A highly sensitive peptide substrate for detecting two A£]-degrading enzymes: Neprilysin and insulin-degrading enzyme

Abstract

Neprilysin has been singled out as the most promising candidate for use in the degradation of A£] as a therapy for Alzheimer's disease. In this study, a quenched fluorogenic peptide substrate containing the first seven residues of the A£] peptide plus a C-terminal Cysteine residue was synthesized to detect neprilysin activity. A fluorophore was attached to the C-terminal Cysteine and its fluorescence was quenched by a quencher linked to the N-terminus of the peptide. When this peptide substrate was degraded by an endopeptidase, fluorescence was produced and proved to be a sensitive detection system for endopeptidase activity. Our results showed that this assay system was extremely sensitive to neprilysin and insulin-degrading enzyme, but insensitive, or much less sensitive, to other A£]-degrading enzymes. As low as 0.1. nM of neprilysin and 0.2. nM of insulin-degrading enzyme can be detected. ? 2010 Elsevier B.V.