PURIFICATION AND CHARACTERIZATION OF PROTEASE INHIBITORS FROM PIGEON PEA (CAJANUS-CAJAN (L) MILLSP) SEEDS

Two protease inhibitors from Cajanus cajan seeds have been purified to homogeneity by trichloroacetic acid (TCA) solubilisation, ion-exchange and gelfiltration chromatography followed by preparative polyacrylamide gel electrophoresis. 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 15 000 and 10 500, respectively. The purified inhibitors in solution were stable to heat at 80 degrees 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.