【作者】 宗万松；

【导师】 刘汝涛；

【作者基本信息】 山东大学 ， 环境工程， 2011， 博士

【摘要】 环境问题是当前最重要的全球性问题之一。环境中的各种污染物对生态环境造成破坏的同时,也严重威胁着人类的健康与生存。外源性环境污染物可以通过呼吸道吸入、由食物链经消化道摄入和皮肤接触等渠道进入生物体内,诱发机体发生环境氧化应激(氧化活性物质(ROS)的过量表达),造成DNA/RNA、蛋白质、多肽、脂类等生物分子的氧化损伤、现已证明环境氧化应激与诸多疾病有关,并且是引起疾病第二位的原因。因此,研究环境污染物诱发机体功能生物大分子的氧化损伤机理,建立其快速、灵敏评价方法有助于人们了解环境污染物的致病机理和全面评价污染物毒性,为相关疾病的早期诊断、预防和治疗提供科学依据。蛋白质是生物体各种功能的直接执行者。当其暴露于氧化应激的环境中时,ROS能够诱发机体蛋白质分子结构的氧化性损伤和功能的消弱／丧失,进而导致诸多疾病的发生。因此,研究环境污染物对蛋白质的氧化损伤作用一直是环境污染与健康领域的热门课题。本论文以分析化学、环境毒理学为背景,结合电化学、高效液相色谱、质谱等实验技术模拟研究了环境氧化应激诱发氨基酸、多肽、蛋白质的氧化损伤机理,并对氧化损伤的评价方法进行了探讨。论文主要包括以下四部分：论文第一章阐述了各类污染物诱发环境氧化应激的途径；归纳了环境氧化应激致氨基酸、多肽、蛋白质生物分子损伤的类型以及蛋白质氧化损伤与相关疾病的相关关系；介绍了蛋白质氧化损伤的分析与评价技术,蛋白质氧化损伤研究进展。在文献综述的基础上分析了目前研究中存在的问题,提出了基于电化学循环伏安、液相色谱-质谱联用技术在模拟研究环境氧化应激诱发氨基酸、多肽、蛋白质的氧化损伤机理的评价方法。论文第二章选择氨基酸、多肽、蛋白质作为电化学氧化损伤的靶分子,从分子水平上模拟研究其相应的界面氧化损伤机理,为机体蛋白质氧化损伤机理的深入研究提供了参考。1)利用电化学循环伏安技术模拟研究了胱氨酸、半胱氨酸在裸金电极上的氧化还原行为,探讨了半胱氨酸的界面氧化损伤机理。研究结果表明(以金电极表面氧化为例),正扫过程中半胱氨酸有两个特征氧化峰,0.65V附近的氧化峰对应于半胱氨酸氧化生成胱氨酸,0.85V附近的氧化峰对应于胱氨酸、半胱氨酸氧化生成CySOxH(x=2、3)；负扫过程半胱氨酸与多晶金形成Au-S键(该过程为失电子过程),还原峰电流明显降低且随半胱氨酸浓度增加而逐步下陷。在此基础上,研究了体系除氧、酸碱度、扫描速率、温度等因素对半胱氨酸在电极上发生电化学氧化损伤过程的影响。该结果对于全面认识氧化应激条件下含硫多肽、蛋白质在生物膜表面的氧化损伤机理及损伤修复提供了新的思路与方法。2)利用电化学循环伏安法模拟研究了还原型谷胱甘肽、氧化型谷胱甘肽在金电极上的界面氧化还原行为。相对位阻较小的半胱氨酸、胱氨酸在金电极表面氧化过程,空间位阻比较大的还原型谷胱甘肽并未发生两步的氧化过程,而是直接氧化生成GSOxH(难以生成氧化性的GSSG)。上述现象与普遍报道的GSH在环境氧化应激条件下生成GSSG的情况并不一致。这可能是由于GSH游离巯基两侧各自存在的一个氨基酸,使得巯基在电极表面的氧化过程存在明显的空间位阻作用。游离巯基之间的距离较大,无法形成二硫键(S-S),因而只能生成GSOxH。该过程一定程度上类似于生物体线粒体膜、细胞膜等部位上发生的生物功能分子的氧化损伤过程,因而对于生物体膜上氧化损伤的研究,乃至相关疾病的研究都具有重要的意义。3)利用电化学循环伏安技术研究了牛胰岛素在裸金电极上的氧化还原行为。通过与磷酸盐缓冲液、胱氨酸和胰岛素所含的其他氨基酸的电化学特征比较,胰岛素的电化学氧化损伤机理被详细阐明：胰岛素分子中的二硫键是其潜在的损伤位点并且其氧化产物为次磺酸(RSOH)、亚磺酸(RSO2H)以及磺酸(RSO3H)类化合物,但相对胱氨酸而言,由于空间位阻作用胰岛素需要更为严苛的条件才能实现氧化；由于胰岛素分子中二硫键S-SCYS6A,CYS11A和s-sCYS20A,CYS19B具有较小的溶剂接触面积而难以氧化。因此,该研究条件下胰岛素的主要氧化位点应为二硫键s-sCys7A-Cys7B。论文的第三章选择多肽、蛋白质氧化损伤生成的羟基、亚砜产物作为准确表述蛋白质(多肽)氧化损伤位点、损伤程度的生物标记物,并利用液相色谱—质谱联用技术及质谱碎片技术(LC-MS、LC-MS/MS)作为主要检测技术,建立其定性、定量研究的新方法。1)利用液相色谱-质谱(LC-MS)、二级质谱(MS/MS)等技术研究了UV/H2O诱发多肽氧化损伤位点、损伤程度及损伤机理,并探讨了以多肽氧化损伤位点作为氧化损伤标志物的可行性。研究证实UV/H2O(模拟环境氧化应激条件)对目标多肽中的FMRF存在明显的氧化损伤且损伤程度与时间正相关,并且LC-MS技术能够实现损伤产物与未损伤多肽的快速分离和准确鉴定。因此,以多肽氧化损伤产物作为直接评价氧化损伤的新型标记物具有潜在优势。本研究不仅拓展了多肽和蛋白质氧化损伤生物标记物的范围,也为阐明多肽等功能分子氧化损伤位点、相应位点的氧化损伤程度以及氧化损伤机理的研究提了供一种准确高效评价的新方法。2)利用高效液相色谱和串联质谱技术建立了以靶蛋白细胞色素C氧化损伤位点和损伤程度作为氧化损伤标志物评价其氧化损伤机理的新策略。液相色谱-质谱联用(LC/MS)和多肽指纹图谱鉴定(MS/MS)技术分别被用于胰蛋白酶解多肽的分离检测和UV/H2O氧化损伤位点的定位研究。结合LC/MS和MS/MS实验结果,可以确定酶解多肽C14AQC(heme)HTVEKK22、C11AQCHTVEK22、E60ETLMEYLENPKK73、M80IFAGIK86、M80IFAGIKK87中的Cys14、Cy17、Met65、Met80残基是主要的氧化损伤位点。上述位点氧化损伤程度与氧化时间的正相关性证实以蛋白质氧化损伤产物作为评价其氧化损伤机理的可行性。3)利用60Co-γ射线辐照牛血红细胞方式,模拟了环境氧化应激条件下过量表达的ROS对牛血红蛋白的氧化损伤作用,通过高效液相色谱和串联质谱技术建立了胞内血红蛋白氧化损伤位点和损伤程度的评价方法。通过比较损伤前后血红蛋白酶解的总离子色谱图及相应组分的MS/MS数据,发现靶分子的氧化损伤位点主要是部分暴露的氨基酸残基(例如α-Phe36、β-Met、β-Trp14)。氧化产物的定量分析表明,各损伤位点的氧化程度与氧化剂量正相关且受到氨基酸残基类型和暴露程度控制。与传统的蛋白羰基标记方法相比,本文所采用的方法为体外模拟条件下以及“体内”氧化应激实际存在时蛋白氧化损伤评价提供有效的技术支持。论文的第四章最后对本论文的各研究部分进行了总结,并分析了蛋白、多肽等靶分子氧化损伤评价方法的优势与不足之处,展望了该领域的发展方向。本研究丰富了环境污染物诱发蛋白、多肽等生物功能分子氧化损伤的评价方法,有助于人们从分子水平了解环境污染物的致病机理和全面评价污染物毒性,为相关疾病的早期诊断、预防和治疗提供了科学依据。更多还原

【Abstract】 Environmental pollution is still one of the most important global problems at present. The environmental contaminants not only create destructions to the world ecology, but also threaten human survival and health seriously. For these contaminants can migrate to organisms through respiratory tract inhalation, digestive tract intake and skin contact, and then induced oxidative stress (forming excess reactive oxygen species, ROS). Under oxidative stress, ROS can destroy the structures of DNA, RNA, proteins, and lipids, hinder their featured functions, subsequently lead to a series of diseases. Therefore, study on the oxidation mechanisms of functional biological macromolecules is very essential for the prevention and treatment of related diseases and the development of related drugs.Proteins are essential parts of organisms and participate in virtually every process (signaling, immune responses, cell cycle, and so on) within life phenomenon. When exposed to environmental oxidative stress conditions, the excessively produced ROS can destroy the integrated structures of proteins, and thus hindrance their diverse physiological functions. Overwhelming evidence indicates that oxidative modification of proteins by reactive oxygen species plays a key role in a number of physiological disorders and diseases. Thus, study on the oxidation mechanisms of proteins has been a hot topic in field of environmental pollution and healthy.In the research, we studied the oxidation mechanisms of partial typical amino acids, peptides, proteins in molecule level by the methods of electrochemical technique, high performance liquid chromatography and mass spectrometry, and the advantages of these methods was discussed.This study has four chapters, just as follows:In chapter one, we describe the pathway of oxidative stress induced by the various types of environmental pollutants, the damage types of amino acids, peptides, proteins induced by oxidative stress, and the relationships between protein oxidation and related disease. We also introduce the experiment techniques and the related research for the evaluation of protein oxidation. Based on literature review, we proposed the the researching aims, meanings, methods and content of our work.In chapter two, amino acids, peptides, proteins were selected as the target molecules of electrochemical oxidation and the corresponding interface oxidation mechanisms of these molecules were clarified at the molecular level.1)A cyclic voltammetry assay was developed for the redox process of cysteine side chain on gold electrode and the oxidative damage mechanisms of cysteine were also proposed. It is demonstrated from the cyclic voltammograms that cysteine has two characteristic oxidative peaks (positive scan). The oxidative peak close-by 0.65V owes to the direct oxidation of -SH in cysteine, forming cystine and the oxidative peak close-by 0.95V owes to the oxidation of cystine and cysteine, forming sulfonic acid and sulfinic acid. The current of reductive peak falls evidently and a current valley expanded with the increase of cysteine (negative scan). The explanation is that cysteine can react with multi-crystal gold, forming Au-S bond and this is an electro-losing process. The influence of soluble oxygen, pH, scan rate, temperature and concentration of cysteine to the oxidative damage of cysteine was also performed. These results will provide a new visual angle for researching on oxidative damage mechanisms of sulfur-containing peptides with proteins and for the control of oxidative damage.2) In the present work, the unusual oxidation process of GSH on an Au electrode was probed by cyclic voltammetry (CV) technique. Voltammetric studies showed that -SH in GSH was the unique target of electro-oxidation by excluding the other potential oxidation sites (amido group, carboxyl group and side-chains of glutamic acid and glycine) and the feeble influence of S-Au interaction. As a result of spatial baffle, GSH was directly oxidized to GSOxH (x=2,3) without forming the intermediate of glutathione disulfide (GSSG). The unusual oxidation process differs from the two-step oxidation processes of cysteine-SH on the Au electrode and the oxidation of -SH in dissolved GSH, but is similar to the biological oxidation of GSH in vivo on biomembranes, where the steric hindrance still exists.3) By using the technique of cyclic voltammetry (CV), we simulated and investigated the oxidative damage of bovine insulin on Au electrode. The experimental results show that there are two anodic peaks for the oxidative damage of bovine insulin, which arise from the oxidation of the exposed disulfide bond, forming sulfenic acid RSOH (1.20V, vs. SCE), sulfinic acid RSO2H and sulfonic acid RSO3H (1.35V, vs. SCE). But due to steric hindrance, the oxidative damage to insulin requires more stringent conditions than that of cystine (free disulfide). Bovine insulin has three disulfide bonds (S-SCYS7A-CYS7B, S-SCYS6A-CYS11A and S-SCYS20A-CYS19B), implying three candidates that can be oxidized in electrochemical processes. S-SCYS6A-CYS11A or S-SCYS20A-CYS19B has small solvent accesible surface areas and can not be oxidized. Thus the damage site within insulin is only S-SCYS7A-CYS7B. These in vitro findings not only demonstrate the applicability of CV in simulating/evaluating the oxidative damage of non-redox proteins but also find two promising candidates (two anodic peaks) for measuring insulin.Biomarkers held both incredible application and significant challenge in probing the oxidation mechanisms of proteins under oxidative stress. In chapter 3, mass spectrometry (MS) coupled with liquid chromatography (LC) was applied to establish a new pipeline to probe the oxidation sites and degrees of peptides and proteins with their oxidative products serving as the biomarkers.1) By using the technique of liquid chromatography and mass spectrometry, we established a new method for evaluating the oxidation site and degree of oxidized peptide, with its oxidative product serving as biomarker. In the three model peptides, peptide FMRF (containing a methionine) was prone to undergoing oxygen addition under UV/H2O2 oxidization, forming a sulfoxide (FM(O)RF) with a stable chromatographic peak separate from the model peptides. The oxidation content of FMRF, expressed as SFM(O)RF/(SFM(O)RF+SFMRF), is positively correlated with oxidation time. Based on sequence analysis of FM(O)RF, the oxidation mechanism (site and extent) of FMRF under UV/H2O2 oxidization was explicitly clarified. By comparing the specific injury to each model peptide, we found that the oxidative products of Met-containing peptides are good biomarkers for OS. This research not only expands the range of biomarkers for OS, but also provides an efficient and accurate method for evaluating oxidation damage to peptides and even proteins.2) Mass spectrometry (MS) coupled with liquid chromatography (LC) was applied to establish a new pipeline to probe the oxidation sites and degrees of horse cytochrome c (HCC) with its oxidative products serving as the biomarkers. Samples of native and UV/H2O2 oxidized HCCs were digested by trypsin and subjected to biomarker discovery using LC/MS and tandem mass spectrometry (MS/MS). Experiment results proved that the main oxidation sites were located at Cys14, Cys17, Met65 and Met80, residues in peptides C14AQC(heme)HTVEK22. C14AQCHTVEK2. E60TLMEYLENPKK73, M80IFAGIK86 and M80FAGIKK87. Quantitation analysis on the oxidized peptides showed the oxidation degrees of target sites had positive correlations with extended oxidation dose and were controlled by residues types and their accessibility to solvent molecules. Being able to provide plentiful information for the oxidation sites and oxidation degrees, the identified oxidized products were feasibility biomarkers for HCC oxidation, compared with the conventional protein carbonyl assay.3) In the work present here, a novel pipeline was established to probe the oxidation mechanisms of bovine hemoglobin (Hb) with its oxidation products served as the biomarkers. Reactive oxygen species generated by 60Coγ-ray source were used as a mimical oxidative stress condition to oxidize Hb in bovine erythrocytes. After Hb extraction and digestion, the oxidized peptides in the tryptic fragments were assigned upon comparison with the total ion chromatography from the control digest. Subsequent tandem mass spectrometry analysis of these peptides proved that oxidations were limited to partial exposed amino acid residues (α-Phe36,β-Met1,β-Trp14, for instance) in Hb. Quantitation analysis on the oxidized peptides showed the oxidation degrees of target sites had positive correlations with the extended oxidation dose and the oxidation processes were controlled by residues types. Compared with the conventional protein carbonyl assay, the identified oxidized products were feasibility biomarkers for Hb oxidation, indicating the proposed biomarker pipeline was able to provide specific and valid information for protein oxidation.Finally, we summarized the research findings of above parts and discussed the future development of the evaluation methods for peptide, protein, and other target molecules. This study has enriched the research on protein oxidation, and provided some reference gists for the toxicities and pathogenesis of environmental pollutantst.更多还原