A reversed-phase HPLC-based dual-parameter assay for the human urinary kallidinogenase enzyme activity

In this work, a dual-parameter quantification protocol was successfully developed for determining the enzymatic activity of human urinary kallidinogenase (KN) using reversed-phase high-performance liquid chromatography (RP-HPLC). KN serves as a hydrolytic enzyme to cleave the substrate S-2266 (H-D-Val-Leu-Arg-pNA). Effective separation of the product, substrate, and KN was achieved under optimized chromatographic conditions. By minimizing matrix interference through HPLC separation and using multi-wavelength detection, both selectivity and sensitivity were significantly improved. Moreover, the dual-parameter quantification method, which simultaneously measured the formation of reaction products and the consumption of substrates to reflect KN enzyme activity, markedly improved the reliability of the determination. This method was successfully applied to the activity assay of actual KN samples. The established method has been demonstrated to be effective and reliable with comparable greenness. It provides a powerful tool for the quantitative monitoring of KN activity in biological products and paves the way for its application in complex biological matrices.