【作者】 邓志鹏； 钟大放； 陈笑艳；

【Author】 DENG Zhi-peng,ZHONG Da-fang,CHEN Xiao-yan Shanghai Institute of Materia Medica,Chinese Academy of Science,Shanghai 210203,China

【机构】 中国科学院上海药物研究所；

【摘要】 目的:研究鱼腥草素与人血清白蛋白的共价结合。方法:将¹⁴C-鱼腥草素与人血清白蛋白的孵化样品经液体闪烁计数器测定放射点数,计算其与蛋白的共价结合量;鱼腥草素修饰人血清白蛋白经链霉蛋白酶水解,采用LC/MS/MS方法鉴定鱼腥草素各修饰的氨基酸片段的结构。结果:¹⁴C-鱼腥草素与人血清白蛋白迅速发生共价结合,结合量为18.2 nmol/mgprotein;鱼腥草素修饰的人血清白蛋白的酶解样品中,发现生成4个氨基酸结合物,质核比分别为m/z 327.2,489.1,492.0和336.9。经过多级质谱分析与合成的标准对照品对照,确认为鱼腥草素与人血清白蛋白中的赖氨酸、精氨酸以及氮端甲硫氨酸残基生成的加合物。结论:放射性同位素标记法和LC/MS/MS方法证明了鱼腥草与人血清白蛋白能够发生共价结合,并对结合位点进行了确定。更多还原

【Abstract】 ABM:To investigate the covalent binding of houttuynin with human serum albumin.METHODS:¹⁴C-houttuynin was incubated with HSA,and the protein-associated radioactivity was determined by Liquid scintillation counting.On the other hand,the modified HSA was digested with Pronase and analyzed by LC/MS/MS method.RESULTS:¹⁴C-labeled houttuynin could quickly and covalently bind with HSA in 1 minute,and the amounts of ¹⁴C-houttuynin bound to protein was 18.2 nmol/mg protein.The proteolysis of houttuynin -modified HSA with Pronase gave four adducts,m/z 327.2 was the Schiff base from the modification of of lysine residues;m/z 489.1 and 492.0 were pyridine-type conjugates from lysine residues and N-terminal methionine;m/z 336.9 was pyrimidine-type conjugate from arginine residues.CONCLUSION:The radiolabeled and LC/MS/MS methods had effectively illuminated that houttuynin could covalently bind with HSA.更多还原