Purification and Characterization of Nk-3FTx: A Three Finger Toxin from the Venom of North East Indian Monocled Cobra.

Snake venom three finger toxins (3FTxs) are a non-enzymatic family of venom proteins abundantly found in elapids. We have purified a 7579.5 ± 0.591 Da 3FTx named as Nk-3FTx from the venom of Naja kaouthia of North East India origin. The primary structure was determined by a combination of N-terminal sequencing and electrospray ionization liquid chromatography-mass spectrometry/mass spectrometry. Biochemical and biological characterization reveal that it is nontoxic to human cell lines and exhibit mild anticoagulant activity when tested on citrated human plasma. Nk-3FTx was found to affect the compound action potential (CAP) and nerve conduction velocity of isolated toad sciatic nerve. This is the first report of a non-conventional 3FTx from Naja kaouthia venom that reduces CAP for its neurotoxic effect. Further studies can be carried out to understand the mechanism of action and to explore its potential therapeutic application.

Snake venomics of monocled cobra (Naja kaouthia) and investigation of human IgG response against venom toxins.

The venom proteome of the monocled cobra, Naja kaouthia, from Thailand, was characterized by RP-HPLC, SDS-PAGE, and MALDI-TOF-TOF analyses, yielding 38 different proteins that were either identified or assigned to families. Estimation of relative protein abundances revealed that venom is dominated by three-finger toxins (77.5%; including 24.3% cytotoxins and 53.2% neurotoxins) and phospholipases A2 (13.5%). It also contains lower proportions of components belonging to nerve growth factor, ohanin/vespryn, cysteine-rich secretory protein, C-type lectin/lectin-like, nucleotidase, phosphodiesterase, metalloproteinase, l-amino acid oxidase, cobra venom factor, and cytidyltransferase protein families. Small amounts of three nucleosides were also evidenced: adenosine, guanosine, and inosine. The most relevant lethal components, categorized by means of a 'toxicity score', were α-neurotoxins, followed by cytotoxins/cardiotoxins. IgGs isolated from a person who had repeatedly self-immunized with a variety of snake venoms were immunoprofiled by ELISA against all venom fractions. Stronger responses against larger toxins, but lower against the most critical α-neurotoxins were obtained. As expected, no neutralization potential against N. kaouthia venom was therefore detected. Combined, our results display a high level of venom complexity, unveil the most relevant toxins to be neutralized, and provide prospects of discovering human IgGs with toxin neutralizing abilities through use of phage display screening.

Anti-inflammatory effects of Neurotoxin-Nna, a peptide separated from the venom of Naja naja atra.

BACKGROUND: Neurotoxin-Nna (NT), an analgesic peptide separated from the venom of Naja naja atra, has reported to have an exceptional specificity to block transmission of the nerve impulse by binding to the α- subunit of the nicotinic acetylcholine receptor in the membrane. However, little information is available on the anti-inflammatory effects of NT. Therefore, the anti-inflammatory activity of Neurotoxin-Nna was investigated in this study. METHODS: The anti-inflammatory effects of NT were evaluated by measuring its influence on several crucial factors in inflammatory pathways, including total antioxidant activity, antinociceptive effects in vivo, nuclear factor kappa B (NF-κB), polymorphonuclear cells (PMN), inducible nitric oxide synthase (iNOS), adhesion molecule (ICAM-1) and tactile hyperalgesia. RESULTS: NT treatment decreased the levels of tumor necrosis factor alpha (TNF-α) and interleukin 1 beta (IL-1ß). NT treatment decreased the total antioxidant status (TAOS) and reduced CFA-induced tactile hyperalgesia in a dose-dependent manner. NT significantly inhibited regulation of NF-kappaB activation and the production of IL-1ß, TNF-α, iNOS and CAM-1. Moreover, NT suppressed infiltration of PMN. CONCLUSIONS: Our results showed that NT reduced CFA-induced tactile hyperalgesia through inhibition inflammatory pathways in experimental inflammatory rats.

Identification of α-cobratoxin in equine plasma by LC-MS/MS for doping control.

Cobra venom (Naja kaouthia) contains a toxin called α-cobratoxin (α-Cbtx). This toxin is a natural protein containing 71 amino acids (MW 7821 Da) with a reported analgesic potency greater than morphine. In 2007, in USA, this substance was found in the barns of a thoroughbred trainer and since then till date, the lack of a detection of this molecule has remained a recurring problem for the horseracing industry worldwide. To solve this problem, the first method for the detection of α-cobratoxin in equine plasma has now been developed. Plasma sample (3 mL) was treated with ammonium sulfate at the isoelectric point of α-Cbtx, and the pellet was dissolved in a phosphate buffer and mixed with methanol for precipitation. The supernatant was then concentrated prior to its extraction on WCX SPE cartridges. The eluate was concentrated with two consecutive filtration steps before the trypsin digestion. The samples were analyzed using a LC-MS/MS Q Exactive instrument at 70,000 resolution on the product ions of the doubly charged precursor of the target peptide ((24)TWCDAFCSIR(33)). The method was validated (n = 18) at 5 µg/L (640 pmol/L) according to the Association of Official Racing Chemists (AORC) requirements. The lower limit of detection was 1 µg/L (130 pmol/L). The present method has made it possible for us to confirm the presence of α-Cbtx in a horse plasma sample 24 h post the administration of α-Cbtx. Thus, the present method provides the first sensitive, specific, and reliable analytical method to confirm the presence of α-Cbtx in equine plasma.

Improved method for the isolation, characterization and examination of neuromuscular and toxic properties of selected polypeptide fractions from the crude venom of the Taiwan cobra Naja naja atra.

An improved chromatographic method was developed to isolate and purify polypeptides and proteins from the crude venom of the Taiwan cobra Naja naja atra. The procedure devised is simple, easy to reproduce, and enables large scale isolation of almost all polypeptides and proteins in this cobra venom. Six pure polypeptide fractions of the venom were isolated and characterized using gel filtration on Sephadex G50 (medium), ion exchange chromatography on SP-Sephadex C25, desalting on Sephadex G25 (fine) and preparative HPLC on a RPC 18 column. The neuromuscular activity of these fractions was tested on the chick biventer cervicis nerve-muscle preparation and their toxicity (LD(50)) was determined after i.v. administration in mice. Their antinociceptive activity was tested in the mouse abdominal test by i.v. application. Two of these polypeptide samples had major physiological effects: one acted as a cardiotoxin causing reversible myocardial contractures with no effect on muscle twitches elicited by nerve stimulation (NS); another was a neurotoxin that blocked muscle contractions in response to NS and exogenously added acetylcholine. The cardiotoxic fraction was identified as CTX I, a well-known cardiotoxin present in this venom, and the neurotoxin was identified as neurotoxin-α with an LD50 in mice of 0.075 mg/kg.

Chromatography, mass spectrometry, and molecular modeling studies on ammodytoxins.

The ammodytoxins (Atxs) are neurotoxic phospholipases which occur in Vipera ammodytes ammodytes (Vaa) snake venom. There are three Atx isoforms, A, B, and C, which differ in only five amino acid positions at the C-terminus but differ substantially in their toxicity. The objective of this study was to establish an analytical method for unambiguous identification of all three isoforms and to use the method to assess a procedure for purification of the most toxic phospholipase, AtxA, from the venom. Isolation procedure for AtxA consisted of isolation of Atx-cross-reactive material (proteins recognized by anti-Atx antibodies), by use of an affinity column, then cation exchange on CIM (Convective Interaction Media) disks. The purification procedure was monitored by means of reversed-phase chromatography (RPC) and mass spectrometry (MS). Although previous cation exchange of the pure isoforms enabled separate elution of AtxA from B and C, separation of AtxA from Atxs mixture was not accomplished. RPC was not able to separate the Atx isoforms, whereas an MS based approach proved to be more powerful. Peptides resulting from tryptic digestion of Atxs which enable differentiation between the three isoforms were successfully detected and their sequences were confirmed by post-source decay (PSD) fragmentation. Separation of Atx isoforms by ion-exchange chromatography is most presumably prevented by Atxs heterodimer formation. The tendency of Atxs to form homodimers and heterodimers of similar stability was confirmed by molecular modeling.

Purification and characterization of a novel antinociceptive toxin from Cobra venom (Naja naja atra).

Snake venoms have demonstrated antinociceptive activity, and certain isolated neurotoxins have demonstrated significant analgesia in animal models. Here we report a novel analgesic toxin which was isolated from Naja naja atra and was given the name 'najanalgesin'. The LD(50) of the crude venom and najanalgesin were 0.89mg/kg and 2.69mg/kg, respectively. We used the writhing test and hot plate test to evaluate the antinociceptive properties of the crude venom and najanalgesin after intraperitoneal (ip) administration. The analgesic mechanism of najanalgesin was also studied. The response latency time was significantly prolonged in the hot plate test after ip administration of the crude venom of Naja naja atra (0.111-0.445mg/kg) in a dose-dependent manner. Najanalgesin (1mg/kg) elicited almost the same antinociceptive effect as that of the crude venom of Naja naja atra at the dose of 0.445mg/kg and remained for 6h after intraperitoneal injection, shown by hot plate test. The percentage of increase in the latency time for the venom and the najanalgesin 3h after drug administration was 96.2% and 112%, respectively. The number of writhes decreased to almost 1/3, 1/6, and 1/12 of the NS (physiological saline) group after intraperitoneal administration of najanalgesin at 0.25, 0.5, and 1.0mg/kg, respectively. Pretreatment with atropine (1mg/kg) or naloxone (3mg/kg) blocked the antinociception of najanalgesin in the hot plate test. Based on the sequence information, najanalgesin is found to be highly homologous with the conventional CTXs (cardiotoxins). To our knowledge, no study had previously reported that a toxin which was homologous with CTXs possessed the antinociceptive activity. Thus, this is the first report that the antinociceptive effect induced by najanalgesin is mediated by cholinergic and opioidergic mechanisms.

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.

Naturally occurring disulfide-bound dimers of three-fingered toxins: a paradigm for biological activity diversification.

Disulfide-bound dimers of three-fingered toxins have been discovered in the Naja kaouthia cobra venom; that is, the homodimer of alpha-cobratoxin (a long-chain alpha-neurotoxin) and heterodimers formed by alpha-cobratoxin with different cytotoxins. According to circular dichroism measurements, toxins in dimers retain in general their three-fingered folding. The functionally important disulfide 26-30 in polypeptide loop II of alpha-cobratoxin moiety remains intact in both types of dimers. Biological activity studies showed that cytotoxins within dimers completely lose their cytotoxicity. However, the dimers retain most of the alpha-cobratoxin capacity to compete with alpha-bungarotoxin for binding to Torpedo and alpha7 nicotinic acetylcholine receptors (nAChRs) as well as to Lymnea stagnalis acetylcholine-binding protein. Electrophysiological experiments on neuronal nAChRs expressed in Xenopus oocytes have shown that alpha-cobratoxin dimer not only interacts with alpha7 nAChR but, in contrast to alpha-cobratoxin monomer, also blocks alpha3beta2 nAChR. In the latter activity it resembles kappa-bungarotoxin, a dimer with no disulfides between monomers. These results demonstrate that dimerization is essential for the interaction of three-fingered neurotoxins with heteromeric alpha3beta2 nAChRs.

Behavioural effects in mice and intoxication symptomatology of weak neurotoxin from cobra Naja kaouthia.

Weak neurotoxins belong to the superfamily of three-finger toxins from snake venoms. In general, weak toxins have a low toxicity and, contrary to other three-finger toxins, their molecular targets are not well characterized: in vitro tests indicate that these may be nicotinic acetylcholine receptors. Here, we report the influence of intraperitoneal and intravenous injections of weak neurotoxin from Naja kaouthia venom on mouse behaviour. Dose-dependent suppression of orientation-exploration and locomotion activities as well as relatively weak neurotropic effects of weak neurotoxin were observed. The myorelaxation effect suggests a weak antagonistic activity against muscle-type nicotinic acetylcholine receptors. Neurotoxic effects of weak neurotoxin were related to its influence on peripheral nervous system. The symptomatology of the intoxication was shown to resemble that of muscarinic agonists. Our data suggest that, in addition to interaction with nicotinic acetylcholine receptors observed earlier in vitro, weak neurotoxin interacts in vivo with some other molecular targets. The results of behavioural experiments are in accord with the pharmacological profile of weak neurotoxin effects on haemodynamics in mice and rat indicating the involvement of both nicotinic and muscarinic acetylcholine receptors.

Purification of a post-synaptic neurotoxic phospholipase A2 from Naja naja venom and its inhibition by a glycoprotein from Withania somnifera.

A post-synaptic neurotoxic phospholipase A(2) (PLA(2)) has been purified from Indian cobra Naja naja venom. It was associated with a peptide in the venom. The association was disrupted using 8 M urea. It is denoted to be a basic protein by its behavior on both ion exchange chromatography and electrophoresis. It is toxic to mice, LD(50) 1.9 mg/kg body weight (ip). It is proved to be post-synaptic PLA(2) by chymographic experiment using frog nerve-muscle preparation. A glycoprotein, (WSG) was isolated from a folk medicinal plant Withania somnifera. The WSG inhibited the phospholipase A(2) activity of NN-XIa-PLA(2,) isolated from the cobra venom, completely at a mole-to-mole ratio of 1:2 (NN-XIa-PLA(2): WSG) but failed to neutralize the toxicity of the molecule. However, it reduced the toxicity as well as prolonged the death time of the experimental mice approximately 10 times when compared to venom alone. The WSG also inhibited several other PLA(2) isoforms from the venom to varying extent. The interaction of the WSG with the PLA(2) is confirmed by fluorescence quenching and gel-permeation chromatography. Chemical modification of the active histidine residue of PLA(2) using p-brophenacyl bromide resulted in the loss of both catalytic activity as well as neurotoxicity of the molecule. These findings suggest that the venom PLA(2) has multiple sites on it; perhaps some of them are overlapping. Application of the plant extract on snakebite wound confirms the medicinal value associated with the plant.

Cloning, overexpression, and characterization of cobrotoxin.

Cobrotoxin (CBTX) is a highly toxic short neurotoxin, isolated from the Taiwan cobra (Naja naja atra) venom. In the present study for the first time we report the cloning and expression of CBTX in high yields (12mg/L) in Escherichia coli. CBTX fused to the IgG-binding domain of protein A (IgG-CBTX) was expressed in the soluble form. The misfolded CBTX portion (of the overexpressed fusion protein) was refolded under optimal redox conditions. The fusion protein (IgG-CBTX) was observed to undergo auto-catalytic cleavage to yield CBTX with additional 5 amino acids upstream of its N-terminal end. The far UV and near UV circular dichroism spectra of the recombinant CBTX were identical to those of the toxin isolated from the crude venom source. Recombinant CBTX was isotope labeled (15N and 13C) and all the resonances ('H, 13C, and 15N) in the protein have been unambiguously assigned. ' H '5N HSQC spectrum of recombinant CBTX revealed that the protein is in a biologically active conformation. 1H-15Nchemical shift perturbation data showed that recombinant CBTX binds to a peptide derived from the alpha7 subunit of the Torpedo acetylcholine receptor (AchR) with high affinity. The AchR peptide is found to bind to residues located at the tip of Loop-2 in CBTX. The results of the present study provide an avenue to understand the structural basis for the high toxicity exhibited by CBTX. In addition, complete resonance assignments in CBTX (reported in this study) are expected to trigger intensive research towards the design of new pharmacological agents against certain neural disorders.

Purification and characterization of a neurotoxin from the venom of Ophiophagus hannah (king cobra).

A neurotoxin, Oh9-1, from the venom of Ophiophagus hannah was isolated by a combination of ion-exchange chromatography and reverse phase HPLC. Amino acid sequence analysis revealed that Oh9-1 consists of 57 amino acids and eight cysteine residues. This protein was mainly constituted with beta-sheet as evidenced by CD spectrum. Oh9-1 inhibited carbachol-induced muscle contraction in an irreversible manner and the dose for achieving 50% inhibition was approximately fourfold that of alpha-bungarotoxin. Since the residues in alpha-neurotoxins closely involve in the binding with acetylcholine receptors are not highly conserved in this toxin molecule, Oh9-1 represents a novel type of neurotoxin structurally distinct from alpha-neurotoxins.

A novel short neurotoxin, cobrotoxin c, from monocellate cobra (Naja kaouthia) venom: isolation and purification, primary and secondary structure determination, and tertiary structure modeling.

A novel short neurotoxin, cobrotoxin c (CBT C) was isolated from the venom of monocellate cobra (Naja kaouthia) using a combination of ion-exchange chromatography and FPLC. Its primary structure was determined by Edman degradation. CBT C is composed of 61 amino acid residues. It differs from cobrotoxin b (CBT B) by only two amino acid substitutions, Thr/Ala11 and Arg/Thr56, which are not located on the functionally important regions by sequence similarity. However, the LD50 is 0.08 mg/g to mice, i.e. approximately five-fold higher than for CBT B. Strikingly, a structure-function relationship analysis suggests the existence of a functionally important domain on the outside of Loop III of CBT C. The functionally important basic residues on the outside of Loop III might have a pairwise interaction with alpha subunit, instead of gamma or delta subunits of the nicotinic acetylcholine receptor (nAChR).

Cobra venom cytotoxin free of phospholipase A2 and its effect on model membranes and T leukemia cells.

Membrane-active toxins from snake venom have been used previously to study protein-lipid interactions and to probe the physical and biochemical states of biomembranes. To extend these studies, we have isolated from Naja naja kaowthia (cobra) venom a cytotoxin free of detectable phospholipase A2 (PLA2). The amino acid composition, pI (10.2), and net charge of the cytotoxin compares well with membrane-active toxins isolated from venoms of other cobras. The cytotoxin, shown by a spin label method, associates with PLA2 in buffers at pH values between 7.0 and 5.0, but not at pH 4.0. It is suggested that cytotoxin and PLA2 (pI close to 4.8) associate electrostatically in the native venom. The effect of the cytotoxin on model phospholipid membranes was studied by EPR of spin probes in oriented lipid multilayers and 1H-NMR of sonicated liposomes. The cytotoxin did not significantly affect the packing of lipids in pure phosphatidylcholine (PC) membranes and in PC membranes containing 10 mol% phosphatidic acid (PA) or cardiolipin (CL). However, the cytotoxin induced an increase in membrane permeability and formation of nonbilayer structures in PC membranes containing 40 mol% of PA or CL. The purified cytotoxin was cytocidal to Jurkat cells, but had little effect on normal human lymphocytes. However, both Jurkat cells and normal lymphocytes were killed equivalently when treated with 10(-9) m PLA2 and 10(-5) m cytotoxin in combination. From its effect on model membranes and Jurkat cells, it is suggested that purified cytotoxin preferentially targets and disrupts membranes that are rich in acidic phospholipids on the extracellular side of the plasma membrane.

A novel neurotoxin, cobrotoxin b, from Naja naja atra (Taiwan cobra) venom: purification, characterization, and gene organization.

A novel neurotoxin, cobrotoxin b, was isolated from Naja naja atra (Taiwan cobra) venom by successive chromatographies on gel filtration and SP-Sephadex C-25 columns. The yield of this novel toxin was 5% of that of cobrotoxin from the same venom. Its neurotoxicity determined as the inhibition of acetylcholine-induced muscle contractions was approximately 50% of that of cobrotoxin. Cobrotoxin b consists of 61 amino acid residues including 8 cysteine residues. Moreover, there are 12 amino acid substitutions between cobrotoxin b and cobrotoxin. The genomic DNA, with a size of 2,386bp, encoding the precursor of cobrotoxin b was isolated from the liver of N. naja atra. The gene consists of three exons separated by two introns. This exon/intron structure is essentially the same as that reported for the cobrotoxin gene. Moreover, the nucleotide sequences of the two neurotoxin genes exhibit 92% identity. These results highly suggest that the cobrotoxin b and cobrotoxin genes are derived from a common ancestor. Comparative analyses of cobrotoxin b and cobrotoxin precursors showed that the protein-coding regions of the exons are more diverse than introns, except for in the signal peptide domain. This indicates that the protein-coding regions may have arised via accelerated evolution. BLAST searches for sequence similarity in the GeneBank databases showed that intron 1 of the cobrotoxin b and cobrotoxin genes encodes a small nucleolar RNA (snoRNA). However, the snoRNA gene is absent from the gene encoding the Laticauda semifasciata erabutoxin c precursor (L. semifasciata and N. naja atra are sea and land snakes, respectively). Since previous studies suggested the potential mobility of snoRNA genes during evolution, we propose that intron insertions or deletions of snoRNA genes occurred with the evolutionary divergence between the sea snake and land snake neurotoxins.

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.

Sequence comparison and computer modelling of cardiotoxins and cobrotoxin isolated from Taiwan cobra.

Six cardiotoxins and one neurotoxin isolated and purified from the Taiwan cobra venom (Naja naja atra) possess distinct pharmacological and biochemical properties despite the existence of a grossly similar tertiary structure among these toxins, i. e., a core consisting of a series of short loops and four disulfide bridges. A systematic structure comparison of these major toxin isoforms was made by the secondary-structure predictions together with computer model-building based on the primary sequences and the established X-ray and NMR structures of one published cardiotoxin isoform and cobrotoxin. It is of interest to find that some defined and subtle differences can be detected upon the superposition of these three-dimensional polypeptide chains, which may reflect the intrinsic differences in the surface hydrophobicity of cardiotoxins and cobrotoxin as revealed by hydropathy profiles of these toxins in one of three major loops. The differences seem to correlate with different inhibitory activities exhibited by cardiotoxins in contrast to the lack of activity by cobrotoxin on protein kinase C (PKC).

Improvement in the binding of cobrotoxin to microtiter plates by glutaraldehyde at neutral pH.

The coating of the wells of microtiter plates with cobrotoxin in Tris (pH 9.8) or PBS (pH 7.2) buffer was assessed by enzyme-linked immunoassay (ELISA). It was found that the poor binding in neutral buffer was improved by adding glutaraldehyde (GA), and the bound amount reached the same extent as that measured with alkali buffer. The optimal concentration of GA was approx. 0.02%. A decrease in optical density was observed with GA concentration higher than 0.02%. This may result from the modification of cobrotoxin by GA, which induced a decrease in the antigenicity of the cobrotoxin as revealed by competitive immunoassay. This study implies that the coating of physiological samples with GA for ELISA may be carried out with physiological buffers without the need to change the buffer to one of alkaline pH.

Structural analysis and comparison of cobrotoxin and cardiotoxins by near-IR Fourier transform Raman spectroscopy.

Venom toxins were isolated from Formosan cobra (Naja naja atra) by cation-exchange chromatography. The near-IR FT-Raman analytical method has been applied to the characterization and classification of the toxin components in their lyophilized forms. Structural analysis and comparison of various purified toxin fractions were made with respect to their amino acid compositions and near-IR Fourier-transform Raman spectra. The results indicate that the major secondary structure of cobra toxins including cobrotoxin and various cardiotoxins is mainly anti-parallel beta-pleated sheet as judged by the Raman signals at 1238 cm-1 (amide III) and 1671 cm-1 (amide I). It is also found that the relative Raman signal intensities of Tyr, Phe, Trp and Met residues in purified toxins correlate very well with the structural data obtained from amino acid analysis. The advantage and improvement of applying the near-IR FT-Raman spectroscopy to the unambiguous classification and comparison of venom toxins are evident and the discrepancies with previous Raman studies on these venom toxins are also revealed and discussed.