A comparative study of the winged bean protease inhibitors and their interaction with proteases

Three protease inhibitors, namely a chymotrypsin inhibitor (WbCI), a trypsi n inhibitor (WbTI) and a chymotrypsin trypsin inhibitor (WbCTI) were purifi ed to homogeneity from the imbibed winged bean (Psophocarpus tetragonolobus (L.) Dc) seeds by affinity chromatography on trypsin and chymotrypsin colu mns. The novelty of the purification lies in exploiting their differential degrees of interaction with their cognate proteases, which results in relat ively higher yields for the three inhibitors as compared to the other metho ds used for purifying any one of them. All have an apparent molecular mass of about 20 kDa as do members of the Kunitz family of protease inhibitors. The amino-terminal sequence of WbCTI is identical to the sequence of the WT I-1 reported earlier by Yamamoto et al. whereas WbTI is a new protein whose N-terminal sequence has no homology with the N-terminal sequence of any kn own proteins. A comparative study of all three inhibitors shows the presenc e of WbTI and WbCTI only in the seeds, whereas the presence of WbCI in othe r tissues has already been documented. Synthesis and degradation during ger mination follow a similar pattern for both the inhibitors. The binding stud ies with cognate proteases show that WbCTI inhibits trypsin more strongly t han chymotrypsin and it never forms a ternary complex even though it binds both proteases. On the other hand. WbTI inhibited only trypsin, in spite of its binding to a chymotrypsin-Sepharose column. The study shows that these protease inhibitors are good candidates for studying the protein-protein i nteraction at the molecular level. (C) 2001 Editions scientifiques et medic ales Elsevier SAS.