草鱼肝胰中CPIs初步分离鉴定及其活性测定  

Separation and identification of CPIs from grass carp hepto-pancreas and its activity assay

作　　者：蒋然然 梁宇[1] 陈治光[1] 杨锦[1] 李新[1] 但静[1] 李冉[1] 钟海霞[1] 李美良[1] 李树红[1] 

机构地区：[1]四川农业大学食品学院,四川雅安625014

出　　处：《食品工业科技》2015年第16期118-123,共6页Science and Technology of Food Industry

基　　金：国家自然科学基金青年科学基金项目(31101249);四川省科技支撑计划项目(2014NZ0003)

摘　　要：首先比较了偶氮酪蛋白（Azocasein）法和荧光合成肽底物法，在监测草鱼肝胰半胱氨酸蛋白酶抑制剂CPIs（cysteineproteinaseinhibitors）活性时的适用性。进而通过凝胶过滤高效液相色谱法、反相酶谱和免疫印迹法，对草鱼肝胰中CPIs的分子量分布及可能的家族类型进行定性鉴定。结果表明在粗提液中加入丝氨酸蛋白酶不可逆抑制剂（40mmol／LPMSF或0.4mmol／LPefablocSC），或经90℃加热5min，仅荧光合成肽底物法可准确地监测到CPIs活性的变化。液相色谱结果显示，草鱼肝胰中存在分子量（98、26、12、5-1ku）不同的CPIs活性部分。但反相酶谱法仅能够判断其中大于20ku的CPIs。而免疫印迹法证明存在约12ku的Cystatin（家族II）类型CPIs，发现小于12ku的显色条带，可能是该蛋白降解形成的低分子量的活性片段。The applicability of the methods of azocasein and fluorescence synthetic peptide in monitoring the inhibitory activity of CPIs（cysteine proteinase inhibitors） from grass carp hepto-pancreas was firstly evaluated. Then the molecular weight distribution and the possible superfamily classification of CPIs were identified by gel filtration HPLC combining with reversed-phase enzyme spectrometry and western blotting methods. The results showed that after adding serine protease inhibitors（40 mmol/L PMSF or 0.4 mmol/L Pefabloc SC） into the crude extract or heating treatment at 90 ℃ for 5 min,the inhibitory activity of CPIs could accurately monitored only by the fluorescence synthetic peptide method. Furthermore,the results of HPLC disclosed that there were several forms of active CPIs with different molecular weight（98,26,12,5~ 1 ku） in grass carp hepto-pancreas. However,only the CPIs more than 20 ku could be determined by the reverse zymography method. In addition, the results from western blotting revealed the existence of CPIs belonging to Cystatin（family II） with the molecular weight about 12 ku and the developing band less than 12 ku was speculated to be the active fragment degraded from the Cystatin protein.

关 键 词：草鱼肝胰 半胱氨酸蛋白酶抑制因子 活性测定 分子量分布 分离鉴定 

分 类 号：TS254.1[轻工技术与工程—水产品加工及贮藏工程]