Snake venom toxins. The amino-acid sequences of three toxins (CM-8, CM-11 and CM-13a) from Naja haje annulifera (Egyptian cobra) venom.

Three toxins (CM-8, CM-11, and CM-13a) were purified from the venom of Naja haje annulifera by gel filtration on Sephadex G-50 and by ion-exchange chromatography on CM-cellulose. Whereas toxin CM-8 and CM-11 comprise 60 amino acid residues, toxin CM-13a contains 61 residues. All three toxins are cross-linked by four intrachain disulphide bridges. The complete amino acid sequences of these toxins have been elucidated. The reduced and S-carboxymethylated toxins were digested with trypsin and chymotrypsin and the peptides purified by ion-exchange chromatography, gel filtration and chromatography or electrophoresis on paper. The Edman procedure, either through the use of the automatic sequencer or by manual manipulation, was employed to obtain the sequence of the intact toxins and the pure peptides. The chymotryptic digests provided the necessary overlapping peptides which allowed the alignment of the tryptic peptides. The properties of the three toxins were compared with those of the cytotoxin group. The toxicities the serological properties, the sequences and the invariant amino acid residues of toxin CM-8 and CM-11 resemble the corresponding properties of the cytotoxin group. The sequence and serological properties of toxin CM-13a show that it is related to the cytotoxin group, but its toxicity is much lower than those encountered in the cytotoxin group.     

Snake venom toxins. The amino acid sequence of three toxins (CM-2e, CM-4a and CM-) from Naja haje annulifera (Egyptian cobra) venom.

Three toxins (CM-2e, CM-4a and CM-7) were purified from the venom of Naja haje annulifera by gel filtration on Sephadex and by ion-exchange chromatography on CM-cellulose. They comprise 60 amino acid residues and are cross-linked by four intrachain disulphide bridges. The complete amino acid sequences of the three toxins have been elucidated. The toxicities, the serological properties, the sequences and the invariant amino acid residues of toxin CM-2e, CM-4a and CM-7 resemble the corresponding properties of the cytotoxin group.     

The amino acid sequence of toxin V II 2, a cytotoxin homologue from banded Egyptian cobra (Naja haje annulifera) venom.

Toxin V II 2 comprises 60 amino acid residues and is cross-linked by four disulphide bridges. The complete amino acid sequence of this toxin was elucidated. The reduced and S-carboxymethylated toxin was digested with trypsin and chymotrypsin and the peptides were purified by ion-exchange chromatography and chromatography or electrophoresis on paper. The Edman procedure, either through the use of the automatic sequenator or by manual manipulation, was employed to obtain the sequence of the intact toxin and the pure peptides. The chymotryptic digest provided the necessary overlapping peptides which allowed the alignment of tryptic peptides. The amino acid sequence of Naja haje annulifera toxin V II 2 shows a high degree of homology with cytotoxin V II 1 of the same venom.     

The complete amino acid sequence of a cardiotoxin from the venom of Naja naja (Cambodian Cobra).

The complete amino acid sequence of a small, basic protein with cardiotoxic activity is described. This toxin, designated Naja naja F8, was isolated from the venom of Naja naja, of Cambodian origin, by gel filtration on Sephadex G-75 followed by gradient ion exchange chromatography on Bio-Rex 70. The cardiotoxin F8, molecular weight 6727 from amino acid composition, consists of 60 amino acids in a single peptide chain cross-linked by four disulfide bridges and is devoid of histidine, tryptophan, and glutamic acid. The chymotryptic and tryptic peptides from the performic acid oxidized toxin were separated by gel filtration on Sephadex G-25 and zone electrophoresis in columns of cellulose powder. The sequence was established by Edman degradation, using the direct phenylthiohydantoin method, and with the aid of carboxypetidase A, and is similar to the consequences reported for other cardiotoxins, cytotoxins, and/or lytic factors from cobra venoms, all of which show considerable homology with the functionally distinct neurotoxins.     

Amino-acid sequence of toxin III of Naja haje.

The complete amino acid sequence of toxin III of Naja haje (72 residues) has been established mainly by use of a protein sequenator (identification of 70 residues). The two C-terminal residues have been determined by digestion with carboxypeptidases A and B. Addition of succinylated protein or peptide greatly improved the performance of the sequenator for the Edman degradation of peptides: on one peptide (39 residues) degradation went to step 34 with a protein program and on two peptides (10 and 13 residues) degradation reached the last amino acid with a peptide program (use of dimethylbenzylamine). Amino acid analysis of tryptic peptides obtained by digestion of the C-terminal cyanogen bromide peptide are in full agreement with the sequence established by automatic degradation. The sequence of toxin III of Naja haje is unique and is very similar to that of Naja nivea alpha (although there are 9 differences), of Naja melanoleuca b (11 differences) and also to that of Naja naja A (18 differences).     

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 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.     

Structure and conformational heterogeneity of the weak toxin from the cobra Naja kaouthia venom

Resonances in the two-dimensional 1H NMR spectra of a weak toxin (WTX) from the venom of cobra Naja kaouthia for all 65 amino acid residues were assigned. The amino acid sequence of WTX, determined by the sequentional assignment of spin systems, was found to be similar to that of the CM-9a toxin from the N. kaouthia venom. Unlike CM-9a, WTX contains an additional Trp36 residue; Lys50 and Tyr52 are interchanged; and there is a Thr residue in place of Arg2. For some residues of WTX, the presence of two components of approximately equal intensities in the spectra was shown, which is explained by the conformational heterogeneity of the polypeptide owing to the cis-trans isomerization of the peptide bond Arg32-Pro33. The data (contacts of the nuclear Overhauser effect, constants of spin-spin coupling of protons, and rates of exchange of amide protons by deuterium of the solvent) made it possible to determine the secondary structure of two forms of WTX, which is characterized by the presence of two antiparallel beta-sheets, one of which consists of two strands (regions 1-5 and 13-17) and the other, of three strands (regions 23-28, 38-43, and 55-59).     

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.     

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.     

The amino acid sequence of protein CM-3 from Dendroaspis polylepis polylepis (black mamba) venom

Protein CM-3 from Dendroaspis polylepis polylepis venom was purified by gel filtration and ion exchange chromatography. It comprises 65 amino acids including eight half-cystines. The complete amino acid sequence of protein CM-3 has been elucidated. The sequence (residues 1-50) resembles that of the N-terminal sequence of the subunits of a synergistic type protein and residues 51-65 that of the C-terminal sequence of an angusticeps type protein. Mixtures of protein CM-3 and angusticeps type proteins showed no apparent synergistic effect, in that their toxicity in combination was no greater than the sum of their individual toxicities.     

Purification, characterization, and partial amino acid sequence of nerve growth factor from cobra venom.

The nerve growth factor (NGF) from Naja naja (cobra) venom has been purified and its structure compared to the NGF from mouse submaxillary gland. A two-step purification procedure has been devised, consisting of a gel filtration step in 1 M acetic acid followed by chromatography of the active pool on carboxymethylcellulose at pH 5. The molecular weight of the native protein was found to be 28000, and this value was reduced by approximately one-half under denaturing conditions. These values are comparable to those obtained for mouse 2.5S or betaNGF. Tryptic peptide maps of S-[14C]carboxymethyl NGF gave the number of labeled peptides expected for a structure composed of two identical or very similar subunits. Thus, the quaternary structures of mouse and cobra NGF are the same. Cyanogen bromide (CNBr) treatment of Naja naja NGF produced three fragments, of which two were purified to homogeneity. These fragments and the whole protein were analyzed in the automated protein Sequencer. The amino-terminal CNBr fragment of the protein was also subjected to digestion by thermolysin and the resultant peptides were purified and characterized. These data plus those from the characterization of the tryptic peptides provided the basis of the construction of a tentative primary structure of Naja naja NGF which is approximately 60% identical with mouse NGF.     

Structural analysis of phospholipase A2 by near-IR Fourier transform Raman spectroscopy.

Venom toxins were isolated from Formosan cobra (Naja naja atra) by cation-exchange chromatography. Most toxin components could be obtained in relatively pure forms by single-step ion-exchange chromatography whereas an extra step of gel permeation was needed for the separation of phospholipase A2 (PLA2) from the major neurotoxic component, i.e. cobrotoxin. The newer near-IR FT-Raman analytical method has been applied to the characterization of PLA2 in their lyophilized forms. Structural analysis of PLA2 and correlation of Raman spectroscopic data with amino acid compositions were made. The results indicate that phospholipase A2 showed the Raman peak at 1659 cm-1 which is characteristic of the alpha-helical structure in this enzyme. 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 application of near-IR FT-Raman techniques in the detection of the microenvironments of the aromatic amino acids such as Tyr and Trp in the native toxins may prove useful in the investigation of the functional properties of various venom toxins.     

Structure and Conformational Heterogeneity of a Weak Toxin from the Cobra Naja kaouthiaVenom

Resonances in the two-dimensional ¹H NMR spectra of a weak toxin (WTX) from the venom of cobra Naja kaouthiafor all 65 amino acid residues were assigned. The amino acid sequence of WTX, determined by the sequential assignment of spin systems, was found to be similar to that of the CM-9a toxin from the N. kaouthiavenom. Unlike CM-9a, WTX contains an additional Trp36 residue; Lys50 and Tyr52 are interchanged; and there is a Thr residue in place of Arg2. For some residues of WTX, the presence of two components of approximately equal intensities in the spectra was shown, which is explained by the conformational heterogeneity of the polypeptide owing to the cis–transisomerization of the peptide bond Arg32–Pro33. The data (contacts of the nuclear Overhauser effect, constants of spin–spin coupling of protons, and rates of exchange of amide protons for deuterium of the solvent) made it possible to determine the secondary structure of two forms of WTX, which is characterized by the presence of two antiparallel -sheets, one of which consists of two strands (regions 1–5 and 13–17) and the other, of three strands (regions 23–28, 38–43, and 55–59).     

Purification, characterization and primary structure of a chymotrypsin inhibitor from Naja atra venom

A chymotrypsin inhibitor, designated NA-CI, was isolated from the venom of the Chinese cobra Naja atra by three-step chromatography. It inhibited bovine alpha-chymotrypsin with a Ki of 25 nM. The molecular mass of NA-CI was determined to be 6403.8 Da by matrix-assisted laser-desorption ionization time-of-flight (MALDI-TOF) analysis. The complete amino acid sequence was determined after digestion of S-carboxymethylated inhibitor with Staphylococcus aureus V8 protease and porcine trypsin. NA-CI was a single polypeptide chain composed of 57 amino acid residues. The main contact site with the protease (P1) has a Phe, showing the specificity of the inhibitor. NA-CI shared great similarity with the chymotrypsin inhibitor from Naja naja venom (identities=89.5%) and other snake venom protease inhibitors.     

舟山眼镜蛇毒CTX1层析分离的流速优化

目的 比较3种流速的CM-Sepharose FF阳离子交换层析柱分离舟山眼镜蛇(Naja naja atra)蛇毒(snake venom,SV)的柱效,为SV的分离纯化提供实验依据.方法 (1)采用3种流速CM-Sepharose FF阳离子交换层析柱分离舟山眼镜蛇蛇毒;(2)反向高效液相法分析各组分的纯度及内标法...     

Production of potent antivenin against cobra venom with conjugated-cobrotoxin.

Cobra venom (Naja naja atra) and its fractions obtained by ammonium sulfate precipitation were subjected to chromatography on CM-Cellulose colum. A highly purified cobrotoxin obtained by the repeated chromatography on preparative CM-Cellulose column was 6.7 times more toxic than the original cobra venom. The toxin was detoxified by a bifunctional reagent, glutaraldehyde, to about 99.8% and utilized for immunization in animals. Mice received 4 weekly immunization with detoxified cobrotoxin and challenged one week after the last injection showed 60% protection in rabbits by immunization with detoxified cobrotoxin reached 360 LD50 neutralizing level against the cobra venom within 30 days. The results indicate that it is feasible and promising to prepare potent antivenin in animals by glutaraldehyde-treated cobrotoxin.     

Disulfide bonds of purothionine, a lethal toxin for yeasts.

Purothionin isolated from commercial wheat flour contained several components and two of them (A-I and A-II) were isolated in pure form by CM-52 column chromatography. Each component contained 45 amino acid residues with a 4 disulfide bonds. Purothionin A-II was digested with trypsin and thermolysin to isolate cystine peptides. These were separated and purified by chromatography on an SP-Sephadex column, and paper electrophoresis and chromatography. A peptide containing a -Cys-Cys- sequence was hydrolyzed with 10 N sulfuric acid. Amino acid compositions and partial sequence studies of the cystine peptides and their performic acid-oxidized peptides revealed the positions of all 4 disulfide bonds in purothionin A-II. They were formed between residues 3 and 39, 4 and 31, 12 and 29, and 16 and 25. The results of a partial study of purothionin A-I are also presented.     

Isolation and biochemical characterization of a fibrinolytic proteinase from Bothrops leucurus (white-tailed jararaca) snake venom

In investigations aimed at characterizing snake venom clot-dissolving enzymes, we have purified a fibrinolytic proteinase from the venom of Bothrops leucurus (white-tailed jararaca). The proteinase was purified to homogeneity by a combination of molecular sieve chromatography on Sephacryl S-200 and ion-exchange chromatography on CM Sepharose. The enzyme called leucurolysin-a (leuc-a), is a 23 kDa metalloendopeptidase since it is inhibited by EDTA. PMSF, a specific serine proteinase inhibitor had no effect on leuc-a activity. The amino acid sequence was established by Edman degradation of overlapping peptides generated by a variety of selective cleavage procedures. Leuc-a is related in amino acid sequence to reprolysins. The protein is composed of 200 amino acid residues in a single polypeptide chain, possessing a blocked NH2-terminus and containing no carbohydrate. The proteinase showed proteolytic activity on dimethylcasein and on fibrin (specific activity=21.6 units/mg and 17.5 units/microg, respectively; crude venom=8.0 units/mg and 9.5 units/microg). Leuc-a degrades fibrin and fibrinogen by hydrolysis of the alpha chains. Moreover, the enzyme was capable of cleaving plasma fibronectin but not the basement membrane protein laminin. Leuc-a cleaved the Ala14-Leu15 and Tyr16-Leu17 bonds in oxidized insulin B chain. The pH optimum of the proteolysis of dimethylcasein by leuc-a was about pH 7.0. Antibody raised in rabbit against the purified enzyme reacted with leuc-a and with the crude venom of B. leucurus. In vitro studies revealed that leuc-a dissolves clots made either from purified fibrinogen or from whole blood, and unlike some other venom fibrinolytic metallopeptidases, leuc-a is devoid of hemorrhagic activity when injected (up to 100 microg) subcutaneously into mice.     

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.