高等学校化学学报

高等学校化学学报

• 研究论文 • 上一篇    下一篇

新型SARS-CoⅤ 3CL蛋白酶荧光多肽底物的设计、制备及其酶动力学研究

万惠新, 陈莉莉, 李欣, 王昕, 胡定宇, 沈旭, 沈竞康   

  1. 中国科学院上海生命科学研究院上海药物研究所国家新药研究重点实验室, 上海 201203
  • 收稿日期:2006-02-10 修回日期:1900-01-01 出版日期:2007-01-10 发布日期:2007-01-10
  • 通讯作者: 沈竞康

Design and Synthesis of a Novel Fluorescent Peptide Substrate of SARS-CoⅤ 3CLpro and Studies on Its Enzymatic Kinetics

WAN Hui-Xin, CHEN Li-Li, LI Xin, WANG Xin, HU Ding-Yu, SHEN Xu, SHEN Jing-Kang   

  1. State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences, Shanghai, 201203, China
  • Received:2006-02-10 Revised:1900-01-01 Online:2007-01-10 Published:2007-01-10
  • Contact: SHEN Jing-Kang

PDF (PC)

被引次数

3

摘要： 以邻氨基苯甲酸(Abz)为荧光发射基团、2, 4-二硝基苯基乙二胺(Eddnp)为荧光猝灭基团, 设计合成了SARS-CoⅤ 3CL蛋白酶的新型荧光多肽底物: H2N-E(Eddnp)STLQSGLK(Abz)-CONH2. 用液相色谱-质谱(LC-MS)联用技术进行了表征, 表明该多肽底物能被SARS-CoⅤ 3CL蛋白酶识别, 并在QS之间被专一性酶解. 另外, 利用该多肽底物的荧光共振能量转移(FRET)特性, 对SARS-CoⅤ 3CL蛋白酶的酶解动力学性质进行了研究, 结果表明, 此荧光多肽底物可以作为荧光探针, 应用于SARS-CoⅤ 3CL蛋白酶活性的测定及其抑制剂的筛选.

关键词: 荧光共振能量转移(FRET), SARS-CoⅤ 3CL蛋白酶, 荧光探针, 液相色谱-质谱

Abstract: A novel fluorescent peptide substrate, H2N-E(Eddnp) STLQSGLK(Abz)-CONH2, based on fluorescence resonance energy transfer(FRET) technique, was designed and synthesized by solid-phase peptide synthesis. Abz/Eddnp was used as the fluorescence donor and quenching pair. The highly fluorescent Abz group was efficiently quenched by energy transfer to the Eddnp group. The fluorescent substrate was specifically hydrolyzed by SARS-CoⅤ 3CLpro between QS with 15-fold increase in fluorescence and the cleavage site was determined by HPLC-MS. The studies on its enzymatic kinetics(Km= 525.5 μmol/L and Kcat=1.29 min-1) elucidate that the synthetic fluorescent peptide substrate is suited to be used as a fluorescent probe in determination of the activity of SARS-CoⅤ 3CLpro and screening assays for its inhibitors.

Key words: Fluorescence resonance energy transfer(FRET), SARS-CoⅤ 3CLpro, Fluorescent probe, LC-MS

中图分类号: 

TrendMD: