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

An assay based on new internally quenched fluorogenic peptide substrates wi th the general structure 4-(4-dimethylaminophenylazo)benzoic acid (DABCYL)G ly-Pro-Ala-Xaa-Leu-Ala-Ile-Gly-5-(2-aminoethylamino)naphthalene-l-sulfonic acid (EDANS), where Xaa = Arg, Lys, has been developed to measure proteolyt ic activity of trypsin and similar proteases, The kinetic parameters for th e tryptic hydrolysis of DABCYL-Gly-Pro-Ala-Arg-Leu-Ala-Ile-Gly-EDANS are K- m = 34 mu M, k(cat) = 40 s(-1), and k(cat)/K-m = 1.17 x 10(6) M-1 s(-1). Th e substrates offer two advantages over common substrates, First they are ve ry sensitive. Applications to chemically modified trypsin and engineered va riants show the ability to detect traces of proteolytic activity. In additi on, these substrates are adapted to the S'-specificity of the investigated protease, These features and the prospect of miniaturization makes the assa y suitable for applications to high-throughput screening, (C) 1998 Academic Press.