Purification, Gene Cloning, Structural And Functional Analysis Of Antioxidant Peptides From Limnonectes Fragilis Skin

Oxygen free radicals is the physiological metabolism product of oxygen breathing animals. It has very strong chemical actibity and can react with almost all substances. In normal physiological process, free radicals are appropriately inhibited, however if the pathological state regulation disorders can cause serious damage to organism.In the long process of evolution, organisms have formed sophisticated defense and protection mechanism. Amphibian especially frogs live in a changeable environment, and their infiltrative and fragile skin is vulnerable to sun radiation. Their skin with respiratory function is susceptible to oxidative damage. Moreover, oxygen free radicals are associated to many diseases, also play important role in the physiological process. However the excessive production of oxygen free radicals can injury organism. After a long period of evolution in the unique survival environment, amphibian skin is endowed with unique antioxidant mechanism, can effectively prevent the oxidative damage to skin.This paper selects Limnonectes fragilis living in long day and muggy area as research material, to study the skin unique antioxidant mechanisms. By ultrafiltration and reverse phase high performance liquid chromatography, two antioxidant peptides with ABTS·+cation radical scavenging activity were isolated and purified from L. fragilis skin secretion, and the ir sequences were:VKRRGQDCIHGFCSD (15amino acid residues) and GQFNDKRWIPFG (12amino acid residues base) by Edman degradation method, belonging to a new antioxidant family, named Frigilin-A1and Frigilin-B2.In order to further analysis their gene sequences and found more antioxidant peptide family, constructed the L. frigilis skin cDNA library. Using conserved antioxidant peptide family gene sequence to design primers, cloned47antioxidant peptide cDNA coding21antioxidant peptides (AOPs), belonging11antioxidant families. From21AOPs, there are14AOPs are totally new.Three antioxidant peptides Frigilin-Al, Frigilin-B2and Odorranain-Q-Lf (APIRMWYMYRKLTDMEPKPVA) were synthesis for their specific sequence. Active detection results fond that they all have no hemolytic and cytotoxic, but has very good antioxidant activity, including the ABTS·+radical cation scavenging activity, DPPH radical scavenging activity, reducing power (Fe3+) activity and lipid peroxidation inhibition activity. From the study Secondary and tertiary structure, we found that3AOPs have regulate structure, and their reducing group exposed to the outside, revealed that free radical scavenging is not by the modes of enzyme action.Finally, we analyzed genetic evolution relation of antioxidant pep tides family, find that AOPs’s different regions (signal, space, and MD nucleotide sequence) have different evolutionary trend, speculating the muture peptides were under positive selection pressure and accelerated evolution trend.