Purification and primary structure determination of two Bowman-Birk type trypsin isoinhibitors from Cratylia mollis seeds

Abstract

Two Bowman-Birk type trypsin inhibitors (CmTI1 and CmTI2) were purified from Cratylia mollis seeds by acetone precipitation, ion exchange, gel filtration and reverse-phase chromatography. CmTI1 and CmTI2, with 77 and 78 amino acid residues, respectively, were sequenced in their entirety and show a high structural similarity to Bowman-Birk inhibitors from other Legummosae. the putative reactive sites of CmTI1 are a lysine residue at position 22 and a tyrosine residue at position 49. Different reactive sites, as identified by their alignment with related inhibitors, were found for CmTI2: lysine at position 22 and leucine at position 49. the dissociation constant K-i of the complex with trypsin is 1.4 nM. the apparent molecular mass is 17 kDa without DDT and 11 kDa with reducing agent and heating. (c) 2005 Elsevier B.V. All rights reserved.