Internally quenched fluorogenic substrates for angiotensin I-converting enzyme
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1999-05-01
Autores
Araujo, M. C.
Melo, R. I.
Del Nery, E.
Alves, MFM
Juliano, M. A.
Casarini, D. E.
Juliano, T.
Carmona, A. K.
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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.
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Journal of Hypertension. Philadelphia: Lippincott Williams & Wilkins, v. 17, n. 5, p. 665-672, 1999.