鲢鱼肝脏中高分子CPIs提取方法的建立及其鉴定

Extraction and Characterization of High-Molecular-Weight CPIs from Silver Carp Liver

研究了不同浓度的丝氨酸蛋白酶抑制剂(PMSF)、热处理条件及pH值对鲢鱼肝脏中高分子半胱氨酸蛋白酶抑制剂(cysteine proteinase inhibitors,CPIs)提取过程中的作用,根据其对CPIs在TSK液相上高分子质量部分的蛋白峰值和比活力(荧光合成肽底物法)的影响,确定了最佳粗提条件为:以含5 mmol/L的苯甲基磺酰氟(phenylmethanesulfonyl fluoride,PMSF),100mmol/L的Tris,3 mmol/L的EDTA(pH 7.5)作为提取缓冲液,而后在pH 8.7碱处理条件下经90℃加热5 min后,再回调到pH 7.0。用该法制备的鲢鱼肝脏CPIs粗提物经制备型Sephacryl S-200分子筛层析,分离得到的高分子活性部分,进一步经过反相酶谱法鉴定得到一种高相对分子质量的活性CPI,推测其可能是与某些蛋白结合形成了复合物,或是高分子CPIs的二聚体形式。

In this study,different concentrations of the serine protease inhibitors phenylmethanesulfonyl fluoride(PMSF),different heat treatment and different pH were conducted to treat on the crude extraction of high-molecular-weight cysteine proteinase inhibitors(CPIs)from Silver carp liver,and we investigated their effect on the protein response values and specific activity of CPIs by HPLC with column of TSK-GEL G2000SW.The optimum extraction conditions were as follows:the condition of extraction buffer is containing 5 mmol/L PMSF,100 mmol/L Tris,and 3 mmol/L EDTA at pH 7.5,followed by pH readjustment to 7.0 after alkali(pH 8.7)treatment at 90℃for 5 min.Then,the high-molecular-weight active CPI was obtained by chromatography on a Sephacryl S-200 column,and it was identified as a high-molecular-weight CPI by the reverse zymography method.Therefore,it may be conjugated with other protein or a dimeric protein.