Purification and characterisation of protease inhibitors from pigeon pea (Cajanus cajan (l) millsp) seeds

Abstract

Two protease inhibitors from Cajanus cajan seeds have been purified to homogeneity by trichloroacetic acid (TCA) solubilisation, ion-exchange and gel-filtration chromatography followed by preparative polyacrylamide gel electro-phoresis. One of the inhibitors, Cajanus trypsin-chymotrypsin inhibitor (CTCI), inhibits both bovine trypsin and chymotrypsin while the other, Cajanus trypsin inhibitor(CTI), inhibits only bovine trypsin. The two inhibitors contained no carbohydrate and had an isoelectric point of 6. CTCI and CTI had average molecular weights of 15000 and 10500, respectively. The purified inhibitors in solution were stable to heat at 80°C for 15 min and pH 7-10. In the pH range 3-5, 80% of the activity was retained. Autoclaving totally destroyed the inhibitor activity. CTCI had two sites for trypsin binding and one site for chymotrypsin binding while CTI had only one site for trypsin binding. The inhibitors were very specific towards mammalian serine proteases and did not inhibit other proteases or serine proteases of bacterial origin.