白眉蝮蛇蛇毒5’-核苷酸酶分离纯化及理化性质研究

发布时间：2018-03-18 09:04

本文选题：白眉蝮蛇　切入点：5　出处：《昆明理工大学》2010年硕士论文　论文类型：学位论文

【摘要】：白眉蝮蛇(Agkistrodon blomhoffii Ussurensis)属于蝰蛇科蝮蛇亚科,是我国剧毒蛇类之一,在国内广泛分布河北、甘肃、上海、辽宁、台湾等省市,在国外朝鲜半岛也有分布。白眉蝮蛇毒已作为生产溶血栓药物“蛇毒溶栓酶”的主要原料,但蛇毒中其他活性组分的分离纯化、生物学特性及其经济价值还未得到全面深入研究。 5’-核苷酸酶(5'-nucleotidase)是催化核苷酸分子中的磷酸键水解的一种特异性水解磷酸酶。5’-核苷酸酶能特异地水解5’-核苷酸酶和5’-脱氧核苷酸酶连接戊糖的5’-磷酸,生成对应的核苷或脱氧核苷和无机磷酸。5’-核苷酸酶是一种常规分子生物学酶试剂,因其活性的变化与很多疾病密切相关,还可以用作许多疾病的诊断对照试剂,比如：肝胆疾病、冠心病、肺癌、消化道肿瘤、甲亢、骨病、喉癌等。5’-核苷酸酶广泛分布于细菌、植物细胞和蛇毒毒液中。1951年Hepple首次从蛇毒中分离纯化出5’-核苷酸酶。至今为止已发现二十八种蛇毒5’-核苷酸酶,但尚未有白眉蝮蛇蛇毒5’-核苷酸酶相关研究报道。建立白眉蝮蛇毒5’-核苷酸酶的分离纯化方法,并对其理化性质进行研究,将为白眉蝮蛇毒的深入开发、积累5’-核苷酸酶基础数据等方面都具有重要意义。本论文采用离子交换DEAE Sephadex A-25柱层析、凝胶过滤SephadexG-75柱层析,从白眉蝮蛇蛇毒分离纯化得到了一个5’-核苷酸酶。采用SDS-聚丙烯酰胺凝胶电泳(SDS-Polyacrylamide Gel Electrophoresis, SDS-PAGE)鉴定了其纯度并计算了其分子量,测定了其比活、最适pH、温度等理化性质,考察了常见金属离子对其活性的影响,还观察了简单糖对其活性的保护作用以及其对ADP诱导的血小板聚集过程的影响。经SDS-PAGE此5’-核苷酸酶分子量为70kDa,用高碘酸硝酸银阿利辛蓝染色法鉴定白眉蝮蛇蛇毒5’-核苷酸酶为糖蛋白。白眉蝮蛇蛇毒5，，-核苷酸酶的最适温度为55℃,最适pH为pH 9.0。以AMP为底物测得纯化所得酶蛋白的5，-核苷酸酶活力为562.44U。从目前的实验结果看来,此5’-核苷酸酶对热稳定酶,耐碱性环境。Cu2+、Fe3+浓度范围为(0.05 mmol/L.0.3mmol/L)能抑制白眉蝮蛇蛇毒5’-核苷酸酶活性,Fe3+强烈抑制白眉蝮蛇蛇毒5’-核苷酸酶活性。Mg2+、Ca2+、K+浓度范围为(0.05 mmol/L.0.3mmol/L)均能促进白眉蝮蛇蛇毒5’-核苷酸酶活性,其中Ca2+促进效果更好。低溶度(0.05 mmol/L.0.25mmol/L)β-巯基乙醇对白眉蝮蛇蛇毒5’-核苷酸酶活性有一定促进作用。低浓度(0.05mmol/L.0.25mmol/L)EDTA对白眉蝮蛇蛇毒5’-核苷酸酶活性有促进作用,而0.3 mmol/L EDTA对白眉蝮蛇蛇毒5’-核苷酸酶有明显抑制作用。可能是因为低溶度EDTA能作为金属螯合剂先与抑制白眉蝮蛇蛇毒5’-核苷酸酶的金属离子结合比如Cu2+、Fe3+,随着EDTA溶度增加与促进白眉蝮蛇蛇毒5’-核苷酸酶金属离子结合比如Mg2+、、Ca2+、K+等。导致白眉蝮蛇蛇毒5’-核苷酸酶活性降低。本文还研究了单糖和双糖对酶蛋白的保护作用,实验中采用了葡萄糖、果糖、海藻糖、蔗糖四种。经测定,葡萄糖、果糖、海藻糖对白眉蝮蛇蛇毒5’-核苷酸酶均具有一定的保护作用。糖对白眉蝮蛇蛇毒5’-核苷酸酶保护作用从未有发现,具体机制尚待研究。白眉蝮蛇蛇毒5’-核苷酸酶对血小板聚集有诱导作用,促进ADP诱导的血小板聚集。白眉蝮蛇蛇毒5’-核苷酸酶可以作为血小板聚集的诱导剂。值得注意的是,这一实验现象与目前文献报道其他蛇毒5’-核苷酸酶活性相反。其机制还待进一步深入探讨。
[Abstract]:Halysussuriensis (Agkistrodon blomhoffii Ussurensis) belongs to the Viperidae Agkistrodon subfamily, is one of China's poisonous snakes, widely distributed in the domestic Hebei, Gansu, Shanghai, Liaoning, Taiwan and other provinces and cities in foreign countries, the Korean Peninsula is also distributed. The main raw material from Agkistrodon halys ussuriensis venom thrombolytic drugs have been produced as "fibrolase", but the venom in the separation of other active components of the purification, biological characteristics and economic value has not been fully investigated.
5 '- nucleotidase (5'-nucleotidase) is the catalytic nucleotide molecule in phosphate bond hydrolysis of a specific phosphatase hydrolysis.5' - nucleotidase can specifically hydrolyze 5 '- and 5' - nucleotidase DNA enzyme linked pentose 5 '- phosphate, generate the corresponding nucleoside or deoxyriboside and inorganic phosphate.5' - nucleotidase is a common molecular biological enzyme reagent, due to changes in its activity is closely related to many diseases, the diagnosis of many diseases can also be used as control agents, such as: liver disease, coronary heart disease, lung cancer, gastrointestinal cancer, hyperthyroidism, bone disease, laryngeal.5 '- nucleotidase widely distributed in bacteria, plants cells and venom.1951 Hepple was first isolated from the venom of 5' - nucleotidase. So far, twenty-eight species have been found from 5 '- nucleotidase, but not the snake venom of Agkistrodon 5' nucleoside acid enzyme Research reports.
A white browed Viper 5 '- nucleotidase purification method, and to study its physicochemical properties, for further development of Agkitrodon acutus venom, the accumulation of 5' - nucleotidase based data and other aspects are of great significance.
This paper adopts DEAE Sephadex A-25 ion exchange chromatography, gel filtration chromatography on SephadexG-75, purified from the snake venom of Agkistrodon a 5 '- nucleotidase. Using SDS- polyacrylamide gel electrophoresis (SDS-Polyacrylamide Gel Electrophoresis, SDS-PAGE) identified its purity and its molecular weight was calculated, the determination of the specific activity, the most optimum pH, temperature and other physical and chemical properties, the effects of metal ions on the activity, also observed the protective effect of simple sugars on the activity and its effects on the platelet aggregation induced by ADP.
The SDS-PAGE of the 5 '- nucleotidase molecular weight is 70kDa, with periodate silver nitrate alcian blue staining to identify the snake venom of Agkistrodon 5' - nucleotidase was a glycoprotein. Halysussuriensis venom 5 - an optimum temperature of 55 DEG C nucleotide enzyme, the optimum pH for pH 9.0. with AMP as substrate measured purification the enzyme 5 - nucleotidase activity was 562.44U. from the current view of the experimental results, the 5 '- nucleotidase of thermostable enzymes, alkaline environment.Cu2+, Fe3+ concentration range (0.05 mmol/L.0.3mmol/L) can inhibit the snake venom of Agkistrodon 5' - nucleotidase activity, Fe3+ strongly inhibited the snake venom of Agkistrodon 5 '- nucleotidase activity.Mg2+, Ca2+, K+ concentration range (0.05 mmol/L.0.3mmol/L) could promote the snake venom of Agkistrodon 5' - nucleotidase activity, of which Ca2+ promotes better. Low solubility (0.05 mmol/L.0.25mmol/L) beta mercaptoethanol on Agkitrodon acutus snake venom 5 'nucleoside Have a certain role in promoting acid enzyme activity. Low concentration of EDTA (0.05mmol/L.0.25mmol/L) on the snake venom of Agkistrodon 5 '- nucleotidase activity promoted, while the 0.3 mmol/L EDTA of snake venom of Agkistrodon 5' - nucleotidase is inhibited. Probably because of the low solubility of EDTA can be used as a metal chelator and inhibition of the snake venom of Agkistrodon 5 '- nucleotidase binding metal ions such as Cu2+, Fe3+, EDTA with the solubility was increased and the promotion of the snake venom of Agkistrodon 5' - nucleotidase binding metal ions such as Mg2+, Ca2+, K+, etc. to the snake venom of Agkistrodon 5 '- nucleotidase activity decreased.
This paper also studied the protective effect of monosaccharide and disaccharide on enzyme, was used in the experiment of glucose, fructose, trehalose, sucrose four. The determination of glucose, fructose, trehalose on the snake venom of Agkistrodon 5 '- nucleotidase has certain protective effect on sugar. Agkitrodon acutus snake venom 5' - nucleotidase protection the role has never found that the specific mechanism remains to be investigated.
The snake venom of Agkistrodon 5 '- nucleotidase on platelet aggregation induced by, promote platelet aggregation induced by ADP. The snake venom of Agkistrodon 5' - nucleotidase as a platelet aggregation inducer. It is worth noting that this phenomenon and the reported other venom 5 '- nucleotidase activity and its mechanism to be opposite. To further study.

【学位授予单位】：昆明理工大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R341

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