Department of Polymer Chemistry, Graduate School of Engineering, Kyoto University, Katsura, Nishikyo-ku, Kyoto 615-8510, Japan.
Bioorg Med Chem. 2012 Jan 1;20(1):96-100. doi: 10.1016/j.bmc.2011.11.026. Epub 2011 Nov 23.
For the quantitative assessment of the glutathione reductase (GR) activity with a (19)F NMR spectroscopy, we developed the heavy metal-free probes based on silica nanoparticles modified with water-soluble perfluorinated dendrimers via the disulfide linkers. Before enzymatic reaction, the molecular rotation of the perfluorinated dendrimers is highly restricted, and the magnitude of (19)F NMR signals from the perfluorinated dendrimers can be suppressed. By the reductive cleavage of the disulfide linkers with the reduced glutathione-mediated enzymatic reaction of GR, perfluorinated dendrimers can be released from the surfaces of the nanoparticles. Consequently, the (19)F NMR signals of perfluorinated dendrimers were recovered. The enzymatic activity of GR was determined from the increase of the magnitude of (19)F NMR signals. Finally, to demonstrate the feasibility of the probe in the presence of miscellaneous molecules under bio-mimetic conditions, the comparison study was executed with the cancer cell lysate. The value determined from our method showed a good agreement with that from the conventional method.
为了用（19）F NMR 光谱法对谷胱甘肽还原酶（GR）活性进行定量评估，我们开发了基于通过二硫键连接体修饰有可水溶的全氟代树状大分子的硅胶纳米粒子的无重金属探针。在酶反应之前，全氟代树状大分子的分子旋转受到高度限制，并且全氟代树状大分子的（19）F NMR 信号的幅度可以被抑制。通过 GR 介导的还原型谷胱甘肽的还原断裂使二硫键断裂，可以将全氟代树状大分子从纳米粒子的表面释放出来。因此，全氟代树状大分子的（19）F NMR 信号得到恢复。通过（19）F NMR 信号幅度的增加来确定 GR 的酶活性。最后，为了在仿生条件下证明探针在存在各种分子的情况下的可行性，在癌细胞裂解物中进行了比较研究。从我们的方法确定的值与传统方法确定的值吻合良好。
