Characterization of partially purified cysteine protease inhibitor from Tetracarpidium conophorum (African walnut)

Abstract

Cysteine protease inhibitors (CPIs) have been known to be present in a variety of seeds of plants, and have been intensively studied as useful tools for potential utilization in pharmacology. This study reports the isolation of CPI from Tetracarpidium conophorum by 65% ammonium sulphate saturation, followed by ion exchange chromatography; further purification was by gel filtration chromatography. The molecular weight of the partially purified protein inhibitor was analyzed by SDS-PAGE to be approximately 20 kDa. The inhibitor had an optimum pH and temperature of 8.0 and 40°C, respectively. The inhibitor competitively inhibited papain with the same Vmax = 71.17´103 µmol/min, Km = 166 µM, and Ki = 53.63 µM. Divalent metal ions such as, Mg2+, Pb2+, Mn2+, Cu2+, Co2+, and Zn2+ had significant effect on inhibitory activity of CPI at concentration as low as 1 mM. Cysteine protease inhibitor of T. conophorum investigated in this study could serve as a template in biotechnology of herbal medicine to arrest the negative modulatory interactions of cysteine proteases in clinical pathogenic expressions.   Key words: Tetracarpidium conophorum, cysteine protease inhibitor, papain, purification, characterization.