Determination Of The D1 Protease Activity And Screening For Lead Compounds Of Inhibitor By Capillary Zone Electrophoresis

Mature D1 protein is the indispensable subunit component in photosystem II of plants, it is the ligand of Mn in Mn4Ca cluster. D1 protease was encoded by the ctpA gene in chloroplasts, containing 389 amino acid residue, molecular weight 45kDa, widely distributed in all kind of plants. Is responsible for processing of precursor D1 protein (pD1) with a short C-terminal extension, transform to biological actively D1 protein. So the CtpA plays an important role in photodamage-repair cycle in photosystemⅡ. Beside this, the content of ctpA in plant is only 1% of that of D1 protein. Therefore, CtpA is considered as a high-efficient herbicidal target in novel herbicide development. It is very important for the methodology establishment of isolation as well as the herbicidal activity screening of novel potential inhibitors based on this novel target.Separation conditions of peptides were optimized as phosphate buffer (50 mmol/ L, pH 3.0) at 18 kV running voltage, with capillary length of 43 cm×75μm (id). Hydrolysis activity of D1 protease and screening for lead compounds of inhibitor was performed by CZE. The results indicate that two lead compounds ITP 21 and ITP 26 show different inhibitory effects of 26% and 13%, respectively, while other compounds have no inhibitory effects on D1 protease activity. This method is simple and convenient, suitable for the screening study on protease's inhibitors.