竹叶青蛇毒磷脂酶A2的分离纯化和性质研究

竹叶青蛇毒磷脂酶Ａ_２的分离纯化和性质研究冯波,吴卫甲,钱嵘,王克夷,周元聪（中国科学院上海生物化学研究所,２０００３１）关键词竹叶青蛇毒,磷脂酶Ａ_２;血小板聚集磷脂酶Ａ。在哺乳动物胰脏和蛇毒毒液中含量较丰富,其中蛇毒磷脂酶Ａ。除能水解甘油磷脂的第二...     

竹叶青属毒蛇的毒理机制与临床治疗新进展

作为中国南方常见毒蛇之一,竹叶青属毒蛇的毒液含有多种生物活性成分。近年来,随着研究的不断深入,竹叶青毒液中各生物活性成分的毒理机制正逐步被阐明。与此同时,竹叶青属毒蛇咬伤的临床治疗技术以及疗效亦在不断发展、提高。本文就近年来对竹叶青属毒蛇毒液活性成分的毒理学研究进展及相应的临床治疗手段等研究资料进行概括与综述。     

蛇毒抗肿瘤作用研究进展

蛇毒含有多种酶类和毒性蛋白,具有广泛的生物学活性。对蛇毒各种组分的研究,发现蛇毒具有抗血栓,止血,镇痛及抗肿瘤的作用。本就其抗肿瘤方面的研究进展进行了综述,介绍了蛇毒的抑瘤机制,抑瘤活性成分的分离纯化,理化特性及研究方向和应用前景。     

蛇毒与细胞因子研究进展

全世界共有蛇类2500余种,其中毒蛇约650余种,估计每年被毒蛇咬伤的人数在30万以上,死亡率约为10％。我国蛇类有160余种,其中毒蛇约有50余种。剧毒、危害剧大的有10种,如眼镜蛇王、金环蛇、眼镜蛇、五步蛇、银环蛇、蝰蛇、蝮蛇、竹叶青、烙铁头、海蛇等,咬伤后能致人于死亡。我国两广地区蛇害严重,每年蛇咬伤的发病率约为25/10000。蛇毒的成分比较复杂．主要由蛋白质、多肽类和多种酶类组成。蛇毒对机体的作用比较复杂,按其有毒成分的毒理作用可分为神经毒、血循环毒和混合毒三类。银环蛇、金环蛇、海蛇的蛇毒主要含神经毒,其主要作用特点为通过多种不同的方式阻断神经一肌肉接头的冲动传递而导致呼吸肌麻痹,是蛇伤致死的主要原因;蝰蛇、五步蛇、烙铁头和竹叶青的蛇毒主要含血循环毒,包括心脏毒、凝血毒、溶血毒、蛋白水解酶、透明质酸酶等;眼镜王蛇等蛇毒属于混合毒,此类蛇毒既含神经毒成分,又含血循环毒成分。     

竹叶青蛇伤的治疗策略研究

目的探讨竹叶青蛇伤后的救治策略,为毒蛇咬伤程序化急诊急救方案提供依据。方法选择55例竹叶青蛇咬伤患者的资料进行分析临床及治疗措施。结果 55例竹叶青蛇咬伤患者按临床分型,血液学变化有统计学意义(P0.01),其中血小板(PLT)、白细胞(WBC)各型比较均有统计学意义(P0.05);竹叶青蛇伤致血液功能障碍主要表现为PLT、纤维蛋白原(Fg)降低,凝血酶原时间(PT)、活化部分凝血活酶时间(APTT)、D-二聚体(DD)和凝血酶时间(TT)延长,甚至出现类DIC。本组危重型患者全部出现类DIC,轻型患者在应用抗蛇毒血清与血液学恢复时间上无统计学意义(P0.05);2例患者在院外行伤口切开排毒出现出血不止,1例在院外诊为"DIC"行肝素抗凝治疗而出现消化道及伤口出血不止。经规范化治疗全部患者均治愈出院。结论按毒蛇伤程序化急诊急救方案治疗竹叶青蛇伤,禁止伤口切开排毒;对重及危重型病例应用同亚科五步蛇、蝮蛇的抗蛇毒血清治疗;对于轻型患者可暂时不用抗蛇毒血清,但应留观,动态观察血液学变化情况;出现类DIC不行抗凝治疗,而是止血、抗纤溶治疗。     

中西医结合救治毒蛇咬伤10例

地处雷州半岛北边的北部湾海岸,眼镜蛇、竹叶青蛇是本地区常见毒蛇之一,被咬伤后若处理不妥易引起肢体伤残。我院自１９９４～１９９８年在无抗蛇毒血清的情况下,采用中西医结合手段治愈眼镜蛇咬伤６例,竹叶青蛇咬伤４例,现总结如下。１　临床资料１１　一般资料　１０例毒蛇咬伤患者中,男３例,女７例,年龄最大６２岁,最小３岁。其中眼镜蛇咬伤６例,竹叶青蛇咬伤４例,均是白天在甘蔗地里劳动被咬伤,夏季在甘蔗地里除草与施肥被眼镜蛇咬伤５例,竹叶青蛇咬伤１例,冬季收甘蔗时被眼镜蛇咬伤１例,竹叶青蛇咬伤３例。１０例患者…     

蛇毒的研究和利用 Ⅰ.我国几种常见毒蛇的蛇毒酶活力的测定

本文对我国几种常见的毒蛇,即五步蛇、蝮蛇、竹叶青蛇、金环蛇、银环蛇、眼镜王蛇以及4种不同产地的眼镜蛇的蛇毒的12种酶活力进行了测定,报告了各活力的数值并对这些结果进行了讨论。     

竹叶青Trimeresurus stejnegeri蛇毒凝血酶样酶的酶学性质研究

本文报道从竹叶青蛇毒中纯化的两个凝血酶样酶(组分Ⅰ和组分Ⅱ)的酶学性质研究。结果表明二者在体外都能使人纤维蛋白原转变为纤维蛋白,具有精氨酸酯酶活性,都能水解凝血酶专一性底物。纤维蛋白平板法证明组分Ⅰ具有激活纤溶作用;组分Ⅱ同时具有激活纤溶和直接纤溶作用;组分Ⅰ和组分Ⅱ能缓慢降解纤维蛋白原的α链,随着作用时间延长,组分Ⅰ还能进一步部分降解纤维蛋白原的β链,活性中心探讨表明,组分Ⅰ的活性中心由丝氨酸蛋白酶部位和糖基部位组成。组分Ⅰ水解BAEE的最适温度为50℃,最适pH为8.6。     

乌梢蛇血清的抗出血因子:一个有前途的抗蛇毒药物原料

用柱层析和聚丙烯酰胺凝胶盘状电泳法,从乌梢蛇血清中分离纯化了一个抗出血因子。用SDS-聚丙烯酰胺凝胶电泳法测得其分子量大约为65 kD;测定了五种蝮亚科蛇毒(尖吻蝮、竹叶青蛇、原矛头蝮、哈扑和短尾蝮)的最小出血剂量和乌梢蛇血清中抗出血因子对这五种蛇毒的抗出血活性;还测定了七种蛇毒(除上述五种毒蛇外,还包括圆斑蝰和银环蛇)的半数致死量,以及抗出血因子对中毒小鼠的治疗作用。结果显示:从乌梢蛇血清中提纯的抗出血因子的抗蛇毒活性,不仅可以抵抗它的捕食者尖吻蝮的蛇毒,而且还可以抵抗具出血活性的其它蛇毒;但它对不具出血活性的银环蛇毒的致死抑制作用不明显。该抗出血因子不仅在体外实验表现出强的中和出血毒素的活性,而且在体内实验中亦表现出对中毒小鼠良好的治疗作用,因而可能成为新的抗蛇毒药物的有前途的原料。乌梢蛇血清对血循毒的中和能力的获得,可能归因于尖吻蝮与乌梢蛇之间捕食与被捕食相互作用的关系。     

竹叶青蛇毒血小板聚集剂的分离纯化及生化特性

经ＳｅｐｈａｄｅｘＧ－７５凝胶过滤和ＦＰＬＣＭｏｎｏＱ阴离子交换柱层析及Ｓｕｐｅｒｏｓｅ－１２凝胶过滤,从竹叶青蛇的蛇毒中纯化到一个能诱导人血小板聚集的均一组分．经ＳＤＳ－聚丙烯酰胺凝胶电泳测定其分子量为６８０００左右．等电点为４．３．测定了它的氨基酸组成,对它活化血小板的作用机理进行了初步研究,结果表明它是一种强的血小板激动剂．     

Snake venom as therapeutic agents: from toxin to drug development

Snake bite injuries and death are socio-medical problems of considerable magnitude. In India a large number of people suffer and die every year due to snake venom poisoning. Snake venom, though greatly feared, is a natural biological resource, containing several components that could be of potential therapeutic value. Use of snake venom in different pathophysiological conditions has been mentioned in Ayurveda, homeopathy and folk medicine. It is well known that snake venom is complex mixture of enzymes, peptides and proteins of low molecular mass with specific chemical and biological activities. Snake venom contains several neurotoxic, cardiotoxic, cytotoxic, nerve growth factor, lectins, disintrigrins, haemorrhagins and many other different enzymes. These proteins not only inflict death to animals and humans, but can also be used for the treatment of thrombosis, arthritis, cancer and many other diseases. An overview of various snake venom components that have prospects in health and diseases are discussed in this review.     

Optical immunoassay for snake venom detection

A sensitive and specific optical immunoassay (OIA) has been developed for snake venom detection. The assay is based on the principle of detection of physical changes in thickness of molecular thin film resulting from specific binding events on an optical silicon chip (SILAS-I, ThermoBioStar, Colorado, USA). The reflection of white light through the thin film results in destructive interference of a particular wavelength of the light from gold to purple-blue depending on the thickness of the thin film formed or the amount of venom in the test sample. A prototype test kit for the simultaneous identification of species and semi-quantitative detection of venoms from four medically important snakes of South Vietnam (Trimeresurus albolabris, Calloselasma rhodostoma, Naja kaouthia and Ophiophagus hannah) has been developed. The kit can detect venom analytes in blood, plasma, urine, wound exudates, blister fluid or tissue homogenates. The efficacy of the test kit in snakebite diagnosis has been demonstrated in experimental envenomations and sample analytes taken from snakebite victims in South Vietnam. This rapid snake venom detection kit based on OIA technique is potentially applicable in the clinics as well as in the field.     

Minor snake venom proteins: Structure,function and potential applications

Snake venoms present a great diversity of pharmacologically active compounds that may be applied as research and biotechnological tools, as well as in drug development and diagnostic tests for certain diseases. The most abundant toxins have been extensively studied in the last decades and some of them have already been used for different purposes. Nevertheless, most of the minor snake venom protein classes remain poorly explored, even presenting potential application in diverse areas. The main difficulty in studying these proteins lies on the impossibility of obtaining sufficient amounts of them for a comprehensive investigation. The advent of more sensitive techniques in the last few years allowed the discovery of new venom components and the in-depth study of some already known minor proteins. This review summarizes information regarding some structural and functional aspects of low abundant snake venom proteins classes, such as growth factors, hyaluronidases, cysteine-rich secretory proteins, nucleases and nucleotidases, cobra venom factors, vespryns, protease inhibitors, antimicrobial peptides, among others. Some potential applications of these molecules are discussed herein in order to encourage researchers to explore the full venom repertoire and to discover new molecules or applications for the already known venom components.     

Characterization of hyaluronidase isolated from Agkistrodon contortrix contortrix (Southern Copperhead) venom

Snake venoms are a rich source of enzymes including many hydrolytic enzymes. Some enzymes such as phospholipase A2, proteolytic enzymes, and phosphodiesterases are well characterized. However many enzymes, such as the glycosidase, hyaluronidase, have not been studied extensively. Here we describe the characterization of snake venom hyaluronidase. In order to determine which venom was the best source for isolation of the enzyme, the hyaluronidase activity of 19 venoms from Elapidae, Viperidae, and Crotalidae snakes was determined. Since Agkistrodon contortrix contortrix venom showed the highest activity, this venom was used for purification of hyaluronidase. Molecular weight was determined by matrix-assisted laser desorption ionization mass spectroscopy and was found to be 59,290 Da. The molecular weight value as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 61,000 Da. Substrate specificity studies indicated that the snake venom enzyme was specific only for hyaluronan and did not hydrolyze similar polysaccharides of chondroitin, chondroitin sulfate A (chondroitin 4-sulfate), chondroitin sulfate B (dermatan sulfate), chondroitin sulfate C (chondroitin 6-sulfate), chondroitin sulfate D, chondroitin sulfate E, or heparin. The enzyme is an endo-glycosidase without exo-glycosidase activity, as it did not hydrolyze p-nitrophenyl-beta-D-glucuronide or p-nitrophenyl-N-acetyl-beta-D-glucosaminide. The main hydrolysis products from hyaluronan were hexa- and tetrasaccharides with N-acetylglucosamine at the reducing terminal. The cleavage point is at the beta1,4-glycosidic linkage and not at the beta1,3-glycosidic linkage. Thus, snake venom hyaluronidase is an endo-beta-N-acetylhexosaminidase specific for hyaluronan.     

Sequence of a cDNA clone encoding the zinc metalloproteinase hemorrhagic toxin e from Crotalus atrox: evidence for signal, zymogen, and disintegrin-like structures.

The sequence of two overlapping cDNA clones for the zinc metalloproteinase hemorrhagic toxin e (also known as atrolysin e, EC 3.4.24.44) from the venom gland of Crotalus atrox, the Western diamondback rattlesnake, is presented. The assembled cDNA sequence is 1975 nucleotides in length and encodes an open reading frame of 478 amino acids. The mature hemorrhagic toxin e protein as isolated from the crude venom has a molecular weight of approximately 24,000 and thus represents the processed product of this open reading frame. From the deduced amino acid sequence, it can be hypothesized that the enzyme is translated with a signal sequence of 18 amino acids, an amino-terminal propeptide of 169 amino acids, a central hemorrhagic proteinase domain of 202 amino acids, and a carboxy-terminal sequence of 89 amino acids. The propeptide has a short region similar to the region involved in the activation of matrix metalloproteinase zymogens. The proteinase domain is similar to other snake venom metalloproteinases, with over 57% identity to the low molecular weight proteinases HR2a and H2-proteinase from the Habu snake Trimeresurus flavoviridis. The carboxy-terminal region, which is not observed in the mature protein, strongly resembles the protein sequence immediately following the proteinase domain of HR1B (a high molecular weight hemorrhagic proteinase from the venom of T. flavoviridis) and the members of a different family of snake venom polypeptides known for their platelet aggregation inhibitory activity, the disintegrins. The cDNA sequence bears striking similarity to a previously reported sequence for a disintegrin cDNA. This report is evidence that this subfamily of venom metalloproteinases is synthesized in a proenzyme form which must be proteolytically activated.(ABSTRACT TRUNCATED AT 250 WORDS)     

烙铁头蛇毒L—氨基酸氧化酶的分离、纯化及其生物学活性

通过DEAESephadexA 5 0阴离子交换柱 ,SephadexG 75分子筛 ,ResourseQ阴离子交换柱三步层析从湖南产的烙铁头蛇毒中分离、纯化得到一个L 氨基酸氧化酶 (TM LAO) ,它由两个非共价的亚基组成 ,每个亚基的分子量为 5 5kD。与台湾产的烙铁头蛇毒L 氨基酸氧化酶分子量 ( 70kD)不同。TM LAO的N末端氨基酸序列是ADNKNPLEECFRETNYEEFLEIAR ,与报道的蝰科的L 氨基酸氧化酶的相似性比眼镜蛇科的要高。TM LAO能抑制大肠杆菌、金黄色葡萄球菌和痢疾杆菌的生长 ,杀死肿瘤细胞以及诱导血小板聚集。这些活性能被过氧化氢酶所抑制 ,说明TM LAO生理学功能主要是通过酶反应产生的过氧化氢 (H2 O2 )介导的     

Plant natural products active against snake bite--the molecular approach

The article surveys the substances identified in plants reputed to neutralize the effects of snake venoms. Protective activity of many of them against the lethal action of the venom of the jararaca (Bothrops jararaca) snake was confirmed by biological assays. It was shown that all belong to chemical classes capable of interacting with macromolecular targets--receptors and enzymes. In a few cases it has been shown that exogenous natural micromolecules can mimic the biological activity of endogenous macromolecules. From the evidence presented, it can be inferred that micromolecules which neutralize the action of snake venoms mechanistically replace endogenous antitoxic serum proteins with venom neutralizing capacity such as produced by some animals.     

菜花烙铁头蛇毒金属蛋白酶Jerdonitin的分离纯化和理化性质

以前从菜花烙铁头蛇毒中分离纯化到Jerdonitin。与其他Ⅱ型蛇毒金属蛋白酶相比,Jerdonitin由金属蛋白酶和去整合素两个结构域组成。但没有检测到其出血和纤维蛋白原降解活性,推测可能高压液相色谱的有机溶液影响了其酶活性。采用不含高压液相色谱柱层析的新分离手段分离得到Jerdonitin。Jerdonitin在还原和非还原SDS—PAGE电泳中分别呈现一条表观分子量为38和36kDa的条带。像其他典型的蛇毒金属蛋白酶一样,Jerdonitin优先降解人纤维蛋白原的alpha链,并且该活性能被EDTA完全抑制,而PMSF对其没有影响。Jerdonitin不诱导小白鼠皮下出血。     

A comparative study of the enzymatic and toxic properties of venoms of the Asian lance-headed pit viper (Genus Trimeresurus)

1. The lethalities, anticoagulant effects, hermorrhagic, thrombin-like enzyme, hyaluronidase, protease, arginine ester hydrolase, 5'-nucleotidase, L-amino acid oxidase, alkaline phosphomonoesterase, phosphodiesterase and phospholipase A activities of twenty-three samples of venoms from twelve species of Asian lance-headed pit vipers (genus Trimeresurus) were examined. 2. The results indicate that notwithstanding individual variations in venom properties, the differences in biological properties of the Trimeresurus venoms can be used for the differentiation of venoms from different species of Trimeresurus. 3. The results also suggest that differences in the biological properties of snake venoms are useful parameters in the classification of snake species. 4. Our results indicate that venoms from the species T. okinavensis exhibited biological properties markedly different from other Trimeresurus venoms examined. This observation supports the recently proposed reclassification of T. okinavensis as a member of the genus Ovophis, rather than the genus Trimeresurus.     

蛇毒素蛋白毕赤酵母表达进展

蛇毒是许多具有独特生物活性的蛋白质与酶的混合物,在基础科学研究和临床上有重大应用价值,但是通过从蛇毒中分离获取活性组分具有局限性。巴斯德毕赤酵母表达系统是最为常用的真核表达系统之一,其真核加工、折叠、翻译后修饰等能力使得所表达的重组蛋白具有与天然蛋白近似的生物活性,因而该系统在富含二硫键或糖基化的蛇毒素蛋白表达中被广为采用。迄今为止,已经有12个属的25种蛇毒素蛋白(包括蛇毒丝氨酸蛋白酶、金属蛋白酶/去整合素、L-氨基酸氧化 酶、C-型凝集素和神经毒素、血管收缩因子、神经生长因子等家族)在毕赤酵母中获得成功表达,蛇毒富半胱氨酸蛋白、缓激肽增强肽(BPP)等至今尚未见酵母表达的报道。毕赤酵母表达蛇毒素蛋白失败的原因可能在于,有关密码子偏爱性、目的基因转录出的RNA二级结构特征、糖基化程度不均一及糖型差异、所表达毒素对酵母细胞的毒性等方面,并对解决的方法进行了讨论。