Development of a new nano arginase HPLC capillary column for the fast screening of arginase inhibitors and evaluation of their binding affinity

A simple and rapid Nano LC method has been developed for the screening of arginase inhibitors. The method is based on the immobilization of biotinylated arginase on a neutravidin functionalized nano HPLC capillary column. The arginase immobilization step performed by frontal analysis is very fast and only takes a few minutes. The miniaturized capillary column of 170 nL (length 5 cm, internal diameter 75 mu m) significantly decreased the required amount of used enzyme (25 pmol). This was of significance importance when working with less available or expensive purified enzyme. Non-selective adsorption of the organic monolith matrix was reduced (<6%) and the arginase efficient yield was high (92%). The resultant affinity capillary columns showed excellent repeatability and long lifetime. The arginase reaction product was achieved within 60 s and the immobilized arginase retained 97% of the initial activity beyond 90 days. This novel approach can thus be used for the fast evaluation of recognition assay induced by a series of inhibitor molecules (caffeic acid phenylamide, chlorogenic acid, piceatannol, nor-NOHA acetate) and plant extracts.

Automated summary

A simple and rapid Nano LC method has been developed for screening arginase inhibitors, using immobilized biotinylated arginase on a neutravidin functionalized nano HPLC capillary column. The miniaturized capillary column significantly decreases the required enzyme amount and achieves high enzyme yield, with reduced non-selective adsorption and excellent repeatability. The immobilized arginase retains high activity over time, making it suitable for recognizing a variety of inhibitor molecules and plant extracts.