用飞行时间质谱研究尿素对重组人肝再生增强因子的修饰作用  

Modification of Recombinant Human Augmenter of Liver Regeneration with Urea Studied by MALDI-TOF Mass Spectrometry

作　　者：潘韵[1] 何国庆[1] 李茹冰 易学瑞 孔祥平 

机构地区：[1]浙江大学生物系统工程与食品科学学院,杭州310029 [2]空军广州医院解放军传染病研究中心分子生物学室,广州510620

出　　处：《生物化学与生物物理学报》2003年第4期355-359,共5页

摘　　要：为研究尿素在变性、复性过程中对重组人肝再生增强因子 (recombinanthumanaugmenterofliverregeneration ,rhALR)的修饰作用及被修饰后蛋白质的结构和生物活性 ,采用基质辅助激光解吸电离飞行时间质谱仪 (MALDI TOF MS)测定蛋白质分子量及以胰蛋白酶酶解后的蛋白质各肽段的分子量 ,验证蛋白质是否被修饰 ,以急性肝损伤动物模型检测被修饰后蛋白质的生物活性。结果包涵体用尿素变性、复性 ,再经纯化获得的rhALR分子量为 30780 ,比理论分子量 30 0 98增加 6 82 ,且含有赖氨酸残基的酶解肽段分子量增加 4 3。采用盐酸胍变性、复性 ,再经纯化获得的rhALR分子量为 30 0 87,与理论值基本吻合。含赖氨酸残基的酶解肽段分子量亦正常。说明尿素可对rhALR蛋白肽链上的赖氨酸残基修饰。活性实验表明虽然尿素分解释放的氰酸盐在变性复性过程中能与蛋白质肽链上赖氨酸的ε氨基结合 ,造成rhALR蛋白质分子量增加 ,但被修饰的rhALR仍能提高四氯化碳致肝损伤小鼠的存活率。To investigate the modification of recombinant human augmenter of liver regeneration (rhALR) by the urea in purification processes and the biological activity of rhALR and modified rhALR, the molecular weight of proteins and tryptic peptides were determined by matrix-assisted laser desorption time of flight mass spectrometry (MALDI-TOF-MS), and the biological activity of rhALR and modified rhALR was also observed by in vivo experiments. A 30 kD homodimer of rhALR was purified under denaturing conditions. The molecular weight of rhALR is 30 780 if urea was used to denature the inclusion bodies; when the denaturant was guanidine hydrochloride, the molecular weight of rhALR was 30 087. The results of MALDI-TOF-MS of digested rhALR that have been modified by urea showed that peptides that contained lysyl were 43 larger than the theoretical value. Proteins purified by different processes were all able to promote the survival rate of CCl 4-intoxicated mice. It could be concluded that cyanate, the cleavage product of urea, could react with the ε-amino group of lysyl in rhALR, and the modified rhALR had the same biological activity as natural rhALR.

关 键 词：重组人肝再生增强因子 尿素 飞行时间质谱 修饰作用 蛋白质 生物活性 包涵体 肝损伤 

分 类 号：Q753[生物学—分子生物学]