| In this paper,the preparation,isolation and purification of the oyster trypsin inhibitor (OTI) are studied systematically,and the anti-tumor activity and some characteristics are also included. This work may provide primary data for further researching on the relationship between the structure and function of OTI,as well as provide a new way for exploiting the anti-tumor medicine.This study includes six parts of content:1.The measurement method of trypsin inhibitory activity is established, basing on the trypsin catalysis hydrolysate of substrate BAEE has the ultraviolet absorbance at 253 nm. The specific inhibitory activity (IUM value) of trypsin inhibitory rate to 50% is choosed as the index of the isolation and purification.2.The raw oyster is dealt with thermal precipitation and ammonium sulfate precipitation,then lyophilized and used as the crude OTI. The yield is 17.98%,and the protein content is 56.51%,and IUM value is 6.581 U/mg. This method is operated easily,cheaply and safely.3.The crude OTI is applied to Sephadex G-75 column,and two peaks are eluted. The first peak A1 has the trypsin inhibitory activity,and the IUM value is 47.43 U/mg. Peak A1 is carried out on trypsin-Sepharose 4B column, and only one combined peak A1B1 displays the activity,and its IUM value is 270.74 U/mg. Before applied to the affinity chromatography,trypsin should be combined to the bromize cyanogens activated Sepharose 4B dry power successfully. Then peak A1B1 is performed on Sephadex G-15 column for desalting,and two peaks are pooled. The second peak A1B1C2 shows the trypsin inhibitory activity,and its IUM value is 299.54 U/mg. Peak A1B1C2 is taken as the OTI. After these operations,the purification fold of the crude OTI is 45.52,and the yield of activity is 19.94%.4.MTT method is employed to determine the anti-tumor activity of OTI, and the anti-tumor medicine 5-Fluorouracil is used as the pharmic comparison simultaneously. The result is that the proliferations of the human lung adenocarcinoma A549 cell and human cervical cancer Hela cell are both inhibited by OTI,and the inhibitory rates are 20.78±2.37 % and 20.68±2.14 %,respectively. At the same time,the effect on A549 cell is better than on Hela cell. And both of the inhibitory rates are about 50% of the same dosage of the 5-Fluorouracil.5.The anti-tumor activity of OTI is purified by HPRPC. OTI is further purified on Zorbax C18 semi-preparative column, and two peaks are provided. The second peak Y2 is the active peak,and the IUM value is 373.31 U/mg. Then the purity of peak Y2 is assessed on Zorbax C18 analytical column,the IUM value of the only peak Y2F is 380.84 U/mg. It is proved that peak Y2F has the high purity,basing on the only peak of Zorbax C18 analytical column. So peak Y2F is selected as the anti-tumor ingredient of OTI and named as OTIA.6.Some characteristics of OTIA are determined. OTIA has the high temperature stability between 20oC and 100oC. Also,it has the high pH stability from pH 2.0 to pH 12.0.The molecular weight of OTIA is estimated by HPSEC on TSK-GEL G3000 PWXL column. As the result,the molecular weight is about 5.036 kDa. The molar ratio of OTIA to trypsin is about 1:2,so OTIA is double-head inhibitor and belongs to Bowman-Birk family.From the inhibition kinetic test of the inhibitor,the Km value of trypsin to substrate BAEE is 1.211 mmol/l, and the inhibiton constant Ki value of OTIA is 1.644×10-2 mmol/l. So OTIA is competetive inhibitor with high inhibitory activity.There are nine kinds of amino acid in OTIA,and the number of the amino acid residues is 40. By comparison,the contents of Glu,Ala and Cys are about 71.20%,and only Cys occupies 35.93% of all the amino acid. From the amino acid composition,OTIA is considered to be acidic inhibitor.
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