Construction Of Mimics With SOD And GPX Activities And Investigation Of Biological Effects

Oxidative stress is a kind of unbalance state between oxidation and antioxidation. Reactive oxidative species (ROS) are harmful to cells when oxidative metabolism products increase or the antioxidative defenses lack in the organism. Lots of researches showed that ROS are related to some physiologic and pathological processes and play an important role in many diseases. ROS have high activity and very strong oxidative ability and they can attack the molecules by the oxidation, and then make the big molecules denaturate, cross-link and breakout, and then lead to the damage of cell structure and function and the injury of the tissue.There are two kinds of the scavengers for free radicals in the organism, namely enzymes (antioxidative enzymes) and non-enzyme compounds. The enzyme scavengers main include SOD, CAT and GPX, and they form the defense system in the organism against free radicals. In addition, many small molecular compounds also have strong antioxidative ability. These compounds are vitamin C, vitamin A,A-tocopherol, reduced glutathione, P -carotene, metal chelate complex, calcium ion antagonists, flavonoid, polyphenol compouns and antioxidant peptides, and so on. They act with antioxidant enzymes to keep free radicals at a normal level of metabolism in the organism.SODs are a class of metalloproteines that are found to be very efficient catalysts of superoxide disproportionationSOI)02T+(V+2H+----- H2O2+ O2and it has been proposed that this activity is their primary function in vivo. The site of reactivity for superoxide,O2-, with Cu,Zn-SOD is the copper ion. Cell damage may also be due to the superoxide itself or, indirectly, even more reactive oxygen species, such as hydroxyl radicals ("OH), formation of which, via the Fenton reaction, is favoured by excess superoxide anion, 02* and is formed as a normal byproduct of aerobic metabolism from a large number of sources, such as cellular respiration, activated polymorphonuclear leukocytes, endothelial cells, and mitochondrial electron flux and it has been demonstrated to be a mediator of ischemia-reperfusion injury, inflammatory diseases and vascular diseases. SOD enzymes are believed to play important roles as antioxidants in accelerating the conversion of superoxide to peroxide, thereby preventing direct reactions of superoxide that may do damage to sensitive targets in the cell. Due to limitations associated with enzyme therapies (short lifetime, high cost, tissue impermeability, and immunogenicity, etc), the stable, non-toxic metal complexes (model compounds) which catalyze the dismutation of Or offer considerable promise and have attracted much attention. Low molecular-weight catalysts that mimic a natural enzymatic function have potential utility for the treatment of diseases characterized* by the overproduction of a deleterious metabolic by-product. Until now, many SOD mimicshave been described, such as copper (II) complexes of peptides, polydentate schiff bases, mixed ligands, and so on.Based on the structure of the active site of Cu, Zn-SOD and of its Chou-Fasman conformation parameters, our group had designed a peptide that consists of 17 amino acids. But we only obtained 16-mer peptide, which is short of arginine by Boc protocol. Now we have synthesized 17-mer peptide by Fmoc protocol, and found that arginine has important effect to the activity of copper (II )-containing 17-mer peptide.GPX is a kind of protein containing selenium. Although the structure of GPX and the biologic effect of selenium have been understood fully, the effect mechanism of GPX needs to be studied further. The GPX mimics can be used to do this. On the other hand, GPX has strong antioxidant ability and it is important for treating and preventing Keshan disease, angiocardiopathy, inflammation and cancer. Due to the limitations associated with native GPX, such as instability, limited availability, big molecular weight and immunogenicity, many scientists have made a great deal of efforts to study the GPX mimics.Due to the lack of the GSH binding site, the early GPX mimics have generally rather low activities. Take ebselen for example, its activity only has 0.99 U/fxmol. Wilson et al. introduced quaternary ammonium salt near to diselenide bridge of GPX mimic to improve the ability to bind GSH by electrostatic attraction to GSH and quaternary ammonium salt can provide proton for reduction of another substrate, H2O2. So the activity of this kind of GPX mimic, dipheyl diselenide compound, can be increased up to 11 U/^mol. Hilvert et al. turned subtilisin to GPX mimic by chemical mutation. The catalytic efficiency of the mimic was 7xlO4 times more than that of dipheyl diselenide compounds using thefavored 3-oxatyl-4-nitryl phenthiol as substrate. But its activity towards GSH, the substrate of native GPX, was rather low. So the key of imitation GPX is to generate a specific binding site for GSH. According to the idea, we have developed several selenium-containing catalytic antibodies. They had high catalytic activities, but there are some drawbacks for use as drugs, such as the big molecular size led to the difficulty for transport in organism and penetration into cells. So we decided to construct a small-molecule GPX mimic with high activity base on the specific combination with substrate and study its enzymatic character and biologic efficacy. We confirmed the biologic efficacy of the GPX mimic in organism through the protective effect of the GPX mimics in the cells.1. A copper-containing 17-mer peptide displays superoxide dismutase activityCopper-containing 17-mer peptide can scavenge superoxide anions and display superoxide dismutase activity. In addition, the Arg residue in the 17-mer peptide is also near the copper site compared with that of 16P, which is short of arginine. The specific activity of the mimic is 216 U/mg. The results show that Arg residue can improve the efficiency of the copper-containing 17-mer peptide. Therefore, we consider that Arg residue could assist the attraction of a negatively charged superoxide anion to the copper center. Thus the arginine residue plays an important, but not an essential role in the catalytic process.2. Construction of selenopeptide to imitate GPXWe obtain a kind of short peptide, 5P, which recognizes GSH. This mimic is characterized with HPLC-MS. Its activity is 11 U/[xmol according to Wilson's ?methods and it has the highest activity among the small-molecule mimics developed so far. The optimal pH for 5P catalyzed reduction of H2O2 by GSH isfound to be 8.94 and the optimal temperature was found to be 43.2°C. The kinetic analysis show that the mechanism of 5P catalyzed reduction of H2O2 by GSH is Ping-Pong mechanism and the values of kcJKmmoi and kcJKmGsn are higher, indicating that 5P has higher affinity with both substrates. In summary, we have successfully synthesized a GPX mimic with high activity, specific binding site for substrate, and small size. These advantages made the novel mimic have very extensive perspective for use in medicine and drugs.3. The biological effects of GPX mimicsWe constructed the H2O2-induced culture hepatocyte cells damage model system and demonstrated the hepatocyte cells had great changes in the damage system. The damages include: lipid peroxidation, DNA fragmentation in the cells and decrease of cell viability . We investigate the protective effects of culture hepatocyte cells by 5P using the damage system, indicating that it can inhibit the increase of the MDA content, the decrease of the cell viability, and the level of DNA fragmentation. 5P also can prevent the leakage of LDH to sustain the well penetration in cell membrane. We also observed the effects of 5P on the level of antioxidative enzymes and GSH in the H2O2 damaged system, indicating that 5P can scavenge free radicals to sustain the function of antioxidative defense system in cells and recover the GSH content to normal level.5P can protect H2O2-induced hepatocyte cells from damage. Therefore, this is a kind of promising antioxidant drug, which prevents H2O2 damage similar to oxidative stress occurred in the organism, thus, the studies for the 5P using the systems provide the theory bases for these GPX mimics to treat the diseases induced by free radicals, such as cataract, angiocardiopathy.