Isolation and characterization of trypsin inhibitors from some Thai legume seeds

Trypsin inhibitors from cultivars of cowpea (Vigna unguiculata (L.) Wasp.), pigeon pea (Cajanus cajan (L.) Millsp.) and bambara groundnuts (Voandzeia subterranea (L.) Thou) grown in Thailand were isolated and characterized. Extraction of seeds with NaCl rendered a higher recovery of trypsin inhibitor than other solvents tested (p<0.05). The extraction time affected the inhibitor recovery (p<0.05). The extraction time of 3 h was optimum for the recovery of trypsin inhibitor from pigeon and bambara groundnuts, whereas I h was optimum for cowpea. Based on inhibitor activity of zones separated by electrophoresis, the molecular mass of the inhibitor from bambara groundnuts was 13 kDa. Two inhibitory bands were observed for cowpea (10 and 18 kDa) and pigeon pea (15 and 25 kDa). Partial purification of inhibitors was achieved by heat-treatment at 90C for 10 min, followed by ammonium sulfate precipitation with 30-65% saturation. The partially purified inhibitors from four seeds were heat stable up to 30 min at 90C at pH 7.0. The activities were also retained over a wide pH range at 25C but were lost when samples were treated with beta-mercaptoethanol prior to electrophoresis.