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

• Published in: Phytochemistry (Oxford) 2006-03, Vol.67 (6), p.545-552
• Main Authors: Paiva, P.M.G., Oliva, M.L.V., Fritz, H., Coelho, L.C.B.B., Sampaio, C.A.M.
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Biological and medical sciences; Bowman-Birk inhibitor purification; Cattle; Chemical constitution; Cratylia mollis; Enzymes; Fabaceae - chemistry; Fundamental and applied biological sciences. Psychology; Hydrogen-Ion Concentration; Leguminosae; Mass Spectrometry; Metabolism; Molecular Sequence Data; Plant physiology and development; Primary sequence; Protein Denaturation; Seeds - chemistry; Sequence Homology, Amino Acid; Temperature; Trypsin inhibitor; Trypsin Inhibitors - chemistry; Trypsin Inhibitors - classification; Trypsin Inhibitors - isolation & purification
• ISSN: 0031-9422; 1873-3700
• DOI: 10.1016/j.phytochem.2005.12.017

The separation of isoinhibitors from Cratylia mollis seeds (CmTI 1 and CmTI 2) by reverse phase chromatography allowed sequence determination and characterization of structural heterogeneity. 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.