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).


METHODS
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.
Araujo
M C
MC
Department of Biophysics, Escola Paulista de Medicina, Universidade Federal de São Paulo, Brazil.
Melo
R I
RI
Del Nery
E
E
Alves
M F
MF
Juliano
M A
MA
Casarini
D E
DE
Juliano
L
L
Carmona
A K
AK
D000806:Angiotensin-Converting Enzyme Inhibitors / Q000494:pharmacology; D000818:Animals; D002216:Captopril / Q000494:pharmacology; D002712:Chlorides / Q000494:pharmacology; D004492:Edetic Acid / Q000494:pharmacology; D006168:Guinea Pigs; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D010455:Peptides / Q000378:metabolism; D007703:Peptidyl-Dipeptidase A / Q000032:analysis* / Q000097:blood; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013379:Substrate Specificity
D000806:Angiotensin-Converting Enzyme Inhibitors; D002712:Chlorides; D010455:Peptides; D004492:Edetic Acid; D002216:Captopril; D007703:Peptidyl-Dipeptidase A