Peptide cleavage assays

Kinetics of PPEP activity was measured in a time course using fluorescent Fluorescence Resonance Energy Transfer (FRET) quenched peptides. FRET peptides consisted of Lys(Dabcyl)-EXXPPXXD-Glu(EDANS), in which X varied between the various tested peptides. Cleavage of FRET peptides by PPEPs was tested in 150 µl PBS containing 50 mM FRET peptide and 2 µg of CD1597 or 500 ng for PPEP-1. Peptide cleavage was measured using the Envision 2105 Multimode Plate Reader. Fluorescence intensity (excitation 350 nm and emission 510 nm) was measured each minute for 1 h, with 10 flashes per measurement.