Internally quenched fluorogenic substrates for angiotensin I-converting enzyme

Internally quenched fluorogenic substrates for angiotensin I-converting enzyme

Author Araujo, M. C. Google Scholar
Melo, R. I. Google Scholar
Del Nery, E. Google Scholar
Alves, MFM Google Scholar
Juliano, M. A. Google Scholar
Casarini, D. E. Google Scholar
Juliano, T. Google Scholar
Carmona, A. K. Google Scholar
Institution Universidade Federal de São Paulo (UNIFESP)
Abstract Objective Development of internally quenched fluorogenic substrates for sensitive and continuous assays of angiotensin I-converting enzyme (ACE).Design We synthesized internally quenched fluorogenic bradykinin-related peptides introducing Abz (ortho-aminobenzoic acid) and EDDnp (N-[2,4-dinitrophenyl]ethylenediamine) at their N- and C-terminal groups, respectively, and these were assayed as ACE substrates. We examined two series of peptides, Abz-GFSPFRX-EDDnp and Abz-GFSPFXQ-EDDnp (X, various amino acids).Methods Hydrolysis of the fluorogenic substrates by ACE was followed by continuous recording of the rising fluorescence (lambda(em) = 420 nm and lambda(ex) = 320 nm), the peptides were obtained by solid-phase synthesis or by classical solution methods.Results Despite of the blocked C-terminal sequences, the internally quenched bradykinin-related peptides were hydrolysed by ACE, the best substrates for plasma guinea pig ACE were Abz-GFSPFRA-EDDnp and Abz-GFSPFFQ-EDDnp, in which the fluorescence appeared after the first cleavage that occurred at R-A and F-Q bond, respectively, This ACE activity was sensitive to NaCl concentration and the optimum pH is greater than 8.0. Measurements of ACE activity with Hip-His-Leu and Abz-GFSPFFQ-EDDnp in the serum of 20 healthy patients correlated closely (r = 0.959), Complete inhibition of the hydrolysis of Abz-GFSPFFQ-EDDnp by human serum was observed with captopril and lisinopril.Conclusions We describe internally quenched fluorogenic substrates for ACE devoid of free C-terminal carboxyl group, They are convenient tools for ACE studies as they permit continuous fluorimetric measurements of the enzymatic activity, even in human serum, J Hypertens 1999, 17:665-672 (C) Lippincott Williams & Wilkins.
Keywords angiotensin I-converting enzyme
converting enzyme assays
internally quenched fluorogenic substrates
Language English
Date 1999-05-01
Published in Journal of Hypertension. Philadelphia: Lippincott Williams & Wilkins, v. 17, n. 5, p. 665-672, 1999.
ISSN 0263-6352 (Sherpa/Romeo, impact factor)
Publisher Lippincott Williams & Wilkins
Extent 665-672
Origin http://dx.doi.org/10.1097/00004872-199917050-00010
Access rights Closed access
Type Article
Web of Science ID WOS:000080447000010
URI http://repositorio.unifesp.br/handle/11600/26076

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