N-V蛋白酶提取过程中沙蚕毒素的检测
摘要
本研究课题组经过数年的研究,运用多种生物化学实验技术,从我国沿海海洋生物沙蚕中分离提取了一种新的蛋白酶,称为N-V蛋白酶。随着药理学、药物代谢动力学和酶学特征等研究的深入,结果表明,该蛋白酶制剂在治疗血栓疾病方面展现了很好的应用前景,有可能开发研究成为一类新药,应用于临床。因此,迫切需要对这一新的蛋白酶的提取过程进行质量监控研究。
本论文首先简要报告了应用盐析及层析等技术从沙蚕体内分离得到N-V蛋白酶的分离纯化工艺,该提取工艺可以有效从沙蚕体内提取出纯度较高的N-V蛋白酶。
以往的研究发现,沙蚕体内存在一种毒素——沙蚕毒素,是一种神经毒素。本论文总结了实验中建立的一套沙蚕毒素的检测方法,并分别对沙蚕原液、N-V蛋白酶提取纯化过程中间产物及成品进行检测,以监测各提取工艺去除毒素的效果。实验证实,此套方法不仅可以定性定量检测N-V蛋白酶成品制剂中的沙蚕毒素,也适用于其它含该物质的样品。方法包括:薄层层析法、高效液相色谱法。方法快速稳定,检测限好,可作为N-V蛋白酶提取纯化制备过程中该相关物质的检测标准。
Summary:
Cardial and cerebral vascular thrombosis disease with its high morbidity will lead to high mortality rates and disability. Clinically, the principal method of clinical treatment is to cut down fibrinogen in the blood in order to reduce deaths and disability and to improve the quality of life. It is an important topic to study and research this kind of drug in the medicine domain. Nereis virens (N-V) proteinase, a kind of proteolytic enzyme in Nereis succinea, had been extracted successfully in our laboratory. N-V proteinase is a kind of aminopeptidase with plasminogen and plasminogen activator activity, which can be developed for defibrase and thrombolytic drugs as a therapeutic approach for heart and brain infarction, for treating and preventing heart and brain thrombus diseases. This thesis reported the purification process of the N-V proteinase.
N-V proteinase is a special kind of protein which uses for medical therapy. The key point of purification is the detection of purity of the protein and pollutants in it. There is a S-N compounds in nereis, known as nereis toxins(NTX), which is often used as a pesticide. NTX is a kind of poisonous impurities that may contain in N-V proteinase. We must detect NTX contained in raw materials, different samples from the course of purification and N-V proteinase.
This paper summarizes the experiments, erected a series of methods that can detect NTX contained in N-V proteinase, and detected different samples from the course of purification. At present, the purification technology of N-V proteinase is more mature. So we use different methods to detect the quantity of NTX contained in proteinase, in order to monitor the effect of extraction process to remove impurities for further improve of the purification technology. And to prepare conditions for the declaration of the new drug. Experimental research:
1. Summary of purification of N-V proteinase.
Salting-out and chromatography were used to purify N-V proteinase. Dialysis was adopted to desalt. And PAGE proves the high purity of N-V proteinase in the course of time.
2. Detection of nereistoxin in N-V proteinase agent
(1) ultraviolet and visible spectrophotometry
UV wavelength scan showed that the maximum absortion of NTX in methanol is 210nm. We detect the different concentrations of the standard solution in this wavelength, and calculated linear regression equation:y=0.0320x+0.0199,n=7,R2=0.9975. The detection limit was 0.125μg/mL.But during the further detecting of N-V proteinase samples, we find there are still some impurities that affect the results, which make them do not match with the facts. So this method can measure the content of NTX in standard substance exactly. But it is not suitable for NTX in N-V proteinase.
(2) thin layer chromatography
We use high-performance thin-layer silica gel G thin layer chromatography plate detection to detect NTX. The developing agent is methanol: acetoacetate: water (5:4:1), the chromogenic agent is calcein-palladium chloride. The detection limit is 0.5μg/mL.After extraction, a great deal of NTX can be detected in the raw materials. The content of NTX in the extract decreased with the continuous extraction process, especially after the second time of chromatography. No NTX is detected in N-V proteinase agent finally. This method is simple. Only UV lamps is needed, and just required few of samples. This method is sensitive and can rapid qualitative analysis of the existence of a small number of NTX, but not specific quantitative tests. It should be combined with other method to test the exact content NTX.
(3)High-performance liquid chromatography
NTX is a kind of free amine with small molecule, which is difficult to retain in the C8 column. So we chose C18 reverse column for detection. According to the characteristics of C18 column, methanol and water were chosen as the mobile phase. And through pre-test, and ultimately determine the column temperature was 20℃, flow rate 1ml/min, detect wavelength 227nm. We detect the different concentrations of the standard solution in this wavelength, and calculated linear regression equation: y = 1.7707x + 22.182, n = 9, R2 = 0.9996. NTX minimum detection limit is 0.06125μg/mL, the accuracy rate was 95.6%—97.4%.
High-performance liquid chromatography is sensitive, rapid, high resolution, reproducible and less demanding on the equipment. It is an effective way to detect NTX in N-V proteinase
Experimental result
N-V proteinase has been purified and extracted after salting-out and chromatography. After extraction, a great deal of NTX can be detected in the raw materials. The content of NTX in the extract decreased with the continuous extraction process, especially after the second time of chromatography. No NTX is detected in N-V proteinase agent finally. The method, thin layer chromatography and high-performance liquid chromatography, can detect NTX in samples fast and sensitively. Particularly high-performance liquid chromatography can detect the quantity of NTX accurately.
This paper summarizes the experiments, erected a series of methods that can detect NTX contained in N-V proteinase We use different methods to detect the quantity of NTX contained in proteinase, in order to monitor the effect of extraction process to remove impurities for further improvement of the purification technology. And to prepare conditions for the clinical application of the new drug.
本论文首先简要报告了应用盐析及层析等技术从沙蚕体内分离得到N-V蛋白酶的分离纯化工艺,该提取工艺可以有效从沙蚕体内提取出纯度较高的N-V蛋白酶。
以往的研究发现,沙蚕体内存在一种毒素——沙蚕毒素,是一种神经毒素。本论文总结了实验中建立的一套沙蚕毒素的检测方法,并分别对沙蚕原液、N-V蛋白酶提取纯化过程中间产物及成品进行检测,以监测各提取工艺去除毒素的效果。实验证实,此套方法不仅可以定性定量检测N-V蛋白酶成品制剂中的沙蚕毒素,也适用于其它含该物质的样品。方法包括:薄层层析法、高效液相色谱法。方法快速稳定,检测限好,可作为N-V蛋白酶提取纯化制备过程中该相关物质的检测标准。
Summary:
Cardial and cerebral vascular thrombosis disease with its high morbidity will lead to high mortality rates and disability. Clinically, the principal method of clinical treatment is to cut down fibrinogen in the blood in order to reduce deaths and disability and to improve the quality of life. It is an important topic to study and research this kind of drug in the medicine domain. Nereis virens (N-V) proteinase, a kind of proteolytic enzyme in Nereis succinea, had been extracted successfully in our laboratory. N-V proteinase is a kind of aminopeptidase with plasminogen and plasminogen activator activity, which can be developed for defibrase and thrombolytic drugs as a therapeutic approach for heart and brain infarction, for treating and preventing heart and brain thrombus diseases. This thesis reported the purification process of the N-V proteinase.
N-V proteinase is a special kind of protein which uses for medical therapy. The key point of purification is the detection of purity of the protein and pollutants in it. There is a S-N compounds in nereis, known as nereis toxins(NTX), which is often used as a pesticide. NTX is a kind of poisonous impurities that may contain in N-V proteinase. We must detect NTX contained in raw materials, different samples from the course of purification and N-V proteinase.
This paper summarizes the experiments, erected a series of methods that can detect NTX contained in N-V proteinase, and detected different samples from the course of purification. At present, the purification technology of N-V proteinase is more mature. So we use different methods to detect the quantity of NTX contained in proteinase, in order to monitor the effect of extraction process to remove impurities for further improve of the purification technology. And to prepare conditions for the declaration of the new drug. Experimental research:
1. Summary of purification of N-V proteinase.
Salting-out and chromatography were used to purify N-V proteinase. Dialysis was adopted to desalt. And PAGE proves the high purity of N-V proteinase in the course of time.
2. Detection of nereistoxin in N-V proteinase agent
(1) ultraviolet and visible spectrophotometry
UV wavelength scan showed that the maximum absortion of NTX in methanol is 210nm. We detect the different concentrations of the standard solution in this wavelength, and calculated linear regression equation:y=0.0320x+0.0199,n=7,R2=0.9975. The detection limit was 0.125μg/mL.But during the further detecting of N-V proteinase samples, we find there are still some impurities that affect the results, which make them do not match with the facts. So this method can measure the content of NTX in standard substance exactly. But it is not suitable for NTX in N-V proteinase.
(2) thin layer chromatography
We use high-performance thin-layer silica gel G thin layer chromatography plate detection to detect NTX. The developing agent is methanol: acetoacetate: water (5:4:1), the chromogenic agent is calcein-palladium chloride. The detection limit is 0.5μg/mL.After extraction, a great deal of NTX can be detected in the raw materials. The content of NTX in the extract decreased with the continuous extraction process, especially after the second time of chromatography. No NTX is detected in N-V proteinase agent finally. This method is simple. Only UV lamps is needed, and just required few of samples. This method is sensitive and can rapid qualitative analysis of the existence of a small number of NTX, but not specific quantitative tests. It should be combined with other method to test the exact content NTX.
(3)High-performance liquid chromatography
NTX is a kind of free amine with small molecule, which is difficult to retain in the C8 column. So we chose C18 reverse column for detection. According to the characteristics of C18 column, methanol and water were chosen as the mobile phase. And through pre-test, and ultimately determine the column temperature was 20℃, flow rate 1ml/min, detect wavelength 227nm. We detect the different concentrations of the standard solution in this wavelength, and calculated linear regression equation: y = 1.7707x + 22.182, n = 9, R2 = 0.9996. NTX minimum detection limit is 0.06125μg/mL, the accuracy rate was 95.6%—97.4%.
High-performance liquid chromatography is sensitive, rapid, high resolution, reproducible and less demanding on the equipment. It is an effective way to detect NTX in N-V proteinase
Experimental result
N-V proteinase has been purified and extracted after salting-out and chromatography. After extraction, a great deal of NTX can be detected in the raw materials. The content of NTX in the extract decreased with the continuous extraction process, especially after the second time of chromatography. No NTX is detected in N-V proteinase agent finally. The method, thin layer chromatography and high-performance liquid chromatography, can detect NTX in samples fast and sensitively. Particularly high-performance liquid chromatography can detect the quantity of NTX accurately.
This paper summarizes the experiments, erected a series of methods that can detect NTX contained in N-V proteinase We use different methods to detect the quantity of NTX contained in proteinase, in order to monitor the effect of extraction process to remove impurities for further improvement of the purification technology. And to prepare conditions for the clinical application of the new drug.
引文
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[3] Temple M., Williamsb S., Johna P., Chaita P., Connollya B. Percutaneous treatment of pediatric thrombosis. European Journal of Radiology, 2005, 53: 14-21.
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[13] Perter B. Thrombolytic therapies; the current state of affairs [J]. J Endovasc Ther,2005,12(2);224-232.
[14]程熠,刘畅,高林,等.极性有机溶剂辅助阴、阳离子交换组台色谱法[J] .中国生化药物杂志,2002,23(4) :163-165.
[15]张云龙,崔佳乐,付海英,等.溶栓素的分离纯化及特性测定[J].中国生化药物杂志,2005,26(1):18-21.
[16]李奇,迟秀梅,石立红,等. N-V蛋白酶体内外纤维蛋白溶解活性的实验研究[J].中国药理学通报,2006,22(8):976~979.
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[18]王昭,李奇,迟秀梅.溶栓素样品精纯方法研究[J].中国老年学杂志. 2005, 25(12): 1502-1503.
[19]王昭.N-V蛋白酶的溶栓机理及酶学特点研究[D].博士论文吉林大学基础医学院生物化学与分子生物学教研室June 2006,94-102.
[20]李奇,洪敏,付海英等.溶栓素抗凝血和抗血栓形成作用的实验研究[J].吉林大学学报(医学版),2004,30(3);401-4.
[21]程熠,张家颖,王之光,等.溶栓索的性质及生物活性研究[J].白求恩医科大学学报,2001;25(5):470-1.
[22]郭尧君.蛋白质电泳实验技术[M],北京:北京科学出版社, 1999:54-115.
[23] Hashimoto Y,Okaichi T. Some chemical properties of nereistoxin [J ] . Ann N Y Acad Sci.1960,17:667-673.
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[29]韩招久,韩昭君.沙蚕毒素类杀虫剂毒理学研究新进展[J].现在农药.2004,3: (6):5-7.
[30]夏利明,何洋.沙蚕毒素杀虫剂重度中毒的临床抢救[J].安徽医药.2004,8: (4):262-263.
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[2] Ginsberg J.S. Antithrombotic therapy in children. Thrombosis Research, 2003, 109: 83
[3] Temple M., Williamsb S., Johna P., Chaita P., Connollya B. Percutaneous treatment of pediatric thrombosis. European Journal of Radiology, 2005, 53: 14-21.
[4]石英.血栓与血栓性疾病的治疗进展.新疆医学,1989,19(4):231- 233.
[5]沈同,王镜岩.生物化学[M].北京:高等教育出版社,1993,131- 133.
[6] Tebbe V, Michels R, Adgey J, et al. Randomized double-blind study comparing saruplase with streptokinase therapy in acute myocardial infarction: the compass equivalence trial. J Am Coll Cardiol, 1998 (31) : 487-493.
[7] Kim DM, Lee SJ, Yoon SK, Byun S M, Specificity role of the streptokinase C-terminal domain in plasminogen activation. Biochemical and Biophysical Research Communications, 2002, 290: 585-588.
[8] McClintock DK, Bell PH. The mechanism of activation of human plasminogen by streptokinase [J]. Biochem Biophys Res Commun, 1971, 43(3): 694-702.
[9]房德兴.组织型纤溶酶原激活因子的分子生物学研究进展.生物工程进展,1993,13(1):1-4.
[10] Richard W S. A fresh look at the molecular pharmacology of plasminogen activators :from theory to test tube to clinical out2 comes [ J ] . Am J Health2Syst Pharm , 1997 , Suppl. 1 , 54(15) :S172S201.
[11] Allan M R. New plasminogen activators :a clinical review[J ] .Clin Cardiol , 1999 , 22 :16521711. [12 ] MD, Thomas K, Nordt, MD, Kadheinz Peter, MD et al. Patency Trials With Reteplase(r-PA):What Do They Tell Us? Am J Cardiol 1996; 78(suppl 12A):16-19
[13] Perter B. Thrombolytic therapies; the current state of affairs [J]. J Endovasc Ther,2005,12(2);224-232.
[14]程熠,刘畅,高林,等.极性有机溶剂辅助阴、阳离子交换组台色谱法[J] .中国生化药物杂志,2002,23(4) :163-165.
[15]张云龙,崔佳乐,付海英,等.溶栓素的分离纯化及特性测定[J].中国生化药物杂志,2005,26(1):18-21.
[16]李奇,迟秀梅,石立红,等. N-V蛋白酶体内外纤维蛋白溶解活性的实验研究[J].中国药理学通报,2006,22(8):976~979.
[17] Yunlong Zhang, Jiayue Cui, Rui Zhang, et al.A novel fibrinolytic serine proteinase from the polychaete Nereis (Neanthes) virens (Sars): Purification and characterization [J]. Biochimie ,2006 doi: 10. 1016/ j.biochi. 2006. 07. 023.
[18]王昭,李奇,迟秀梅.溶栓素样品精纯方法研究[J].中国老年学杂志. 2005, 25(12): 1502-1503.
[19]王昭.N-V蛋白酶的溶栓机理及酶学特点研究[D].博士论文吉林大学基础医学院生物化学与分子生物学教研室June 2006,94-102.
[20]李奇,洪敏,付海英等.溶栓素抗凝血和抗血栓形成作用的实验研究[J].吉林大学学报(医学版),2004,30(3);401-4.
[21]程熠,张家颖,王之光,等.溶栓索的性质及生物活性研究[J].白求恩医科大学学报,2001;25(5):470-1.
[22]郭尧君.蛋白质电泳实验技术[M],北京:北京科学出版社, 1999:54-115.
[23] Hashimoto Y,Okaichi T. Some chemical properties of nereistoxin [J ] . Ann N Y Acad Sci.1960,17:667-673.
[24] Hagiwara H, Numata M, Konishi K, et al. Synthesis of nereistoxin and related compoundsⅠ[J ].Chem Pharm Bull (Tokyo),1965 ,13:253-260.
[25]蒋义国.农药多噻烷纯品的制备与鉴定[J].广州医学院报,1996,24(4):63-66.
[26]孙玉善.海洋天然有机物资源化学(六).中国海洋药物,1988,7(4),38
[27] Hitoshi Aoshima, Kenzi Hori, Akinori Yamamoto. Acetycholine receptor-controlled ion translocation caused by phenyltrimethylammonium and nereistoxin:simple estimation of equilibrium constants of the minimal model [J]. Biochem. 1987, 101:347-355.
[28]王绪卿等.农药杀虫双的代谢毒理学研究[J].环境化学,1997,16:(2):58-59.
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