Amino acid sequence and circular dichroism of Indian cobra (Naja naja naja) venom acidic phospholipase A2

The full amino acid sequence of the acidic phospholipase A2 from Indian cobra (Naja naja naja) venom was determined and its tertiary structure examined by circular dichroism (CD). The sequence was aligned with other sequences of secreted phospholipase A2 from snakes of the genus Naja, using the progressive alignment method of Feng and Doolittle (J. Mol. Evol. (1987) 25, 351-360). The primary sequence of Naja naja naja phospholipases A2 shows up to 85% identity with the other acidic Naja phospholipase A2. CD studies indicate a 40-50% alpha-helical content in a tertiary structure which resists denaturation at high temperature, with or without chaotropic salts.     

Amino acid sequence of cytotoxin IIa isolated from the venom of the Indian cobra (Naja naja)

A cytotoxic basic polypeptide, designated as cytotoxin IIa, was purified to homogeneous state from the venom of the Indian cobra (Naja naja) by a combination of gel filtration on Sephadex G-50, CM-cellulose chromatography, and fast protein liquid chromatography. Cytotoxin IIa is a single polypeptide consisting of 60 amino acid residues with four intramolecular disulfide linkages. The toxin showed high cytotoxicity toward Yoshida sarcoma and ascites hepatoma cells as did cytotoxins I and II isolated from the same venom. Analysis of the amino acid sequence revealed that cytotoxin I, IIa, and II are highly homologous in their primary structures and that cytotoxin IIa differs from cytotoxin I only in having Phe 25 and Val 52 in place of Tyr 25 and Glu 52 residues.     

Amino acid sequence of a less-cytotoxic basic polypeptide (LCBP) isolated from the venom of the Indian cobra (Naja naja)

A less-cytotoxic polypeptide, designated as LCBP, was isolated from the venom of Naja naja by gel filtration on Sephadex G-50 followed by CM-cellulose chromatography. The cytotoxicity toward Yoshida sarcoma cells and lethal toxicity toward mice of LCBP were both one order of magnitude lower than that of cytotoxins and that of toxin A, respectively. LCBP is a single polypeptide consisting of 61 amino acid residues with four intramolecular disulfide linkages, and the amino acid sequence is the same as that of cardiotoxin-like basic polypeptide (CLBP) isolated from the venom of Naja naja atra. This is the first time that the same polypeptides were isolated from different cobra venoms.     

Fibrinogenolytic toxin from Indian monocled cobra (Naja kaouthia) venom

A fibrinogenolytic toxin of molecular weight 6.5 kDa has been purified from the venom of Indian monocled cobra (Naja kaouthia) by repeated cation exchange chromatography on CM-sephadex C-50. The purified toxin did not show any phospholipase activity but was mildly hemolytic on human erythrocytes. This toxin, called Lahirin, cleaved fibrinogen in a dose- and time-dependent manner. The digestion process apparently started with the A alpha chain, and gradually other lower-molecular-weight chains were also cleaved to low-molecular-weight peptides. The fibrinolytic activity was completely lost after treatment with ethylene di-amine tetra acetic acid (EDTA). However, exposure to 100 degree C for 1 min or pre-treatment with phenyl methyl sulfonyl fluoride (PMSF) did not affect the fibrinolytic activity. Cleavage of di-sulphide bonds by beta-mercaptoethanol or unfolding the protein with 4 M urea caused complete loss of activity of pure Lahirin.     

Amino acid sequence of nerve growth factor purified from the venom of the Formosan cobra Naja naja atra

Nerve growth factor (NGF) was isolated from the venom of the Formosan cobra (Naja naja atra). The amino acid sequence was determined by a combination of conventional methods. The total number of amino acid residues was 116, giving a molecular mass of 13,057 Da. The sequence was identical with that deduced from the nucleotide sequence of an NGF cDNA from the venom gland of Naja naja siamensis, reported by Selby et al. [J. Neurosci. Res., 18, 293-298 (1987)].     

Partial sequence of hemoglobin from cobra (Naja naja naja)

Hemoglobin from the cobra snake, Naja naja naja, was isolated and its chains separated on a CM-cellulose column. The separation profile revealed an and two chains having the molar proportions of []₂,[ ₁]₁,[ ₂]₁. The N-terminal amino acid sequence of the intact chains and of the CNBr peptides were carried out. The ₂ chain was found to be heterogeneous comprising a minor component amounting to 11%. This later showed changes at two positions 9 and 14 in the first 30 residues sequenced.     

Localization of the five disulfide bridges in toxin B from the venom of the Indian cobra (Naja naja)

By degradation with acid protease and thermolysin the five disulfide bridges in toxin B from the venom of the Indian cobra have been localized. Toxin B consists of 71 amino acid residues and the five intramolecular disulfide bridges link half cystine residues 3 and 20, 14 and 41, 26 and 30, 45 and 56, and 57 and 62.     

Neutralization of cobra venom by cocktail antiserum against venom proteins of cobra (Naja naja naja)

Naja naja venom was characterized by its immunochemical properties and electrophoretic pattern which revealed eight protein bands (14 kDa, 24 kDa, 29 kDa, 45 kDa, 48 kDa, 65 kDa, 72 kDa and 99 kDa) by SDS-PAGE in reducing condition after staining with Coomassie Brilliant Blue. The results showed that Naja venom presented high lethal activity. Whole venom antiserum or individual venom protein antiserum (14 kDa, 29 kDa, 65 kDa, 72 kDa and 99 kDa) of venom could recognize N. naja venom by Western blotting and ELISA, and N. naja venom presented antibody titer when assayed by ELISA. The neutralization tests showed that the polyvalent antiserum neutralized lethal activities by both in vivo and in vitro studies using mice and Vero cells. The antiserum could neutralize the lethal activities in in-vivo and antivenom administered after injection of cobra venom through intraperitoneal route in mice. The cocktail antiserum also could neutralize the cytotoxic activities in Vero cell line by MTT and Neutral red assays. The results of the present study suggest that cocktail antiserum neutralizes the lethal activities in both in vitro and in vivo models using the antiserum against cobra venom and its individual venom proteins serum produced in rabbits.     

Effect of modification of tyrosine residues in cytotoxin-1 from Indian cobra venom. (Naja naja naja)

The role of tyrosine residues in the biological activity of cytotoxin-1 was evaluated using N-bromo succinimide. N-bromo succinimide effected the oxidation of tyrosine residues in cytotoxin-1 with an increase in absorption at 260 nm. N-chloro succinimide was ineffective in the oxidation of tyrosine residues in the toxin. Oxidation of a single tyrosine residue (at 3.50 equivalents of N-bromo succinimide/mole of the toxin) resulted in complete loss of lethal activity of the toxin. The lytic activity of the toxin (lysis of erythrocytes) remained uneffected even after three of the four tyrosine residues in the toxin were oxidised.     

Characterization of a cytotoxin-like basic protein from the cobra (Naja naja naja) venom

A cytotoxin-like basic protein has been isolated from the venom of the nominate race of cobra (Naja naja naja from Pakistan) by a single step of high-performance liquid chromatography. The primary structure was determined and consists of 62 amino acid residues in a single polypeptide chain. It is highly similar to that of the cytotoxin-like basic proteins isolated from other Naja species, but differs in two of the SS-loop structures from that of cytotoxins.     

Amino acid sequence of cardiotoxin-analogue I from the venom of Naja naja atra.

Cardiotoxin(CTX)-analogue I was obtained in a yield of about 5.3% from the venom of $\text{naja naja atra}$ by gel filtration on Sephadex G-50, followed by CM-cellulose column chromatography. When injected intraperitoneally in mice, its LD₅₀ was 2.8 (2.45–3.19) μg/g body weight. This toxin was cytotoxic to Yoshida sarcoma cells and produced contracture of the skeletal muscle as did CTX. The amino acid sequence of CTX-analogue I exhibits a high degree of homology with that of CTX from the same venom but differs in 13 positions.     

Antigenic relationships between human and cobra complement factors C3 and cobra venom factor (CVF) from the Indian cobra (Naja naja)

The presence of a factor immunologically related to cobra venom factor (CVF) was demonstrated in serum and plasma from the Indian cobra (Naja naja kaoutia). The factor was purified from cobra plasma by affinity chromatography on an anti-CVF gel and was found to consist of a protein composed of two polypeptide chains similar in size to those of human C3. With use of immunoblotting technique, common antigenic determinants were found in the smaller chain of the prepared material and the beta-chain of human C3; the larger chain may display antigenic determinants present in the alpha-chain of human C3. These findings suggest that this molecule represents the C3 of the cobra complement system. Common antigenic determinants were also demonstrated in the alpha-chain of CVF and the beta-chains of human and cobra C3. No reactions were observed between the beta- and gamma-chains of CVF and any antiserum against human C3 or its subunits. Upon immunodiffusion analysis, cobra serum was found to contain a factor besides C3 sharing antigens specific for CVF, while cobra C3 was antigenically deficient compared to CVF. This suggests that cobra C3 physiologically is degraded to a molecule very similar to or identical with CVF.     

Amino acid sequence of a cardiotoxin-like basic polypeptide (CLBP) with low cytotoxic activity isolated from the venom of the Formosan cobra (Naja naja atra)

A cardiotoxin-like basic polypeptide, designated as CLBP, was isolated from the venom of Naja naja atra by gel filtration on Sephadex G-50 followed by CM-cellulose chromatography. The cytotoxicity toward Yoshida sarcoma cells and lethal toxicity toward mice of CLBP were both one-order lower than those of cardiotoxins and cobrotoxin, respectively. CLBP is a single polypeptide consisting of 61 amino acid residues with four intramolecular disulfide linkages. The amino acid sequence of CLBP shows a high degree of homology with those of cardiotoxins from the same venom, but differs in the 19 to 23 positions.     

Variation in biochemical and pharmacological properties of Indian cobra (Naja naja naja) venom due to geographical distribution

Indian cobra (Naja naja naja) venom obtained from three different geographical regions was studied in terms of electrophoretic pattern, biochemical and pharmacological activities. SDS-PAGE banding pattern revealed significant variation in the protein constituents of the three regional venoms. The eastern venom showed highest indirect hemolysis and hyaluronidase activity. In contrast, western and southern venoms were rich in proteolytic activity. All the three regional venoms were devoid of p-tosyl-L-arginine methyl ester hydrolysing activity. The eastern venom was found to be most lethal among the three regional venoms. The lethal potency varied as eastern > western > southern regional venoms. In addition, all the three regional venoms showed marked variations in their ability to induce symptoms/signs of neurotoxicity, myotoxicity, edema and effect on plasma coagulation process. Polyclonal antiserum prepared against the venom of eastern region cross-reacted with both southern and western regional venoms, but varied in the extent of cross-reactivity by ouchterlony immunodiffusion and ELISA.