Isolation and characterization of a trypsin-chymotrypsin inhibitor from the seeds of green lentil (Lens culinaris)

Abstract

A Bowman-Birk type trypsin-chymotrypsin inhibitor was isolated from seeds of the legume green lentil (Lens culinaris) by means of affinity chromatography on Affi-gel blue gel, ion exchange chromatography on Q-Sepharose, ion exchange chromatography by fast protein liquid chromatography (FPLC) on Mono Q and Mono S, and gel filtration by FPLC on Superdex 75. The trypsin-chymotrypsin inhibitor was bound on the first three types of chromatographic media. It appeared as a single 16-kDa peak in gel filtration and a single 16-kDa band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The trypsin inhibitory activity of the inhibitor was sensitive to the reducing agent dithiothreitol. It was completely abrogated after treatment with 10 mM dithiothreitol for 20 minutes. The protease inhibitor did not exert any inhibitory effect on hepatoma (Hep G2) and breast cancer (MCF 7) cell lines. There was no suppressive action on several fungal species including Botrytis cinerea, Fusarium oxysporum and Mycosphaerella arachidicola. It slightly inhibited the activity of HIV-1 reverse transcriptase, with an IC50 of 30 mM.