Design and synthesis of fluorogenic trypsin peptide substrates based on resonance energy transfer

An assay based on new internally quenched fluorogenic peptide substrates with the general structure 4-(4-dimethylaminophenylazo)benzoic acid (DABCYL)-Gly-Pro-Ala-Xaa-Leu-Ala-Ile-Gly-5-(2-aminoethylamino++ +)naphtha lene-1-sulfonic acid (EDANS), where Xaa = Arg, Lys, has been developed to measure proteolytic activity of trypsin and similar proteases. The kinetic parameters for the tryptic hydrolysis of DABCYL-Gly-Pro-Ala-Arg-Leu-Ala-Ile-Gly-EDANS are Km = 34 microM, kcat = 40 s-1, and kcat/Km = 1.17 x 10(6) M-1 s-1. The substrates offer two advantages over common substrates. First they are very sensitive. Applications to chemically modified trypsin and engineered variants show the ability to detect traces of proteolytic activity. In addition, these substrates are adapted to the S'-specificity of the investigated protease. These features and the prospect of miniaturization makes the assay suitable for applications to high-throughput screening.