Immunoaffinity chromatography purification and characterisation of pea trypsin inhibitors

Abstract

Trypsin inhibitors from pea (Pisum sativum (L) cultivar Progreta grown in Denmark) have been isolated and shown to consist of at least nine pea proteinase inhibitors (PPI) with inhibitor activity toward both trypsin and chymotrypsin. The isoelectric points of the inhibitors were in the range 4.9–7.8. From this PPI mixture at least four inhibitors were isolated by immunoaflinity chromatography on a column containing immobilised monoclonal antibody (mAb) with inhibitor specificity. The PPI isolated by immunoaffinity chromatography were further separated by HPLC, and subsequent SDS-PAGE analysis showed molecular weights for four of the PPI in the range 11.3–14.2 kD. Their pI were determined by isoelectric focusing, mAbs were used for immunochemical characterisation and their amino acid composition showed a high (14.7–21%) content of cysteine. Tryptophan was not present in any of the isolated PPI. The data now obtained support the resemblance of PPI with inhibitors of the Bowman-Birk class and the differences in immunochemical properties of the various PPI indicate that pea has at least two gene loci coding for the inhibitors.