Purification and characterization of protease inhibitor from rice bean (Vigna umbellata T.) seeds

A Protease inhibitor from seeds of rice bean has been purified to apparent homogeneity as judged by native-PAGE with about 29% recovery using ammonium sulfate fractionation, ion exchange chromatography on DEAE-cellulose, and gel filtration through Sephadex G-100, The purified preparation with molecu lar weight of 16.8 kD was found to be a monomer as revealed by SDS-PAGE und er reducing and non-reducing conditions, The purified inhibitor in solution was stable upto 40 degrees C. However, it lost its activity gradually and was completely inactive when heated at: 100 degrees C for 2 h and 125 degre es C for 1 h, Heating at 125 degrees C changed the conformation of the inhi bitor as was evident from the altered UV spectrum compared to that of nativ e, It had two pH optima at pH 6.0 and at 10.0 and was stable over a wide ra nge of pH (pH 3.0 to 10.0). It lost its activity on exposure to P-mercaptoe thanol, indicating the role of S-S linkages in maintaining the three dimens ional structure of the protein inhibitor. The inhibitor was completely inac tive towards papain, while it inhibited pepsin only slightly. Trypsin and c hymotrypsin were inhibited upto the extent of 60% and 30%, respectively. Tr ypsin inhibition was of non competitive type with dissociation constant for the enzyme-inhibitor complex in the region of 2.07 mg ml(-1), The rice bea n inhibitor appears to be of Bowman-Birk type as it has molecular weight lo wer than that generally observed for Kunitz type inhibitors and seems to be double headed - a characteristic specific of Bowman - Birk type inhibitor.