【作者】 于斐；

【导师】 屈凌波； 李建军；

【作者基本信息】 郑州大学 ， 分析化学， 2004， 硕士

【摘要】 在生物体中，蛋白质是必不可少的生命物质，在生命的运动和发展延续中起着重要的作用。有关蛋白质构象、组成结构、以及与小分子相互作用的机理，是目前生命科学、化学和临床医学中共同关注和感兴趣的课题。近年来，由于医药学和生命科学的发展，研究小分子，尤其是药物和具有药效的小分子与蛋白质的相互作用机理过程，对药代动力学的认识和开发新药有极其重要的意义。 本文用荧光法研究了两种黄酮（7-羟基黄酮、5，7-二羟基黄酮）及其磷酰化后的产物（7-羟基磷酰化黄酮、5，7-二羟基磷酰化黄酮）在液态环境中和溶菌酶、肌红蛋白以及牛血清白蛋白的相互作用情况。结果显示磷酰化黄酮均能够和溶菌酶、肌红蛋白和牛血清白蛋白发生弱相互作用，与黄酮相比磷酰化后的黄酮对蛋白质更具亲和力。并根据荧光猝灭双倒数图计算了磷酰化黄酮与溶菌酶、肌红蛋白和牛血清白蛋白之间的结合常数，分别为：溶菌酶与7-羟基磷酰化黄酮的结合常数为：k_20℃=1.68×10⁴L／mol，k_37℃=1.06×10⁴L／mol；溶菌酶与5，7-二羟基磷酰化黄酮的结合常数为：k_20℃=1.9×10⁴L／mol，k_37℃=1.7×10⁴L／mol；肌红蛋白与7-羟基磷酰化黄酮的结合常数为：k_20℃=1.42×10⁴；k_37℃=1.13×10⁴；肌红蛋白与5，7-二羟基磷酰化黄酮的结合常数为：k_20℃=5.13×10⁴L／mol，k_37℃=4.5×10⁴L／mol；牛血清白蛋白与5，7-二羟基磷酰化黄酮的结合常数为：k_20℃=5.29×10⁴L／mol和k_37℃=3.74×10⁴；牛血清白蛋白与7-羟基磷酰化黄酮的结合常数为：k_20℃=3.3×10⁴，k_37℃=2.9×10⁴L／mol。所有实验证明随着温度的升高，磷酰化后的黄酮与三种蛋白质的结合常数逐渐降低，说明了两者之间形成了复合物，所有的荧光猝灭过程都为静态猝灭。本文还根据F（?）ster能量传递原理计算出两种磷酰化后的黄酮与三种蛋白质的结合距离，分别为：溶菌酶与7-羟基磷酰化黄酮：r=5.70nm；溶菌酶与5，7-二羟基磷酰化黄酮：r=5.23nm；肌红蛋白与7-羟基磷酰化黄酮r=4.7nm；肌红蛋白与5，7-二羟基磷酰化黄酮：r=4.31nm：牛血清白蛋白与7-羟基磷酰化黄酮：r=4.13nm；牛血清白蛋白与5，7-二羟基磷酰化黄酮：r=3.69nm。并根据热力学参数确定了两种磷酰化后的黄酮与蛋白质之间的作用力类型为电荷作用力。郑州大学硕士学位论文由实验结果可以得出以下结论:1，与黄酮相比，磷酞化后的7一经基黄酮和磷酞化后的5，7一二轻基黄酮的确对 蛋白质更具亲和力。2.由实验数据可以看出黄酮经过磷酞化改造后的活性增强，而且随着磷酞化基 团的增多与蛋白的亲和性更好。3.本文所用的方法对研究小分子与蛋白在液相中的相互作用情况是一种有效、 可靠的方法。更多还原

【Abstract】 Fluorescence method was used to study the interactions between lysozyme, myglobal, and bovine serum albumin and tow phosphorylated flavonoids and two flavonoids . The results showed that the phosphorylated flavonoids could form non-covalent complexes with lysozyme, myglobal, and bovine serum albumin while the flavonoids cannot. The results showed higher binding affinity between the proteins and the phosphorylated flavonoids than flavonoids did. The association constants of lysozyme, myglobal, and bovine serum albumin and the phosphorylated flavonoids were determined from a double reciprocal Lineweaver-Burk plot. The binding constants are foot:=1-68 104 L/mol and k3TC =1.06 104 L/mol (lysozyme-Diethyl Flavone-7-yl Phosphate), respectively. And so on. Experiments demonstrated that the higher the temperature were, the lower the slops of quenching curve of proteins were in presence of different amounts of the phosphorylated flavonoids. It was confirmed that the combinations for the phosphorylated flavonoids with the three proteins were single static quenching processes. According to the Foster dipole-dipole energy transfer, the distance to be measured between the phosphorylated flavonoids and tryptophane. From thermo dynamical coordination it could be judged that the binding power between the phosphorylated flavonoids and, myglobal, and bovine serum albumin were static electric power.Fluorescence in this paper provides good information about the phosphorylated flavonoids-proteins interactions. Moreover, the rapidity of data acquisition and the relative simplicity of interpretation make fluorescence a powerful tool for screening numerous small molecule-protein complexes.更多还原