New Methods For The Evaluation Of Protein (Peptide) Oxidation Induced By Environmental Oxidative Stress

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 60Co?-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 (?-Phe36,?-Met1,?-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.