The Study On The Oxidative Damage Assessment Of Four Amino Acids In Different Environmental Conditions

In normal physiological conditions, reactive oxygen species (ROS) like hydrogen peroxide and hydroxyl radical could be generated in the course of biological metabolism. If a large amount of ROS exist in vivo or the antioxidant function of organism decreased, the oxidation system and antioxidation system would be out of balance and oxidative stress would be caused. ROS can react directly or indirectly with amino acid thus change the structure or cause oxidative damage of protein, influence the functional expression of protein and cause a series of diseases. So the study on the biomarker of protein damage caused by oxidation stress and research on the examination method are helpful to investigate the mechanism of protein oxidative damage which could be used in the prevention and treatment of related diseases and the development of related drugs.Electrochemical technique is used in the study of tyrosine electrochemical behavior on electrode and high performance liquid chromatography-tandem mass spectrometry technique is used in the study of oxidative damage degree and sites of angiotensin and myoglobin under the effect of hydroxyl radical. It studied that the oxidation of tyrosine, phenylalanine, methionine and isoleucine in different surrounding conditions. The assessment methods of the oxidative damage of the four amino acid are also established. The dissertation consists of the following three parts.In the first part, it was studied that the electrochemical behavior of tyrosine on glassy carbon electrode utilizing electrochemical cyclic voltammetry. The results showed that there appeared an anode peak on the cyclic voltammetry spectra of tyrosine without a cathode peak. The peak potential located near 0.9V. So it was considered that the electrochemical oxidative damage reaction of tyrosine is irreversible. It was also studied that the effect factor of tyrosine electrochemical behavior on glassy carbon electrode in this part. From the deoxygenation experiment result, dissolved oxygen has obvious effect. With vacuum deaeration, the potential of the oxidative peak shifts negatively and the current increases slightly. With scanning times increased, the current of oxidative peak decreases gradually, so it was obtained that weak adsorption of electroactive plasmid was existed. The current of oxidative peak and v1/2 have a linear relationship in which R=0.9982. The oxidative peaks potential shift negatively and the current decrease with p H increase. When the temperature increases, the oxidative peaks shift in the negative direction and their current gradually increase. The concentration of tyrosine has little effect on the oxidative peak potential. But with the concentration increases, the current shows increasing trend.In the second part, it was studied that the oxidative damage degree and sites of AngⅠ,ⅡandⅢin the reaction with hydroxyl radicals which generated from Fenton reaction utilizing liquid chromatography-mass spectrometry technology. The results proved that the oxidative damage degree increased with longer reaction time. The Ang I oxidative damage degree are 0,1.73,1.93,4.29,4.88 (%) respectively corresponding to the 0,2,4,8,16min reaction time. The AngⅡare 0,2.16,2,17,3.03,5.42 (%) and AngⅢare 0,1.45,1.82,3.33,5.52 (%) respectively. Tyrosine and phenylalanine are all oxidized by hydroxyl radical in three angiotensins. The specific oxidation product could be used as biomarker of angiotensin oxidative damage by ROS.In the third part, the oxidative damage degree and sites of myoglobin caused byγ-ray irradiation was studied with liquid chromatography-mass spectrometry technology. The experimental results showed that the oxidative damage degree was going to a deeper level with the increase of irradiation doses. Choose the enzymolysis product of myglobin V17EADIAGHGQEVLIR31 for example, its oxidative damage degree are 0%,3.8%,8.3%,13.4%,20.9% respectively corresponding to the 0,10, 20,40, 100Gyγirradiation doses. From the MS/MS results, it was obtained that methionine and isoleucine were oxidized in myoglobin. The oxidative product could be used as biomarker of myoglobin oxidative damage.