Purification and characterization of trypsin inhibitor from seeds of faba bean (Vicia faba L.)

A trypsin inhibitor from seeds of faba bean (Vicia faba L.) was purified to near homogeneity as judged by native-PAGE with about 11% recovery using am monium sulphate fractionation, ion-exchange chromatography on DEAE-cellulos e and gel filtration through Sephadex G-100. The inhibitor had a molecular weight of 18 kD as determined by SDS-PAGE and Sephadex G-100. The inhibitor inhibited trypsin and chymotrypsin to the extent of 48 and 12%, respective ly. The inhibtion was of noncompetitive type with dissociation constant for the enzyme inhibitor complex in the region of 0.07 mg.ml(-1). The inhibtor was stable between pH 4 and 5. It completely lost its activity when heated at 125 degreesC for 1 h or at 100 degreesC for 2 h. The inhibtor also lost its activity on exposure to 2-mercaptoethanol. Based on these properties, it could be concluded that Vicia faba trypsin inhibitor belongs to Bowman-B irk type of inhibitors, as it has molecular weight lower than generally obs erved for Kunitz type inhibitors.