Caesalpinia bonduc serine proteinase inhibitor CbTI–2: Exploring the conformational features and antimalarial activity

Abstract

Seeds of tropical legumes posses a repertoire of proteinase inhibitors (PI) and the current study highlights some structural/functional features of a strong serine PI from the seeds of Caesalpinia bonduc (CbTI–2). Following purification, N-terminal sequence of CbTI–2 revealed over 40% similarity with a few serine PIs of Caesalpinioideae subfamily. Upon exposure to metal ions and ionic/non ionic surfactants, CbTI–2 showed immense variation in the levels of antitryptic activity. Exposure of CbTI–2 to 1,4-Dithiothreitol, Guanidinium HCl, H2O2 and Dimethyl sulfoxide led to a steady loss of inhibitory activity. Chemical modification of amino acids suggested an arginine as the active site residue. Circular Dichroism spectrum of native CbTI–2 revealed an unordered state. Secondary structure composition of CbTI–2 following exposure to extreme conditions (heat, acidic/alkaline environment, Guanidine hydrochloride and DTT) showed considerable perturbations that caused severe loss of antiproteolytic activity. DLS studies yielded a hydrodynamic radius of ∼2.2 nm for CbTI–2 and also reconfirmed 1:1 stoichiometry for the trypsin-CbTI–2 complex. Initial studies indicated CbTI–2 to be a potent antiplasmodial agent by being highly toxic towards growth, schizont rupture process and erythrocytic invasion of Plasmodium falciparum.