Prolidase is the only enzyme capable of cleaving imidodipeptides containing C-terminal proline (Pro) or hydroxyproline and plays a crucial role in several physiological processes such as wound healing and cell proliferation. Here, we developed a new method to determine prolidase activity. This method is based on a novel fluorescence (FL) reaction selective for N-terminal glycine (Gly)-containing peptides using 3,4-dihydroxyphenylacetic acid (3,4-DHPAA). The 3,4-DHPAA can selectively react with Gly–Pro, the substrate for prolidase, and the prolidase activity is measured by monitoring the decrease in FL intensities. The prolidase activities in fibroblasts and HeLa cells were successfully measured by the proposed method. Compared with classical Chinard’s method, our method does not require any caustic acids, pre-incubation to activate the enzyme, and heating for reaction with the detection reagent. The proposed method enables facile and specific measurement for biogenic prolidase activity.
