鲢鱼肝脏中高分子CPIs提取方法的建立及其鉴定

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），100 mmol/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