval, widened QRS complex and complete A-V block. When tested on isolated rat right atria, OHV A-PLA 2 (10–20 μg/ml) produced a positive chronotropic effect. When tested on isolated rat left atria or papillary muscle preparations, OHV A-PLA 2 (2.5–20 μg/ml) caused positive inotropic effect, followed by contracture. The positive inotropic effects could be abolished by high Ca 2+ and enhanced by low Ca 2+; both treatments accelerated contracture. The contracture could be inhibited in Mn 2+ (5 mM)-containing medium and abolished by Ca 2+-free bath solution containing 1 mM EDTA. The cardiotoxic action of OHV A-PLA 2 was not influenced by verapamil, tetrodotoxin, propranolol, phentolamine, atropine or indomethacin. It is suggested that the cardiotoxic effects of OHV A-PLA 2 may result from increasing intracellular levels of Ca 2+.

Sequence Characterization of a Novel α-Neurotoxin from the King Cobra (Ophiophagus hannah) Venom

Several postsynaptic neurotoxins (α-neurotoxins) with distinct pharmacological and biochemical properties were isolated and purified from the King cobra venom (Ophiophagus hannah) by employing sequentially preparative-scale cation-exchange chromatography on SP-Sephadex C-25 coupled with gel filtration and reversed-phase HPLC. The complete sequence of one neurotoxin was determined by N-terminal Edman degradation with the automatic pulsed-liquid phase sequencer on some peptide fragments generated from the endopeptidases, i. e. trypsin, S. aureus V8 protease and lysyl endopeptidase. This novel neurotoxin is a basic polypeptide of pI 9.05, consisting of 72 amino-acid residues with 10 cysteine residues. It is found to share about 60 % sequence homology with Toxins a and b isolated from the same venom and the well established α-bungarotoxin, a major postsynaptic toxic ligand for acetylcholine receptor isolated from Bungarus multicinctus. The characterized α-neurotoxin molecules were also shown to bind specifically with nicotinic acetylcholine receptors of the electric eel, Torpedo californica.

Purification and sequence characterization of various α-neurotoxins from the king cobra (Ophiophagus hannah) venom

Purification and sequence characterization of various α-neurotoxins from the king cobra (Ophiophagus hannah) venom

Bites by the King Cobra (<italic>Ophiophagus hannah</italic>) in Myanmar: Successful Treatment of Severe Neurotoxic Envenoming

Three patients bitten by the world's largest species of venomous snake, the king cobra (Ophiophagus hannah), were observed in Myanmar (Burma). All three were involved in the famous snake dance in Yangon (Rangoon) Zoological Gardens. One patient showed no signs of envenoming despite a sustained bite, another developed only signs of local envenoming, but in a third there was severe neurotoxic envenoming requiring mechanical ventilation for 64 1/2 hours, episodes of hypotension and massive swelling of the bitten limb. This patient showed some signs of recovery before delayed treatment with specific antivenom. It is possible that all three patients had some immunity to king cobra venom resulting from traditional 'immunization' achieved by scratching venom into the skin. The literature on king cobra bites is reviewed and recommendations given for antivenom and ancillary treatments.

Substrate specificity of king cobra ( Ophiophagus hannah) VENOM l-amino acid oxidase

1. 1. Substrate specificity of purified king cobra ( Ophiophagus hannah) venom l-amino acid oxidase was investigated. 2. 2. The enzyme was highly specific for the l-enantiomer of amino acid. Effective oxidation of l-amino acid by the enzyme requires the presence of a free primary α-amino group but the α-carboxylate group is not as critical for the catalysis. 3. 3. The enzyme was very active against l-Lys, l-Phe, l-Leu, l-Tyr, l-Tryp, l-Arg, l-Met, l-ornithine, l-norleucine and l-norvaline and moderately active against l-His, l-cystine and l-Ileu. Other l-amino acids were oxidized slowly or not oxidized. 4. 4. The data suggest the presence of a side chain binding site in the enzyme, and that the binding site comprises at least five ‘subsites’: the hydrophobic subsites a, b and c; and the two ‘amino’ binding subsites d and e. Subsite b appears to be able to accommodate two methylene/methyl carbons.

Effect of king cobra venom on α2-macroglobulin and proteases in human blood plasma

Normal human blood plasma showed hydrolytic activities on several synthetic substrates for proteases, the most effective being H-D-Ile-Pro-Arg-p-nitroanilide, H-D-Pro-Phe-Arg-p-nitroanilide and H-D-Val-Leu-Arg-p-nitroanilide. When plasma was preincubated for 12 h at 37°C, there was no significant alteration of the hydrolytic activities. On incubation for 12 h with king cobra venom (2 μg for 0.1 ml plasma), there was considerable decrease in the activities and complete abolition of the protease binding capacity of α2-macroglobulin. On chromatography on Sephadex G-200, α2-macroglobulin activity and bulk of the protease activity of normal plasma were eluted in the void volume region. A minor protease peak was eluted with aVe/Vo value of 2.5. With venom treated plasma, there was no decrease with this peak. The major protease peak and α2-macroglobulin activity were drastically reduced. Chromatography on red Sepharose showed that all the α 2 − acroglobulin activity and bulk of the protease activity in normal plasma were bound to the column. In venom treated plasma there was marked reduction in the bound fraction. The data suggest that cobra venom proteases directly or through proteases generated in plasmain situ causes limited cleavage of α2-macroglobulin as well as α2-macroglobulin bound proteases, inactivating them.

Preparation of antibodies to king cobra (Ophiophagus hannah) venom hemorrhagin and investigation of their cross-reactivity

N.-H. Tan, M. N. Saifuddin and M. I. Nik Jaafar. Preparation of antibodies to king cobra ( Ophiophagus hannah) venom hemorrhagin and investigation of their cross-reactivity. Toxicon 28, 1355–1359, 1990.—Hannahtoxin, the major hemorrhagin purified from king cobra ( Ophiophagus hannah) venom, elicits hemorrhages in rabbits but not in mice. Two antisera against hannahtoxin were prepared: one raised against purified hannahtoxin, while the other was raised against glutaraldehyde cross-linked and detoxified hannahtoxin. The antisera were refined by pepsin digestion and ammonium sulfate precipitation. They are of approximately equal potency in their ability to neutralize the hemorrhagic activity of king cobra venom in rabbits. The antisera did not form a precipitin line with venom of snakes of the Viperidae family nor neutralize hemorrhages elicited in mice by any of these venoms. However, when the hemorrhagic activity was assayed in rabbits, both antisera were able to abolish the hemorrhages elicited by all of the venoms tested. These results suggest that hannahtoxin displays few epitopes in common with hemorrhagins of viperid venoms, except those involved in the neutralization of hemorrhagic activity in rabbits. The epitopes of viperid venom hemorrhagins involved in the neutralization reaction in rabbits are different from those in mice.

Purification and characterization of two acidic phospholipase A 2 enzymes from king cobra ( Ophiophagus hannah) snake venom

1. 1. The two major phospholipase A 2 enzymes (OHPLA-DE1 and OHPLA-DE2) of king cobra ( Ophiophagus hannah) venom have been purified to electrophoretic homogeneity. 2. 2. The isoelectric points of OHPLA-DE1 and OHPLA-DE2 were 3.81 and 3.89, respectively and the M w s were 14,000 and 15,000, respectively, as estimated by Sephadex G-75 gel filtration chromatography; and 14,000 as estimated by SDS-PAGE. 3. 3. The enzymes were not lethal to mice at a dosage of 10 μg/g body wt by i.v. route. Both phospholipase A 2 enzymes, however, exhibited moderate edema-inducing and anti-coagulant activities. 4. 4. Bromophenacylation of the enzymes reduced the enzymatic activity drastically but did not affect the edema-inducing activity of the enzymes.

Enzymatic and toxic properties of Ophiophagus hannah (king cobra) venom and venom fractions

Nget-Hong Tan and M. N. Saifuddin Hj. Enzymatic and toxic properties of Ophiophagus hannah (king cobra) venom and venom fractions. Toxicon 27, 689–695, 1989.—Some enzymatic activities and toxic properties of four samples of Ophiophagus hannah (king cobra) venom were investigated. There is little intraspecific variation in enzyme contents, protein composition and toxic properties of the venom. The venom does not exhibit hemolytic or edema-inducing activity but is characterized by an exceptionally high alkaline phosphomonoesterase activity. DEAE-Sephacel ion exchange chromatography and Sephadex G-75 gel filtration chromatography of the venom indicate that the major lethal toxins are the low mol.wt, non-enzymatic basic proteins. Venom fractions exhibiting high enzymatic activities apparently do not play an important role in the lethality in mice of Ophiophagus hannah venom.