Purification procedure for the isolation of a P-I metalloprotease and an acidic phospholipase A 2 fromBothrops atrox snake venom

Detalhes bibliográficos
Autor(a) principal: Menaldo,Danilo L.
Data de Publicação: 2015
Outros Autores: Jacob-Ferreira,Anna L., Bernardes,Carolina P., Cintra,Adélia C. O., Sampaio,Suely V.
Tipo de documento: Artigo
Idioma: eng
Título da fonte: The Journal of venomous animals and toxins including tropical diseases (Online)
Texto Completo: http://old.scielo.br/scielo.php?script=sci_arttext&pid=S1678-91992015000100337
Resumo: Background Snake venoms are complex mixtures of inorganic and organic components, mainly proteins and peptides. Standardization of methods for isolating bioactive molecules from snake venoms is extremely difficult due to the complex and highly variable composition of venoms, which can be influenced by factors such as age and geographic location of the specimen. Therefore, this study aimed to standardize a simple purification methodology for obtaining a P-I class metalloprotease (MP) and an acidic phospholipase A2 (PLA 2 ) from Bothrops atroxvenom, and biochemically characterize these molecules to enable future functional studies.Methods To obtain the toxins of interest, a method has been standardized using consecutive isolation steps. The purity level of the molecules was confirmed by RP-HPLC and SDS-PAGE. The enzymes were characterized by determining their molecular masses, isoelectric points, specific functional activity and partial amino acid sequencing.Results The metalloprotease presented molecular mass of 22.9 kDa and pI 7.4, with hemorrhagic and fibrin(ogen)olytic activities, and its partial amino acid sequence revealed high similarity with other P-I class metalloproteases. These results suggest that the isolated metalloprotease is Batroxase, a P-I metalloprotease previously described by our research group. The phospholipase A 2 showed molecular mass of 13.7 kDa and pI 6.5, with high phospholipase activity and similarity to other acidic PLA2 s from snake venoms. These data suggest that the acidic PLA2 is a novel enzyme from B. atrox venom, being denominated BatroxPLA 2 .Conclusions The present study successfully standardized a simple methodology to isolate the metalloprotease Batroxase and the acidic PLA 2 BatroxPLA2 from the venom of B. atrox, consisting mainly of classical chromatographic processes. These two enzymes will be used in future studies to evaluate their effects on the complement system and the inflammatory process, in addition to the thrombolytic potential of the metalloprotease.
id UNESP-11_bd015537d5947e09a4146d63c17018be
oai_identifier_str oai:scielo:S1678-91992015000100337
network_acronym_str UNESP-11
network_name_str The Journal of venomous animals and toxins including tropical diseases (Online)
repository_id_str
spelling Purification procedure for the isolation of a P-I metalloprotease and an acidic phospholipase A 2 fromBothrops atrox snake venomSnake venomsBothrops atroxToxinsMetalloproteasePhospholipase A2IsolationCharacterizationChromatographyBackground Snake venoms are complex mixtures of inorganic and organic components, mainly proteins and peptides. Standardization of methods for isolating bioactive molecules from snake venoms is extremely difficult due to the complex and highly variable composition of venoms, which can be influenced by factors such as age and geographic location of the specimen. Therefore, this study aimed to standardize a simple purification methodology for obtaining a P-I class metalloprotease (MP) and an acidic phospholipase A2 (PLA 2 ) from Bothrops atroxvenom, and biochemically characterize these molecules to enable future functional studies.Methods To obtain the toxins of interest, a method has been standardized using consecutive isolation steps. The purity level of the molecules was confirmed by RP-HPLC and SDS-PAGE. The enzymes were characterized by determining their molecular masses, isoelectric points, specific functional activity and partial amino acid sequencing.Results The metalloprotease presented molecular mass of 22.9 kDa and pI 7.4, with hemorrhagic and fibrin(ogen)olytic activities, and its partial amino acid sequence revealed high similarity with other P-I class metalloproteases. These results suggest that the isolated metalloprotease is Batroxase, a P-I metalloprotease previously described by our research group. The phospholipase A 2 showed molecular mass of 13.7 kDa and pI 6.5, with high phospholipase activity and similarity to other acidic PLA2 s from snake venoms. These data suggest that the acidic PLA2 is a novel enzyme from B. atrox venom, being denominated BatroxPLA 2 .Conclusions The present study successfully standardized a simple methodology to isolate the metalloprotease Batroxase and the acidic PLA 2 BatroxPLA2 from the venom of B. atrox, consisting mainly of classical chromatographic processes. These two enzymes will be used in future studies to evaluate their effects on the complement system and the inflammatory process, in addition to the thrombolytic potential of the metalloprotease.Centro de Estudos de Venenos e Animais Peçonhentos (CEVAP/UNESP)2015-01-01info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersiontext/htmlhttp://old.scielo.br/scielo.php?script=sci_arttext&pid=S1678-91992015000100337Journal of Venomous Animals and Toxins including Tropical Diseases v.21 2015reponame:The Journal of venomous animals and toxins including tropical diseases (Online)instname:Universidade Estadual Paulista (UNESP)instacron:UNESP10.1186/s40409-015-0027-6info:eu-repo/semantics/openAccessMenaldo,Danilo L.Jacob-Ferreira,Anna L.Bernardes,Carolina P.Cintra,Adélia C. O.Sampaio,Suely V.eng2015-09-24T00:00:00Zoai:scielo:S1678-91992015000100337Revistahttp://www.scielo.br/jvatitdPUBhttps://old.scielo.br/oai/scielo-oai.php||editorial@jvat.org.br1678-91991678-9180opendoar:2015-09-24T00:00The Journal of venomous animals and toxins including tropical diseases (Online) - Universidade Estadual Paulista (UNESP)false
dc.title.none.fl_str_mv Purification procedure for the isolation of a P-I metalloprotease and an acidic phospholipase A 2 fromBothrops atrox snake venom
title Purification procedure for the isolation of a P-I metalloprotease and an acidic phospholipase A 2 fromBothrops atrox snake venom
spellingShingle Purification procedure for the isolation of a P-I metalloprotease and an acidic phospholipase A 2 fromBothrops atrox snake venom
Menaldo,Danilo L.
Snake venoms
Bothrops atrox
Toxins
Metalloprotease
Phospholipase A2
Isolation
Characterization
Chromatography
title_short Purification procedure for the isolation of a P-I metalloprotease and an acidic phospholipase A 2 fromBothrops atrox snake venom
title_full Purification procedure for the isolation of a P-I metalloprotease and an acidic phospholipase A 2 fromBothrops atrox snake venom
title_fullStr Purification procedure for the isolation of a P-I metalloprotease and an acidic phospholipase A 2 fromBothrops atrox snake venom
title_full_unstemmed Purification procedure for the isolation of a P-I metalloprotease and an acidic phospholipase A 2 fromBothrops atrox snake venom
title_sort Purification procedure for the isolation of a P-I metalloprotease and an acidic phospholipase A 2 fromBothrops atrox snake venom
author Menaldo,Danilo L.
author_facet Menaldo,Danilo L.
Jacob-Ferreira,Anna L.
Bernardes,Carolina P.
Cintra,Adélia C. O.
Sampaio,Suely V.
author_role author
author2 Jacob-Ferreira,Anna L.
Bernardes,Carolina P.
Cintra,Adélia C. O.
Sampaio,Suely V.
author2_role author
author
author
author
dc.contributor.author.fl_str_mv Menaldo,Danilo L.
Jacob-Ferreira,Anna L.
Bernardes,Carolina P.
Cintra,Adélia C. O.
Sampaio,Suely V.
dc.subject.por.fl_str_mv Snake venoms
Bothrops atrox
Toxins
Metalloprotease
Phospholipase A2
Isolation
Characterization
Chromatography
topic Snake venoms
Bothrops atrox
Toxins
Metalloprotease
Phospholipase A2
Isolation
Characterization
Chromatography
description Background Snake venoms are complex mixtures of inorganic and organic components, mainly proteins and peptides. Standardization of methods for isolating bioactive molecules from snake venoms is extremely difficult due to the complex and highly variable composition of venoms, which can be influenced by factors such as age and geographic location of the specimen. Therefore, this study aimed to standardize a simple purification methodology for obtaining a P-I class metalloprotease (MP) and an acidic phospholipase A2 (PLA 2 ) from Bothrops atroxvenom, and biochemically characterize these molecules to enable future functional studies.Methods To obtain the toxins of interest, a method has been standardized using consecutive isolation steps. The purity level of the molecules was confirmed by RP-HPLC and SDS-PAGE. The enzymes were characterized by determining their molecular masses, isoelectric points, specific functional activity and partial amino acid sequencing.Results The metalloprotease presented molecular mass of 22.9 kDa and pI 7.4, with hemorrhagic and fibrin(ogen)olytic activities, and its partial amino acid sequence revealed high similarity with other P-I class metalloproteases. These results suggest that the isolated metalloprotease is Batroxase, a P-I metalloprotease previously described by our research group. The phospholipase A 2 showed molecular mass of 13.7 kDa and pI 6.5, with high phospholipase activity and similarity to other acidic PLA2 s from snake venoms. These data suggest that the acidic PLA2 is a novel enzyme from B. atrox venom, being denominated BatroxPLA 2 .Conclusions The present study successfully standardized a simple methodology to isolate the metalloprotease Batroxase and the acidic PLA 2 BatroxPLA2 from the venom of B. atrox, consisting mainly of classical chromatographic processes. These two enzymes will be used in future studies to evaluate their effects on the complement system and the inflammatory process, in addition to the thrombolytic potential of the metalloprotease.
publishDate 2015
dc.date.none.fl_str_mv 2015-01-01
dc.type.driver.fl_str_mv info:eu-repo/semantics/article
dc.type.status.fl_str_mv info:eu-repo/semantics/publishedVersion
format article
status_str publishedVersion
dc.identifier.uri.fl_str_mv http://old.scielo.br/scielo.php?script=sci_arttext&pid=S1678-91992015000100337
url http://old.scielo.br/scielo.php?script=sci_arttext&pid=S1678-91992015000100337
dc.language.iso.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv 10.1186/s40409-015-0027-6
dc.rights.driver.fl_str_mv info:eu-repo/semantics/openAccess
eu_rights_str_mv openAccess
dc.format.none.fl_str_mv text/html
dc.publisher.none.fl_str_mv Centro de Estudos de Venenos e Animais Peçonhentos (CEVAP/UNESP)
publisher.none.fl_str_mv Centro de Estudos de Venenos e Animais Peçonhentos (CEVAP/UNESP)
dc.source.none.fl_str_mv Journal of Venomous Animals and Toxins including Tropical Diseases v.21 2015
reponame:The Journal of venomous animals and toxins including tropical diseases (Online)
instname:Universidade Estadual Paulista (UNESP)
instacron:UNESP
instname_str Universidade Estadual Paulista (UNESP)
instacron_str UNESP
institution UNESP
reponame_str The Journal of venomous animals and toxins including tropical diseases (Online)
collection The Journal of venomous animals and toxins including tropical diseases (Online)
repository.name.fl_str_mv The Journal of venomous animals and toxins including tropical diseases (Online) - Universidade Estadual Paulista (UNESP)
repository.mail.fl_str_mv ||editorial@jvat.org.br
_version_ 1748958539982831616