Purification And Some Properties Of A Trypsin Inhibitor From Cassia Obtusifolia L.

A trypsin inhibitor named COTI was purified from the seeds of Cassia obtusifolia L. by extracting with distilled water twice, heating at 65℃, precipitation with (NH₄)₂SO₄, trypsin-sepharose-4B affinity chromatography and Sephadex G-75 gel filtration. A series study of COTI showed that the inhibitor had a relative molecular weight of 19 kD by SDS-PAGE.Thermal stability test shows, COTI has good thermal stability, which held definite inhibitory activity at 100℃for 80 min. Both Active-staining Gelatin-PAGE and BAPNA methods testified around 50% residues of COTI activity was seen with 2 m mol/L DTT for 120min at 37℃. These results suggest that the presence of disulfide linkages is critical for maintaining the native structure and function of the inhibitor. However, COTI which is stabile still had some activity after reduction by 2 m mol/L DTT.Inhibition kinetic curve of COTI suggests, COTI is a competitive inhibitor, and its KI is 5.091×10^-8 mol/LBoth double-immuno diffusion and western blotting proved that we had prepared a good antibody with great immunity. These results suggested that we had gained the active purified COTI of 19 KD successfully.The weights of Pieris rapae.L had decreased rapidly after being feeded on Brassica rape leaves contained purified COTI. Further analysis showed that the serine proteases activities of the distilled supernatant from midgut tissues of Pieris rapae.L were inhibited by the purified COTI . The results above suggest that COTI has obviously inhibitory effect on Pieris rapae.L and it is available for the transgenetic crops cultivation.