Purification And Partial Characterization Of Trypsin Inhibitor From Phaseolus Vulgaris

Trypsin inhibitor is a kind of nature insecticide and plays important roles in transgenic plants against insects. It is also a good model for studying protein structure and function. And trypsin inhibitor has an important regulating action in physiology and has tremendous possibilities in inhibiting transfer of cancer cells. However, it is significant to seek new trypsin inhibitor with high activity.A trypsin inhibitor (PVTI) was isolated and purified from Phaseolus vulgaris, and partial characterization of PVTI was studied. The experiment results were as follows:1. An effective plan of crude extract has been worked out. The crude extract was obtained by defating with ethyl ether, extracting with a pH4.0 buffer of dilute acid, heating at 70 ℃, 15min, and precipitating with 40-70% of (NH₄)₂SO₄.2. The trypsin inhibitor was purified further from the crude extract by DEAE Sepharose Fast Flow and gel filtration chromatography. PAGE and IEF showed a single zone. The molecular weight of PVTI was 59kD by gel filtration chromatography. The result of SDS-PAGE indicated that there were three subunits in protein molecular of PVTI. The molecular weight of those subunits was respectively 34kDa, 16kDa and 15kDa. And the isoelectric point of the inhibitor was 5.25. PVTI belonged to irreversible inhibitor according to method of kinetic.3. UV absorption platform of PVTI was between 250nm to 280nm. It indicated that there was a few of aromatic amino acid in PVTI.4. We studied the inhibitor activity and molecular conformation of PVTI under conditions of different temperature, reducer and denaturant. The results were as follows. The tryptophan in nature PVTI located in hydrophobic site of the protein molecular. Residual activity of PVTI was 70 percent after treatment at 100 ℃ 60min. So PVTI was a protein of thermal stability. Under small concentration of DTT, PVTI inhibitor activity lost. It was supposed that disulfide bond existed on...