Internally quenched fluorogenic substrates for angiotensin I-converting enzyme

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.