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

Two Bowman-Birk type trypsin inhibitors (CmTI(1) and CmTI(2)) were purified from Cratylia mollis seeds by acetone precipitation, ion exchange, gel filtration and reverse-phase chromatography. CmTI(1) and CmTI(2), 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 Leguminosae. The putative reactive sites of CmTI(1) 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 CmTI(2): 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.