鲢鱼卵高分子质量CPI-I的纯化与鉴定

Purification and Characterization of High Molecular Weight Caspase Inhibitor CPI-I from Silver Carp Eggs

以鲢鱼卵为材料制备半胱氨酸蛋白酶抑制因子(CPIs)的粗提浓缩液,进而经Q Sepharose Fast Flow阴离子层析和Sephacryl S-200分子筛层析,获得部分纯化了的CPI-I。以偶氮酪蛋白(azocasein)法监测粗提液及层析纯化中CPIs的抑制活性。层析过程中,以灵敏度更高的荧光合成肽底物(Z-Phe-Arg-MCA)法监测洗脱物的CPIs抑制活性的热稳定性。最终鲢鱼卵CPI-I被纯化了72倍,酶回收率为10.25%。利用Sephacryl S-200分子筛层析Marker标准曲线以及反相酶谱法,初步判断CPI-I的分子质量为89kD。高碘酸-Schiff试剂(PAS)染色法证明该蛋白为糖蛋白。CPI-I能够抑制鲢鱼组织蛋白酶L,且具有显著的热稳定性。

In this study, crude extract concentrate from silver carp eggs was prepared and purified by Q Sepharose Fast Flow anionic chromatography and subsequent Sephacryl S-200 molecular sieve chromatography to obtain partially purified casepase inhibitor （CPI） named as CPI-I. Azocasein assay was used to determine the inhibitory activity of crude extract concentrate and CPI-I. In addition, the heat stability of CPI-I activity was evaluated using the fluorescent peptide Z-Phe-Arg-MCA as substrate. The purification factor of CPI-I relative to silver carp egg homogenate was 72, and the recovery rate was 10.25%. Based on the molecular weight standard curve obtained from Sephacryl S-200 molecular sieve chromatography and the results of reverse zymographic analysis, the molecular weight of CPI-I was preliminarily identified as 89 kD. The results of periodic acid-Schiff （PAS） staining demonstrated that CPI-I was a glycoprotein. CPI-I could inhibit cathepsin L from silver carp meat and showed remarkable heat stability.