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

Abstract

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 μm) 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 bya series of inhibitor molecules (caffeic acid phenylamide, chlorogenic acid, piceatannol, nor-NOHA acetate) and plant extracts.