白眉蝮蛇蛇毒纤溶酶的分离纯化

目的用白眉蝮蛇蛇毒分离纯化纤溶酶。方法利用离子交换层析、亲和层析、分子筛层析技术进行分离纯化纤溶酶。结果得到了电泳纯的纤溶酶,其相对分子质量为24000。结论该工艺可以快速地分离纯化白眉蝮蛇蛇毒纤溶酶：     

五步蛇毒纤溶组分FⅨcaⅠ的分离纯化和生物活性的测定

目的从五步蛇(安徽产)毒分离纯化一种具有纤溶活性的组分FⅨcaⅠ,并研究其理化性质和生物活性。方法应用DEAE-Sephadex A-50阴离子交换层析、Sephadex G-75凝胶层析、Chelating Sepharose Fast Flow金属离子螯合亲和层析和Sephadex G-50凝胶层析四步分离纯化目的组分FⅨcaⅠ;纤维蛋白平板法和SDS-PAGE测定FⅨcaⅠ的生物活性;通过小鼠皮下注射不同剂量的FⅨcaⅠ,测量皮下出血斑的面积并求出最小出血剂量。结果从五步蛇毒分离纯化的FⅨcaⅠ组分为单体蛋白,相对分子量23 ku,等电点为4.8。FⅨcaⅠ可降解纤维蛋白和纤维蛋白原,优先降解α链,呈时效、量效关系。蛇毒纤溶酶FⅨcaⅠ最小出血剂量为54.9μg,为非出血性蛇毒纤溶酶。结论从五步蛇(安徽产)毒分离纯化的FⅨcaⅠ是一种分子量较小,比较稳定,纤溶活性强,可直接降解纤维蛋白且非出血性的新蛇毒纤溶酶。     

蝮蛇毒纤溶酶的分离纯化及性质研究

目的 :寻找一种分离纯化蝮蛇毒纤溶酶的工艺并研究其理化性质。方法 :采用DEAE SepharoseCL 6B和HeparinCL 6B层析方法 ,从蝮蛇毒中分离纯化纤溶酶。结果 :蝮蛇毒纤溶酶经HPLC为单一峰 ,等电聚焦电泳为一条带 ,其等电点为 4.5 5 ,经SDS 聚丙烯酰胺凝胶电泳测得分子量为 2 9.4kD。该酶对热不稳定 ,在 pH6～ 9时稳定 ,氨基酸组成分析表明含酸性氨基酸较多。结论 :用此工艺可制得高纯度的蝮蛇毒纤溶酶。     

蝰蛇(缅甸亚种)毒促凝—纤溶双相活性组分FⅥbb的分离纯化及生物活性

目的从蝰蛇(缅甸亚种)毒分离纯化一种促凝—纤溶双相活性组分FⅥbb,并研究其理化性质和生物活性。方法应用CM-Sephadex C-50阳离子交换层析Sephadex G-150(超细)凝胶过滤层析Chelating Sepharose Fast Flow金属离子螯合亲和层析分离纯化蛇毒,反相HPLC检测组分FⅥbb纯度,采用MALDI质谱测定法测定分子量,以发色底物法、纤维平板法和SDS-PAGE测定FⅥbb的酶学特征和生物活性。结果从蝰蛇(缅甸亚种)毒分离得到的促凝—纤溶双相活性组分FⅥbb为单体蛋白,相对分子质量为59 138,最适温度为40℃,最适pH为10.0,为金属蛋白酶。结论应用CM-Sephadex C-50阳离子交换层析和Chelating Sepha-rose Fast Flow金属离子螯合亲和层析等方法可以从蝰蛇(缅甸亚种)毒纯化出促凝—纤溶双相活性组分FⅥbb,具有促凝—纤溶双相活性。     

蝮蛇毒纤溶酶的分离纯化及其制剂的临床应用

目的:采用凝胶电泳法分离纯化长白山白眉蝮蛇毒纤溶酶并将其制剂应用于临床。方法:采用DEAE- Sepharose CL-6B和Heparin CL-6B层析方法,从蝮蛇毒中分离纯化纤溶酶,通过临床应用分析成品制剂的治疗效果。结果:蝮蛇毒纤溶酶经HPLC为单一峰,等电聚焦电泳为一条带,其等电点为4．55,经SDS-聚丙烯酰胺凝胶电泳测得分子量为29．4kD,将其成品制剂应用于临床,结果表明有效减少了缺血性脑血管病血栓的形成,使缺血部位迅速恢复功能。     

蚯蚓纤溶酶的亲和层析纯化及部分性质

用牛血纤溶酶原Ｓｅｐａｒｏｓｅ４Ｂ亲和层析方法从蚯蚓粗提物丙酮粉中分离纯化一种纤维蛋白溶解酶。该酶为糖蛋白，含糖量约为１．４３％，Ｍｒ为３００００。实验结果表明此酶具有直接溶解纤维蛋白和激活纤溶酶原的间接溶解纤维蛋白的双重作用，并对人血凝块有明显的溶解作用。１％ＰＭＳＦ对酶有显著的抑制作用，说明此蚯蚓纤溶酶为典型的丝氨酸蛋白酶类酶。     

原矛头蝮蛇毒中一个新颖双链纤溶酶的分离纯化与性质研究

目的从原矛头蝮蛇毒中寻找新型纤溶酶,对其理化性质与生物学活性进行研究,以了解其在蛇伤中的作用与毒性机制,并评估其潜在的应用价值。方法采用蛋白层析技术分离纯化获得目标蛋白,检测其分子量、等电点、肽指纹图谱、多种蛋白水解活性、抗补体活性、出血活性、水肿活性及对流血时间的影响。结果通过阴离子交换层析、凝胶过滤层析、亲和层析从原矛头蝮蛇毒中纯化出一个酸性纤溶酶PMSP-A,它是由两条等电点分别为5.7与6.1的非均等肽链共价结合而成。SDS-PAGE和凝胶过滤测定其分子量分别为26.1 ku与25.3 ku。肽指纹图谱分析表明PMSP-A与黄绿烙铁头蛇毒中的血液凝固结合因子有部分序列吻合。PMSP-A能够依次降解纤维蛋白原的Bβ、Aα链,该活性能被PMSF、1,10-phenanthroline抑制,EDTA、EGTA、SBTI不能抑制其活性。PMSP-A具有纤维蛋白、精氨酸酯水解活性,没有偶氮酪蛋白水解活性。它还具有抗补体活性。动物实验表明,PMSP-A明显延长小鼠尾静脉流血时间,能诱导小鼠足趾轻度水肿,无皮下出血活性。结论 PMSP-A是一个新颖的原矛头蝮蛇毒双链丝氨酸蛋白酶,具有多种影响机体的生物学活性。     

一种具有纤溶活性的蛋白酶的分离纯化及性质

目的分离纯化BacillussubtilisJin2 1发酵产生的具有纤溶活性的蛋白酶 ,并对其性质进行研究。方法通过硫酸铵分级盐析 ,SerineSepharose 2B和SephacrylS— 2 0 0色谱柱进行分离纯化 ,用SDS PAGE测定纯度 ,SDS PAGE和凝胶层析测定相对分子质量 ,纤维蛋白平板法测定酶活力。结果分离得到了电泳纯的酶 ,该酶的相对分子质量为 2 8kD ,等电点约为 8 6 ,最适 pH为 7 5 ,最适温度为 4 0℃ ,在 4 0℃以下较稳定 ,超过 5 0℃时酶活力开始下降 ;金属离子Mn2 + 对酶有明显的激活作用 ,而Zn2 + 对酶有抑制作用 ,二异丙基磷酰氟 (DFP)和苯甲基磺酰氟 (PMSF)完全抑制酶活性。结论该酶是一种丝氨酸蛋白酶 ,与纳豆激酶相似 ,有望开发成为一种新型口服溶栓药物     

刺桐胰蛋白酶抑制剂的高效表达、纯化、亲和介质的制备及其在r-PA纯化中的应用

刺桐胰蛋白酶抑制剂(ETI)是一种丝氨酸蛋白酶抑制剂,能够特异性结合胰蛋白酶及组织型纤溶酶原激活剂,抑制其活性。因此ETI可以作为配体与固定相偶联制备亲和层析介质,用于tPA(组织型纤溶酶原激活剂)及其突变体的纯化。利用基因工程方法构建了ETI的高表达工程菌株,诱导表达后目的蛋白占总蛋白的39.6%,经包涵体变性、复性、纯化,制备了纯度高于96%的ETI样品,收率达到了63.5%。利用纯化的ETI,偶联制备了亲和层析介质,偶联率达到99%。用制备的ETI亲和层析介质纯化瑞替普酶(r-PA),获得的r-PA样品纯度高于95%,生物学活性高于5×105U/mg。本研究为ETI的进一步生产及应用奠定了基础。     

蛇毒纤溶酶原激活剂的研究进展

蛇毒是含有多种生物活性蛋白质和多肽的混合物,其中一些蛋白可作用于人的凝血系统。蛇毒纤溶酶原激活剂是具独特活性的蛇毒丝氨酸蛋白酶,具有较长的半衰期,可专一性地裂解人Glu-纤溶酶原的Arg^561-Val^562肽键,使之变成有活性的纤溶酶,从而发挥溶栓作用。该文主要对蛇毒纤溶酶原激活剂的结构特点和作用机制进行了阐述,旨在为新型溶栓剂的研究提供方向。     

Antithrombotic effect of a novel tissue plasminogen activator from the venom of Gloydius brevicaudus viper

OBJECTIVE To investigate the thrombolytic and antiplatelet effects of a novel plasminogen activator from the venom of Gloydius brevicaudus viper(GBV-PA)in vitro and in vivo.METHODS Thrombolytic experiments were performed in rabbit models of ear vein thrombosis and carotid artery thrombosis,and in dog model of acute cerebral infarction.Inhibition of thrombus formation was evaluated in rat inferior vena cava thrombosis model and ferric chloride-induced arterial thrombosis.In vitro,we assayed the antithrombotic effect of GBV-PA on rabbit blood clots,euglobulin lysis time(ELT)of rabbit plasma,and on ADP-induced platelet aggregation.RESULTS GBV-PA intravenous administ ration significantly reduced vascular recanalization times of rabbit ear veins thrombosis and thrombus weight of rabbit carotid artery thrombosis.The arterial recanalization rates were dose-and time-dependently improved after administration of GBV-PA in canine acute cerebral infarction model.Thrombus length and weight was significantly reduced by GBV-PA both in rat inferior vena cava and ferric chloride-induced arterial thrombosis models.Thrombus formation of blood of rabbits they were administrated with GBV-PA was also inhibited.GBV-PA radically reduced plasma ELT of rabbit′s blood clots.ADP-induced platelet aggregation was inhibited by GBV-PA in a dose-dependent manner with a half-maximal inhibitory concentration of 19.9μg·mL-1.CONCLUSION This study demonstrates that GBV-PA is a thrombolytic and antiplatelet agent.It has significant antithrombotic effects on various in vitro and in vivo experimental models of thrombosis.The mechanisms that underline its antithrombotic effects were related to GBV-PA′s capabilities of increasing fibrinolytic activities and inhibition of platelet aggregation.     

Hydrophilic interaction/cation-exchange chromatography for the purification of synthetic peptides from closely related impurities: serine side-chain acetylated peptides

Abstract: Mixed-mode hydrophilic interaction/cation-exchange chromatography (HILIC/CEC) is a novel HPLC technique which has excellent potential for peptide separations. Separations by HILIC/CEC are carried out by subjecting peptides to linear increasing salt gradients in the presence of high levels of acetonitrile, which promotes hydrophilic interactions overlayed on ionic interactions with the cation-exchange matrix. Complex peptide mixtures produced by solid-phase synthesis are a frequently encountered and challenging purification problem. In the present study a two-step protocol, consisting of HILIC/CEC followed by RPC, was required for the successful purification of a 21-residue synthetic amphipathic α-helical peptide from serine side-chain acetylated impurities, with HILIC/CEC proving to be highly sensitive to subtle differences in hydrophilicities between the acetylated peptides and the desired product. Investigation of the three potential sites of serine acetylation through solid-phase synthesis of acetylated analogues of the desired peptide (peptides of the same sequence and secondary structure, but acetylated at different positions on the hydrophilic face of the α-helix) demonstrated that acetylation was occurring at different sites on the peptide. HILIC/CEC was able to take advantage of very subtle changes in environment around the acetylation sites and thus effect a separation of these analogues not achievable by RPC or CEC alone.     

Discreplasminin,a plasmin inhibitor isolated from <Emphasis Type="Italic">Tityus discrepans</Emphasis> scorpion venom

Tityus discrepans venom (TdV) produces digestive hemorrhages, disseminated intravascular coagulation, alveoli fibrin deposition and/or prothrombin and partial thromboplastin time alterations in humans. T. discrepans venom presents an in vitro tissue plasminogen activator-like (tPA-like), fibrino(geno)lytic and plasmin inhibitory activities. The plasmin inhibitor, called discreplasminin, was isolated from TdV. Discreplasminin has a pI of 8.0 and a relative molecular weight of <6,000 Da. Discreplasminin and aprotinin strongly inhibited plasmin activity and moderately tPA activity, while epsilon amino caproic acid (EACA) moderately inhibited both enzymes. In presence and absence of fibrin, the plasmin generation by tPA was completely inhibited by aprotinin and discreplasminin. EACA in the absence of fibrin partially inhibited plasmin generation (37%); however, it produced a total inhibition of plasmin generation on a fibrin surface. The tPA-clot lysis assay showed that discreplasminin acts like aprotinin inducing a slight delay in lysis time and lysis rate; in contrast, EACA presented a total inhibitory effect on fibrin lysis. These results suggest that discreplasminin presents an anti-fibrinolytic mechanism similar to aprotinin. Discreplasminin probably interacts with the active sites of plasmin and tPA. The presence of discreplasminin and other similar components in scorpion venom could partially explain the generalized fibrin deposition which was found previously in rams.     

溶栓素的分离纯化及特性测定

目的研究一种高效分离纯化溶栓素的方法并测定其pI和相对分子质量(Mr)。方法采用阴、阳离子交换色谱等技术分离纯化溶栓素并通过对缓冲体系的pH值、离子强度的调节及对初始供试品pH值的调节来优化纯化过程,以等电聚焦聚丙烯酰胺凝胶电泳(IEF PAGE)和SDS PHGE方法分别测定纯品的pI和Mr。结果溶栓素达到简捷、高效分离纯化的目的,电泳鉴定为一条蛋白质带,并测定溶栓素的pI为4 .5 0、Mr 为35 10 0。结论溶栓素的纯化工艺达到高纯度纯化程度,为进一步研究溶栓素奠定了基础     

Soluble expression of active recombinant human tissue plasminogen activator derivative (K2S) in Escherichia coli

Context: The kringle 2 plus serine protease domains (K2S) of human tissue plasminogen activator (tPA) is an efficacious thrombolytic drug, which has been used to treat heart attacks and strokes by breaking up the clots that cause them. It has nine disulfide bridges, which are needed for proper folding and be the bottleneck in improving the production in the Escherichia coli system. So far, few reports have described the production of soluble active K2S from E. coli.

Objective: To achieve high-level expression of active K2S in the E. coli system.

Materials and methods: The DNA fragment coding for K2S was fused with the E. coli disulfide isomerase DsbC. The constructed fusion protein was expressed in E. coli, and then purified with the Ni²⁺-chelating affinity chromatography. K2S was released by cleavage with Factor Xa protease, and the thrombolytic activity was determined using the fibrin plate assay.

Results: The fusion protein DsbC-K2S was found in the culture supernatant of recombinant E. coli as a soluble form of ~40%. The result of fibrinolysis fibrin plate assay showed that the purified recombinant K2S exhibited significant fibrinolysis activity in vitro.

Discussion and conclusion: These works provided a novel approach for the production of active K2S in E. coli without the requirements of in vitro refolding process, and might establish a significant foundation for the following production of K2S.     

Isolation of human thrombin by affinity chromatography with silicate to be used in the preparation of biological glue

At present the preparations of fibrin glue used a thrombin from equine or bovine origin. In order to remove the problems of antigenicity we developed a method of purification from acidified plasma. We used one affinity chromatography on benzamidine-Spherodex and compared three methods of elution: non specific (sodium chloride gradient) and biospecific competitors (arginin methylester or benzamidin). The yield is evaluated between 64 and 84% and the purification factor close to 160. The obtained thrombin is better than animal thrombin in the preparations of fibrin glue.     

Purification and biochemical characterization of F II(a), a fibrinolytic enzyme from Agkistrodon acutus venom.

A fibrinolytic enzyme, F II(a), was isolated from Agkistrodon acutus venom by ion-exchange chromatography and gel filtration. F II(a) consisted of a single polypeptide chain with a molecular weight of 26,000 and an isoelectric point of 4.6. F II(a) was shown to solubilize fibrin and fibrinogen. F II(a) cleaved, primarily, the alpha chain of fibrinogen and fibrin followed by the beta chain, while the gamma chain was minimally affected. Thus, the enzyme was an alpha,beta-fibrinogenase. The cleavage pattern of fibrinogen clearly varied from plasmin cleavage of the same molecule. In vivo, F II(a) had no influence on the rat's tissue-type plasminogen activator and plasminogen activator inhibitor-1 activities in plasma. At the dosage of 5mg/kg, histological examination of heart, liver and lung tissue showed no hemorrhage. F II(a) is an enzyme that hydrolyzed fibrin directly without hemorrhagic activity.     

Chromatographic analysis of toxic phosphylated oximes (POX): a brief overview

Poisoning with organophosphorus compounds (OP), e.g. pesticides and nerve agents, causes inhibition of acetylcholinesterase (AChE) by phosphylation of the active site serine residue. Consequently, accumulation of stimulating acetylcholine in the synaptic cleft induces cholinergic crisis which ultimately may lead to death. For standard causal therapy, enzyme reactivators are administered representing oxime derivatives of quarternary pyridinium compounds, e.g. pralidoxime (2-PAM), obidoxime and HI 6. The mechanism of action includes removal of the phosphyl moiety by a nucleophilic attack of the oximate molecule substituting the enzyme and forming a phosphylated oxime (POX). POX is produced in stoichiometric amounts of reactivated enzyme and exhibits a significantly enhanced toxicity (inhibition rate constant) when compared to the parent OP. However, stability of POX under physiological conditions appears to be highly limited. Nevertheless, the presence of POX reveals a potential critical issue for both therapeutic efficacy in vivo and pharmacokinetic and pharmacodynamic (PK-PD) modelling based on cholinesterase activity data. Detailed characterization represents an important need for elaboration of the entire oxime pharmacology.Nevertheless, reports on POX toxicity and analysis are quite rare and may therefore be indicative of the challenge of POX analysis. This review provides a concise overview of chromatographic approaches applied to POX separation. Chromatography represents the key technology for POX purification and quantification in kinetic in vitro studies using buffers and biological fluids. Applications based on reversed-phase chromatography (RPC), ion pair chromatography (IPC) and an affinity approach as well as thin layer chromatography (TLC) are discussed and novel applications and data are presented.     

Antibiotic A10255 (thioplabin) enhances fibrin binding and activation of plasminogen

Three thiopeptide metabolites that enhance fibrin binding of plasminogen were isolated from a culture of Streptomyces sp. R1401. A combination of spectroscopic analyses revealed that these compounds were identical with the antibiotic A10255B, E and G. These agents enhanced fibrin binding of plasminogen and plasminogen/urokinase-mediated fibirinolysis at concentrations of 5 to approximately 20 microM. A10255B reversibily increased urokinase-catalyzed activation of plasminogen by lowering Km, while the agent did not enhance urokinase activity when substrates other than plasminogen were used, indicating that the agent affects plasminogen to increase its affinity to urokinase. A smaller but significant increase in activation was also observed when conformationally relaxed plasminogen derivatives such as Lys-plasminogen and mini-plasminogen were used. Two related thiopeptide antibiotics with a C-terminal amide had no effect on plasminogen activation, suggesting a role of the terminal carboxyl of the A10255 molecule in activity.