Internally quenched fluorogenic substrates for angiotensin I‐converting enzyme
作者:
Mauricio Araujo,
Robson Melo,
Elaine Nery,
Marcio Alves,
Maria Juliano,
Dulce Casarini,
Luiz Juliano,
Adriana Carmona,
期刊:
Journal of Hypertension
(OVID Available online 1999)
卷期:
Volume 17,
issue 5
页码: 665-672
ISSN:0263-6352
年代: 1999
出版商: OVID
关键词: angiotensin I-converting enzyme;converting enzyme assays;internally quenched fluorogenic substrates
数据来源: OVID
摘要:
ObjectiveDevelopment of internally quenched fluorogenic substrates for sensitive and continuous assays of angiotensin I-converting enzyme (ACE).DesignWe synthesized internally quenched fluorogenic bradykinin-related peptides introducing Abz (ortho-aminobenzoic acid) and EDDnp (N-[2,4-dinrtrophenyl]-ethylenediamine) at their N- and C-terminal groups, respectively, and these were assayed as ACE substrates. We examined two series of peptides, Abz-GFSPFRXEDDnp and Abz-GFSPFXQ-EDDnp (X, various amino acids).MethodsHydrolysis of the fluorogenic substrates by ACE was followed by continuous recording of the rising fluorescence (λem= 420 nm and λex= 320 nm). The peptides were obtained by solid-phase synthesis or by classical solution methods.ResultsDespite 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-GFSPFFQEDDnp, 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-QFSPFFQ-EDDnp in the serum of 20 healthy patients correlated closelyr= 0.959). Complete inhibition of the hydrolysis of Abz-GFSPFFQ-EDDnp by human serum was observed with captopril and lisinopril.ConclusionsWe 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.
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