N-V蛋白酶的定量方法系统研究与效价测定
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摘要
本论文报告了N-V蛋白酶定量测定法的系统研究及效价的测定。为进一步研究此种该酶的特性、评价制剂质量标准提供了实验方法和理论依据。
首先采用全波长扫描法对N-V蛋白酶进行分析,在此基础上,采用凯氏定氮法,紫外吸收法,Lowry法以及考马斯亮蓝法测定N-V蛋白酶制剂的蛋白含量;以尿激酶为标准,采用琼脂糖-纤维蛋白法测定N-V蛋白酶效价。
实验结果如下:
全波长扫描制剂呈现典型单一峰,样品在280nm和260nm处的吸光度值:A_(260)=0.14149;A_(280)=0.27289;A_(280)/A_(260)=1.928,其数值大于1.8,因此可以确定,N-V蛋白酶制剂纯度合格,没有或极少有核酸或核苷酸污染。由于制剂的蛋白质纯度是合格的,因此根据凯氏定氮法测出的N-V蛋白酶的含量,结果是准确的,可以以此为根据评估其他测定方法的准确度和优缺点。
采用凯氏定氮法测得的结果(5.164±0.013)g·L~(-1),考马斯亮蓝法得到蛋白含量为(5.150±0.002)g·L~(-1),紫外吸收法(A_(280)-A_(260))测得蛋白含量的结果为(5.202±0.001)g·L~(-1),而Lowry法测得的结果为(1.168±0.011)g·L~(-1),紫外吸收法(A215-A225)测得的结果为(6.206±0.021)g·L~(-1)。紫外吸收法(A_(280)-A_(260))和考马斯亮蓝法测出的蛋白含量,与凯氏法结果相近,适合本制剂的浓度测定,前者可作为初步定量的依据,后者则是测定N-V蛋白酶含量的首选。而紫外吸收法(A215-A225)和Lowry法也测定出蛋白含量,但是结果与凯氏法结果差异比较大。尤其是Lowry法结果明显偏小。这两种方法不适合此N-V蛋白酶的含量测定。
以尿激酶标准品为参照物,测得每毫克N-V蛋白酶的效价,相当于3000IU尿激酶。综上,本实验基本完成了该蛋白酶定量方法研究以及效价的测定,有利于对其进行深入的研究。
Thromboembolic disease has become the number one enemy of the human health, which have seriously affected people's life expectancy and the quality of life. Cardiovascular and cerebrovascular diseases each year have brought a heavy burden to patients and their families and even the society, People have put more and more attentions to this phenomenon. In order to resolve such difficult problems, the antithrombotic drugs and fibrinolytic drugs are applied mainly, Fibrinolytic drugs have been considered one of the best treatments. At present, the focus of the study are mainly in the thrombolytic enzyme therapy and thrombosis research. The first generation of the thrombolytic drug representative are Streptokinase SK and Urokinase UK,The main features are very strong with the efficacy of fibrin dissolution and side effects, but weak with the specificity fibrinolysis and difficult to control the dosage . The second-generation thrombolytic drugs are representative by tissue-type plasminogen activator (t-PA) .The main feature is that it includs fibrinolytic specificity in some extent, but it still has a very strong side effects, and dosage is difficult to control, and the half-life is shorter. The third-generation thrombolytic agent are mainly improved products for molecular biology produced by the means of protein engineering , but most of them are at an experimental stage.At present, all these thrombolytics exist some defects,needing to develop a new type safe medicine.
Jilin University, Professor Hongmin group of Biochemistry Department spent several years to study a N-V protease, which is abstracted from Nereis.It is a proteolytic enzyme. N-V protease is able to hydrolyze fibrinogen and fibrin significantly both in vivo and in vitro. It is not only a plasminogen activator, but also can dissolve the fibrin thrombosis directly, There are also other advantages such as taking effect quickly, without accumulation in the body,less side effects, It does show a very good application prospects in the treatment of thrombotic diseases. This enzyme has been found in our country to apply for patent, application Number.is 02144828.0, and the public Number is 1,500,873.It also have landed at Swiss prot protein database.The landing Number is P83433. Now we have formed a whole set of purification process, and the purity of extracted products have achieve he provisions of the standard drugs of the Ministry of Health.In order to declare this new drug,we should standardize the concentration and titration which are required in the quality standard.
This thesis reports on the concentration of N-V protein enzyme, as well as the titration. Further improve the index declared in the drug quality standard.
First of all, the N-V protein enzyme is prepared for a full wavelength scan, the result shows that N-V protein enzyme has only a typical single-peak .The absorbance value at 280nm and 260nm is as follow:A260=0.14149; A280=0.27289; A280/A260 = 1.928.The absorbance ratio of pure protein A280/A260 is about 1.8, if the ratio is less than 1.8 It shows that there is a high concentration of nucleic acids and nucleotides.The result of N-V protein enzyme in A280/A260 was 1.9 which proves that this protein is in high purity.This can rule out the impurities of nucleic acid or nuclear the effects of guanosine monophosphate. Our full-wavelength detection for the assay of N-V protein enzyme provides a fundamental basis.
N-V protein enzyme is determined under the basic findings above. Quantitative methods have different principle, different sensitivity and different interfering factors, so not all methods are suitable to a particular protein. Therefore, a specific protein, should have the exact assay of the appropriate method. National Pharmacopoeia provides three kinds of protein concentration assays, respectively Kjeldahl,Lowry and Biuret. In this experiment, Kjeldahl method, UV absorption method, Lowry method and the Coomassie brilliant blue method are used to the N-V protein enzyme. Because of the purity of the N-V protein enzyme is qualified, therefore other methods can be measured according to Kjeldahl method .The results are accurate, which used to evaluate the advantages and disadvantages. The results are as follows: Kjeldahl Method: (5.164±0.013)g·L~(-1); UV absorption: (5.202±0.001)g·L~(-1) (A280-A260), (6.206±0.021)g·L~(-1) (A215-A225) ; Coomassie brilliant blue method: (5.150±0.002)g·L~(-1); Lowry method: (1.168±0.011)g·L~(-1). UV absorption (A280-A260) and Coomassie brilliant blue method measured are similar with the result of Kjeldahl method .They are both suit for assay.The former can be used as the basic method for initial quantitative, while the latter is the best for N-V protein enzyme. And UV absorption (A215-A225) and Lowry method also measured the concentration, but the results have large differences with Kjeldahl method relatively. In particular, the result Lowry method are significantly smaller. These two methods are not suitable for this assay of N-V protein enzyme.
Titer is one of the important qualitive index in the declaration of new drugs. In this experiment, agarose - fibrin plate method is used for the titer of the N-V protein enzyme. Agarose - fibrin plate are made of the agarose, fibrinogen (fibrinogen) and thrombin (thrombin). Agarose acts as a support material, fibrinogen, prothrombin, respectively, are coagulation factorⅠ,Ⅱt o participate in vivo coagulation process, and also the process of thrombosis. N-V protein enzyme is a dissolution, we have chosen Urokinase as a standard. Urokinase thrombolytic drugs commonly used for clinical. They all can be dissolved on the fibrin plate to form circle. Measurement of dissolved then circle the diameter of the vertical length of two to urokinase standard logarithm of the number of units for the abscissa, vertical diameter of the product of two logarithmic ordinate to do for the standard curve regression curve was Y=0.273X-0.220,R2=0.994, and then measured the diameter of the sample into the standard curve calculation of NV protease. The result is that titer of every mg N-V protease is equivalent to 3000IU Urokinase
首先采用全波长扫描法对N-V蛋白酶进行分析,在此基础上,采用凯氏定氮法,紫外吸收法,Lowry法以及考马斯亮蓝法测定N-V蛋白酶制剂的蛋白含量;以尿激酶为标准,采用琼脂糖-纤维蛋白法测定N-V蛋白酶效价。
实验结果如下:
全波长扫描制剂呈现典型单一峰,样品在280nm和260nm处的吸光度值:A_(260)=0.14149;A_(280)=0.27289;A_(280)/A_(260)=1.928,其数值大于1.8,因此可以确定,N-V蛋白酶制剂纯度合格,没有或极少有核酸或核苷酸污染。由于制剂的蛋白质纯度是合格的,因此根据凯氏定氮法测出的N-V蛋白酶的含量,结果是准确的,可以以此为根据评估其他测定方法的准确度和优缺点。
采用凯氏定氮法测得的结果(5.164±0.013)g·L~(-1),考马斯亮蓝法得到蛋白含量为(5.150±0.002)g·L~(-1),紫外吸收法(A_(280)-A_(260))测得蛋白含量的结果为(5.202±0.001)g·L~(-1),而Lowry法测得的结果为(1.168±0.011)g·L~(-1),紫外吸收法(A215-A225)测得的结果为(6.206±0.021)g·L~(-1)。紫外吸收法(A_(280)-A_(260))和考马斯亮蓝法测出的蛋白含量,与凯氏法结果相近,适合本制剂的浓度测定,前者可作为初步定量的依据,后者则是测定N-V蛋白酶含量的首选。而紫外吸收法(A215-A225)和Lowry法也测定出蛋白含量,但是结果与凯氏法结果差异比较大。尤其是Lowry法结果明显偏小。这两种方法不适合此N-V蛋白酶的含量测定。
以尿激酶标准品为参照物,测得每毫克N-V蛋白酶的效价,相当于3000IU尿激酶。综上,本实验基本完成了该蛋白酶定量方法研究以及效价的测定,有利于对其进行深入的研究。
Thromboembolic disease has become the number one enemy of the human health, which have seriously affected people's life expectancy and the quality of life. Cardiovascular and cerebrovascular diseases each year have brought a heavy burden to patients and their families and even the society, People have put more and more attentions to this phenomenon. In order to resolve such difficult problems, the antithrombotic drugs and fibrinolytic drugs are applied mainly, Fibrinolytic drugs have been considered one of the best treatments. At present, the focus of the study are mainly in the thrombolytic enzyme therapy and thrombosis research. The first generation of the thrombolytic drug representative are Streptokinase SK and Urokinase UK,The main features are very strong with the efficacy of fibrin dissolution and side effects, but weak with the specificity fibrinolysis and difficult to control the dosage . The second-generation thrombolytic drugs are representative by tissue-type plasminogen activator (t-PA) .The main feature is that it includs fibrinolytic specificity in some extent, but it still has a very strong side effects, and dosage is difficult to control, and the half-life is shorter. The third-generation thrombolytic agent are mainly improved products for molecular biology produced by the means of protein engineering , but most of them are at an experimental stage.At present, all these thrombolytics exist some defects,needing to develop a new type safe medicine.
Jilin University, Professor Hongmin group of Biochemistry Department spent several years to study a N-V protease, which is abstracted from Nereis.It is a proteolytic enzyme. N-V protease is able to hydrolyze fibrinogen and fibrin significantly both in vivo and in vitro. It is not only a plasminogen activator, but also can dissolve the fibrin thrombosis directly, There are also other advantages such as taking effect quickly, without accumulation in the body,less side effects, It does show a very good application prospects in the treatment of thrombotic diseases. This enzyme has been found in our country to apply for patent, application Number.is 02144828.0, and the public Number is 1,500,873.It also have landed at Swiss prot protein database.The landing Number is P83433. Now we have formed a whole set of purification process, and the purity of extracted products have achieve he provisions of the standard drugs of the Ministry of Health.In order to declare this new drug,we should standardize the concentration and titration which are required in the quality standard.
This thesis reports on the concentration of N-V protein enzyme, as well as the titration. Further improve the index declared in the drug quality standard.
First of all, the N-V protein enzyme is prepared for a full wavelength scan, the result shows that N-V protein enzyme has only a typical single-peak .The absorbance value at 280nm and 260nm is as follow:A260=0.14149; A280=0.27289; A280/A260 = 1.928.The absorbance ratio of pure protein A280/A260 is about 1.8, if the ratio is less than 1.8 It shows that there is a high concentration of nucleic acids and nucleotides.The result of N-V protein enzyme in A280/A260 was 1.9 which proves that this protein is in high purity.This can rule out the impurities of nucleic acid or nuclear the effects of guanosine monophosphate. Our full-wavelength detection for the assay of N-V protein enzyme provides a fundamental basis.
N-V protein enzyme is determined under the basic findings above. Quantitative methods have different principle, different sensitivity and different interfering factors, so not all methods are suitable to a particular protein. Therefore, a specific protein, should have the exact assay of the appropriate method. National Pharmacopoeia provides three kinds of protein concentration assays, respectively Kjeldahl,Lowry and Biuret. In this experiment, Kjeldahl method, UV absorption method, Lowry method and the Coomassie brilliant blue method are used to the N-V protein enzyme. Because of the purity of the N-V protein enzyme is qualified, therefore other methods can be measured according to Kjeldahl method .The results are accurate, which used to evaluate the advantages and disadvantages. The results are as follows: Kjeldahl Method: (5.164±0.013)g·L~(-1); UV absorption: (5.202±0.001)g·L~(-1) (A280-A260), (6.206±0.021)g·L~(-1) (A215-A225) ; Coomassie brilliant blue method: (5.150±0.002)g·L~(-1); Lowry method: (1.168±0.011)g·L~(-1). UV absorption (A280-A260) and Coomassie brilliant blue method measured are similar with the result of Kjeldahl method .They are both suit for assay.The former can be used as the basic method for initial quantitative, while the latter is the best for N-V protein enzyme. And UV absorption (A215-A225) and Lowry method also measured the concentration, but the results have large differences with Kjeldahl method relatively. In particular, the result Lowry method are significantly smaller. These two methods are not suitable for this assay of N-V protein enzyme.
Titer is one of the important qualitive index in the declaration of new drugs. In this experiment, agarose - fibrin plate method is used for the titer of the N-V protein enzyme. Agarose - fibrin plate are made of the agarose, fibrinogen (fibrinogen) and thrombin (thrombin). Agarose acts as a support material, fibrinogen, prothrombin, respectively, are coagulation factorⅠ,Ⅱt o participate in vivo coagulation process, and also the process of thrombosis. N-V protein enzyme is a dissolution, we have chosen Urokinase as a standard. Urokinase thrombolytic drugs commonly used for clinical. They all can be dissolved on the fibrin plate to form circle. Measurement of dissolved then circle the diameter of the vertical length of two to urokinase standard logarithm of the number of units for the abscissa, vertical diameter of the product of two logarithmic ordinate to do for the standard curve regression curve was Y=0.273X-0.220,R2=0.994, and then measured the diameter of the sample into the standard curve calculation of NV protease. The result is that titer of every mg N-V protease is equivalent to 3000IU Urokinase
引文
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[4] 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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[2] Yang T.Y., Chung K.J., Huang T.L., Kung C.T. Massive pulmonary embolism in a young patient on clozapine therapy. The Journal of Emergency Medicine, 2004, 27(1): 27-29.
[3] Ginsberg J.S. Antithrombotic therapy in children. Thrombosis Research, 2003, 109: 83.
[4] Temple M., Williamsb S., Johna P., Chaita P., Connollya B. Percutaneous treatment of pediatric thrombosis. European Journal of Radiology, 2005, 53: 14-21.
[5]石英.血栓与血栓性疾病的治疗进展.新疆医学,1989,19(4):231-233.
[6]沈同,王镜岩.生物化学[M].北京:高等教育出版社,1993,131- 133.
[7] Segel G.B., Francis C.W. Anticoagulant Proteins in Childhood Venous and Arterial Thrombosis: A Review. Blood Cells, Molecules, and Diseases, 2000, 26(5) October: 540-560.
[8] Kane KK. Fibrinolysis [J]. Ann Clin Lab Sci, 1984, 14(6): 443-449.
[9] Collen D, Lijnen HR. The fibrinolytic system in man [J]. Crit Rev Oncol Hematol, 1986, 4(3): 249-301.
[10] Kluft C. The fibrinolytic system and thrombotic tendency [J]. Pathophysiol Haemost Thromb, 1998,33(5-6): 425-429.
[11]杨晓光译.纤维蛋白溶解的展望[J].国外医学输血及血液学分册,1980, 2:96.
[12] Hillis L D , Borer J , Brauqwald E , et al. High dose intra2venous strep- tokinase for acute myocardial infarction :preliminaryresults of a multicenter trial [J]. J Am Coll Cardiol,1985,6:95729621.
[13] 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.
[14] McClintock DK, Bell PH. The mechanism of activation of human plasminogen by streptokinase [J]. Biochem Biophys Res Commun, 1971, 43(3): 694-702.
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