AbstractA synthetic collagenase substrate containing the internal peptide sequence—Gly‐Gly‐Pro‐Leu‐Gly‐Pro‐Pro‐Gly‐Pro—has been synthesized, with an N‐terminus 4‐((4‐(dimethylamino)phenyl)azo)‐benzoyl (DABCYL) group and C‐terminus 5‐[2‐(acetamido)ethylamino] naphthalene‐1‐sulfonic acid (AEDANS) moiety resulting in internal quenching of AEDANS fluorescence. Peptide bond hydrolysis results in a large increase in fluorescence at 490 nm upon excitation at 336 nm. The substrate is cleaved exclusively by Clostridium histolyticum collagenase and is completely resistant to attack by proteases like thermolysin, proteinase K, and trypsin. Km and Vmax values for substrate hydrolysis by collagenase have been determined, establishing the peptide as one of the best binding substrates for the enzyme. MALDI mass spectrometry using a derivative of the substrate establishes that the sites of cleavage lie within the collagen like domain. The CD spectrum of an analog peptide lacking the donor and acceptor groups reveals spectral features that are reminiscent of weak polyproline structures. © 2008 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 90: 131–137, 2008.This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com