A continuous fluorescence resonance energy transfer angiotensin I-converting enzyme assay.
Angiotensin I-converting enzyme (ACE) is involved in various physiological and physiopathological conditions; therefore, the measurement of its catalytic activity may provide essential clinical information. This protocol describes a sensitive and rapid procedure for determination of ACE activity using fluorescence resonance energy transfer (FRET) substrates containing o-aminobenzoic acid (Abz) as the fluorescent group and 2,4-dinitrophenyl (Dnp) as the quencher acceptor. Hydrolysis of a peptide bond between the donor/acceptor pair generates fluorescence that can be detected continuously, allowing quantitative measurement of the enzyme activity. The FRET substrates provide a useful tool for kinetic studies and for ACE determination in biological fluids and crude tissue extracts. An important benefit of this method is the use of substrates selective for the two active sites of the enzyme, namely Abz-SDK(Dnp)P-OH for N-domain, Abz-LFK(Dnp)-OH for C-domain and Abz-FRK(Dnp)P-OH for somatic ACE. This methodology can be adapted for determinations using a 96-well fluorescence plate reader.
Carmona
Adriana K
AK
Department of Biophysics, Escola Paulista de Medicina, Universidade Federal de São Paulo, SP, Brazil.
Schwager
Sylva L
SL
Juliano
Maria A
MA
Juliano
Luiz
L
Sturrock
Edward D
ED
D000818:Animals; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D007703:Peptidyl-Dipeptidase A / Q000378:metabolism*; D011817:Rabbits
D007703:Peptidyl-Dipeptidase A