A high performance liquid chromatography assay for monitoring proprotein convertase activity

A rapid HPLC assay was developed for monitoring the activity of the two proprotein convertases, PACE-4 and furin. Six novel peptide substrates were synthesized containing the minimal PC recognition sequence (Arg-X-X-Arg), as well as tryptophan residue(s) for easy detection. Four of the peptides were cleaved by both PCs and their kinetic parameters determined. Two peptides were not cleaved but were shown to be good negative controls although not inhibitors of either PC. In addition, inhibition curves were plotted and IC50 values calculated for PACE-4 and furin in the presence of two polyarginine peptides, hexa and deca-D-arginine.