A continuous fluorescent assay for the determination of plasma and tissue angiotensin I-converting enzyme activity
Autor(a) principal: | |
---|---|
Data de Publicação: | 2005 |
Outros Autores: | , , , , , , |
Tipo de documento: | Artigo |
Idioma: | eng |
Título da fonte: | Repositório Institucional da UNIFESP |
Texto Completo: | http://repositorio.unifesp.br/handle/11600/2524 http://dx.doi.org/10.1590/S0100-879X2005000600007 |
Resumo: | A continuous assay using internally quenched fluorescent peptides with the general sequence Abz-peptidyl-(Dnp)P-OH (Abz = ortho-aminobenzoic acid; Dnp = 2,4-dinitrophenyl) was optimized for the measurement of angiotensin I-converting enzyme (ACE) in human plasma and rat tissues. Abz-FRK(Dnp)P-OH, which was cleaved at the Arg-Lys bond by ACE, was used for the enzyme evaluation in human plasma. Enzymatic activity was monitored by continuous recording of the fluorescence (lambdaex = 320 nm and lambdaem = 420 nm) at 37ºC, in 0.1 M Tris-HCl buffer, pH 7.0, with 50 mM NaCl and 10 µM ZnCl2. The assays can be performed directly in the cuvette of the fluorimeter and the hydrolysis followed for 5 to 10 min. ACE measurements in the plasma of 80 healthy patients with Hip-His-Leu and with Abz-FRK(Dnp)P-OH correlated closely (r = 0.90, P < 0.001). The specificity of the assay was demonstrated by the complete inhibition of hydrolysis by 0.5 µM lisinopril or captopril. Abz-FRK(Dnp)P-OH cleavage by ACE was monitored in rat lung, kidney, heart, and liver homogenates in the presence of a cocktail of inhibitors containing trans-epoxy-succinyl-L-leucylamido-(4-guanido)-butene, pepstatin, phenyl-methylsulfonyl fluoride, N-tosyl-L-phenylalanyl-chloromethyl ketone, and N-tosyl-lysyl-chloromethyl ketone to prevent undesirable hydrolysis. ACE activity in lung, heart and kidney homogenates, but not in liver homogenates, was completely abolished by 0.5 µM lisinopril or captopril. The advantages of the method are the procedural simplicity and the high sensitivity providing a rapid assay for ACE determinations. |
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Alves, Marcio Fernando Madureira [UNIFESP]Araujo, Mauricio de Campos [UNIFESP]Juliano, Maria Aparecida [UNIFESP]Oliveira, Edilamar Menezes deKrieger, Jose EduardoCasarini, Dulce Elena [UNIFESP]Juliano, Luiz [UNIFESP]Carmona, Adriana Karaoglanovic [UNIFESP]Universidade Federal de São Paulo (UNIFESP)Universidade de São Paulo (USP)2015-06-14T13:31:34Z2015-06-14T13:31:34Z2005-06-01Brazilian Journal of Medical and Biological Research. Associação Brasileira de Divulgação Científica, v. 38, n. 6, p. 861-868, 2005.0100-879Xhttp://repositorio.unifesp.br/handle/11600/2524http://dx.doi.org/10.1590/S0100-879X2005000600007S0100-879X2005000600007.pdfS0100-879X200500060000710.1590/S0100-879X2005000600007WOS:000230125300007A continuous assay using internally quenched fluorescent peptides with the general sequence Abz-peptidyl-(Dnp)P-OH (Abz = ortho-aminobenzoic acid; Dnp = 2,4-dinitrophenyl) was optimized for the measurement of angiotensin I-converting enzyme (ACE) in human plasma and rat tissues. Abz-FRK(Dnp)P-OH, which was cleaved at the Arg-Lys bond by ACE, was used for the enzyme evaluation in human plasma. Enzymatic activity was monitored by continuous recording of the fluorescence (lambdaex = 320 nm and lambdaem = 420 nm) at 37ºC, in 0.1 M Tris-HCl buffer, pH 7.0, with 50 mM NaCl and 10 µM ZnCl2. The assays can be performed directly in the cuvette of the fluorimeter and the hydrolysis followed for 5 to 10 min. ACE measurements in the plasma of 80 healthy patients with Hip-His-Leu and with Abz-FRK(Dnp)P-OH correlated closely (r = 0.90, P < 0.001). The specificity of the assay was demonstrated by the complete inhibition of hydrolysis by 0.5 µM lisinopril or captopril. Abz-FRK(Dnp)P-OH cleavage by ACE was monitored in rat lung, kidney, heart, and liver homogenates in the presence of a cocktail of inhibitors containing trans-epoxy-succinyl-L-leucylamido-(4-guanido)-butene, pepstatin, phenyl-methylsulfonyl fluoride, N-tosyl-L-phenylalanyl-chloromethyl ketone, and N-tosyl-lysyl-chloromethyl ketone to prevent undesirable hydrolysis. ACE activity in lung, heart and kidney homogenates, but not in liver homogenates, was completely abolished by 0.5 µM lisinopril or captopril. The advantages of the method are the procedural simplicity and the high sensitivity providing a rapid assay for ACE determinations.Universidade Federal de São Paulo (UNIFESP) Escola Paulista de Medicina Departamento de BiofísicaUniversidade Federal de São Paulo (UNIFESP) Escola Paulista de Medicina Departamento de MedicinaUniversidade de São Paulo Faculdade de Medicina Instituto do CoraçãoUniversidade de São Paulo Escola de Educação Física e Esporte Laboratório de Bioquímica da Atividade MotoraUNIFESP, EPM, Depto. de BiofísicaUNIFESP, EPM, Depto. de MedicinaSciELO861-868engAssociação Brasileira de Divulgação CientíficaBrazilian Journal of Medical and Biological ResearchAngiotensin-converting enzyme activityFluorometric assayRat tissue angiotensin- converting enzymeHuman plasma angiotensin-converting enzymeA continuous fluorescent assay for the determination of plasma and tissue angiotensin I-converting enzyme activityinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/articleinfo:eu-repo/semantics/openAccessreponame:Repositório Institucional da UNIFESPinstname:Universidade Federal de São Paulo (UNIFESP)instacron:UNIFESPORIGINALS0100-879X2005000600007.pdfapplication/pdf636697${dspace.ui.url}/bitstream/11600/2524/1/S0100-879X2005000600007.pdf057c129c9efce415132792af33a24cdfMD51open accessTEXTS0100-879X2005000600007.pdf.txtS0100-879X2005000600007.pdf.txtExtracted texttext/plain30983${dspace.ui.url}/bitstream/11600/2524/2/S0100-879X2005000600007.pdf.txte5c8666b2d9051098c9d78c7ca076890MD52open access11600/25242021-09-29 11:23:43.256open accessoai:repositorio.unifesp.br:11600/2524Repositório InstitucionalPUBhttp://www.repositorio.unifesp.br/oai/requestopendoar:34652023-05-25T12:23:24.011630Repositório Institucional da UNIFESP - Universidade Federal de São Paulo (UNIFESP)false |
dc.title.en.fl_str_mv |
A continuous fluorescent assay for the determination of plasma and tissue angiotensin I-converting enzyme activity |
title |
A continuous fluorescent assay for the determination of plasma and tissue angiotensin I-converting enzyme activity |
spellingShingle |
A continuous fluorescent assay for the determination of plasma and tissue angiotensin I-converting enzyme activity Alves, Marcio Fernando Madureira [UNIFESP] Angiotensin-converting enzyme activity Fluorometric assay Rat tissue angiotensin- converting enzyme Human plasma angiotensin-converting enzyme |
title_short |
A continuous fluorescent assay for the determination of plasma and tissue angiotensin I-converting enzyme activity |
title_full |
A continuous fluorescent assay for the determination of plasma and tissue angiotensin I-converting enzyme activity |
title_fullStr |
A continuous fluorescent assay for the determination of plasma and tissue angiotensin I-converting enzyme activity |
title_full_unstemmed |
A continuous fluorescent assay for the determination of plasma and tissue angiotensin I-converting enzyme activity |
title_sort |
A continuous fluorescent assay for the determination of plasma and tissue angiotensin I-converting enzyme activity |
author |
Alves, Marcio Fernando Madureira [UNIFESP] |
author_facet |
Alves, Marcio Fernando Madureira [UNIFESP] Araujo, Mauricio de Campos [UNIFESP] Juliano, Maria Aparecida [UNIFESP] Oliveira, Edilamar Menezes de Krieger, Jose Eduardo Casarini, Dulce Elena [UNIFESP] Juliano, Luiz [UNIFESP] Carmona, Adriana Karaoglanovic [UNIFESP] |
author_role |
author |
author2 |
Araujo, Mauricio de Campos [UNIFESP] Juliano, Maria Aparecida [UNIFESP] Oliveira, Edilamar Menezes de Krieger, Jose Eduardo Casarini, Dulce Elena [UNIFESP] Juliano, Luiz [UNIFESP] Carmona, Adriana Karaoglanovic [UNIFESP] |
author2_role |
author author author author author author author |
dc.contributor.institution.none.fl_str_mv |
Universidade Federal de São Paulo (UNIFESP) Universidade de São Paulo (USP) |
dc.contributor.author.fl_str_mv |
Alves, Marcio Fernando Madureira [UNIFESP] Araujo, Mauricio de Campos [UNIFESP] Juliano, Maria Aparecida [UNIFESP] Oliveira, Edilamar Menezes de Krieger, Jose Eduardo Casarini, Dulce Elena [UNIFESP] Juliano, Luiz [UNIFESP] Carmona, Adriana Karaoglanovic [UNIFESP] |
dc.subject.eng.fl_str_mv |
Angiotensin-converting enzyme activity Fluorometric assay Rat tissue angiotensin- converting enzyme Human plasma angiotensin-converting enzyme |
topic |
Angiotensin-converting enzyme activity Fluorometric assay Rat tissue angiotensin- converting enzyme Human plasma angiotensin-converting enzyme |
description |
A continuous assay using internally quenched fluorescent peptides with the general sequence Abz-peptidyl-(Dnp)P-OH (Abz = ortho-aminobenzoic acid; Dnp = 2,4-dinitrophenyl) was optimized for the measurement of angiotensin I-converting enzyme (ACE) in human plasma and rat tissues. Abz-FRK(Dnp)P-OH, which was cleaved at the Arg-Lys bond by ACE, was used for the enzyme evaluation in human plasma. Enzymatic activity was monitored by continuous recording of the fluorescence (lambdaex = 320 nm and lambdaem = 420 nm) at 37ºC, in 0.1 M Tris-HCl buffer, pH 7.0, with 50 mM NaCl and 10 µM ZnCl2. The assays can be performed directly in the cuvette of the fluorimeter and the hydrolysis followed for 5 to 10 min. ACE measurements in the plasma of 80 healthy patients with Hip-His-Leu and with Abz-FRK(Dnp)P-OH correlated closely (r = 0.90, P < 0.001). The specificity of the assay was demonstrated by the complete inhibition of hydrolysis by 0.5 µM lisinopril or captopril. Abz-FRK(Dnp)P-OH cleavage by ACE was monitored in rat lung, kidney, heart, and liver homogenates in the presence of a cocktail of inhibitors containing trans-epoxy-succinyl-L-leucylamido-(4-guanido)-butene, pepstatin, phenyl-methylsulfonyl fluoride, N-tosyl-L-phenylalanyl-chloromethyl ketone, and N-tosyl-lysyl-chloromethyl ketone to prevent undesirable hydrolysis. ACE activity in lung, heart and kidney homogenates, but not in liver homogenates, was completely abolished by 0.5 µM lisinopril or captopril. The advantages of the method are the procedural simplicity and the high sensitivity providing a rapid assay for ACE determinations. |
publishDate |
2005 |
dc.date.issued.fl_str_mv |
2005-06-01 |
dc.date.accessioned.fl_str_mv |
2015-06-14T13:31:34Z |
dc.date.available.fl_str_mv |
2015-06-14T13:31:34Z |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/article |
format |
article |
status_str |
publishedVersion |
dc.identifier.citation.fl_str_mv |
Brazilian Journal of Medical and Biological Research. Associação Brasileira de Divulgação Científica, v. 38, n. 6, p. 861-868, 2005. |
dc.identifier.uri.fl_str_mv |
http://repositorio.unifesp.br/handle/11600/2524 http://dx.doi.org/10.1590/S0100-879X2005000600007 |
dc.identifier.issn.none.fl_str_mv |
0100-879X |
dc.identifier.file.none.fl_str_mv |
S0100-879X2005000600007.pdf |
dc.identifier.scielo.none.fl_str_mv |
S0100-879X2005000600007 |
dc.identifier.doi.none.fl_str_mv |
10.1590/S0100-879X2005000600007 |
dc.identifier.wos.none.fl_str_mv |
WOS:000230125300007 |
identifier_str_mv |
Brazilian Journal of Medical and Biological Research. Associação Brasileira de Divulgação Científica, v. 38, n. 6, p. 861-868, 2005. 0100-879X S0100-879X2005000600007.pdf S0100-879X2005000600007 10.1590/S0100-879X2005000600007 WOS:000230125300007 |
url |
http://repositorio.unifesp.br/handle/11600/2524 http://dx.doi.org/10.1590/S0100-879X2005000600007 |
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eng |
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eng |
dc.relation.ispartof.none.fl_str_mv |
Brazilian Journal of Medical and Biological Research |
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info:eu-repo/semantics/openAccess |
eu_rights_str_mv |
openAccess |
dc.format.none.fl_str_mv |
861-868 |
dc.publisher.none.fl_str_mv |
Associação Brasileira de Divulgação Científica |
publisher.none.fl_str_mv |
Associação Brasileira de Divulgação Científica |
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reponame:Repositório Institucional da UNIFESP instname:Universidade Federal de São Paulo (UNIFESP) instacron:UNIFESP |
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Repositório Institucional da UNIFESP |
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