The use of Fluorescence Resonance Energy Transfer (FRET) peptides for measurement of clinically important proteolytic enzymes.
Proteolytic enzymes have a fundamental role in many biological processes and are associated with multiple pathological conditions. Therefore, targeting these enzymes may be important for a better understanding of their function and development of therapeutic inhibitors. Fluorescence Resonance Energy Transfer (FRET) peptides are convenient tools for the study of peptidases specificity as they allow monitoring of the reaction on a continuous basis, providing a rapid method for the determination of enzymatic activity. Hydrolysis of a peptide bond between the donor/acceptor pair generates fluorescence that permits the measurement of the activity of nanomolar concentrations of the enzyme. The assays can be performed directly in a cuvette of the fluorimeter or adapted for determinations in a 96-well fluorescence plate reader. The synthesis of FRET peptides containing ortho-aminobenzoic acid (Abz) as fluorescent group and 2, 4-dinitrophenyl (Dnp) or N-(2, 4-dinitrophenyl)ethylenediamine (EDDnp) as quencher was optimized by our group and became an important line of research at the Department of Biophysics of the Federal University of São Paulo. Recently, Abz/Dnp FRET peptide libraries were developed allowing high-throughput screening of peptidases substrate specificity. This review presents the consolidation of our research activities undertaken between 1993 and 2008 on the synthesis of peptides and study of peptidases specificities.
Carmona
Adriana K
AK
Departamento de Biofísica, Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, SP, Brasil. adriana@biofis.epm.br
Juliano
Maria Aparecida
MA
Juliano
Luiz
L
D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D015260:Neprilysin / Q000378:metabolism*; D010447:Peptide Hydrolases / Q000737:chemistry / Q000378:metabolism*; D010455:Peptides / Q000378:metabolism*; D007703:Peptidyl-Dipeptidase A / Q000378:metabolism*; D013379:Substrate Specificity
D010455:Peptides; D010447:Peptide Hydrolases; D007703:Peptidyl-Dipeptidase A; D015260:Neprilysin