【作者】 张鸿伟； 王凤山；

【Author】 ZHANG Hong-wei, WANG Feng-shan*(Institute of Biochemical and Biotechnological Drug, School of Pharmaceutical Science, Shandong University, Jinan 250012, China)

【机构】 山东大学生化与生物技术药物研究所；

【摘要】 目的研究铜锌超氧化物歧化酶(Cu,Zn-Superoxide dismutase,Cu,Zn-SOD)经低分子肝素(Low molecular weight heparin,LMWH)化学修饰前后蛋白分子二级结构的变化。方法使用KBr法、溶剂挥发成膜法和液体膜法测定Cu,Zn-SOD和LMWH修饰后结合物(LWMH-SOD)的傅立叶转换红外(FTIR)谱,并利用傅立叶去卷积和二阶求导技术对获得谱图的酰胺Ⅰ带(1700～1600cm-1)进行计算机分峰和图谱拟合来分析不同物理状态下Cu,Zn-SOD和LMWH-SOD酶蛋白分子的二级结构,同时,运用圆二色谱(CD谱)技术对修饰前后酶蛋白分子的二级结构变化进行佐证分析。结果FTIR和CD分析结果表明,LMWH-SOD与Cu,Zn-SOD相比,酶蛋白分子二级结构中α-螺旋构象比例上升,β-构象成分比例接近且在两者中均为主要构象成分。结论Cu,Zn-SOD和LMWH-SOD结合物二级结构中均以β-构象为优势构象,使用LMWH进行适当的化学修饰,主要是影响酶蛋白分子二级结构中的α-螺旋构象,使其含量上升,对酶蛋白优势构象同时也是构成酶分子活性中心的β-构象的影响微弱。更多还原

【Abstract】 OBJECTIVE To study the difference in secondary structure of enzyme molecule between Cu,Zn-Superoxide dismutase (Cu,Zn-SOD) and low molecular weight heparin (LMWH) chemically modified Cu,Zn-SOD (LMWH-SOD conjugate). METHODS In the states of powder, solvent-dried thin film and D2O solution after H/D exchange, conformation of Cu,Zn-SOD and LMWH-SOD were analized by Fourier transform infrared (FTIR) spectroscopy with its self-deconvolution and derivative resolution enhancement as well as curve-fitting procedures on amide Ⅰband (1700～1600cm-1). Circular dichroism (CD) analysis gave a comparison on secondary structure changes of zymoprotein obtained by FTIR determination. RESULTS FTIR and CD analysis showed that secondary structure of zymoprotein, presented in LMWH-SOD, gave an increase in α-helix content but similar β-conformation content to that of Cu,Zn-SOD, also the β-conformation is the primary conformation for both. CONCLUSION β-conformation is dominant in secondary structure of Cu,Zn-SOD and LMWH-SOD. Considerately chemical modification with LMWH raises α-helix content in secondary structure of zymoprotein, while little to dominant β-conformation, which also serves as the activity center of zymoprotein.更多还原