一种新型日本刺沙蚕纤溶酶的分离、特性及质量标准
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摘要
本论文报告了从一种海洋生物日本刺沙蚕体内分离纯化出一种高纯度、单一组分的新型纤溶酶,命名为日本刺沙蚕纤溶酶,英文名为NJF。日本刺沙蚕纤溶酶在体外有较强的溶解纤维蛋白和纤维蛋白原作用,是一种具有潜在临床应用前景的新型溶栓剂。
通过酶的初提、硫酸铵盐析、Phenyl Sepharose 6 FF疏水层析、DEAE Sepharose FF阴离子离子交换层析和Sephacryl S-100 HR凝胶过滤层析,获得日本刺沙蚕纤溶酶,其在SDS-PAGE和常规PAGE电泳中为单一条带,用高效液相凝胶过滤色谱法检测其纯度不低于95%。
日本刺沙蚕纤溶酶是一条单链蛋白,分子量在28-32kDa,等电点为4.4。其N末端存在封闭现象,通过MALDI-TOF-TOF串联质谱进行De Novo测序,获得三个可信肽段的32个氨基酸序列,登陆到UniProt数据库中,获得登录号为P86330。通过氨基酸序列比对,证明其是一种新发现的蛋白质。
日本刺沙蚕纤溶酶的酶学性质:酶活性能被PMSF完全抑制,是一种典型的丝氨酸蛋白酶;Hg2+能够强烈的抑制蛋白酶的活性,Cu2+具有较弱的抑制作用,而其它金属离子对酶活性没有明显的作用;具有较好的热稳定性和pH稳定性,在40～80°C和pH7～11范围内表现出良好的稳定性,最适温度为60°C,最适pH为9.0;特异发色底物为凝血酶发色底物S-2238,其水解活性为11.2 mmol/min/mg;能够直接溶解纤维蛋白,起主要作用,也具有较弱的激酶活性;最先水解纤维蛋白原的Aα链,然后是Bβ,最后水解γ链。
日本刺沙蚕纤溶酶的部分质量标准:采用常规PAGE电泳法和高效液相凝胶过滤色谱法检测的纯度均不低于95%;使用鲎试剂检测细菌内毒素和家兔法检测热原,结果均符合标准;以S-2238为底物检测的比活力不少于10,000 U/mg;以尿激酶标准品为标准采用琼脂糖-纤维蛋白平板法检测的比活力不少于50,000 IU/mg。
通过对日本刺沙蚕纤溶酶的分子特性、酶学性质和部分质量标准进行系统性研究,为以后的临床前研究和将来的临床应用奠定基础。
Cardiovascular diseases(CVDs) are the leading cause of morbidity in the worldwide. According to World Health Organization statistics, about 17.1million people died from CVDs in 2004, which represented 30% of all global deaths. By 2030, almost 23.6 million people will die from CVDs, mainly from heart disease and stroke. Intravascular thrombosis due to fibrin aggregation in arteries is one of the main causes of CVDs.
Currently, thrombolytic therapy is the the most extensive and most effective method for clinical treatment of thrombotic disease. The major thrombolytic agents availabl for clinical application are plasminogen activators, such as urokinase, streptokinase and tPA (tissue-type plasminogen activator). Despite their widespread use, all these agents have undesirable side effects, and are also relatively expensive. Therefore, the search for other novel thrombolytic agents from various sources is very necessary. Many organisms are important sources of thrombolytic agents.
Neanthes japonica (Iznka) is a marine invertebrate and widely distributed in the north coast of China and the coast of Japan. It is rich in protein, so it is the high-quality food of sea water creatures, such as fish, shrimp and crab and the good bait for fishing in the sea as well. In this stuty, we isolated and purified a novel fibrinolytic enzyme from a marine invertebrate, Neanthes japonica (Iznka), named NJF, and studied its molecular characteristics, enzymatic properties and partial quality standards systematicly in this paper. These studies laid the foundation for pre-clinical researches and clinica application in the future. The main achievements in our investigations are now showed as follows.
1. NJF was achieved by a combination of several isolation and purification methods using shredded autolysis, ammonium sulfate salting out, hydrophobic interaction chromatography by Phenyl Sepharose 6 FF, anion exchange chromatography by DEAE Sepharose FF and gel-filtration chromatography by Sepharcyl S-100 High Resolution. The enzyme was purified 2390-fold with a final yield of 16% after these purification steps. The purified NJF was shown as a single band in SDS–PAGE and Native-PAGE, and the purity of NJF determined by HPLC gel-filtration was 95.2%.
2. NJF consisted of a single polypeptide chain with a molecular weight of 28–32 kDa, which was determined by MALDI-TOF mass spectrum and SDS–PAGE. The isoelectric point of NJF determined by isoelectric focusing electrophoresis (IEF) was 4.4.
3. Purified enzyme was resistant to Edman degradation chemistry for determination of N-terminal sequence, indicating that the N-terminus of NJF is blocked. In further studies, we have obtained the amino acid sequences of three peptides by MALDI-TOF-TOF mass spectrometry and De Novo sequencing. The amino acid sequences of the three peptides are RGVTDHLYN-NH2, RSPGWL-NH2, and RSQVDGVMWDLGDLLGA-NH2. We reported these amino acid sequences of three peptides data in the UniProtKB/Swiss-Prot Knowledgebase and obtained the accession number is P86330. Sequences blast by using standard protein-protein BLAST (blastp) in NCBI protein databases, showed that these amino-acid sequences are different from that of other known proteins. These results suggest that NJF is a novel fibrinolytic enzyme.
4. The protease activity was inhibited by PMSF, but not by EDTA, EGTA,β- Mercaptoe- thanol , Iodoacetate , Indoacetamide and Pepstatin A. This result sugest that NJF is a serine protease. However, other serine protease inhibitors, including aprotinin, benzamidine and trypsin inhibitor had no obvious effect on proteolytic activity, indicating that NJF is a unique serine protease.
5.The protease activity was inhibited completely by Hg2+ ion, and inhibited slightly by Cu2+ ion. However, other metal ions including Na+, K+, Li+,Ca2+, Zn2+, Co2+, Mg2+, Fe2+, Fe3+, Al3+ ions have no obvious effect on the enzyme activity. These results suggest that the molecular structure of NJF may exist one or more disulfide bonds.
6. The effect of temperature and pH on enzyme activity indicated that NJF has better thermal stability and pH stability. The enzyme activity was stable between 40°C and 80°C, and in the pH range of 7-11. Maximum activity of enzyme was observed at 60°C and pH 9.0. These results indicated that the NJF may be used for prevention and treatment of cardiovascular and cerebrovascular diseases by administered orally or intravenously in the future, and has potential clinical application. Because the resulting pH values are around alkaline, NJF can be classified into alkaline protease.
7. Using azocasein as substrate, the enzyme reaction velocity is rapidly increased as substrate congcentration increased between 0.2-4mg/ml. When substrate congcentration is approximately 8 mg/ml, the enzyme reaction velocity reaches its maximum, but when the substrate congcentration higher than 8 mg/ml, the enzyme reaction velocity is not increased. The enzyme dynamic parameters calculated according to double reciprocal plot are as follows: Km=2.359, Vmax=1.845.
8. The study of specific substrate was measured spectrophotometrically, using chromo- genic protease substrates such as S-2238 (H-D-Phe-Pip-Arg-pNA for thrombin), S-2251 (H-D-Val-Leu-Lys-pNA for plasmin and streptokinase-activated plasminogen) and S-2444 (pyro-Glu-Gly-Arg-pNA for urokinase). NJF exhibited a higher degree of specificity for the substrate S-2238 (H-D-Phe-Pip-Arg- pNA), which was 11.2 mmol/min/mg. NJF had strong hydrolytic activity for S2444 and S2251, which suggested that the enzyme acted on arginine and lysine residue at P1 position of the chromogenic substrate. NJF acted on arginine and lysine residue, which similar to trypsin.
9. Two methods of agarose - fibrin plate was used to determine whether NJF has the kinase activity. The results of two methods were showed that NJF was a plasmin-like protease which can directly degrade the fibrin, which play a major role, and it has the weaker kinase activity also. Its fibrinolytic activity was more strong than plasmin.
10. The fibrinogenolytic activity of NJF analyzed by SDS-PAGE showed that the Aα-chain was completely in a very short time (1 min), and Bβ-chain was completely degraded within 10 min. Theγ-chain was the last subunit to be hydrolysed by NJF, and was completely degraded in 1 h. Fibrinogenolytic activity of NJF can reduce blood viscosity, increase blood flow velocity and improve microcirculation, inhibiting thrombus formation and further increased. NJF may be used for prevention and treatment of cardiovascular and cerebrovascular diseases in the future, and has potential clinical application.
11. The purity of the purified NJF was tested using Native-PAGE analyzed by BandScan 5.0, which was 97.4%. The purity of the purified NJF was 96.74% by HPLC gel-filtration chromatography. The mechanism of two determined methods is different, so their results can reflect the true purity of NJF. The results determined by the two methods are consistent, and the purity of the different batches of purified NJF is 95% at least.
12. The check of bacterial endotoxin test used Limulus amoebocyte lysate and the check of pyrogen using rabbits for the purified NJF. The results of two methods are in line with the reqirements of Chinese Pharmacopoeia. These results laid the foundation for pre-clinical researches and clinica application in the future.
13. The specific activity of NJF was tested by the chromogenic substrate method and the agarose - fibrin plate method using Urokinase standard as standard. The specific activity of NJF was not less than 10,000 U/mg tested by the chromogenic substrate method ,and 50,000 IU/mg Urokinase units determined by the agarose - fibrin plate method.
通过酶的初提、硫酸铵盐析、Phenyl Sepharose 6 FF疏水层析、DEAE Sepharose FF阴离子离子交换层析和Sephacryl S-100 HR凝胶过滤层析,获得日本刺沙蚕纤溶酶,其在SDS-PAGE和常规PAGE电泳中为单一条带,用高效液相凝胶过滤色谱法检测其纯度不低于95%。
日本刺沙蚕纤溶酶是一条单链蛋白,分子量在28-32kDa,等电点为4.4。其N末端存在封闭现象,通过MALDI-TOF-TOF串联质谱进行De Novo测序,获得三个可信肽段的32个氨基酸序列,登陆到UniProt数据库中,获得登录号为P86330。通过氨基酸序列比对,证明其是一种新发现的蛋白质。
日本刺沙蚕纤溶酶的酶学性质:酶活性能被PMSF完全抑制,是一种典型的丝氨酸蛋白酶;Hg2+能够强烈的抑制蛋白酶的活性,Cu2+具有较弱的抑制作用,而其它金属离子对酶活性没有明显的作用;具有较好的热稳定性和pH稳定性,在40～80°C和pH7～11范围内表现出良好的稳定性,最适温度为60°C,最适pH为9.0;特异发色底物为凝血酶发色底物S-2238,其水解活性为11.2 mmol/min/mg;能够直接溶解纤维蛋白,起主要作用,也具有较弱的激酶活性;最先水解纤维蛋白原的Aα链,然后是Bβ,最后水解γ链。
日本刺沙蚕纤溶酶的部分质量标准:采用常规PAGE电泳法和高效液相凝胶过滤色谱法检测的纯度均不低于95%;使用鲎试剂检测细菌内毒素和家兔法检测热原,结果均符合标准;以S-2238为底物检测的比活力不少于10,000 U/mg;以尿激酶标准品为标准采用琼脂糖-纤维蛋白平板法检测的比活力不少于50,000 IU/mg。
通过对日本刺沙蚕纤溶酶的分子特性、酶学性质和部分质量标准进行系统性研究,为以后的临床前研究和将来的临床应用奠定基础。
Cardiovascular diseases(CVDs) are the leading cause of morbidity in the worldwide. According to World Health Organization statistics, about 17.1million people died from CVDs in 2004, which represented 30% of all global deaths. By 2030, almost 23.6 million people will die from CVDs, mainly from heart disease and stroke. Intravascular thrombosis due to fibrin aggregation in arteries is one of the main causes of CVDs.
Currently, thrombolytic therapy is the the most extensive and most effective method for clinical treatment of thrombotic disease. The major thrombolytic agents availabl for clinical application are plasminogen activators, such as urokinase, streptokinase and tPA (tissue-type plasminogen activator). Despite their widespread use, all these agents have undesirable side effects, and are also relatively expensive. Therefore, the search for other novel thrombolytic agents from various sources is very necessary. Many organisms are important sources of thrombolytic agents.
Neanthes japonica (Iznka) is a marine invertebrate and widely distributed in the north coast of China and the coast of Japan. It is rich in protein, so it is the high-quality food of sea water creatures, such as fish, shrimp and crab and the good bait for fishing in the sea as well. In this stuty, we isolated and purified a novel fibrinolytic enzyme from a marine invertebrate, Neanthes japonica (Iznka), named NJF, and studied its molecular characteristics, enzymatic properties and partial quality standards systematicly in this paper. These studies laid the foundation for pre-clinical researches and clinica application in the future. The main achievements in our investigations are now showed as follows.
1. NJF was achieved by a combination of several isolation and purification methods using shredded autolysis, ammonium sulfate salting out, hydrophobic interaction chromatography by Phenyl Sepharose 6 FF, anion exchange chromatography by DEAE Sepharose FF and gel-filtration chromatography by Sepharcyl S-100 High Resolution. The enzyme was purified 2390-fold with a final yield of 16% after these purification steps. The purified NJF was shown as a single band in SDS–PAGE and Native-PAGE, and the purity of NJF determined by HPLC gel-filtration was 95.2%.
2. NJF consisted of a single polypeptide chain with a molecular weight of 28–32 kDa, which was determined by MALDI-TOF mass spectrum and SDS–PAGE. The isoelectric point of NJF determined by isoelectric focusing electrophoresis (IEF) was 4.4.
3. Purified enzyme was resistant to Edman degradation chemistry for determination of N-terminal sequence, indicating that the N-terminus of NJF is blocked. In further studies, we have obtained the amino acid sequences of three peptides by MALDI-TOF-TOF mass spectrometry and De Novo sequencing. The amino acid sequences of the three peptides are RGVTDHLYN-NH2, RSPGWL-NH2, and RSQVDGVMWDLGDLLGA-NH2. We reported these amino acid sequences of three peptides data in the UniProtKB/Swiss-Prot Knowledgebase and obtained the accession number is P86330. Sequences blast by using standard protein-protein BLAST (blastp) in NCBI protein databases, showed that these amino-acid sequences are different from that of other known proteins. These results suggest that NJF is a novel fibrinolytic enzyme.
4. The protease activity was inhibited by PMSF, but not by EDTA, EGTA,β- Mercaptoe- thanol , Iodoacetate , Indoacetamide and Pepstatin A. This result sugest that NJF is a serine protease. However, other serine protease inhibitors, including aprotinin, benzamidine and trypsin inhibitor had no obvious effect on proteolytic activity, indicating that NJF is a unique serine protease.
5.The protease activity was inhibited completely by Hg2+ ion, and inhibited slightly by Cu2+ ion. However, other metal ions including Na+, K+, Li+,Ca2+, Zn2+, Co2+, Mg2+, Fe2+, Fe3+, Al3+ ions have no obvious effect on the enzyme activity. These results suggest that the molecular structure of NJF may exist one or more disulfide bonds.
6. The effect of temperature and pH on enzyme activity indicated that NJF has better thermal stability and pH stability. The enzyme activity was stable between 40°C and 80°C, and in the pH range of 7-11. Maximum activity of enzyme was observed at 60°C and pH 9.0. These results indicated that the NJF may be used for prevention and treatment of cardiovascular and cerebrovascular diseases by administered orally or intravenously in the future, and has potential clinical application. Because the resulting pH values are around alkaline, NJF can be classified into alkaline protease.
7. Using azocasein as substrate, the enzyme reaction velocity is rapidly increased as substrate congcentration increased between 0.2-4mg/ml. When substrate congcentration is approximately 8 mg/ml, the enzyme reaction velocity reaches its maximum, but when the substrate congcentration higher than 8 mg/ml, the enzyme reaction velocity is not increased. The enzyme dynamic parameters calculated according to double reciprocal plot are as follows: Km=2.359, Vmax=1.845.
8. The study of specific substrate was measured spectrophotometrically, using chromo- genic protease substrates such as S-2238 (H-D-Phe-Pip-Arg-pNA for thrombin), S-2251 (H-D-Val-Leu-Lys-pNA for plasmin and streptokinase-activated plasminogen) and S-2444 (pyro-Glu-Gly-Arg-pNA for urokinase). NJF exhibited a higher degree of specificity for the substrate S-2238 (H-D-Phe-Pip-Arg- pNA), which was 11.2 mmol/min/mg. NJF had strong hydrolytic activity for S2444 and S2251, which suggested that the enzyme acted on arginine and lysine residue at P1 position of the chromogenic substrate. NJF acted on arginine and lysine residue, which similar to trypsin.
9. Two methods of agarose - fibrin plate was used to determine whether NJF has the kinase activity. The results of two methods were showed that NJF was a plasmin-like protease which can directly degrade the fibrin, which play a major role, and it has the weaker kinase activity also. Its fibrinolytic activity was more strong than plasmin.
10. The fibrinogenolytic activity of NJF analyzed by SDS-PAGE showed that the Aα-chain was completely in a very short time (1 min), and Bβ-chain was completely degraded within 10 min. Theγ-chain was the last subunit to be hydrolysed by NJF, and was completely degraded in 1 h. Fibrinogenolytic activity of NJF can reduce blood viscosity, increase blood flow velocity and improve microcirculation, inhibiting thrombus formation and further increased. NJF may be used for prevention and treatment of cardiovascular and cerebrovascular diseases in the future, and has potential clinical application.
11. The purity of the purified NJF was tested using Native-PAGE analyzed by BandScan 5.0, which was 97.4%. The purity of the purified NJF was 96.74% by HPLC gel-filtration chromatography. The mechanism of two determined methods is different, so their results can reflect the true purity of NJF. The results determined by the two methods are consistent, and the purity of the different batches of purified NJF is 95% at least.
12. The check of bacterial endotoxin test used Limulus amoebocyte lysate and the check of pyrogen using rabbits for the purified NJF. The results of two methods are in line with the reqirements of Chinese Pharmacopoeia. These results laid the foundation for pre-clinical researches and clinica application in the future.
13. The specific activity of NJF was tested by the chromogenic substrate method and the agarose - fibrin plate method using Urokinase standard as standard. The specific activity of NJF was not less than 10,000 U/mg tested by the chromogenic substrate method ,and 50,000 IU/mg Urokinase units determined by the agarose - fibrin plate method.
引文
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