Complement-inhibiting acidic factors from the venom of Central Asian cobra Naja naja oxiana

Two anticomplementic factors isolated from the venom of the Central Asian cobra Naja naja oxiana by chromatography on DEAE-Sepharose CL-6B and subsequent gel filtration on Sephacryl S-200 were studied. Of these, five factors (CFA-Ia, CFA-Ib, CFA-Ic, CFA-IIa and CFA-IIb), CFA-Ib had been characterized earlier, while CFA-Ia was assigned to a previously identified H-CoF factor. It was shown that CFA-Ic has a molecular mass of 3900 Da; its content in the venom amounts to 2.6 mg/g of dry venom. This factor inhibits the classical pathway of C3 convertase formation abrogating the C2 component activation by subcomponent C1s [Ki = (2.5 +/- 0.8).10(-7) M]. CFA-IIa and CFA-IIb are present in the venom in very low amounts (2 mg/g) and have Mr of 5700 and 3200 Da, respectively. The complement-inhibiting action was studied for a more active CFA-IIa. Factor CFA-IIa was shown to inactivate the native component of C2 with a rate constant, k, of (2.7 +/- 0.2).10(3) s-1M-1 (37 degrees C, pH 7.4). CFA-IIa had no effect on C2 and C2a within their complexes with C4b.     

Major complement inhibiting factors from the venom of the Central Asian cobra Naja naja oxiana

The properties of two anticomplementic factors isolated by CM-Sepharose chromatography from the basic non-adsorbed on DEAE-Sepharose fraction of the Central Asian cobra Naja naja oxiana venom, were studied. Of these three factors (CFB-I, CFB-II and CFB-III) the latter had been characterized earlier. CFB-I was shown to be a protein with an N-terminal Asp and a molecular mass of about 39 kDa (data from gel chromatography); its content in the venom is 3.6 mg/g of dry venom. The protein inhibits mainly the classical pathway of the complement activation, being bound to component C4 (Ki = 9 nM). CFB-I seems to be analogous to the CI inhibitor from the venom of the Naja haje cobra. An analysis of the N-terminal sequence of CFB-II showed it to be identical to the earlier characterized cytotoxin I. CFB-I inhibits the formation of C3 convertase with Ki = 2.2-2.8 microM by way of binding to C4b and thus interfering with the component C2 sorption.     

L-Amino acid oxidase from Naja naja oxiana venom

A new l-amino acid oxidase (LAAO) was isolated from the Central Asian cobra Naja naja oxiana venom by size exclusion, ion exchange and hydrophobic chromatography. The N-terminal sequence and the internal peptide sequences share high similarity with other snake venom l-amino acid oxidases, especially with those isolated from elapid venoms. The enzyme is stable at low temperatures (− 20 °C, − 70 °C) and loses its activity by heating at 70 °C. Specific substrates for the isolated protein are l-phenylalanine, l-tryptophan, l-methionine and l-leucine. The enzyme has antibacterial activity inhibiting the growth of Gram-positive (Bacillus subtilis) and Gram-negative (Escherichia coli) bacteria. N. naja oxiana LAAO dose-dependently inhibited ADP- or collagen-induced platelet aggregation with IC₅₀ of 0.094 μM and 0.036 μM, respectively. The antibacterial and anti-aggregating activity was abolished by catalase.     

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.     

Proton-nuclear-magnetic-resonance study of the conformation of neurotoxin II from Middle-Asian cobra (Naja naja oxiana) venom.

A proton nuclear magnetic resonance (NMR) study at 100 and 300 MHz of neurotoxin II from the venom of Middle-Asian cobra Naja naja oxiana has been performed in 2H2O and H2O solutions. By means of chemical modification and double resonance all the aromatic residue resonances have been assigned. From the NMR titration curves, pK values of histidine 4 and histidine 31 residues have been determined. For one of the two neighbouring tryptophan residues pH dependence (in the 2-8-pH range) of the chemical shifts of indole protons has been revealed. According to the different sensitivity of the linewidth of indole NH resonances to pH in H2O solution, the accessibility of each of the tryptophan residues has been estimated. Temperature dependence has been observed for the linewidth of the aromatic resonances of the tyrosine 24 residue. Deuterium exchange rates have been measured for amide protons as well as for C(2)H histidine resonances. The NMR data obtained have allowed the conclusions to be made that the two histidine residues and one of the tryptophan residues should be localized on the surface of the protein globule, that arginine residues should be present in the environment of histidine 4, that histidine 31 and the buried tryptophan are possibly localized in close spatial proximity and that the side chain of tyrosine 24 is buried within the protein globule.     

Interaction of central Asian cobra (Naja naja oxiana Eichwald) venom cytotoxins with phospholipase D

Effect of cytotoxins from the venom of Naja naja oxiana Eichwald on the hydrolytic function of phospholipase D has been further analysed. Cytotoxins in the absence of Ca2+ activated the enzyme, whereas in its presence they inhibited it. Inhibition is shown to be related to the interaction of cytotoxins with the enzyme which blocks the absorption of the enzyme at the surface of the substrate phase. Synergism in the action of cytotoxin and phospholipase D was not noticed.     

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.     

A new low molecular-weight protein effector of human complement from the venom of the Central Asian cobra Naja naja oxiana

Six protein effectors of human complement were isolated from the whole venom of the Central Asian cobra Naja naja oxiana. Three of them have acidic properties and molecular weights of 61 000, 5000 and 3000, and the rest are basic proteins with molecular weights of 54 000, 9000 and 7000. Two low molecular weight basic proteins CFB-II and CFB-III are isolated in as high amounts as 115 and 85 mg per g of dry venom. All the effectors inhibit the classical pathway of complement activation and, with the exception of CFB-II and CFB-III, the alternative pathway. The latter, on the contrary, enhances the alternative pathway of activation. N-Terminal sequence determination for CFB-III demonstrated its identity to the earlier characterized cytotoxin II. The action of CFB-III on the classical pathway of complement activation consists in the component C4 inactivation. A mechanism for the CFB-III activation of the alternative pathway is proposed implying the CFB-III induced transformation of the C3 component into a C3b-like one producing a soluble C3 convertase.     

A factor from the venom of the Central Asiatic cobra Naja naja oxiana, which inactivates component C4 of human complement

An acid glycoprotein (mol. m. 60 kDa) containing 6 sialic acid residues and N-terminal Thr was isolated from the venom of the central asian cobra Naja naja oxiana. The protein has an anticomplementary activity selectively inactivating of the C4 component of the human complement. This factor (CFA-Ib) binds C4 with Ki = 0.27 +/- 0.13 microM and then irreversible inactivates it with a rate constant k = 0.75 +/- 0.25 min-1. Membrane bound C4b restores its ability of CFA-Ib binding. This binding hinders component C2 sorption on C4b and C3 convertase formation.     

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.     

Crystallization and preliminary x-ray diffraction study of neurotoxin-I from Naja naja oxiana venom

Crystals of the neurotoxin-I (NTX-I) from the venom of the middle Asian cobra Naja naja oxiana have been grown by vapour diffusion and dialysis methods. The crystals belong to space group P2(1)2(1)2 with dimension of a = 25.19 A, b = 75.59 A, c = 36.09 A and diffract to 1.9 A resolution. The asymmetric unit contains one molecule (Vm = 2.2 A/Da). Using the molecule of alpha-cobratoxin (CTX) as a starting model for NTX-I structure determination coordinates of C alpha atoms of the NTX-I molecule were obtained and the position of NTX-I in the unit cell was derived.     

Evaluating the Importance of Environmental Variables on Spatial Distribution of Caspian cobra Naja oxiana(Eichwald, 1831) in Iran

Over recent years, the population of Caspian cobra Naja oxiana has declined in its distribution range in Iran due to habitat destruction and overhunting. Consequently, their small and isolated populations in fragmented landscapes are facing genetic and demographic threats. Evaluating the spatial distribution pattern of Naja oxiana, identifying core habitat patches and improving landscape connectivity among the patches have a significant role in the long-term survival of the species. This study predicts the spatial distribution map of the Caspian cobra considering the factors affecting the predictive power of the distribution models, including sampling bias in presence points, correct selection of background locations, and input model parameters. The sampling bias in presence points was removed using spatial filtering. Several models were run using 19 environmental variables that eventually led to the selection of the effective habitat variables and best MaxEnt distribution model. We also used an ensemble model(EM) of habitat suitability methods to predict the potential habitats of the species. Topographical roughness, shrublands, average annual precipitation, and sparse rangeland with a density of ≤ 20% had the most effect on the spatial distribution of Caspian cobra. The evaluation of models confirmed that the EM has more predictive performance than MaxEnt in predicting the distribution of Naja oxiana.     

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.     

Cytoarchitectonic pattern of the hypothalamus in the cobra,Naja naja

Summary The distribution and cytoarchitectonic pattern of the magno- and parvocellular hypothalamic nuclei of the cobra, Naja naja, are described at the light-microscopic level. With respect to their tinctorial affinity to paraldehyde fuchsin (AF) as a representative of the Gomori-type of stains, the magnocellular neurons belong to the AF-positive and the parvocellular neurons to the AF-negative elements. In addition to the supraoptic and paraventricular nuclei proper, two accessory aggregations of magnocellular neurons, the nucleus retrochiasmaticus and nucleus circularis, can be identified. Although in a peculiar location, they may be regarded as subunits of the supraopticoparaventricular neurosecretory complex. As many as 22 AF-negative nuclear areas are identified in the hypothalamus of the cobra. The nucleus periventricularis hypothalami of earlier authors is subdivided into several circumscribed neuronal complexes. The nucleus arcuatus, nucleus hypothalamicus lateralis and nucleus lateralis recessus infundibuli are well developed. An attempt is made to interpret the significance of these nuclei on a comparative and phylogenetic basis.On leave from the Department of Zoology, Nagpur University, Nagpur, India     

Spatial structure of the basic chain of the neurotoxin I molecule from the venom of cobra Naja naja oxiana and its crystal packing

The structure of the alpha-carbon chain was solved by molecular replacement method at 2.7 A resolution. Neurotoxin I (NTX-I) is one of the main protein components purified from the venom of the central asian cobra Naja naja oxiana. NTX-I is known to bind specifically to acetylcholine receptors thus preventing the transmission of the neuroconductivity signal from synapses to muscles. NTX-I crystals were grown either by vapour diffusion or dialysis methods using specially prepared microdialysis cell. The intensities of reflections from native NTX-I crystals were measured in the range of 38.0-2.1 A-1 by omega-scan method with a Syntex P21 diffractometer operated in automatic regime. To determine the position and mode of packing of NTX-I molecule in unit cell program packages MERLOT and BLANC were applied running on a NORD-500 computer.     

Specificity of synthetic polyribonucleotides hydrolysis by endoribonuclease from cobra (Naja oxiana) venom

An analysis of kinetic differences in homopolyribonucleotides hydrolysis by cobra venom endoribonuclease was carried out. It was concluded that the rate and intensity of hydrolysis as well as the length of the linear parts of the kinetic curves are correlated with the content of the nucleotide units with the C3'-endo conformation in the substrates. The structure factor was shown to predominate in some cases over the temperature factor. Protamine sulfate inhibits the enzyme by blocking its phosphodiether bonds. Study on the effects of divalent metal ions demonstrated the possibility that the enzyme-Me2+ complex is functionally active and that the ion-free polyribonucleotides are true substrates.     

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.