Isolation and characterization of hemorrhagic toxin from the venom of Trimeresurus elegans

1. Hemorrhagic toxin from the venom of Trimeresurus elegans was purified in a homogeneous form using gel filtration and ion exchange chromatographies. 2. Hemorrhagic toxin possessed hemorrhagic and TAME (tosyl-L-arginine methyl ester) hydrolytic activities. These activities were inhibited when hemorrhagic toxin was incubated with benzamidine or N-bromosuccinimide. 3. Its mol. wt was 28,500 and the isoelectric point was 7.4. 4. This toxin contains ca 1.5 mol Ca per mol of protein.     

Isolation and biochemical characterization of hemorrhagic toxin f from the venom of Crotalus atrox (western diamondback rattlesnake)

Hemorrhagic toxin f (HT-f) was isolated from Crotalus atrox (Western Diamondback Rattlesnake) venom by a five-step purification procedure. Homogeneity was established by the formation of a single band in acrylamide gel electrophoresis, isoelectric focusing, and sodium dodecyl sulfate (SDS)-electrophoresis. HT-f has a molecular weight of 64,000 and contains 572 amino acid residues. It contains 1 mol of zinc per mol of protein. Zinc is essential for both hemorrhagic and proteolytic activities. HT-f possesses proteolytic activity hydrolyzing the Val-Asn, Gln-His, Leu-Cys, His-Leu, Ala-Leu, and Tyr-Leu bonds of oxidized insulin B chain. HT-f did not coagulate fibrinogen to fibrin, yet it did hydrolyze the gamma chain of fibrinogen without affecting either the A alpha or B beta chains. This is the first time that a hemorrhagic toxin was shown to have fibrinogenase activity. HT-f was shown to differ immunologically from other hemorrhagic toxins such as HT-a and HT-c. HT-f also possesses lethal toxicity. When zinc was removed the apo-HT-f lost its lethal toxicity. HT-f produced not only local hemorrhage in the skin and muscle, but also produced systemic hemorrhage in internal organs such as the intestine, kidney, lung, heart, and liver.     

Hemorrhagic protease from the venom of Calloselasma rhodostoma.

1. A hemorrhagic protease I (HP-I) was isolated from Calloselasma rhodostoma venom by Sephadex G-75, DEAE-Sephacel and Q-Sepharose column chromatographies. 2. Homogeneity was established by the formation of a single band in acrylamide gel electrophoresis. 3. HP-I has a molecular weight of 34,800 and possesses hemorrhagic and proteolytic activities. Both activities are inhibited by ethylenediaminetetraacetic acid (EDTA), 1,10-phenanthroline, ethyleneglycolbis-(beta-aminoethyl ether) N,N'-tetraacetic acid (EGTA), and tetraethylenepentamine (TEP). However neither soybean trypsin inhibitor nor p-chlorobenzoic acid (PCMB) were found to have any effect. 4. The toxin contains 311 amino acid residues and exhibits an isoelectric point of 4.5. 5. The A alpha chain of fibrinogen was cleaved first, followed later by the B beta chain.     

Biochemical characterization of hemorrhagic toxin from crotalus viridis viridis (prairie rattlesnake) venom

Hemorrhage, necrosis and edema are some of the effects often observed following snake bites. This paper reports studies on the isolation and biological properties of hemorrhagic toxin from Crotalus viridis viridis (Prairie rattlesnake) venom. A hemorrhagic toxin was isolated from C. v. viridis venom by Sephadex G-50, DEAE-Sephacel and Q-Sepharose column chromatographies.The hemorrhagic toxin from C. v. viridis venom was shown to be homogenous as demonstrated by a single band on polyacrylamide gel electrophoresis and immunodiffusion. Its molecular weight was approximately 54,000 dallons, and it contained 471 amino acid residues. The toxin possessed hemorrhagic activity with a minimum hemorrhagic dose (MHD) of 0.11 μ g, and hydrolytic activity on dimethylcasein, casein, azocasein, azoalbumin, azocoll and hide powder azure. Hemorrhagic and casein hydrolytic activities were inhibited by EDTA, o-phenanthroline or dithiothreitol. The toxin contained 1 mole of zinc per mole of protein and zinc is essential for both hemorrhagic and proteolytic activities. Hemorrhagic toxin possessed hydrolytic activity on the B-chain of insulin, which cleaves His(5)-Leu(6), His(10)-Leu(11), Ala(14)-Leu(15), Tyr(16)-Leu(17) and Phe(24)-Phe(25) bonds. This toxin also hydrolyzed Aα and Bβ chains of fibrinogen. Intramuscular injections of hemorrhagic toxin caused an increase of creatine phosphokinase activity in mice serum from 50.3 mU/ml to 1133 mU/ml. A toxin isolated from C. v. viridis venom was shown to have strong hemorrhagic activity. Partial characterization is reported for this major hemorrhagic toxin in C. v. viridis venom.     

Isolation and characterization of horridum toxin with arginine ester hydrolase activity from Heloderma horridum (beaded lizard) venom

A hemorrhagic toxin with lethal and arginine ester hydrolytic activities was isolated from Heloderma horridum (beaded lizard) venom by Sephadex G-75, DEAE-Sephacel, and Q-Sepharose column chromatography. The hemorrhagic toxin was shown to be homogeneous as demonstrated by a single band on acrylamide gel electrophoresis and immunodiffusion. Its molecular weight is approximately 31,000 with an isoelectric point of 3.9. Hemorrhagic, lethal, and benzoyl-L-arginine ethyl ester hydrolytic activities of this preparation were inhibited by diisopropyl fluorophosphate (DFP), N-bromosuccinimide, and beta-mercaptoethanol, suggesting that serine, tryptophan, and disulfide bonds are involved in these activities. Also there was an increase in creatine kinase activity in mice serum which is an indicator that the toxin is involved in muscle damage. This protein was stable to heat and pH ranges between 2 and 11. The Michaelis constant (Km), for benzoyl-L-arginine ethyl ester, and inhibition constant (Ki), for DFP, were found to be 6.9 X 10(-3) and 1.93 X 10(-4) M, respectively.     

Presence of zinc in proteolytic hemorrhagic toxin isolated from Agkistrodon acutus venom

1. Hemorrhagic toxin (Ac1-proteinase) was isolated from the venom of Agkistrodon acutus (Formosa) and the zinc content was determined (1.15 mol/mol protein). The results we extensively compared with hemorrhagic toxin e of Crotalus atrox venom (U.S.A.). Comparable results were obtained for zinc content, hemorrhagic and proteolytic activities for native hemorrhagic toxins from two different venoms. It is of interest that the zinc content of hemorrhagic toxins is identical even though the venoms are obtained from snakes inhabiting totally different continents. 2. Zinc content, hemorrhagic and proteolytic activities were compared before and after the removal of zinc. It was found that both hemorrhagic and proteolytic activities disappeared upon removal of the zinc. 3. Zinc content of all hemorrhagic toxins with proteolytic activity reported so far were also compared and it is concluded that regardless of their geographic origin, zinc is present in venom hemorrhagic toxins on a unimolar basis. 4. When zinc is removed there is a drastic change in the low-frequency region of the Raman spectrum suggesting the presence of a zinc ligand co-ordination. The decrease of alpha-helical content and increase of random coil are reflected in the amide I and III bands of Raman spectroscopy after the removal of zinc. Increase of random coil and the loss of zinc are probably responsible for the loss of hemorrhagic and proteolytic activities.     

Isolation and characterization of hemorrhagic factors a and b from the venom of the Chinese habu snake (Trimeresurus mucrosquamatus)

Hemorrhagic factors a and b were isolated from Trimeresurus mucrosquamatus venom by Sephadex G-100, CM-Sephadex C-50 and DEAE-Sephacel column chromatographies. The hemorrhagic factors were homogeneous, as established by a single band on acrylamide gel electrophoresis, isoelectric focusing and sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Molecular weights of 15 000 and 27 000 were found for hemorrhagic factors a and b, respectively. Factor a possesses proteolytic activity hydrolyzing the His(10)-Leu(11), Tyr(16)-Leu(17) and Arg(22)-Gly(23) bonds of oxidized insulin B chain, whereas, factor b hydrolyzed only the Ala(14)-Leu(15) bond. Hemorrhagic activity of these hemorrhagic factors was inhibited by ethylenediaminetetraacetic acid, 1,10-phenanthroline or p-chloromercuribenzoate, but not by soybean trypsin inhibitor or diisopropyl fluorophosphate. The hemorrhagic factors were injected into the skin of the back of albino rabbits, and the minimum hemorrhagic dose of factors a and b was 1.7 and 2.3 micrograms, respectively. These purified hemorrhagic factors were not lethal at 15 micrograms/g in mice. Factor a hydrolyzed the B beta chain of fibrinogen, while factor b hydrolyzed the A alpha chain. Hemorrhagic factor a was shown to differ immunologically from factor b. Factors a and b produced systemic hemorrhage in internal organs such as the heart and stomach of mice. Moreover, factor b produced hemorrhage in the liver.     

Isolation and characterization of hemorrhagic factors a and b from the venom of the Chinese habu snake (Trimeresurus mucrosquamatus)

Hemorrhagic factors a and b were isolated from Trimeresurus mucrosquamatus venom by Sephadex G-100, CM-Sephadex C-50 and DEAE-Sephacel column chromatographies. The hemorrhagic factors were homogeneous, as established by a single band on acrylamide gel electrophoresis, isoelectric focusing and sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Molecular weights of 15 000 and 27 000 were found for hemorrhagic factors a and b, respectively. Factor a possesses proteolytic activity hydrolyzing the His(10)-Leu(11), Tyr(16)-Leu(17) and Arg(22)-Gly(23) bonds of oxidized insulin B chain, whereas, factor b hydrolyzed only the Ala(14)-Leu(15) bond. Hemorrhagic activity of these hemorrhagic factors was inhibited by ethylenediaminetetraacetic acid, 1,10-phenanthroline or p-chloromercuribenzoate, but not by soybean trypsin inhibitor or diisopropyl fluorophosphate. The hemorrhagic factors were injected into the skin of the back of albino rabbits, and the minimum hemorrhagic dose of factors a and b was 1.7 and 2.3 μg, respectively. These purified hemorrhagic factors were not lethal at 15 μg/g in mice. Factor a hydrolyzed the Bβ chain of fibrinogen, while factor b hydrolyzed the Aα chain. Hemorrhagic factor a was shown to differ immunologically from factor b. Factors a and b produced systemic hemorrhage in internal organs such as the heart and stomach of mice. Moreover, factor b produced hemorrhage in the liver.     

Comparative study of three proteinases from the venom of the Chinese habu snake (Trimeresurus mucrosquamatus)

Three immunochemically distinct proteinases (P-1, 2 and 3) devoid of hemorrhagic activity were isolated from the lyophilized venom of Trimeresurus mucrosquamatus using column chromatography on Sephadex G-100, CM-Sephadex C-50, DEAE-Sephacel, CM-Cellulose and Bio-Rex 70. By these procedures, about 7.6, 7.3 and 8.2 mg of purified P-1, 2 and 3 may be obtained from 1 g of crude venom, respectively. The purified proteinases 1-3 were homogeneous by disc electrophoresis on polyacrylamide gel at pH 4.3, isoelectric focusing and by the presence of one precipitin line on immunodiffusion. The isoelectric point of P-1 was 8.1; P-2, 9.2; P-3, 9.8. The molecular weights of proteinases 1-3 were determined to be 23,000, 23,500 and 23,000, by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, respectively. The purified proteinases 1-3 possessed caseinolytic and fibrinogenolytic activities. These activities were inhibited when the proteinases were incubated with the metal chelators ethylenediaminetetraacetic acid (EDTA), 1,10-phenanthroline or cysteine, but not with egg white trypsin inhibitor (EWTI) or soybean trypsin inhibitor (SBTI). P-1 cleaved the B beta-chain of fibrinogen first and then the A alpha-chain, whereas P-2 and 3 cleaved the A alpha-chain first and then the B beta-chain. However, these three proteinases did not hydrolyze the gamma-chain.     

Proteolytic specificity of hemorrhagic toxin b from Crotalus atrox (western diamondback rattlesnake) venom

In our effort to identify the proteolytic specificity of various hemorrhagic toxins isolated from western diamondback rattlesnake venom, hemorrhagic toxin b was isolated in homogeneous form by previously published methods. Hemorrhagic toxin b hydrolyzed glucagon, producing six fragments. The proteolytic sites were identified as Thr(5)-Phe(6), Thr(10)-Ser(11), Asp(15)-Ser(16), Asp(21)-Phe(22) and Try(25)-Leu(26). When oxidized insulin B chain was used, proteolysis occurred at four sites: Asn(3)-Gln(4), His(10)-Leu(11), Tyr(16)-Leu(17) and Gly(23)-Phe(24). The proteolytic specificity of hemorrhagic toxin b is quite different from those of the nonvenom proteases such as thermomycolin, aspergillopeptidase c, alkaline protease from Aspergillus flavus, elastase, subtilisin and papain.     

Purification and biochemical characterization of atroxase, a nonhemorrhagic fibrinolytic protease from western diamondback rattlesnake venom

Crotalus atrox venom contains a variety of proteases which render fibrinogen incoagulable and solubilize fibrin. One of these proteases was purified by using ion-exchange and gel permeation liquid chromatography. The protease, called atroxase, consists of a single nonglycosylated polypeptide chain with a molecular weight of 23,500 and an isoelectric point of pH 9.6. Amino acid analysis indicates atroxase to contain 206 residues with no sulfhydryl groups. Metal analysis found zinc and potassium at 1 mol/mol of protein, and calcium at 0.3 mol/mol of protein. Proteolytic activity is inhibited by ethylenediaminetetraacetate and alpha 2-macroglobulin. Maximal proteolytic activity occurs at pH 9.0 and 55 degrees C. Proteolytic specificity, using oxidized insulin B chain, is similar to that of several hemorrhagic toxins found within the same venom, yet atroxase shows no hemorrhagic activity and exhibits low lethality when tested on Swiss Webster mice. Atroxase, an A alpha, B beta fibrinogenase, cleaves the A alpha chain of fibrinogen first followed by the B beta chain and shows no effect on the gamma chain. The nonspecific action of the enzyme results in the extensive hydrolysis of fibrinogen which releases a variety of fibrinopeptides. Fibrin solubilization appears to occur primarily from the hydrolysis of alpha-polymer and unpolymerized alpha and beta chains. Although crude venom induces platelet aggregation, atroxase demonstrated no ability to induce or inhibit aggregation.     

Two toxins from the venom of Trimeresurus tokarensis

1. Two toxins, Tokaratoxin-1 (TT-1) and Tokaratoxin-2 (TT-2), were isolated from the venom of Trimeresurus tokarensis using gel filtration on a Sephadex G-100 column, followed by chromatography on DEAE-Sephacel and carboxymethyl-cellulose. TT-1 possessed both hemorrhagic and proteolytic activities. However, TT-2 did not show hemorrhagic activity. 2. Homogeneity was established by the formation of a single band in acrylamide gel electrophoresis, isoelectric focusing and SDS-PAGE. 3. Molecular weights of TT-1 and TT-2 were 71,000 and 25,400, respectively. Although TT-2 is a basic protein, TT-1 is an acidic protein. 4. Biological activities of TT-1 and TT-2 were inhibited by EDTA, EGTA and o-phenanthroline, suggesting that the toxins are metalloproteins. Atomic absorption analyses indicated that TT-1 contains 2.79 mol Ca/mol protein and TT-2 contains 1.04 mol Ca/mol protein and 1.07 mol Zn/mol protein, respectively. 5. The two toxins degraded the A alpha and B beta chains of fibrinogen. 6. TT-1 induced necrosis in addition to its hemorrhagic activity while TT-2 induced necrosis only.     

Characterization of Ac3-proteinase from the venom of Agkistrodon acutus (hundred-pace snake)

Ac3-Proteinase from the venom of Agkistrodon acutus was isolated in a homogeneous form by a previously published method. Ac3-Proteinase possessed lethal, hemorrhagic, caseinolytic, azocaseinolytic, dimethylcaseinolytic and hide powder azure hydrolytic activities. These activities were inhibited when Ac3-Proteinase was incubated with the metal chelators ethylenediaminetetraacetic acid (EDTA), ethyleneglycol-bis-(beta-aminoethyl ether)-N,N'-tetraacetic acid (EGTA), tetraethylenepentamine (TEP), 1,10-phenanthroline, phosphoramidon or beta-mercaptoethanol. The toxin also hydrolyzed the oxidized A and B chains of both insulin and fibrinogen. The cleavage sites in the oxidized B chain of insulin were identified as His(10)-Leu(11), Ala(14)-Leu(15), Tyr(16)-Leu(17) and Phe(24)-Phe(25). The A alpha chain of fibrinogen was digested first followed by hydrolysis of the B beta chain. Toxicological and biochemical properties of Ac3-Proteinase were investigated further and are reported in this paper.     

Characterization of Ac1-Proteinase from the venom of Agkistrodon acutus (Hundred-pace snake) from Taiwan

1. Ac1-Proteinase from the venom of Agkistrodon acutus was isolated in a homogeneous form by a previously published method. 2. Ac1-Proteinase possessed lethal, hemorrhagic, caseinolytic, azocaseinolytic, azoalbumin hydrolytic and hide powder azure hydrolytic activities. 3. The toxin also hydrolyzed the oxidized B chain of insulin and fibrinogen. The cleavage sites in the oxidized B chain of insulin were identified as Ala(14)-Leu(15) and Tyr(16)-Leu(17). The A alpha chain of fibrinogen was digested. 4. Biological properties of Ac1-Proteinase were investigated further and are reported in this paper.     

Purification and characterization of the hemorrhagic factor II from the venom of the Bushmaster snake (Lachesis muta muta)

Hemorrhagic factor II (LHF-II) was isolated from Lachesis muta muta (Bushmaster snake) venom using column chromatographies on Sephadex G-100, CM-Sepharose CL-6B and two cycles on Sephadex G-50. This preparation was devoid of phospholipase A2 as well as of the enzymes active on arginine synthetic substrates (TAME and BAPNA) which are present in the crude venom. LHF-II was homogeneous by SDS-polyacrylamide gel electrophoresis, immunodiffusion and immunoelectrophoresis. Also, a single symmetrical boundary with a value of 2.59 S was obtained by ultracentrifugation. LHF-II contains 180 amino acid residues, has a molecular weight of 22,300, and an isoelectric point of 6.6. It contains one gatom zinc and two gatoms calcium per mol protein. The hemorrhagic factor possesses proteolytic activity toward various substrates such as, casein, dimethylcasein, hide powder azure, fibrinogen and fibrin. It hydrolyzes selectively the A alpha-chain of fibrinogen, leaving the B beta- and gamma-chains unaffected. LHF-II is activated by Ca2+ and inhibited by Zn2+. The hemorrhagic as well as the proteinase activity is inhibited by cysteine and by metal chelators such as EDTA, EGTA and 1,10-phenanthroline. Inhibitors of serine proteinases such as phenylmethanesulfonyl fluoride (PMSF) and soybean trypsin inhibitor (SBTI) have no effect on the hemorrhagic factor.     

尖吻蝮蛇蛇毒出血毒素DaHT-3的纯化与性质

被尖吻蝮蛇咬伤会引起严重的出血,对蛇毒出血毒素的研究有利于治疗蛇伤出血药物的筛选.通过SephadexG-75,DEAE-SephadexA-50,SephadexG-200和两次PBE聚焦层析,从尖吻蝮蛇(Dienagkistrodonacutus)蛇毒中纯化到一个分子量为56000的出血毒素(DaHT-3),经氨基酸组成测定计算,由487个氨基酸残基组成.此成分在SDS-PAGE上显示出一条均一的蛋白染色带,用等电聚焦电泳测定,其pI为5.50.该出血成分的最小出血剂量是2.6μg,具有蛋白水解酶活力,其活力为3.68,但没有精氨酸酯酶和磷脂酶A2活力.用红外光谱仪研究DaHT-3在溶液中酰胺I带的吸收谱,该毒素含有31.8%的α螺旋、56.1%的β折叠和12.1%的转角;当加入EDTA螯合剂去除金属离子后,它们的α螺旋、β折叠、转角和无规卷曲分别变为11%、26.4%、46.2%和16.5%,而出血活力和蛋白水解酶活力均丧失,表明该出血毒素是金属蛋白酶     

Purification and partial characterization of stonustoxin (lethal factor) from Synanceja horrida venom.

1. The lethal factor of the stonefish (Synanceja horrida) venom, designated as the stonustoxin, was purified to homogeneity by a two-step procedure on Sephacryl S-200 High Resolution (HR) gel permeation and DEAE Bio-Gel A anion exchange chromatography. 2. Stonustoxin has a native mol. wt of 148,000 and an isoelectric point of 6.9. 3. SDS-polyacrylamide gel electrophoresis revealed two subunits (designated alpha and beta) with mol. wts of 71,000 and 79,000, respectively. 4. The amino acid composition of both subunits and the N-terminal amino acid sequence of the beta subunit were also determined. 5. Purified stonustoxin had an LD50 of 0.017 microgram/g which is 22-fold more potent than that of the crude venom. 6. The toxin exhibited potent haemolytic activity in vitro and edema-inducing activity with a minimum edema dose (MED) of 0.15 micrograms in mouse paw. The edema effect was not antagonized by diphenhydramine.     

Characterization of two hemorrhagic zinc proteinases, toxin c and toxin d, from western diamondback rattlesnake (Crotalus atrox) venom

Two hemorrhagic proteinases from Crotalus atrox venom, hemorrhagic toxin c (Ht-c) and hemorrhagic toxin d (Ht-d), were characterized and compared to one another. The two toxins are zinc metalloproteinases which both have molecular weights of 24,000. Their isoelectric points are slightly acidic, Ht-c being the more basic of the two with an isoelectric point of 6.2, whereas Ht-d has an isoelectric point of 6.1. Only minor differences were found in the amino acid compositions of the two toxins. The toxins were both demonstrated to be hemorrhagic, using an in vivo assay, and also proteolytic. Prior treatment of the hemorrhagic proteinases with ethylenediaminetetraacetic acid and o-phenanthroline eliminated both the hemorrhagic and the proteolytic activities. Aprotinin and phenylmethylsulfonyl fluoride had no effect upon these activities. The pH optimum of the proteolysis by Ht-c and Ht-d on hide powder azure as the substrate was between pH 8 and pH 9. The circular dichroism spectra for Ht-c and Ht-d appear almost identical with respect to minima positions and elipticities, indicative of very similar solution structures for the two enzymes. Antiserum raised in mice against Ht-c was assayed on double-diffusion Ouchterlony plates for cross-reactivity with other hemorrhagic toxins from C. atrox venom. From this experiment it was concluded that the two hemorrhagic proteinases Ht-c and Ht-d share identical antigenic structures. This was corroborated by tryptic mapping of the two toxins. Only one major difference was observed from the maps. In the case of Ht-c, it was determined that an aspartate was substituted by an alanine when compared to Ht-d. From these characterization studies we conclude that Ht-c and Ht-d are isoenzymes with only very minor differences in their structures.     

Chemical properties and amino acid composition of beta1-bungarotoxin from the venom of Bungarus multicinctus (Formosan banded krait)

beta-Bungarotoxin purified from the venom of Bungarus multicinctus (Formosan banded krait) contained no carbohydrate and behaved as a homogeneous protein on polyacrylamide gel electrophoresis at pH 4.1 and SDS-polyacrylamide gel electrophoresis without 2-mercaptoethanol treatment. Its molecular weight and isoelectric point were estimated to be about 21,000 by gel filtration and about 9.5 by isoelectric focusing, respectively. The toxin treated with the reducing agent was split into two polypeptide chains as revealed by SDS-polyacrylamide gel electrophoresis and their molecular weights were calculated to be about 13,000 and 7,000. The two polypeptide chains (the large one named the A chain and the small one the B chain) were isolated by gel filtration after reduction of disulfide bonds in the toxin followed by alkylation. The A chain contained 120 amino acid residues including 13 half-cystines and the B chain 60 residues including 7 half-cystines. The two chains were supposed to link by disulfide bond(s) in the intact toxin which contained no free sulfhydryl groups. The N-terminal residues of the A and B chains were asparagine and arginine and the C-terminal ones were glutamine and proline, respectively, in accordance with the results of the terminal analyses of the intact toxin.     

A mammal toxin derived from the venom of a chactoid scorpion

1. It has been shown that the low toxicity to mammals (LD50 of about 200 mg per kg mice body weight) of the chactoid scorpion venom Scorpio maurus palmatus (Scorpionidae) is due to a single low molecular weight basic protein. 2. This compound was purified by the aid of gel filtration and ion exchange column chromatography, possessed about 80% of the mice lethality of the crude venom with an increase of about 60 fold in its specific toxicity. 3. It is composed of 32 amino acids (mol. wt = 3478) and devoid of isoleucine, leucine, phenylalanine, histidine and tryptophan. 4. The unique amino acid composition of the present toxin is compared to those of the well known buthoid scorpion venom mammal toxins and some other toxins derived from the same venom. 5. It is the first chemically characterized chactoid toxin.