A continuous fluorescence resonance energy transfer angiotensin I-converting enzyme assay

A continuous fluorescence resonance energy transfer angiotensin I-converting enzyme assay

Autor Carmona, Adriana K. Google Scholar
Schwager, Sylva L. Google Scholar
Juliano, Maria A. Google Scholar
Juliano, Luiz Google Scholar
Sturrock, Edward D. Google Scholar
Instituição Univ Cape Town
Universidade Federal de São Paulo (UNIFESP)
Resumo Angiotensin I-converting enzyme (ACE) is involved in various physiological and physiopathological conditions; therefore, the measurement of its catalytic activity may provide essential clinical information. This protocol describes a sensitive and rapid procedure for determination of ACE activity using fluorescence resonance energy transfer ( FRET) substrates containing o-aminobenzoic acid (Abz) as the fluorescent group and 2,4-dinitrophenyl (Dnp) as the quencher acceptor. Hydrolysis of a peptide bond between the donor/acceptor pair generates fluorescence that can be detected continuously, allowing quantitative measurement of the enzyme activity. the FRET substrates provide a useful tool for kinetic studies and for ACE determination in biological fluids and crude tissue extracts. An important benefit of this method is the use of substrates selective for the two active sites of the enzyme, namely Abz-SDK( Dnp) P-OH for N-domain, Abz-LFK( Dnp)-OH for C-domain and Abz-FRK(Dnp) P-OH for somatic ACE. This methodology can be adapted for determinations using a 96-well fluorescence plate reader.
Idioma Inglês
Data de publicação 2006-01-01
Publicado em Nature Protocols. London: Nature Publishing Group, v. 1, n. 4, p. 1971-1976, 2006.
ISSN 1754-2189 (Sherpa/Romeo, fator de impacto)
Publicador Nature Publishing Group
Extensão 1971-1976
Fonte http://dx.doi.org/10.1038/nprot.2006.306
Direito de acesso Acesso restrito
Tipo Artigo
Web of Science WOS:000251155500038
Endereço permanente http://repositorio.unifesp.br/handle/11600/28656

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