电化学法测定超氧化歧化酶活性的研究

【作者】 李玲霞；

【导师】 邹洪；

【作者基本信息】 首都师范大学 ， 分析化学， 2005， 硕士

【摘要】 在生命活动的代谢过程中不断产生各种氧自由基,超氧阴离子自由基（O₂^-）和羟基自由基（HO-）是具有代表性的重要的活性氧自由基,它们与生物体的衰老、心脑血管病、风湿性关节炎、癌症等许多病理都有重要的关系。生物体中一些具有抗氧化活性的酶可以控制生物体中自由基的量,如超氧化歧化酶（SOD）和过氧化物酶（PO）。众所周知一些非酶活性的物质也具有抗氧化活性,如维生素、中草药和黄酮类化合物等。近年来,从生物体中清除过量的自由基,以及寻找一些能清除自由基的抗氧化剂倍受重视,因此,SOD活性测定和抗氧化剂清除自由基的能力的研究显得尤为重要。本文建立了快速简便测定SOD活性的四种方法,论文包括以下四部分: 1、介绍了活性氧自由基与疾病、超氧化歧化酶和超氧阴离子自由基之间的关系。综述了近年来国内外超氧化歧化酶活性测定及中草药清除活性氧自由基的发展和应用。 2、在Na₂CO₃-NaHCO₃（pH8.9）缓冲溶液中,SDS对钯（Ⅱ）与8-羟基喹啉-5-磺酸（8-OXSA）络合物峰电流有明显的增敏作用,其反应机理与电极表面生成的O₂^-有关。由于超氧化歧化酶（SOD）可催化O₂^-发生歧化反应,使钯-8-OXSA-SDS体系的峰电流降低,本文依此建立了测定SOD活性的方法,并研究了几种常见的黄酮类化合物清除O₂^-的作用。 3、盐酸羟胺在碱性水溶液中发生自氧化反应,可产生O₂^-,自身被氧化为亚硝酸根离子,在酸性介质中SCN^-催化亚硝酸根离子与二苯胺或二苯胺-4-磺酸反应生成具有电活性的亚硝基化合物,在-0.67V和-0.60V处有一波形很好的二阶极谱峰。在一定条件下,极谱峰电流的大小与亚硝酸根离子的浓度呈线性关系。SOD可使O₂^-发生歧化反应,体系中加入SOD,使生成的亚硝酸根离子的量减小,由此建立了通过检测亚硝酸根离子的生成量来测定超氧化歧化酶活性的电化学方法。测定了酶活力曲线,并研究了常见的黄酮类化合物对O₂^-的清除作用。 4、邻苯三酚在碱性条件下,可以发生自氧化反应,产生O₂^-,其自氧化产物在-0.96V和-1.44V（SCE）有两个还原波,用极谱法研究了邻苯三酚在更多还原

【Abstract】 There is no doubt that oxygen species occur in biological systems. Superoxide anion radical（O2 ） and hydroxyl radical （HO） are typical ones.They are presumed to play an important role in a number of biological phenomena such as aging, oronary heart disease rheumatoid arthritis, cancer and other pathological conditions. These free radical are controlled in biological systems by some enzymes possessing antioxidant activities such as superoxide dismutase（SOD） and peroxidase （PO）. It is well known that non-enzymatic substances such as vitamins, Chinese Traditional and Herbal Drugs （CTHD） and flavonoids, also scavenge free radical. Recently, the importance of removing excessive active oxygen species from living organisms is becoming increasing recognized, together with a growing interest in finding antioxidants that can scavenge the free radicals. Hence the determination of SOD activity and the study of scavenging activity of the antioxidants are necessary. Here we present four simple and rapid methods for the determination of SOD activity. This thesis research includes four parts and the main results could be expressed as follows:1. The relation between active oxygen radical and illness, superoxide dismutase and superoxide anion radical were introduced. The recent advance of determination of SOD activity and scavenging active oxygen radical by CTHD in the world were summarized.2. In the buffer solution of Na₂CO₃-NaHCO₃（pH 8.9）, the current of the palladium（ II） -8-hydroxyquinoline-5-sulfonic acid （8-OXSA） system with an anionic surfactant , sodium dodecyl sulfate（SDS） was increased markedly compared to that obtained without SDS. The reaction mechanism was related to the formation of superoxide anions radical （O₂ ）. Polaragraphic determination of superoxide dismutase activity and the study of scavenging of superoxide anions by flavonoids was described which is based on that superoxide dismutase（SOD） catalyzed the dismutatiom of superoxide radical and reduced the peak current of the 8-OXSA-SDS system .3. In an alkaline medium , hydroxylammoniumchloride reacted with oxygen to form superoxide radical and was oxided to nitrite ion. At low pH with thiocyanate as catalyst, nitrite ion reacted rapidly and quantitatively with diphenylamine or diphenylamine-4-sulfonic acid toform nitrate complex with electrochemical activity, which gave sensitive second-order derivative reduction wave with the peak potentials of -0.67V and -0.60V（SCE） . Under the certain conditions the peak current was directly proportional to the concentration of nitrite ion. Superoxide dismutase enzyme catalyzed the dismutation of superoxide radical（O₂ ）into H₂O₂ and O₂,The system with SOD inhibited the formation of nitrite ion. The electrochemical method for determination of SOD activity which was based on the detection of the formation of nitrite ion during autoxidation of hydroxylammoniumchloride was described. The curve of the SOD activity was detected. The method has been applied into the research of scavenging activity of some flavonoids.4. In a weakly alkaline medium , pyrogallol reacted with oxygen to form superoxide radical and several intermediate products which gave two waves and the peak potentials were - 0. 96V and -1.44V （SCE） . In Tris-HCl （pH8.30） buffer solution, the mechanism of the autoxidation of pyrogallol and the optimum experimental conditions were investigated. Superoxide dismutase enzyme catalyzed the dismutation reaction of superoxide radical so that dicreased the ratio of pyrogallol autoxidation, which could determinate superoside dismutase enzyme activity by detect the chang of the ratio of the pyrogallol autoxidation in with different SOD .A simple and sensitive electrochemical method was discribed. The method has been applied into the research of scanvenging activity of some flavonoids .The results indicated that the sensitivity of determination of SOD activity in -0.96V was better than in -1.44V.The results of four methods were summarized .The mechanism of scavenging active oxygen radicals by flavonoids , th更多还原