New strategy to design fluorescent substrates of carboxypeptidases using a combination of dansylated peptides and albumin

Abstract

A new strategy to design a fluorescent substrate for carboxypeptidases (CPs) was devised using dansylated sarcosine (DS) and albumin as a fluorophore and signal amplifier, respectively. The fluorescent substrate was designed by attaching an amino acid or peptide, which acts as the recognition unit for CP, to the C-terminus of DS. In the presence of the target CP, the low-fluorescence substrate is hydrolyzed to a strongly fluorescent DS by binding with albumin. As a proof concept, Dansyl-Sar-Lys-Pro (DS-KP) and Dansyl-Sar-Arg (DS-R) were developed as fluorescent substrates for the angiotensin-converting enzyme and carboxypeptidase B, respectively. The CP assay system was verified to be capable of measuring the activities of both dipeptidyl-CP and mono-CP and the inhibition efficiency of various CP inhibitors, confirming its applicability as a high-throughput screening method for numerous inhibitor candidates.