Purification of a proteolytic enzyme from the venom os Bothrops brazili sanke and estudy of its activity on fibrinogen
A proteolitic enzyme was purified from Bothrops brazili peruvian snake venom using Sephadex G-100 followed by CM-Sephadex C-50, in both two cases with 0.05M ammonium acetate pH 7,0. The enzyme was purified 3,2 fold with 52,5% of yield and by gel filtration the enzyme showed 18 000 of molecular weight, while the PAGE-SIDS showed only one protein band of 22 000 in the presence of mercaptoethanol and 20 300 under nonreducing conditions which suggests that the enzyme has a single polypeptide chain with disulfide bond. The enzyme hydrolizes fibrinogen, fibrin, casein and albumin, but not hemoglobin and mioglobin. The enzyme is a Aa-fibrinogenase because preferentially hydrolizes the Aa chain of the fibrinogen molecule. This activity is inhibited by EDTA but not by PMSF, TLCK, iodoacetate and pepstatin, suggests that is a metalloproteinase, however the ions Ca++, Mg++ and Zn++ cannot reactivate the inhibition by EDTA. Finally the enzyme is stable up to 45 °C and in its effect on fibrin the enzyme is to attack a chain rapidly.
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Universidad Nacional Mayor de San Marcos, Facultad de Ciencias Biológicas
2000
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oai:ojs.csi.unmsm:article67282020-05-26T14:52:39Z Purification of a proteolytic enzyme from the venom os Bothrops brazili sanke and estudy of its activity on fibrinogen Purificación de una enzima proteolítica del veneno de Bothrops brazili y estudio de su actividad sobre fibrinógeno Azañero, María Escobar, Enrique Yarlequé, Armando Enzyme fibrinogenase venom Bothrops brazili. Enzima fibrinogenasa veneno Bothrops brazili A proteolitic enzyme was purified from Bothrops brazili peruvian snake venom using Sephadex G-100 followed by CM-Sephadex C-50, in both two cases with 0.05M ammonium acetate pH 7,0. The enzyme was purified 3,2 fold with 52,5% of yield and by gel filtration the enzyme showed 18 000 of molecular weight, while the PAGE-SIDS showed only one protein band of 22 000 in the presence of mercaptoethanol and 20 300 under nonreducing conditions which suggests that the enzyme has a single polypeptide chain with disulfide bond. The enzyme hydrolizes fibrinogen, fibrin, casein and albumin, but not hemoglobin and mioglobin. The enzyme is a Aa-fibrinogenase because preferentially hydrolizes the Aa chain of the fibrinogen molecule. This activity is inhibited by EDTA but not by PMSF, TLCK, iodoacetate and pepstatin, suggests that is a metalloproteinase, however the ions Ca++, Mg++ and Zn++ cannot reactivate the inhibition by EDTA. Finally the enzyme is stable up to 45 °C and in its effect on fibrin the enzyme is to attack a chain rapidly. Se ha aislado una enzima proteolítica del veneno de la serpiente peruana Bothrops brazili, por cromatografía en Sephadex G-100 y CM-Sephadex C-50, con buffer acetato de amonio 0,05M pH 7,0. La enzima fue purificada 3,2 veces con un rendimiento de 52,5% y el peso molecular calculado por filtración en gel fue de 18 000, mientras que la PAGE-SDS permitió observar una sola banda proteica de 22 000 daltons en condiciones reductoras y de 20 300 daltons en condiciones no reductoras, determinándose que la enzima es de una sola cadena polipeptídica con al menos un enlace disulfuro. La enzima hidroliza fibrinógeno, fibrina, caseína y albúmina, pero no hemoglobina ni mioglobina. Por su acción sobre el fibrinógeno, es una Aa-fibrinogenasa ya que hidroliza primero la cadena Aa del fibrinógeno y luego la cadena Bb. Esta actividad es inhibida por EDTA pero no por PMSF, TLCK, iodoacetato y pepstatin, indicando que se trata de una metaloproteinasa; sin embargo, los iones Ca++, Mg++ y Zn++, no restablecen la actividad. Finalmente, la enzima es estable hasta los 45 °C y en su acción sobre fibrina, la hidrólisis se da preferencialmente sobre la cadena a. Universidad Nacional Mayor de San Marcos, Facultad de Ciencias Biológicas 2000-06-19 info:eu-repo/semantics/article info:eu-repo/semantics/publishedVersion info:eu-repo/semantics/article info:eu-repo/semantics/article application/pdf https://revistasinvestigacion.unmsm.edu.pe/index.php/rpb/article/view/6728 10.15381/rpb.v7i1.6728 Revista Peruana de Biología; Vol. 7 No. 1 (2000); 55 - 66 Revista Peruana de Biología; Vol. 7 Núm. 1 (2000); 55 - 66 1727-9933 1561-0837 spa https://revistasinvestigacion.unmsm.edu.pe/index.php/rpb/article/view/6728/5973 Derechos de autor 2000 María Azañero, Enrique Escobar, Armando Yarlequé https://creativecommons.org/licenses/by-nc-sa/4.0 |
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| author |
Azañero, María Escobar, Enrique Yarlequé, Armando |
| spellingShingle |
Azañero, María Escobar, Enrique Yarlequé, Armando Purification of a proteolytic enzyme from the venom os Bothrops brazili sanke and estudy of its activity on fibrinogen |
| author_facet |
Azañero, María Escobar, Enrique Yarlequé, Armando |
| author_sort |
Azañero, María |
| title |
Purification of a proteolytic enzyme from the venom os Bothrops brazili sanke and estudy of its activity on fibrinogen |
| title_short |
Purification of a proteolytic enzyme from the venom os Bothrops brazili sanke and estudy of its activity on fibrinogen |
| title_full |
Purification of a proteolytic enzyme from the venom os Bothrops brazili sanke and estudy of its activity on fibrinogen |
| title_fullStr |
Purification of a proteolytic enzyme from the venom os Bothrops brazili sanke and estudy of its activity on fibrinogen |
| title_full_unstemmed |
Purification of a proteolytic enzyme from the venom os Bothrops brazili sanke and estudy of its activity on fibrinogen |
| title_sort |
purification of a proteolytic enzyme from the venom os bothrops brazili sanke and estudy of its activity on fibrinogen |
| description |
A proteolitic enzyme was purified from Bothrops brazili peruvian snake venom using Sephadex G-100 followed by CM-Sephadex C-50, in both two cases with 0.05M ammonium acetate pH 7,0. The enzyme was purified 3,2 fold with 52,5% of yield and by gel filtration the enzyme showed 18 000 of molecular weight, while the PAGE-SIDS showed only one protein band of 22 000 in the presence of mercaptoethanol and 20 300 under nonreducing conditions which suggests that the enzyme has a single polypeptide chain with disulfide bond. The enzyme hydrolizes fibrinogen, fibrin, casein and albumin, but not hemoglobin and mioglobin. The enzyme is a Aa-fibrinogenase because preferentially hydrolizes the Aa chain of the fibrinogen molecule. This activity is inhibited by EDTA but not by PMSF, TLCK, iodoacetate and pepstatin, suggests that is a metalloproteinase, however the ions Ca++, Mg++ and Zn++ cannot reactivate the inhibition by EDTA. Finally the enzyme is stable up to 45 °C and in its effect on fibrin the enzyme is to attack a chain rapidly. |
| publisher |
Universidad Nacional Mayor de San Marcos, Facultad de Ciencias Biológicas |
| publishDate |
2000 |
| url |
https://revistasinvestigacion.unmsm.edu.pe/index.php/rpb/article/view/6728 |
| work_keys_str_mv |
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