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.
Department of Biophysics, Escola Paulista de Medicina, Universidade Federal de São Paulo, Brazil.
M C
MC
Araujo
R I
RI
Melo
E
E
Del Nery
M F
MF
Alves
M A
MA
Juliano
D E
DE
Casarini
L
L
Juliano
A K
AK
Carmona
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