HPLC-based kinetics assay for monitoring prolidase activity

This short communication features a validated HPLC-DAD kinetic assay for monitoring prolidase (EC 3.4.13.9) activity, a peptidase responsible for cleaving dipeptides with a C-terminal L-proline or hydroxyproline. Prolidase plays an essential role in various physiological processes, including wound healing, inflammation, angiogenesis, and cell proliferation. Inhibition of prolidase could be beneficial for conditions with abnormal collagen turnover, such as cancer, fibrotic diseases, and certain skin disorders. The newly validated, cost-effective HPLC-DAD method provides real-time analysis of prolidase activity by measuring the cleavage rate of Gly-L-Pro, the substrate with the highest affinity for the enzyme. The assay quantifies the release of the aminoacid glycine (Gly) in nanomoles per minute per milligram of protein, offering insights into prolidase's catalytic behavior with or without inhibitors. Cbz-Pro, a well-known competitive inhibitor, was used to study enzyme inhibition. Our results confirm that Cbz-Pro inhibits prolidase competitively, with a K_i value of 90.4 µM. Kinetic parameters, including Michaelis-Menten constant (K_m), maximum reaction velocity (V_max), and catalytic rate constant (k_cat), were determined through standard graphical methods (Michaelis-Menten, Lineweaver-Burk, and Dixon plots). The set-up HPLC-DAD methodology represents a fairly simple approach for qualitative and quantitative analysis, useful to effectively elucidate prolidase's kinetic profile and the inhibitory potential of Cbz-Pro, demonstrating its suitability for routine screening of novel prolidase inhibitors in research applications, overcoming the inconsistent results of colorimetric assays.