湖南尖吻蝮蛇毒两个出血毒素的纯化和理化性质

经ＳｅｐｈａｄｅｘＧ－７５凝胶过滤,ＱＡＥ－ＳｅｐｈａｄｅｘＡ－５０和ＣＭ－Ｓｅｐｈａｄｅｘ Ｃ－２５离子交换层析的步骤,从湖南产尖吻蝮蛇毒中纯化出两个出血毒素。ＳＤＳ－ＰＡＧＥ测得分子量均为２３．５ｋＤ,ＩＥＦ－ＰＡＧＥ测得等电点分别为５．６和５．２,两者具有相似的氨基酸组成,其中酸性氨基酸分别占２３％和２４％。ＤａＨＴ－１和ＤａＨＴ－２的最小出血剂量（ＭＨＤ）分别为０．５μｇ和０．８μｇ。都具     

系统感染TMV的番茄叶胞外β-1,3-葡聚糖酶

系统感染ＴＭＶ （ｔｏｂａｃｃｏ ｍ ｏｓａｉｃ ｖｉｒｕｓ）的番茄（Ｌｙｃｏｐｅｒｓｉｃｏｎ ｅｓｃｕｌｅｎｔｕｍ Ｍｉｌｌ．）叶胞外蛋白提取液经冰冻干燥浓缩、－ ２０℃丙酮沉淀、ＣＭ－Ｓｅｐｈａｄｅｘ Ｃ－２５离子交换层析、ＤＥＡＥ－Ｓｅｐｈａｄｅｘ Ａ－２５离子交换层析和Ｓｅｐｈａｄｅｘ Ｇ－７５凝胶层析纯化,获得ＰＡＧＥ均一的β－１,３－葡聚糖酶．ＳＤＳ－ＰＡＧＥ证明,它包含分子量为３６ ｋＤ 和２７ ｋＤ的两个同工酶．以昆布多糖为底物,酶的最适ｐＨ 在４．８—５．２之间,在ｐＨ ４—８稳定;酶的最适温度在３０—４０℃之间,在４０℃保温１ｈ 后酶活性不变;Ｋｍ 值为９．２ ｍ ｇ／ｍ Ｌ．在系统感染ＴＭＶ 的番茄叶胞外蛋白提取液中,有分子量为２２ ｋＤ、２７ ｋＤ和３６ ｋＤ的３个β－１,３－葡聚糖酶同工酶     

与光系统Ⅱ颗粒结合的对DTT敏感的蛋白酶的纯化和性质

菠菜光系统Ⅱ颗粒的１ｍｏｌ／ＬＮａＣｌ抽提液经ｂｕｔｙｌ－Ｔｏｙｏｐｅａｒｌ６５０Ｍ疏水层析和ＤＥＡＥ－ＳｅｐｈａｄｅｘＡ－５０柱层析分离得一种对ＤＴＴ敏感的蛋白酶。用ＳＤＳ－ＰＡＧＥ和Ｓｕｐｅｒｏｓｅ１２凝胶过滤测得该酶的分子量为３７ｋＤ,其为单体酶。该酶可降解１８ｋＤ和２４ｋＤ水溶性蛋白质,分别产生１３．２ｋＤ、１２ｋＤ、１０．５ｋＤ和２３ｋＤ、２２ｋＤ、２０ｋＤ片段,最适ｐＨ为８．０,对ＮａＣｌ不敏感,对ＤＴＴ和β－Ｍｅ敏感。抑制实验表明该酶不属丝氨酸类蛋白酶。     

甲基单胞菌Methylomonas sp.GYJ3中甲烷单加氧酶还原酶组分的纯化和性质

Ｍｅｔｙｌｏｍｏｎａｓｓｐ．ＧＹＪ３菌的甲烷单加氧酶（ＭＭＯ）粗酶提取液经ＤＥＡＥ－ＳｅｐｈａｒｏｓｅＣＬ－６Ｂ阴离子交换层析、ＳｅｐｈａｄｅｘＧ－１００凝胶过滤层析和ＤＥＡＥ－ＴＳＫｇｅｌＨＰＬＣ分离纯化出ＭＭＯ还原酶组分．经ＨＰＬＣ分析,纯度大于９５％,纯化倍数为４．４,加入至ＭＭＯ羟基化酶和调节蛋白Ｂ的体系中表现比活为２２８ｎｍｏｌ环氧丙烷每分钟毫克蛋白．ＳＤＳ－ＰＡＧＥ电泳表明还原酶由一种亚基组成,分子量４２ｋＤ．ＩＣＰ－ＡＥＳ测定还原酶的Ｆｅ含量为１．８３ｍｏｌＦｅ每ｍｏｌ蛋白．ＵＶ－Ｖｉｓ光谱表明还原酶除２８０ｎｍ蛋白质特征峰外在４６０ｎｍ有最大吸收峰,且Ａ２８０ｎｍ／Ａ４６０ｎｍ为２．５０,与其它黄素一铁硫蛋白相似,推测还原酶可能含一个ＦＡＤ辅基和Ｆｅ２Ｓ２中心．在厌氧条件下,还原酶能够和ＮＡＤＨ作用,ＵＶ－Ｖｉｓ光谱分析表明还原酶４６０ｎｍ处特征吸收峰消失,说明在ＭＭＯ催化过程中还原酶接受ＮＡＤＨ的电子．ＤＥＡＥ－ＳｅｐｈａｒｏｓｅＣＬ－６Ｂ阴离子交换层析分离出调节蛋白Ｂ,部分纯化的调节蛋白Ｂ的分子量大约在２０ｋＤ,它能够提高ＭＭＯ比活性４０倍,ＭＭＯ还原酶和调节蛋白Ｂ单独存在时不具有ＭＭＯ     

广东眼镜王蛇毒酸性磷脂酶A2的分离纯化及抗血小板聚集作用

目的 研究眼镜王蛇毒中酸性磷酸脂酶Ａ２对血小板的作用。方法 采用ＣＭ－Ｓｅｐｈａｄｅｓｘ Ｃ－２５Ｓｅｐｈａｄｅｘ Ｇ－７５,ＤＥＡＥ－Ｓｅｐ「ｈａｄｅｘＡ－２５,Ｓｅｐｈａｄｅｘ Ｇ－７５多步柱层析法,聚丙烯酰胺产胶电泳,酸性磷脂酶Ａ２酶活性测定;血小板聚集实验。累进要从粗中分离纯化出一酸性磷脂酶Ａ２单体,分子量为１４ＫＤ;等电点ＰＩ３．８;ＰＬＡ２比值２０μｍｏｌ．ｍｇ＾－１．ｍｉｎ＾－１;小     

百合巯基蛋白酶抑制剂的纯化及部分性质研究

百合的鳞茎中含有一种对木瓜蛋白酶有强抑制作用的巯基蛋白酶抑制剂．百合的鳞茎经浸取加热处理,木瓜蛋白酶偶联的Ｓｅｐｈａｒｏｓｅ４Ｂ柱亲和层析和ＳｅｐｈａｄｅｘＧ－１００分子筛层析,可获得在ＰＡＧＥ和ＳＤＳ－ＰＡＧＥ均为单一蛋白带的百合巯基蛋白酶抑制剂（ＣＰＩ）．此ＣＰＩ为单链蛋白,含有０．３０７％的中性糖;Ｎ端氨基酸为Ｉｌｅ;ＳＤＳ－ＰＡＧＥ测得亚基分子量为１２０００;ＳｅｐｈａｄｅｘＧ－１００测得分子量为１２５００．百合ＣＰＩ在１００℃内和ｐＨ２～１２范围内非常稳定;对木瓜蛋白酶的抑制属竞争性抑制类型,其Ｋｉ值为１．１５×１０~（－９）ｍｏｌ／Ｌ,对木瓜蛋白酶的抑制摩尔比为８．５：１．     

地衣芽孢杆菌β－甘露聚糖酶的纯化及酶学性质

地衣芽孢杆菌（Ｂａｃｉｌｌｕｓｌｉｃｈｅｎｉｆｏｒｍｉｓ）ＮＫ－２７菌株发酵产生的β－甘露聚糖酶（β－ｍａｎｎａｎａｓｅ）经硫酸铵盐析沉淀,两次ＤＥＡＥ纤维素和ＳｅｐｈａｄｅｘＧ－１００离子交换柱层析以及制备ＰＡＧＥ筹步骤,获得了凝胶电泳均一的样品。用ＳＤＳ－凝胶电泳测得纯化后的β－甘露聚糖酶分子量为２６ｋＤ,用凝胶聚焦电泳测得等电点ＰＩ为５．０。酶反应的最适ｐＨ为９．０,最后温度为６０℃,稳定ｐＨ为６．０—９．０,稳定温度为４０℃。金属离子中Ｍｇ￣（２＋）、Ｃａ￣（２＋）、Ｆｅ￣（２＋）、Ｎｉ￣（２＋）对该酶有一定的激活作用;而Ｓｎ￣（２＋）、Ｚｎ￣（２＋）、Ａｌ￣（３＋）、Ａｇ￣＋和Ｈｇ￣（２＋）对该酶有强烈的抑制作用。ＮＫ－２７菌株的β－甘露聚糖酶对魔芋葡萄甘露聚糖和角豆胶半乳甘露聚糖的Ｋｍ值分别为７．１４和５．５６ｍｇ·ｍｌ￣（－１）;Ｖ＿（ｍａｘ）分别为２００．５３和１５７．４５μｍｏｌ·ｍｇ￣（－１）·ｍｉｎ￣（－１）。     

湖南尖吻蝮蛇毒出血毒素的圆二色性和化学修饰

用远紫外ＣＤ谱研究了湖南产尖吻蝮蛇毒的两个出血毒素（ＤａＨＴ－１、ＤａＨＴ－２）的溶液构象,计算得ＤａＨＴ－１的α螺旋、β折叠、无规卷曲的含量分别为３６．９％、３５．５％、２７．６％;ＤａＨＴ－２的α螺旋、β折叠、无规卷曲分别为２３．４％、３１．３％、４５．３％。随ｐＨ的增大或减小,峰位蓝移,酸性条件下的变化比碱性条件下的变化大。构象单元含量计算表明：α螺旋减少,无规卷曲增多,β折叠基本未变。温度和ｐＨ对ＣＤ谱的影响相似,５０℃时峰位蓝移,α螺旋减少,无规卷曲增多．ＥＤＴＡ对ＣＤ谱影响显著,０．０２ｍｏｌ／ＬＥＤＴＡ便导致两个出血毒素呈极度的无序状态。ＥＤＴＡ完全抑制,半胱氨酸部分抑制,胰蛋白酶不影响它们的出血活性。     

水稻巯基蛋白酶抑制剂的纯化及其性质研究

水稻的糠皮和胚经生理盐水浸取、离心后的上清液加热至８０℃处理１０ｍｉｎ,离心获得的上清液调ｐＨ至８．０,得到沉淀。沉淀溶解于０．０１ｍｏｌ／ＬＨＣｌ,经透析冷冻干燥得水稻巯基蛋白酶抑制剂（ＣＰＩ）粗品;粗品再经ＤＥＡＥ－Ｓｅｐｈａｒｏｓｅ柱线性离子梯度洗脱和ＳｅｐｈａｄｅｘＧ－１００柱分子筛层析,即可获得在ＰＡＧＥ、ＳＤＳ－ＰＡＧＥ和ＨＰＬＣ上均为单一蛋白带的ＣＰＩ样品。经上述步骤,ＣＰＩ可被纯化５８倍。经ＳｅｐｈａｄｅｘＧ－１００和ＳＤＳ－ＰＡＧＥ测定其分子量均为１２０００,Ｎ末端氨基酸为Ｐｒｏ,等电点５．６．水稻ＣＰＩ经１００℃处理１０ｍｉｎ后,其抑制活性无任何变化,在ｐＨ２．０～９．０之间,活性也不发生改变,但ｐＨ在９．０以上,其活性逐渐下降,水稻ＣＰＩ对木瓜蛋白酶是一种高亲和性的抑制剂,它对木瓜蛋白酶和无花果蛋白酶有强抑制作用,对菠萝蛋白酶仅有弱抑制作用,但对胰蛋白酶则全无抑制作用;其抑制类型属竞争性抑制剂类型,Ｋ＿ｉ值约３．５×１０￣（－８）ｍｏｌ／Ｌ对木瓜蛋白酶的抑制摩尔比约为１：１。     

长白山白眉蝮蛇蛇毒磷脂酶A2的分离和初步表征

东北长白山白眉蝮蛇（ＡｇｋｉｓｔｒｏｄｏｎｂｌｏｍｈｏｆｆｉＵｓｕｒｅｎｓｉｓ）蛇毒经ＤＥＡＥＳｅｐｈａｄｅｘＡ５０离子交换层析柱,连续３步ＳｅｐｈａｄｅｘＧ７５凝胶过滤柱得到了磷脂酶Ａ２（ＰＬＡ２）的纯品。ＳＤＳ聚丙烯酰胺凝胶电泳（ＳＤＳＰＡＧＥ）以及基质辅助激光解析电离飞行时质谱（ＭＡＬＤＩ／ＴＯＦ／ＭＳ）表征为单一蛋白,其准确分子量为（１４．００８±０．００７）ｋＤ。最适ｐＨ范围８．０～９．０,最适的反应温度为４５℃。在溶液中有多聚体的存在。     

Biochemical characterization of hemorrhagic toxins with fibrinogenase activity isolated from Crotalus ruber ruber venom

Three hemorrhagic toxins with proteolytic activity were isolated from the venom of Crotalus ruber ruber (red rattlesnake). Molecular weights of HT-1, HT-2, and HT-3 were 60,000, 25,000, and 25,500, respectively. Although HT-3 was a basic protein, HT-1 and HT-2 were slightly acidic proteins. Total amino acid residues were 482,207, and 221 for HT-1, HT-2, and HT-3, respectively. Protease activity of all the toxins was inhibited in the presence of EDTA or o-phenanthroline, suggesting that the toxins are metalloproteins. Analyses for various metals by inductively coupled plasma-atomic emission spectrometry indicated that sodium, potassium, zinc, and calcium atoms were present in significant quantities. With all three toxins, there was roughly 1 mol of zinc to 1 mol of protein; the results for calcium were not consistent. All three hemorrhagic toxins degraded the A alpha chain of fibrinogen, while HT-1 also degraded the B beta chain. Although fibrinogen was degraded by the three toxins, no clots were observed, indicating that the proteolytic specificities of the three toxins were different from those of thrombin. The hemorrhagic toxins increased creatine kinase activity in mice serum, indicating muscle damage, which was substantiated by histological examination.     

Hemorrhagic toxins from Western diamondback rattlesnake (Crotalus atrox) venom: isolation and characterization of five toxins and the role of zinc in hemorrhagic toxin e.

Five previously unknown hemorrhagic proteins, designated hemorrhagic toxins a,b,c,d, and e, were isolated from the venom of the western diamondback rattlesnake (Crotalus atrox). Molecular weights of hemorrhagic toxins a-e were determined to be 68 000, 24 000, 24 000, 24 000, and 25 700, respectively, by sodium dodecyl sulfate-phosphate gel electrophoresis using various polyacrylamide gel concentrations. Amino acid composition showed a total of 636, 200, 213, 214, and 219 amino acids for hemorrhagic toxins a-e, respectively. All the hemorrhagic toxins were found to lose their hemorrhagic activities with the metal chelators ethylenediaminetetraacetic acid and 1, 10-phenanthroline. All the hemorrhagic toxins were found to contain approximately 1 mol of zinc/mol of toxin, and they were all demonstrated to be proteolytic when dimethylcasein and dimethylhemoglobin were used as substrates. When zinc was removed from hemorrhagic toxin e with 1,10-phenanthroline, both both the proteolytic and hemorrhagic activities were equally inhibited. When the apohemorrhagic toxin e thus produced was incubated with zinc, the hemorrhagic and proteolytic activities were regenerated to the same extent. CD, UV, and Raman spectroscopy were used to study the structure of native hemorrhagin toxin e as well as the structural changes caused by zinc removal. From CD spectroscopy the native toxin was estimated to consist of 23% alpha helix, 6% beta structure, and 71% random-coil conformation. When over 90% of the zinc was removed, the alpha-helix content dropped from 23 to 7%.     

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.     

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.     

Substrate specificities and inhibition of two hemorrhagic zinc proteases Ht-c and Ht-d from Crotalus atrox venom

The proteolytic specificities of two zinc hemorrhagic toxins (Ht-c and Ht-d), isolated from Crotalus atrox venom, were investigated by using the oxidized B chain of bovine insulin and synthetic peptide substrates. The enzymes cleaved the Ala14-Leu15 bond of the insulin B chain most rapidly and the Tyr16-Leu17 slightly more slowly. The His5-Leu6, His10-Leu11, and Gly23-Phe24 bonds were also cleaved but at considerably slower rates. In order to assess the substrate length preferences of the enzymes, peptide analogs of the B chain about the Ala14-Leu15 bond were synthesized ranging in length from four to seven residues. The heptapeptide NH2-Leu-Val-Glu-Ala-Leu-Tyr-Leu-COOH was the best peptide substrate tested with the other peptides having decreasing kcat/Km values with decreasing length. The tetrapeptide NH2-Ala-Leu-Tyr-Leu-COOH was not cleaved by the enzymes. Furthermore, this peptide was shown to serve as a competitive inhibitor of the toxins. The N-acetylated pentapeptides and hexapeptides, synthesized to probe the active site environment of the enzymes, were significantly better substrates than their unacetylated counterparts. The toxins had the highest kcat/Km values for the acetylated peptide Ac-Val-Ala-Leu-Leu-Ala-COOH. The data suggest that the toxins may indeed have extended substrate-binding sites, which may accommodate at least six amino acid residues. The best substrate examined thus far for the toxins is the fluorogenic peptide analog 2-aminobenzoyl-Ala-Gly-Leu-Ala-4-nitrobenzylamide, suggestive of similarities between the toxins and mammalian collagenases as well as thermolysin. Mechanisms for inhibition of the enzymes were investigated using amino acid hydroxamates, chloromethyl esters, phosphoramidon and the peptide NH2-Ala-Leu-Tyr-Leu-COOH. All of these inhibitors had Ki values in the 10(-4) M range.     

Isolation of minax toxins from the venom of the scorpion Buthus minax and their metabolic effects

Two neurotoxins, minax toxins 1 and 2, were isolated from venom of the scorpion Buthus minax from the Sudan. Molecular weights of 7000 and 6800 and 66 and 62 amino acids were found for minax toxins 1 and 2, respectively. Both toxins contain four disulfide bonds, 1 mol each of phenylalanine, histidine, and tryptophan, no free sulfhydryl groups, and no methionine. Both minax toxins 1 and 2 are basic polypeptides with isoelectric points of 8.2 and 9.0, respectively. There is a significant increase in the calcium content of rat hearts envenomated with minax toxins 1 and 2 or crude venom. This confirms earlier electron microscopic findings of calcium deposits in the heart following scorpion envenomation. There is a concomitant decrease in the calcium and phosphorus content of rat serum following envenomation. It seems that neither scorpion toxins nor scorpion venoms affect the mineral metabolism of the bone. The present investigation indicates that scorpion toxins have not only a neurotoxic action but also broader biological effects such as mineral metabolism.     

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     

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.     

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.