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题名	大绿臭蛙皮肤抗氧化肽antioxidin-RL的分离纯化及结构和功能研究
作者	刘存宝
学位类别	博士
答辩日期	2012-05
授予单位	中国科学院研究生院
授予地点	北京
导师	赖仞
关键词	抗氧化肽 自由基清除 机理 青蛙皮肤
其他题名	Purification, structure and function of antioxidant peptide antioxidin-RL from frog (Odorrana livida) skin
学位专业	动物学
中文摘要	能够有效清除来自于生存环境或者自身代谢产物的有毒活性物质对所有的生物机体来说都非常重要。对有氧呼吸生物来说，自由基特别是活性氧自由基如超氧阴离子自由基、过氧化氢自由基等的形成是需氧呼吸代谢过程中不可避免的副产物，正常生理状况下线粒体完成有氧呼吸的过程中，会有1-2%的氧气转化为超氧阴离子自由基，并由此产生一系列破坏性极强的氧化自由基如羟基自由基，通过脂质过氧化、蛋白质变性或者对遗传物质DNA的损伤对细胞或组织造成严重破坏。在漫长的进化过程中，生物机体演化出各种清除体内自由基的方式，主要分为两大类：第一类是非基因编码的代谢产物，如尿酸、维生素C、维生素E、胡萝卜烯类、谷胱甘肽、硫辛酸、辅酶Q等；第二类是基因编码的大分子抗氧化酶类，如超氧化物歧化酶、过氧化氢酶、过氧化物酶、硫氧还蛋白还原酶系统和谷胱甘肽酶系统等。 我们实验室以长日照、强紫外线环境生活的高原蛙类－滇蛙（Rana pleuraden）为研究对象，发现滇蛙皮肤具有极强的氧化自由基清除能力，并从其皮肤分泌液中发现了一系列由基因编码且可分泌表达的小分子抗氧化多肽，这种新型的抗氧化系统也被称为“第三套抗氧化系统”。此类抗氧化小肽通常由15-30个氨基酸组成，由基因编码产生，其前体约为60-70个氨基酸，前体被加工后释放成熟的抗氧化多肽于皮肤分泌液中，发挥抗氧化功能。 在本研究中，我们选取了以一种广泛分布于我国陕西、四川、云南、贵州、安徽、浙江、江西、湖南、福建、广东、广西、海南和香港等地的大绿臭蛙（Odorrana livida），用以研究其皮肤内的抗氧化小肽，并试图探究其抗氧化机理。 通过两步分离纯化过程，一步Sephadex G-50凝胶分子筛层析，一步C18反相高效液相色潽层析，通过ABTS自由基清除法追踪抗氧化活性，我们从大绿臭蛙皮肤分泌液中纯化得到了一种自由基清除能力很强的抗氧化小肽，命名为antioxidin-RL。Edman降解法测得其氨基酸序列为AMRLTYNRPCIYAT，ESI-MS质谱分子量分析测得其表观分子量为1672.02，ExPASy Compute Mw/pI tool (http://www.expasy.ch/tools/pi toll.html) 计算antioxidin-RL的理论等电点为9.31。 根据Edman降解法测定的氨基酸序列设计的正向简并引物及后续的特异性反向引物，我们从构建的大绿臭蛙皮肤cDNA文库中PCR扩增得到了编码antioxidin-RL前体的cDNA序列（GenBank accession number EU294118.1）。该序列由306个核苷酸组成，由此推测出来的antioxidin-RL前体由63个氨基酸组成，前体包括信号肽、成熟肽和间隔肽段，前体中存在双碱性氨基酸残基位点(-K42R43和K58R59-)，为丝氨酸类蛋白酶切割位点，用以切割释放成熟小肽。 通过对抗氧化小肽antioxidin-RL对超氧阴离子自由基、羟基自由基、DPPH自由基和ABTS自由基的清除实验结果分析发现，antioxidin-RL存在超氧阴离子自由基和羟基自由基清除能力，ABTS自由基清除方法是一种比较合适小肽抗氧化机理研究的实验方法；通过抗氧化小肽antioxidin-RL对金属离子螯合活性实验、还原力测定实验结果分析，发现antioxidin-RL存在很好的还原活性，但是不螯合金属离子；圆二色谱分析和核磁共振实验结果表明，抗氧化小肽antioxidin-RL及其突变体在水溶液环境下不具有规则的二级结构，提示antioxidin-RL不是通过酶作用的方式进行自由基的清除。 抗氧化小肽antioxidin-RL清除ABTS自由基的反应动力学表明，该抗氧化小肽有很强的ABTS自由基清除能力，且随着自由基浓度的增加清除能力急剧增加；pH值对抗氧化肽清除ABTS自由基的影响实验结果表明，抗氧化小肽在机体pH环境下活性最强，且提示小肽中唯一存在的半胱氨酸残基Cys10可能具有重要限速作用；巯基封闭实验表明，半胱氨酸残基被碘乙酰胺封闭后，小肽清除ABTS自由基的能力急剧下降，突变体实验进一步证明了Cys10是antioxidin-RL快速清除ABTS自由基的关键氨基酸。 通过分子筛凝胶Sephadex-G25的分离，我们成功获取了antioxidin-RL与ABTS形成的紫色络合物，抗氧化肽突变体实验的全波长扫描结果和核磁共振结果也证明了其存在。全波长扫描的结果和核磁共振结果还说明，Tyr6和Tyr12是形成紫色络合物的关键氨基酸；当半胱氨酸残基突变为Gly后，Tyr6和Tyr12的存在同样能赋予抗氧化小肽突变体ABTS自由基清除能力，但此时的抗氧化速度较慢。
英文摘要	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.
语种	中文
公开日期	2012-06-07
内容类型	学位论文
源URL	[http://159.226.149.42:8088/handle/152453/6966]
专题	昆明动物研究所_动物毒素室
推荐引用方式 GB/T 7714	刘存宝. 大绿臭蛙皮肤抗氧化肽antioxidin-RL的分离纯化及结构和功能研究[D]. 北京. 中国科学院研究生院. 2012.

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