Imitation Of Glutathione Peroxidase Using Selenium-containing 5-mer Peptide And Investigation Of Its Biological Effects

Selenium, as an essential trace element in organism, mainly functions as the constituent of selenoenzymes. Glutathione peroxidase (GPX) is a well-known selenoenzyme that works as an antioxidant and catalyzes the reduction of harmful peroxide by glutathione and protects cells against oxidative damage. Because many diseases are related to oxidative stress, GPX is an ancient foe of many diseases. Antioxidants are very useful for biological bodies, and considerable effort has been spent to find compounds that could imitate the properties of GPX. Although many GPX mimics have been made, they possess serious disadvantages: low activity, low solubility in water, and, in some cases, toxicity. In order to overcome these drawbacks, we have proposed a new strategy of imitating GPX. First, a receptor with a substrate binding site is generated. Next, a catalytic group is incorporated into the receptor near the substrate binding site, allowing the catalytic group access to the functional group of the substrate, and finally a highly efficient enzyme mimic is obtained.1. Construction of selenium-containing 5-mer peptideWe design and obtain a kind of short peptide identifying GSH with solid-phase synthesis technology based on the active center of native GPX. This mimic is characterized with HPLC-MS. Its activity is 11 U/μmol according to Wilson's methods and it has the highest activity relative to its molecular weight compared with other small molecular mimics. The optimal pH and temperature of Se-5P catalyzing the reduction of H₂O₂ by GSH are found to be 8.94 and 43.2℃respectively. In addition, Se-5P shows similar kinetic behavior as natural GPXs, that is they both abide the Ping-Pong mechanism. And the values of kcat/KmH2O2 and kcat/KmGSH of Se-5P are higher compared with natural enzyme, indicating that Se-5P has higher affinity with both substrates. In summary, we have successfully synthesized a GPX mimic with small size, high activity and specific binding site for substrates. These advantages made the novel mimic have very extensive perspective for use in medicine and drugs.2. The biological effects of selenium-containing 5-mer peptideWe construct the Vc/Fe²⁺ ROS-induced mitochondria damage model system, H2O2-induced culture hepatocyte cells damage model system, and Wistar rats hepatic ischemia-reperfusion injury model system. Demonstrating the damaged mitochondria, hepatocyte cells and hepatic tissues have great changes in the three systems. The damages include: the extent of mitochondria swelling is increased, lipid peroxidation is occurred with dose-dependent, penetration of the cell membrane decrease, DNA in the cells fragmentations and the cell viability decreases, decrement of GPX activity, oxygen radical-induced apoptosis. We investigate the protective effects of mitochondria, cultured hepatocyte cells and hepatic tissues by Se-5P using the three damage systems, indicating that they can inhibit the swelling of mitochondria, the increase of the MDA content, and the decrease of the cell viability. They also can prevent the leakage of LDH to sustain the well penetration in cell membrane, regulate the expressions of apoptotic protein Bcl-2 and Bax to inhibit apoptosis. These results indicate that Se-5P can scavenge free radicals to sustain the function of antioxidative defense system in cells. The studies for the Se-5P using the three systems provide the theory bases for this GPX mimic to treat the diseases induced by free radicals, such as cataract, angiocardiopathy and cancers.