Agacutase体外水解牛纤维蛋白原机制的研究(英文)

Agacutase是自尖吻蝮蛇蛇毒中分离出的新的具有止血功能的蛇毒类凝血酶,它能够将纤维蛋白原转化为纤维蛋白单体。通过SDS-PAGE和ELISA方法,我们研究了Agacutase体外水解牛纤维蛋白原的分子机制。实验结果显示,Agacutase仅仅水解牛纤维蛋白原的α亚基并释放血纤肽A,而对牛纤维蛋白原的β和/或γ亚基没有影响。研究表明Agacutase属于血纤肽A类(FP-A类型)的类凝血酶。     

黑眉蝮蛇蛇毒纤溶酶的纯化及其性质研究

目的从黑眉蝮蛇蛇毒中纯化出无出血毒的纤溶酶。方法用DEAE-Sepharose FF阴离子交换树脂、CM-sepharose FF阳离子交换树脂和Sephacryl S-100凝胶过滤树脂三步分离方法,从黑眉蝮蛇蛇毒中纯化出一种纤溶酶活性成分。结果经SDS-聚丙烯酰胺凝胶电泳检测为单一蛋白质,相对分子质量为31 800,小鼠皮下注射无出血反应。该酶与纤维蛋白原保温15 min能迅速水解Bβ(β)链,随后缓慢水解Aα(α)链,而对γ(γ-γ)链无影响。结论此工艺可以快速纯化黑眉蝮蛇蛇毒纤溶酶。     

尖吻蝮蛇毒和蝮蛇毒凝血酶样酶促凝的协同作用

尖吻蝮蛇毒和蝮蛇毒凝血酶样酶促凝的协同作用魏文利１，孙家钧，陈家树（中山医科大学药理教研室，广州５１００８９，中国）关键词尖吻蝮蛇；蝮蛇；蛇毒；凝血酶类；血液凝固目的：尖吻蝮蛇（ＤＡ）和蝮蛇（ＡＨ）蛇毒凝血酶样酶（ＴＬＥ）对促凝作用的协同效果．方法：...     

降纤酶研究进展

降纤酶(Defibrase)是从尖吻蝮蛇Agkistrodon acutus和长白山白眉蝮蛇Agkistrodon halys ussuriensis蛇毒中提取分离得到的一种类凝血酶。具有显著的去纤、降粘、溶栓等作用,广泛地应用于临床治疗和预防心脑血管血栓性疾病。对降纤酶的生化性质与结构、生产制备、药理作用与临床应用以及质量分析与控制等方面的研究进行了综述。     

大剂量尖吻蝮蛇凝血酶对家兔血浆纤维蛋白原的影响

目的探讨大剂量尖吻蝮蛇凝血酶对家兔血浆纤维蛋白原的影响,为临床应用提供参考。方法家兔耳缘静脉注射不同剂量的尖吻蝮蛇凝血酶,并于给药后不同时间检测其血浆纤维蛋白原含量。结果0.2～1.0U·kg-1剂量的尖吻蝮蛇凝血酶可导致家兔血浆纤维蛋白原明显下降(P<0.05),下降速度及下降幅度与剂量成正相关。剂量≥0.4U·kg-1时家兔血浆纤维蛋白原开始出现耗竭,剂量≥0.8U·kg-1时所有家兔血浆纤维蛋白原均出现耗竭。结论大剂量尖吻蝮蛇凝血酶具有明显降低家兔血浆纤维蛋白原的作用。     

蛇毒对神经疾病的治疗作用

蛇毒中90％-95％是蛋白质，包括毒素，非毒性蛋白，酶及活性多肽。蛇毒的非蛋白成份包括金属离子，无机阴离子和有机小分子化合物。生物活性物质大部分是蛋白质。在蛇毒含有的酶中，应用较广泛的是类凝血酶，目前我国研制的清栓酶，蕲蛇酶等蛇毒酶制剂，分别取自于白眉蝮蛇、尖吻蝮蛇等。国外则多从马来西亚红口蝮蛇及南美洲矛头蝮蛇中提取。这些酶制剂可引起血浆纤维蛋白原浓度降低，表现为抗凝效应，抑制血栓形成过程，降低血液粘度，从而改善血液循环。     

白唇竹叶青蛇(Trimeresurus albolabris)毒降纤酶的分离纯化以及性质

目的从白唇竹叶青蛇(T.albolabris)毒中分离纯化无出血作用的降纤活性组分,探讨其理化性质及部分生物功能。方法用DEAE-SephadexA-25,SephadexG-100和CM-SephadexC-50三步色谱法进行分离纯化。SDS-PAGE和HPLC鉴定其纯度和相对分子质量,平板法测定其降纤活性。结果从白唇竹叶青蛇毒中分离纯化获得单一的降纤组分,能迅速水解纤维蛋白原或纤维蛋白原Aα链,缓慢水解Bβ链,而对γ链无作用,SDS-PAGE鉴定其相对分子质量为56000。EDTA能抑制其纤维蛋白原水解活性,而PMSF、β-巯基乙醇对其活性无影响,提示该组分为单链α金属蛋白酶。结论从白唇竹叶青蛇毒中分离纯化得到1种无出血作用且降纤活性强的新蛇毒降纤酶。     

大剂量尖吻蝮蛇凝血酶对家兔血浆纤维蛋白原的影响

目的 探讨大剂量尖吻蝮蛇凝血酶对家兔血浆纤维蛋白原的影响,为临床应用提供参考。方法 家兔耳缘静脉注射不同剂量的尖吻蝮蛇凝血酶,并于给药后不同时间检测其血浆纤维蛋白原含量。结果 0.2~1.0 U·kg^-1剂量的尖吻蝮蛇凝血酶可导致家兔血浆纤维蛋白原明显下降(P<0.05),下降速度及下降幅度与剂量成正相关。剂量≥0.4 U·kg^-1时家兔血浆纤维蛋白原开始出现耗竭,剂量≥0.8 U·kg^-1时所有家兔血浆纤维蛋白原均出现耗竭。结论 大剂量尖吻蝮蛇凝血酶具有明显降低家兔血浆纤维蛋白原的作用。     

长白山白眉蝮蛇血凝酶的分离纯化研究

摘 要 目的：建立一种分离纯化长白山白眉蝮蛇血凝酶的方法。方法: 先后采用分子筛层析法、离子交换色谱法、肝素 Sepharose亲和层析法从长白山白眉蝮蛇蛇毒中分离纯化得到一种具有有凝血活性的酶成分,并采用SDS-Page法、RP-HPLC法测定其纯度,凝胶电泳法(SDS-Page)考察其对牛纤维蛋白原的作用方式、高效空间排阻色谱法（HPSEC）测定其相对分子质量,等电聚焦电泳分析法（IEF）测定其等电点,Lowry法测定其蛋白浓度。结果: 从长白山白眉蝮蛇蛇毒中分离纯化了一种凝血酶成分,SDS-Page显示为一条带,HPLC得到单一的色谱峰,该酶只作用于纤维蛋白原的α链,其相对分子质量为32.2 kD,等电点为5.21,此酶具有体外凝血活性。结论:该方法可用于长白山白眉蝮蛇血凝酶的分离纯化。     

尖吻蝮蛇毒小分子多肽的分离及抗血小板聚集作用

目的从尖吻蝮蛇毒中分离纯化一种抗血小板聚集小分子多肽,研究其理化性质以及对ADP、胶原、凝血酶诱导的血小板聚集反应的影响。方法经SephadexG-75凝胶过滤,超滤,DEAE-SepharoseCL-6B离子交换层析法分离纯化蛇毒组分,采用高效液相鉴定纯度,用SDS-凝胶电泳(SDS-PAGE)测定其分子量,用比浊法测定其抗血小板聚集活性。结果从尖吻蝮蛇毒中分离相对分子质量约为7862u等电点为4.29的组分,该组分能抑制由ADP、胶原、凝血酶诱导的血小板聚集并成剂量依赖性。结论此法成功地从尖吻蝮蛇毒中纯化出抗血小板聚集组分。该组分与去整合素比较相似,可能属于去整合素家族。     

Cross-neutralization of thrombin-like enzymes in snake venoms by polyvalent antivenoms

and . Cross-neutralization of thrombin-like enzymes in snake venoms by polyvalent antivenoms. Toxicon 27, 1397–1399, 1989.—Five polyvalent antivenoms (Crotalidae; Orient, North, Central and South Africa) were tested for their ability to neutralize the thrombin-like activity of snake venoms (Bitis gabonica, Agkistrodon acutus, Bothrops asper, B. atrox, Crotalus adamanteus). Considerable cross-neutralization was observed. Anti-coagulase antibodies were isolated from an antivenom by affinity chromatography using a purified enzyme from Bitis gabonica venom. These antibodies neutralized the activity of most snake venom coagulant enzymes.     

长白山白眉蝮蛇蛇毒中类凝血酶的分离纯化方法研究

目的:研究长白山白眉蝮蛇蛇毒中类凝血酶的简单分离纯化方法。方法:采用DEAE-Sephadex A-25及Sephadex G-25层析的方法,比较二者对长白山白眉蝮蛇蛇毒中类凝血酶简单分离纯化的效果。结果:从长白山白眉蝮蛇蛇毒中分离出类凝血酶,SDS-PAGE电泳显示为一条带,分子量大约为35.5kDa,达到电泳纯。理化性质研究表明,此类凝血酶具有体外凝血活性,体外凝血酶比活力为12.57IU.mg-1,用N-苯甲酰-L-精氨酸乙酯盐酸盐测得该酶的精氨酸酯酶比活力为137.65IU.mg-1。用蛋白酶抑制剂和乙二胺四乙酸对该酶进行抑制实验,结果表明该酶属于丝氨酸蛋白酶,而不是金属蛋白酶。结论:本方法可用于长白山白眉蝮蛇蛇毒中类凝血酶的分离纯化。     

~(125)I尖吻蝮蛇毒凝血样酶在兔体内的药物动力学

~(125)Ⅰ标记尖吻蝮蛇毒凝血样酶,放化纯95.5％和比放射性188.7 MBq/mg。 兔iv ~(125)Ⅰ-TLE后,血放射性—时间曲线符合二室模型,算出药物动力学参数。组织分布以肾、胆、肝的放射性最高。     

Characterization of a thrombin-like serine protease, Kangshuanmei, isolated from the venom of a Chinese snake, Agkistrodon halys brevicaudus stejneger

An enzyme, referred to as Kangshuanmei, was isolated from the venom of the Chinese snake Agkistrodon halys brevicaudus stejneger by gel filtration chromatography followed by affinity chromatography. Kangshuanmei is composed of a single polypeptide chain with a molecular weight of approximately 34,000, estimated by SDS-PAGE. The enzyme hydrolyzed both benzoyl-arginine ethyl ester and H-D-Phe-Pip-Arg-p-nitroanilide, specific substrates for thrombin. The protease activity of Kangshuanmei was inhibited by 4-(2-aminoethyl)-benzensulfonyl fluoride, but was not affected by EDTA. The enzyme acted on human fibrinogen to form a fibrin clot and released three fragments. These fragments were shown to be fibrinopeptide A, fibrinopeptide B, and the Bbeta1-42 peptide of fibrinogen, respectively. These results indicate that Kangshuanmei is a thrombin-like serine protease with coagulant activity. However, the enzyme did not induce activation of blood coagulation factor XIII, unlike thrombin. Moreover, antithrombin-III, the specific thrombin inhibitor in plasma, had no inhibitory effect on the thrombin-like amidolytic activity of Kangshuanmei. The N-terminal amino acid sequence of the enzyme up to 50 residues was determined by a peptide sequencer. The N-terminal sequence of Kangshuanmei was highly homologous to most thrombin-like serine proteases from the venom of the snakes of the crotalidae family.     

液相色谱分离纯化五步蛇毒凝血酶样酶

目的：改进分离工艺，找到液相色谱分离纯化五步蛇毒中凝血酶样酶的最佳方案。方法：五步蛇毒依次经SuperdexG75凝胶过滤、DEAE．SephroseF．F阴离子交换和SuperdexG30凝胶过滤，通过改变洗脱液pH值、流速、Tris浓度、盐浓度及上样量来考察对分离纯化五步蛇毒中凝血酶样酶的影响。结果：Superdex75凝胶过滤色谱分离纯化受洗脱液流速、Tris浓度、盐浓度、pH值、上样量的影响；DEAE—SephroseF．FN离子交换色谱受盐浓度的影响：SuperdexG30凝胶过滤色谱受上样量的影响。三步分离纯化后获得一种五步蛇毒凝血酶样酶，分子量约23kD。结论：依次经SuperdexG75、DEAE—SephroseF．F和SuperdexG30凝胶，通过改变洗脱液流速、Tris浓度、盐浓度、pH值、上样量等能得到最佳分离条件．最终可获得五步蛇毒凝血酶样酶的纯品。     

Immunological properties of the fibrinolytic enzyme (fibrolase) from southern copperhead (Agkistrodon contortrix contortrix) venom and its purification by immunoaffinity chromatography

An antibody to the fibrinolytic enzyme in southern copperhead venom was produced by immunizing rabbits with chromatographically purified enzyme. The antibody was purified from rabbit blood by ammonium sulfate fractionation and protein-A affinity chromatography. The purified antibody reacted only with the fibrinolytic enzyme in southern copperhead venom as demonstrated by immunodiffusion and immunoelectrophoresis. Western immunoblotting revealed that several snake venoms, including Agkistrodon piscivorus conanti, Crotalus atrox, Crotalus basiliscus basiliscus, and Bothrops asper, cross-reacted with the antibody to varying degrees. However, Deinagkistrodon acutus showed no cross-reaction. Immobilized antibody has been used, in combination with molecular sieve chromatography, to purify the fibrinolytic enzyme from southern copperhead venom. In this two-step purification procedure, the enzyme was purified in good yield within two days. The specific activity of the enzyme purified by the immunoaffinity chromatography procedure is comparable with that of enzyme purified by a four-step chromatographic procedure. The mol. wt of the purified enzyme is approximately 23,000-24,000 as determined by SDS-PAGE. Interestingly, the enzyme purified by this two-step immunoaffinity chromatography procedure possesses virtually no hemorrhagic activity.     

过碘酸钠的体内抗蛇毒作用

用体内毒素中和试验方法研究ＮａＩＯ４的抗蛇毒作用。结果发现：分别以２００μｇ五步蛇毒、７０μｇ蝮蛇毒和４０μｇ眼镜蛇毒对小白鼠进行攻击，４６．７５３ｍｍｏｌ·Ｌ－１的ＮａＩＯ４０．１ｍｌ对小白鼠有非常明显的保护作用（Ｐ＜０．０１）。提示ＮａＩＯ４对多种蛇毒均具有较强的抗蛇毒作用。