Enzymatic glutaredoxin-dependent method to determine glutathione and protein S-glutathionylation using fluorescent eosin-glutathione.
Identifieur interne : 000172 ( Main/Exploration ); précédent : 000171; suivant : 000173Enzymatic glutaredoxin-dependent method to determine glutathione and protein S-glutathionylation using fluorescent eosin-glutathione.
Auteurs : Lucia Coppo [Suède] ; Fernando T. Ogata [Suède] ; Sebastin M. Santhosh [Suède] ; Teodor Sventelius [Suède] ; Arne Holmgren [Suède]Source :
- Analytical biochemistry [ 1096-0309 ] ; 2019.
Descripteurs français
- KwdFr :
- MESH :
- analyse : Glutathion.
- composition chimique : Glutarédoxines.
- métabolisme : Glutarédoxines, Glutathion.
- Cellules cultivées, Fluorescence, Humains.
English descriptors
- KwdEn :
- MESH :
- chemical , analysis : Glutathione.
- chemical , chemistry : Glutaredoxins.
- chemical , metabolism : Glutaredoxins, Glutathione.
- Cells, Cultured, Fluorescence, Humans.
Abstract
Glutathione is an abundant low-molecular-weight thiol, up to 10 mM in mammalian cells, and exists in three major forms: reduced sulphydryl (GSH), glutathione disulfide (GSSG) or bound to Cys residues in proteins (PSSG). The ratio GSH/GSSG has been used as an indicator of the cells redox level but this parameter can also be estimated by the quantification of PSSG. In fact, PSSGs have the advantage of being more stable than GSSG. Here we present a highly sensitive fluorescent-based method for detection of low concentrations of glutathione in complex samples such as cell lysates, tissues and plasma. The method is based on our previously described protocol to study Glutaredoxin (Grx) activity. The whole procedure was optimized to measure the fluorescence increase of the di-eosin-glutathione disulfide (Di-E-GSSG) reduced by Grx in the presence of Glutathione Reductase and NADPH, keeping GSH as the limiting factor to drive the reaction. The methods to selectively measure PSSG are expensive and not widely accessible, therefore we optimized our glutaredoxin protocol to quantify this post-translational modification using common laboratory equipments. Overall, our method has simplicity and rapidity combined with high sensitivity as its main advantages; therefore, it may be particularly suitable for large-scale clinical studies.
DOI: 10.1016/j.ab.2018.12.021
PubMed: 30597126
Affiliations:
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Electronic address: lucia.coppo@ki.se.</nlm:affiliation><country xml:lang="fr">Suède</country><wicri:regionArea>Department of Medical Biochemistry and Biophysics, Karolinska Institutet, SE-17177, Stockholm</wicri:regionArea><placeName><settlement type="city">Stockholm</settlement><region nuts="2">Svealand</region></placeName></affiliation></author><author><name sortKey="Ogata, Fernando T" sort="Ogata, Fernando T" uniqKey="Ogata F" first="Fernando T" last="Ogata">Fernando T. 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Santhosh</name><affiliation wicri:level="3"><nlm:affiliation>Department of Medical Biochemistry and Biophysics, Karolinska Institutet, SE-17177, Stockholm, Sweden.</nlm:affiliation><country xml:lang="fr">Suède</country><wicri:regionArea>Department of Medical Biochemistry and Biophysics, Karolinska Institutet, SE-17177, Stockholm</wicri:regionArea><placeName><settlement type="city">Stockholm</settlement><region nuts="2">Svealand</region></placeName></affiliation></author><author><name sortKey="Sventelius, Teodor" sort="Sventelius, Teodor" uniqKey="Sventelius T" first="Teodor" last="Sventelius">Teodor Sventelius</name><affiliation wicri:level="3"><nlm:affiliation>Department of Medical Biochemistry and Biophysics, Karolinska Institutet, SE-17177, Stockholm, Sweden.</nlm:affiliation><country xml:lang="fr">Suède</country><wicri:regionArea>Department of Medical Biochemistry and Biophysics, Karolinska Institutet, SE-17177, Stockholm</wicri:regionArea><placeName><settlement type="city">Stockholm</settlement><region nuts="2">Svealand</region></placeName></affiliation></author><author><name sortKey="Holmgren, Arne" sort="Holmgren, Arne" uniqKey="Holmgren A" first="Arne" last="Holmgren">Arne Holmgren</name><affiliation wicri:level="3"><nlm:affiliation>Department of Medical Biochemistry and Biophysics, Karolinska Institutet, SE-17177, Stockholm, Sweden. Electronic address: arne.holmgren@ki.se.</nlm:affiliation><country xml:lang="fr">Suède</country><wicri:regionArea>Department of Medical Biochemistry and Biophysics, Karolinska Institutet, SE-17177, Stockholm</wicri:regionArea><placeName><settlement type="city">Stockholm</settlement><region nuts="2">Svealand</region></placeName></affiliation></author></analytic><series><title level="j">Analytical biochemistry</title><idno type="eISSN">1096-0309</idno><imprint><date when="2019" type="published">2019</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>Cells, Cultured (MeSH)</term><term>Fluorescence (MeSH)</term><term>Glutaredoxins (chemistry)</term><term>Glutaredoxins (metabolism)</term><term>Glutathione (analysis)</term><term>Glutathione (metabolism)</term><term>Humans (MeSH)</term></keywords><keywords scheme="KwdFr" xml:lang="fr"><term>Cellules cultivées (MeSH)</term><term>Fluorescence (MeSH)</term><term>Glutarédoxines (composition chimique)</term><term>Glutarédoxines (métabolisme)</term><term>Glutathion (analyse)</term><term>Glutathion (métabolisme)</term><term>Humains (MeSH)</term></keywords><keywords scheme="MESH" type="chemical" qualifier="analysis" xml:lang="en"><term>Glutathione</term></keywords><keywords scheme="MESH" type="chemical" qualifier="chemistry" xml:lang="en"><term>Glutaredoxins</term></keywords><keywords scheme="MESH" type="chemical" qualifier="metabolism" xml:lang="en"><term>Glutaredoxins</term><term>Glutathione</term></keywords><keywords scheme="MESH" qualifier="analyse" xml:lang="fr"><term>Glutathion</term></keywords><keywords scheme="MESH" qualifier="composition chimique" xml:lang="fr"><term>Glutarédoxines</term></keywords><keywords scheme="MESH" qualifier="métabolisme" xml:lang="fr"><term>Glutarédoxines</term><term>Glutathion</term></keywords><keywords scheme="MESH" xml:lang="en"><term>Cells, Cultured</term><term>Fluorescence</term><term>Humans</term></keywords><keywords scheme="MESH" xml:lang="fr"><term>Cellules cultivées</term><term>Fluorescence</term><term>Humains</term></keywords></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">Glutathione is an abundant low-molecular-weight thiol, up to 10 mM in mammalian cells, and exists in three major forms: reduced sulphydryl (GSH), glutathione disulfide (GSSG) or bound to Cys residues in proteins (PSSG). The ratio GSH/GSSG has been used as an indicator of the cells redox level but this parameter can also be estimated by the quantification of PSSG. In fact, PSSGs have the advantage of being more stable than GSSG. Here we present a highly sensitive fluorescent-based method for detection of low concentrations of glutathione in complex samples such as cell lysates, tissues and plasma. The method is based on our previously described protocol to study Glutaredoxin (Grx) activity. The whole procedure was optimized to measure the fluorescence increase of the di-eosin-glutathione disulfide (Di-E-GSSG) reduced by Grx in the presence of Glutathione Reductase and NADPH, keeping GSH as the limiting factor to drive the reaction. The methods to selectively measure PSSG are expensive and not widely accessible, therefore we optimized our glutaredoxin protocol to quantify this post-translational modification using common laboratory equipments. Overall, our method has simplicity and rapidity combined with high sensitivity as its main advantages; therefore, it may be particularly suitable for large-scale clinical studies.</div></front></TEI><pubmed><MedlineCitation Status="MEDLINE" Owner="NLM"><PMID Version="1">30597126</PMID><DateCompleted><Year>2019</Year><Month>12</Month><Day>06</Day></DateCompleted><DateRevised><Year>2019</Year><Month>12</Month><Day>17</Day></DateRevised><Article PubModel="Print-Electronic"><Journal><ISSN IssnType="Electronic">1096-0309</ISSN><JournalIssue CitedMedium="Internet"><Volume>568</Volume><PubDate><Year>2019</Year><Month>03</Month><Day>01</Day></PubDate></JournalIssue><Title>Analytical biochemistry</Title><ISOAbbreviation>Anal Biochem</ISOAbbreviation></Journal><ArticleTitle>Enzymatic glutaredoxin-dependent method to determine glutathione and protein S-glutathionylation using fluorescent eosin-glutathione.</ArticleTitle><Pagination><MedlinePgn>24-30</MedlinePgn></Pagination><ELocationID EIdType="pii" ValidYN="Y">S0003-2697(18)30803-0</ELocationID><ELocationID EIdType="doi" ValidYN="Y">10.1016/j.ab.2018.12.021</ELocationID><Abstract><AbstractText>Glutathione is an abundant low-molecular-weight thiol, up to 10 mM in mammalian cells, and exists in three major forms: reduced sulphydryl (GSH), glutathione disulfide (GSSG) or bound to Cys residues in proteins (PSSG). The ratio GSH/GSSG has been used as an indicator of the cells redox level but this parameter can also be estimated by the quantification of PSSG. In fact, PSSGs have the advantage of being more stable than GSSG. Here we present a highly sensitive fluorescent-based method for detection of low concentrations of glutathione in complex samples such as cell lysates, tissues and plasma. The method is based on our previously described protocol to study Glutaredoxin (Grx) activity. The whole procedure was optimized to measure the fluorescence increase of the di-eosin-glutathione disulfide (Di-E-GSSG) reduced by Grx in the presence of Glutathione Reductase and NADPH, keeping GSH as the limiting factor to drive the reaction. The methods to selectively measure PSSG are expensive and not widely accessible, therefore we optimized our glutaredoxin protocol to quantify this post-translational modification using common laboratory equipments. Overall, our method has simplicity and rapidity combined with high sensitivity as its main advantages; therefore, it may be particularly suitable for large-scale clinical studies.</AbstractText><CopyrightInformation>Copyright © 2018. Published by Elsevier Inc.</CopyrightInformation></Abstract><AuthorList CompleteYN="Y"><Author ValidYN="Y"><LastName>Coppo</LastName><ForeName>Lucia</ForeName><Initials>L</Initials><AffiliationInfo><Affiliation>Department of Medical Biochemistry and Biophysics, Karolinska Institutet, SE-17177, Stockholm, Sweden. Electronic address: lucia.coppo@ki.se.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Ogata</LastName><ForeName>Fernando T</ForeName><Initials>FT</Initials><AffiliationInfo><Affiliation>Department of Medical Biochemistry and Biophysics, Karolinska Institutet, SE-17177, Stockholm, Sweden.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Santhosh</LastName><ForeName>Sebastin M</ForeName><Initials>SM</Initials><AffiliationInfo><Affiliation>Department of Medical Biochemistry and Biophysics, Karolinska Institutet, SE-17177, Stockholm, Sweden.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Sventelius</LastName><ForeName>Teodor</ForeName><Initials>T</Initials><AffiliationInfo><Affiliation>Department of Medical Biochemistry and Biophysics, Karolinska Institutet, SE-17177, Stockholm, Sweden.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Holmgren</LastName><ForeName>Arne</ForeName><Initials>A</Initials><AffiliationInfo><Affiliation>Department of Medical Biochemistry and Biophysics, Karolinska Institutet, SE-17177, Stockholm, Sweden. Electronic address: arne.holmgren@ki.se.</Affiliation></AffiliationInfo></Author></AuthorList><Language>eng</Language><PublicationTypeList><PublicationType UI="D016428">Journal Article</PublicationType><PublicationType UI="D013485">Research Support, Non-U.S. Gov't</PublicationType></PublicationTypeList><ArticleDate DateType="Electronic"><Year>2018</Year><Month>12</Month><Day>28</Day></ArticleDate></Article><MedlineJournalInfo><Country>United States</Country><MedlineTA>Anal Biochem</MedlineTA><NlmUniqueID>0370535</NlmUniqueID><ISSNLinking>0003-2697</ISSNLinking></MedlineJournalInfo><ChemicalList><Chemical><RegistryNumber>0</RegistryNumber><NameOfSubstance UI="C516005">GLRX protein, human</NameOfSubstance></Chemical><Chemical><RegistryNumber>0</RegistryNumber><NameOfSubstance UI="D054477">Glutaredoxins</NameOfSubstance></Chemical><Chemical><RegistryNumber>GAN16C9B8O</RegistryNumber><NameOfSubstance UI="D005978">Glutathione</NameOfSubstance></Chemical></ChemicalList><CitationSubset>IM</CitationSubset><MeshHeadingList><MeshHeading><DescriptorName UI="D002478" MajorTopicYN="N">Cells, Cultured</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D005453" MajorTopicYN="Y">Fluorescence</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D054477" MajorTopicYN="N">Glutaredoxins</DescriptorName><QualifierName UI="Q000737" MajorTopicYN="N">chemistry</QualifierName><QualifierName UI="Q000378" MajorTopicYN="Y">metabolism</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D005978" MajorTopicYN="N">Glutathione</DescriptorName><QualifierName UI="Q000032" MajorTopicYN="Y">analysis</QualifierName><QualifierName UI="Q000378" MajorTopicYN="N">metabolism</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D006801" MajorTopicYN="N">Humans</DescriptorName></MeshHeading></MeshHeadingList><KeywordList Owner="NOTNLM"><Keyword MajorTopicYN="Y">Di-eosin-glutathione</Keyword><Keyword MajorTopicYN="Y">Fluorescence assay</Keyword><Keyword MajorTopicYN="Y">Glutathione</Keyword><Keyword MajorTopicYN="Y">Protein S-glutathionylation</Keyword></KeywordList></MedlineCitation><PubmedData><History><PubMedPubDate PubStatus="received"><Year>2018</Year><Month>08</Month><Day>07</Day></PubMedPubDate><PubMedPubDate PubStatus="revised"><Year>2018</Year><Month>10</Month><Day>18</Day></PubMedPubDate><PubMedPubDate PubStatus="accepted"><Year>2018</Year><Month>12</Month><Day>27</Day></PubMedPubDate><PubMedPubDate PubStatus="pubmed"><Year>2019</Year><Month>1</Month><Day>1</Day><Hour>6</Hour><Minute>0</Minute></PubMedPubDate><PubMedPubDate PubStatus="medline"><Year>2019</Year><Month>12</Month><Day>18</Day><Hour>6</Hour><Minute>0</Minute></PubMedPubDate><PubMedPubDate PubStatus="entrez"><Year>2019</Year><Month>1</Month><Day>1</Day><Hour>6</Hour><Minute>0</Minute></PubMedPubDate></History><PublicationStatus>ppublish</PublicationStatus><ArticleIdList><ArticleId IdType="pubmed">30597126</ArticleId><ArticleId IdType="pii">S0003-2697(18)30803-0</ArticleId><ArticleId IdType="doi">10.1016/j.ab.2018.12.021</ArticleId></ArticleIdList></PubmedData></pubmed><affiliations><list><country><li>Suède</li></country><region><li>Svealand</li></region><settlement><li>Stockholm</li></settlement></list><tree><country name="Suède"><region name="Svealand"><name sortKey="Coppo, Lucia" sort="Coppo, Lucia" uniqKey="Coppo L" first="Lucia" last="Coppo">Lucia Coppo</name></region><name sortKey="Holmgren, Arne" sort="Holmgren, Arne" uniqKey="Holmgren A" first="Arne" last="Holmgren">Arne Holmgren</name><name sortKey="Ogata, Fernando T" sort="Ogata, Fernando T" uniqKey="Ogata F" first="Fernando T" last="Ogata">Fernando T. Ogata</name><name sortKey="Santhosh, Sebastin M" sort="Santhosh, Sebastin M" uniqKey="Santhosh S" first="Sebastin M" last="Santhosh">Sebastin M. Santhosh</name><name sortKey="Sventelius, Teodor" sort="Sventelius, Teodor" uniqKey="Sventelius T" first="Teodor" last="Sventelius">Teodor Sventelius</name></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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