| 其他摘要 | The ability to eliminate toxic chemicals either from the environment or metabolic products is essential for the survival of all organisms. Formation of free radicals and reactive oxygen species (ROS) such as superoxide anion radical (.O2-) and hydroxyl (.OH) is an unavoidable consequence of aerobic metabolism during respiration. Evidence suggests that mitochondria convert 1-2% of the oxygen consumed into .O2- under normal physiological conditions and turn them into series of very destructive ROS like hydroxyl. These active radicals may cause damage to cells or tissues easily by lipid peroxidation, denaturation of proteins or nucleic acid damage with severe consequence on overall metabolism. In the long history of evolution, organisms survived have developed a lot of strategies to eliminate these dangerous radicals, which could be summarized as two systems, one group include non-gene coded metabolic products such as uric acid, ascorbic acid, α-tocopherol, carotenes, glutathione, lipoic acid, ubiquinol, another group are gene-coded antioxidant enzymes, such as superoxide dismutase, catalase, peroxidase, thioredoxin systems and glutathione systems. In our previous work, our lab has done some research on Rana pleuraden (a frog live in the long sunshine sub-tropical plateau environments with intensive ultraviolet ray) skin, and found series of gene-coding, expressive secreted antioxidant peptides which named as “the third antioxidant system”. These gene-coded antioxidant peptides contain 15-30 amino acids. The precursor of these peptides are cleaved by serine proteinase and released as mature antioxidant peptides into skin secretions to protect the skin. In this research, we choose Odorrana livida (a frog widely distributed in Shangxi, Sichuan, Yunnan, Guizhou, Anhui, Zhejiang, Jiangxi, Hunan, Fujian, Guangdong, Guangxi, Hainan and Hongkong) to study the antioxidant peptides in its skin, and try to find out how the antioxidant peptides work. Two protein separation steps, including molecular sieve chromatography by Sephadex-G50 and reversed phase high performance liquid chromatography by C18 column were adopted for the separation of frog skin secretions. The antioxidant activities were tracked by ABTS radical scavenging method during these processes. A powerful antioxidant peptide named antioxidin-RL was discovered. Its amino acid sequence is determined as AMRLTYNRPCIYAT by automated Edman degradation and the observed molecular monoisotopic mass is 1672.02 by ESI-MS analysis. Its Isoelectric point is 9.31 by the calculation of ExPASY Compute Mw/pI tool (http://www.expasy.ch/tools/pi toll.html). Using one degenerate sense primer designed according to the amino sequences determined by Edman degradation and then a specific antisense primer designed according to the nucleotide sequence got from the former PCR, cDNA sequence of antioxindin-RL was cloned from the skin cDNA library of Rana livida (GenBank accession number EU294118.1). The cDNA sequence of antioxidin-RL contains 306 nucleotides and deduced precursor is composed of 63 amino acids, including the signal peptide, mature peptide and spacer peptide region. There are two di-basic cutting sites (-K42R43- and –K58R59-) for trypsin-like proteases to hint the location of the mature peptide. Analysis of radical scavenging activities on superoxide anion, hydroxyl, DPPH radical and ABTS radical by antioxidin-RL showed that it could eliminate superoxide anion and hydroxyl, but ABTS radical scavenging test may be a more suitable method for the study of its radical scavenging mechanisms. Chelating effects on ferrous ions and reducing power tests showed that antioxidin-RL cannot chelate metal ions but do have powerful reducing ability. Circular dichoroism spectroscopy and nuclear magnetic resonance analysis showed that antioxidin-RL and its mutants didn’t take a regular secondary structure in water environments, which implied that antioxidin-RL cannot work like antioxidant enzymes. The reaction kinetics of antioxidin-RL on ABTS radical scavenging showed that it has a powerful effect on ABTS elimination and this effect grows up rapidly when the radicals increase. Tests of pH showed that antioxidin-RL are most powerful at pH 8.0, which implied that antioxidin-RL are most effective under physiological conditions and Cys10 may play an important role for the rapid elimination of ABTS radicals. Rapid loss of ABTS radical ability after alkylation of cysteine plus the mutant experiments assured that Cys10 is responsible for the rapid elimination of ABTS radicals. By molecular sieve chromatography using Sephadex-G25, we separated the purple complex formed by antioxidin-RL and ABTS. Wavescan and NMR experiments of antioxidin-RL and its mutants also provided evidence for its existence. Wavescan and NMR experiments also proved that Tyr6 and Tyr12 are key amino acids for the formation of this purple product. When Cys10 were turned into Gly, Tyr6 and Tyr12 still give these peptides the ability to eliminate ABTS radicals, but at a much slower rate. |
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