【作者】 王晓光；

【导师】 罗贵民；

【作者基本信息】 吉林大学 ， 生物化学与分子生物学， 2006， 硕士

【副题名】A Novel GPX Mimic

【摘要】 谷胱甘肽过氧化酶(GPX)利用硒代半胱氨酸(Sec)作为它的催化官能团,通过谷胱甘肽(GSH)催化还原氢过氧化物,从而保护细胞免遭氧化损伤。我们从该酶催化的本质-底物结合和分子内催化出发,利用化学和生物学的方法和原理,制备出具有较高GPX活力的模拟酶6A, 6A’-咪唑-6B, 6B’-二碲桥联-β-环糊精。由于咪唑基的引入,改变了环糊精空腔的微环境,增大了极性,进而增大了与底物的结合能力,增强酶的催化效率。我们测定了模拟物对线粒体膨胀度和脂质过氧化两个指标的影响,证明此模拟物能消除体系中过多的H2O2。同时,我们通过实验证明咪唑基的引入使模拟物具备清除羟自由基的能力,使我们的模拟物的应用范围更加广泛,为以后研究多功能酶模拟物奠定了基础。更多还原

【Abstract】 Reactive oxygen species (ROS) are products of the normal metabolicactivities of aerobic living organism and are produced in response to variousstimuli. Under normal conditions, there is a balance between the production ofROS and their destruction. In certain pathogenic states the production of ROS isenhanced and the excess ROS damage biomacromolecules, therefore it cuasesROS-mediated diseases. In order to scavenge ROS, the living organism has severallines of defense system, including enzymatic and non-enzymatic action.Glutathione peroxidase (GPX) was the first mammalian enzyme characterized asselenoprotein. As a primary enzyme with antioxidant activity, it catalyses thereduction of harmful peroxides by glutathione and protects the cell membranefrom oxidative damage. Consequently, it has the potential to cure variousROS-related diseases such as reperfusion injury, inflammatory process, age-relateddiseases, neuronal apoptosis, cancer and cataract. Due to the limitations associatedwith native GPX (instability, limited availability, immunogenicity, costs ofproduction, and proteolysis digestion), scientists have made a great deal of effortsto study the structure-activity correlation of native GPX, prepared many GPXmimics and studied the biological effects of GPX mimics.In order to overcome the shortcomings of the previous GPX mimics whichare without substrate binding sites, we prepared 6A, 6A’-imidazole-6B, 6B’-tellurium bridged β-cyclodextrin(6-ImTeCD) using the cavity of CD as substratebinding site and ditellurium bond as catalytic group. Also we introduce imidazolegroup to regulate the hydrophobic microenvironment of CD. Elemental analysis,IR, 13C NMR and X-ray photoelectron spectroscopy were used to characterize6-ImTeCD. The GPX activity of 6-ImTeCD with H2O2 were determined to be 9.6U/μmol, which is higher than Ebselen and 6-TeCD. And the GPX activities of6-ImTeCD catalyzed reduction of CuOOH and t-BuOOH were found to be 6.8 and14.1 U /μmol, respectively. It is found that pH and temperature had a effect on theactivity of 6-ImTeCD. Its optimal pH and optimal temperature were 8.95 and 45℃ ,respectively. Detailed kinetic study of 6-ImTeCD was undertaken. Saturationkinetics was observed for both H2O2 and GSH. Double reciprocal plots of theinitial velocity versus the concentration of substrates were a family of parallel lines,consistent with a Ping-Pong mechanism involving at least one covalent enzymeintermediate.In the living organism, most of the ROS is produced by mitochondria, so weconstructed the ferrous sulfate/ascorbate-induced mitochondria damage modelsystem to evaluate the biological effects of the mimic. The swelling, lipid peroxide(MDA content) of mitochondria were chosen as measures to determine theprotective effect on mitochondria by the mimic. The mimic can reduce theswelling of mitochondria during damage and decrease the level of MDAaccumulation. These data show that the mimic has great antioxidant activity. Suchmimic may result in better clinical therapies for disease mediated by ROS.We also studied the scavenging ·OH by 6-ImTeCD using Rhodamine B,whichshowed a color change when it interact with ·OH. The results showed that themimic can scavebge ·OH effectively and protect the living cells.This mimic as well as the others derived from cyclodextrin has the advantageof low molecular weight, good solubility and stability. It also has great antioxidantactivity. So it should be a promising prodrug.更多还原