Purification and characterization of anticomplement factor (cobra venom factor) from the Naja naja atra venom

Anticomplement factor (cobra venom factor) from the venom of Naja naja atra was purified by means of successive chromatography on DEAE-cellulose, Sephadex G-200 and Sepharose CL-6B. The purified anticomplement factor was homogeneous as judged by polyacrylamide discontinuous gel electrophoresis at pH 9.4. The yield from 3.0 g of the crude venom was approx. 28 mg. The molecular weight was estimated to be about 156 000 by SDS-polyacrylamide gel electrophoresis. The isoelectric point was about 5.2. SDS-polyacrylamide gel electrophoresis of the anticomplement factor in the presence of dithiothreitol demonstrated that the molecule possesses three different polypeptide chains cross-linked covalently to one another by disulfide bridge(s). By SDS-polyacrylamide gel electrophoresis, the molecular weight of each subunit was determined to be approx. 77000, 47500 and 29 000, respectively. All subunits were stained with Coomassie brilliant blue G-250 and periodate-Schiff reagent, indicating these subunits to be glycoprotein. Distribution of the anticomplement factor in various snake venoms, which shows cross-reactivity against the anti-Naja naja atra anticomplement factor antiserum, was examined. From the results, all venoms belonging to cobra family in the Elapidae tested so far were found to contain such cross-reactivity.     

Isolation and characterization of a lethal phospholipase A2 (NN-IVb1-PLA2) from the Indian cobra (Naja naja naja) venom.

Indian cobra (Naja naja naja) venom is reported to contain multiple forms of phospholipase A2. Only a couple of them have been isolated and characterized. A lethal phospholipase A2 (NN-IVb1-PLA2) from Naja naja naja venom has been purified in three steps involving CM-Sephadex C-25, Sephadex G-50 and rechromatography on CM-Sephadex C-25 columns. It is a basic protein with pl value between 7-7.5 and has molecular weight between 11,000-11,500. The LD50 of NN-IVb1-PLA2 is 1.2 mg/K g body weight. It induces neurotoxic symptoms in the experimental mice and is devoid of myotoxic, anticoagulant, edema inducing and direct hemolytic activities.     

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.     

Isolation and characterization of an acidic lethal phospholipase Az from Malayan cobra (Naja naja sputatrix) venom

An acidic, lethal phospholipase Az was purified to electrophoretic homogeneity from the venom of the Malayan cobra (Naja naja sputatrix). The enzyme has an isoelectric point of 5.58, a molecular weight of 12000, and a medium lethal dose (LD50) of 0.86 micrograms/g in mice by intravenous injection. The enzyme also exhibited weak anticoagulant and edema-forming activities. The amino acid composition of the enzyme is similar to those of other cobra venom phospholipases Az.     

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.     

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.     

INN-toxin, a highly lethal peptide from the venom of Indian cobra (Naja naja) venom-Isolation, characterization and pharmacological actions

A novel toxic polypeptide, INN-toxin, is purified from the venom of Naja naja using combination of gel-permeation and ion-exchange chromatography. It has a molecular mass of 6951.6Da as determined by MALDI-TOF/MS and the N-terminal sequence of LKXNKLVPLF. It showed both neurotoxic as well as cytotoxic activities. INN-toxin is lethal to mice with a LD(50) of 1.2mg/kg body weight. IgY raised in chicks against basic peptide pool neutralized the toxicity of INN-toxin. INN-toxin did not inhibit cholinesterase activity. It is toxic to Ehrlich ascites tumor (EAT) cells, but it is not toxic to leukocyte culture. The toxin appears to be specific in its mode of action. Interaction of N-bromosuccinamide (NBS) with the peptide resulted in the modification of tryptophan residues and loss of lethal toxicity of INN-toxin.     

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.     

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.     

Cardiotoxin of the Indian cobra(Naja naja) is a pyrophosphatase

An inorganic pyrophosphatase has been purified to apparent homogeniety from Indian cobra(Naja naja) venom, with a ten-fold increase in specific activity. The enzyme activity is intrinsic to a protein fraction in the venom which is normally termed cardiotoxin, cobramine, cytotoxin and so on. The enzyme shows a lowK _m (70 μI) and high heat stability. The enzyme was active against sodium pyrophosphate; it also hydrolyses a few mononucletides and sugar phosphates at much lower rates. The physiological significance of inorganic pyrophosphatase in venom is discussed.     

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.     

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.     

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.     

Isolation and characterization of nerve growth factor from the venom of Naja naja atra.

Nerve growth factor was isolated from the venom of Naja naja atra by ion exchange and gel permeation chromatography and was found to be homogeneous by disc gel electrophoresis. The molecular weight was estimated to be approximately 20,000 by gel filtration and 22,000 by ultracentrifugation. This protein, which showed an isoelectric point of pH 7.02, probably consists of two subunits of equal molecular weight which are held together or interact with each other noncovalently. The biological activity survives treatment by a number of proteolytic enzymes, such as trypsin [EC 3.4.21.4], chymotrypsin [EC 3.4.21.1], and pepsin [EC 3.4.23.1].     

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.