Partial Purification and Characterization of Fibrinolytic Enzymes from Yellow Mealworm
Abstract Two proteins with fibrinolytic activity were partially purified from yellow mealworm (Tenebrio molitor) by ammonium sulfate precipitation between 30 and 70% saturation, gel filtration on Sephacryl-S200-HR, ion exchange chromatography on DEAE-Sepharose-FF and metal chelate on Cu–HiTrap–IMAC–FF, but the enzymes had not been completely separated from each other. The two partially purified fibrinolytic enzymes were designated as TMFE-I and TMFE-II (Tenebrio molitor fibrinolytic enzyme) with molecular weights of 27.5 and 24.9 kDa by SDS-PAGE individually. The partially purified solution of TMFE-I and TMFE-II was considerably stable in the range of pH 5–10 and characterized by pH optimum of the enzymatic activity at 8.0. Thermal stability of TMFE was excellent at 45°C and below. The KM value was 0.26 mM for amidolysis of Bz–Arg–pNA. According to inhibitor analysis by fibrin plate method, phenylmethylsulfonyl fluoride and tosyl-lysine chloromethyl ketone inactivated TMFE almost completely, but trans-(epoxysuccinyl)-l-leucylamino-4-guanidinobutane (E-64) and EDTA had little effect on their fibrinolytic activity. According to metal ion analysis by fibrin plate method, the effect of metal ions on activity of TMFE showed a great difference. $ Na^{+} $, $ K^{+} $ and $ Zn^{2+} $ had little effect on the activity of TMFE. $ Mg^{2+} $ and $ Cu^{2+} $ showed inhibition effect on the fibrinolytic activity of TMFE, but $ Ca^{2+} $ increased the fibrinolytic activity of TMFE at final concentration varying from 0 to 30 mM..
| Medienart: |
Artikel |
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| Erscheinungsjahr: |
2012 |
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| Erschienen: |
2012 |
| Enthalten in: |
Zur Gesamtaufnahme - volume:18 |
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| Enthalten in: |
International journal of peptide research and therapeutics - 18(2012), 2 vom: 23. Feb., Seite 153-161 |
| Sprache: |
Englisch |
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| Beteiligte Personen: |
Huang, Ming Xing [VerfasserIn] |
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| Links: |
Volltext [lizenzpflichtig] |
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| BKL: | |
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| Themen: |
Fibrinolytic enzyme |
| Anmerkungen: |
© Springer Science+Business Media, LLC 2012 |
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| doi: |
10.1007/s10989-012-9288-x |
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| funding: |
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| Förderinstitution / Projekttitel: |
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OLC2067151177 |
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| 520 | |a Abstract Two proteins with fibrinolytic activity were partially purified from yellow mealworm (Tenebrio molitor) by ammonium sulfate precipitation between 30 and 70% saturation, gel filtration on Sephacryl-S200-HR, ion exchange chromatography on DEAE-Sepharose-FF and metal chelate on Cu–HiTrap–IMAC–FF, but the enzymes had not been completely separated from each other. The two partially purified fibrinolytic enzymes were designated as TMFE-I and TMFE-II (Tenebrio molitor fibrinolytic enzyme) with molecular weights of 27.5 and 24.9 kDa by SDS-PAGE individually. The partially purified solution of TMFE-I and TMFE-II was considerably stable in the range of pH 5–10 and characterized by pH optimum of the enzymatic activity at 8.0. Thermal stability of TMFE was excellent at 45°C and below. The KM value was 0.26 mM for amidolysis of Bz–Arg–pNA. According to inhibitor analysis by fibrin plate method, phenylmethylsulfonyl fluoride and tosyl-lysine chloromethyl ketone inactivated TMFE almost completely, but trans-(epoxysuccinyl)-l-leucylamino-4-guanidinobutane (E-64) and EDTA had little effect on their fibrinolytic activity. According to metal ion analysis by fibrin plate method, the effect of metal ions on activity of TMFE showed a great difference. $ Na^{+} $, $ K^{+} $ and $ Zn^{2+} $ had little effect on the activity of TMFE. $ Mg^{2+} $ and $ Cu^{2+} $ showed inhibition effect on the fibrinolytic activity of TMFE, but $ Ca^{2+} $ increased the fibrinolytic activity of TMFE at final concentration varying from 0 to 30 mM. | ||
| 650 | 4 | |a Fibrinolytic enzyme | |
| 650 | 4 | |a Serine protease | |
| 650 | 4 | |a Metal chelate | |
| 650 | 4 | |a Thrombolytic agent | |
| 700 | 1 | |a Ye, Yun |4 aut | |
| 700 | 1 | |a Chen, Ya Xiong |4 aut | |
| 700 | 1 | |a Han, Ya Li |4 aut | |
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