High performance liquid chromatography assay of bacterial collagenase

The activity of bacterial collagenase Clostridiopeptidase A was estimated using a labelled synthetic peptide, 4-phenylazobenzyloxycarbonyl-L-Pro-L-Leu-Gly-L-Pro-D-Arg, as substrate. The N-protected dipeptide obtained after enzymatic hydrolysis of Leu-Gly peptide bond was quantified by reversed-phase, high-performance liquid chromatography using 4-phenylazobenzyloxycarbonyl-L-Pro-L-Phe as internal standard. The time dependence of the appearance of the hydrolysis product and the dependence of rates of hydrolysis on collagenase concentration were linear. Kinetic parameters for collagenase were determined to test the suitability of the described procedure.