Mensuration Primary Structure And Character Of Trypsin Inhibitor From Onobrychis Saliva

The Trypsin Inhibitor from Onobrychis Saliva in Xinjiang(OSTI) was found in 1998. It is a kind of protein that can inhibit trypsin intensively. Based on the primary study on it, the study on primary structure and character of OSTI was done in this paper. The research content and results are as follows:1. Isolation and purification of OSTI. Salting-out with (NH4)2SO4, separating by FPLC Mono S, and obtaining purified OSTI by PAGE. Mensuration of molecular weigh of OSTI is around 22,000Da by SDS-PAGE. 2. Researching of OSTI'S character. Taking BAPNA as the substrate, mensurating inhibit type and mole inhibitory ratio under the optimum conditions, the results of research indicate that OSTI has effect of irreversible inhibition on trypsin, and the mole ratio is about 1:2. Modifing Arginine residue of OSTI with Cyclohexanedione, and the result shows that Arginine is an active center of OSTI. Modifing Lysine residue of OSTI with Maleic anhydride, and the result indicates that Lysine is also an active site of OSTI. The residue modification tests show that the active centers of OSTI are Arginine and Lysine.3. Mensuration of the primary structure of OSTI.The sample is provided by our lab and accomplished by the Walter and Eliza Hall Institute for Medical Research of Australia. Using the method of MS to mensurate the primary structure of OSTI. The details are as follows: Tranfering the PAGE gel to nitrate membrane, using Trypsin and V8 to chip OSTI separately, obtaining the purified peptide with Hplc, then mensurating the sequence of peptide segment, finding out the overlap sequence from the different segments, piecing them together to get the whole OSTI sequence finally.The primary structure of OSTI is made of 203 amino acid residues, the end of N is Met, the end of C is Ala. Referred to protein database, and it is found that the homologizaion of the sequence between OSTI and Kunitz-type Trypsin Inhibitor KTI2-Soybn is 88%. Therefore, OSTI is a new member belonging to the Kunitz family. The primary structure of OSTI has established foundation for the further study on its gene and application.