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辣根过氧化物酶与一氧化氮作用机理及其在一氧化氮测定中的应用

Binding Mechanism of Horseradish Peroxidase to Nitric Oxide and Its Application to Nitric Oxide Determination

作者: 专业:分析化学 导师:周杰 年度:2010 学位:硕士  院校: 山东农业大学

Keywords

Nitric oxide, Horseradish peroxidase, Binding constant, Non-competitive inhibition, Second-order derivative spectrometry

        本文利用紫外吸收光谱法研究了一氧化氮(NO)与辣根过氧化物酶(HRP)的结合,并在此基础上通过二阶导数光谱法建立了一种检测NO的新方法。在无氧的0.2mol/L pH 7.4磷酸盐缓冲溶液中,用紫外分光光度计法研究了HRP与NO的结合。根据HRP与NO结合平衡方程,推导出一种评定结合常数的模型方程,并计算出结合常数为(1.55±0.06)×104L/mol,说明在无氧条件下,HRP与NO能形成稳定的复合物。在H2O2和NO存在下,根据HRP催化氧化愈创木酚的初始反应速率证明了NO对HRP的抑制类型,由Lineweaver-Burk法确定为明显的非竞争性抑制并建立了抑制机理。由动力学机理求得结合常数为(5.22±0.06)×104L/mol。两种方法求得的结合常数几乎相等,进一步说明了非抑制竞争机理是合理的。这种由乒乓机理催化双底物的非竞争抑制模型也同样适合NO对其它金属酶相互作用的研究。根据HRP与NO之间的反应,用二阶导数光谱法建立了一种检测NO的新方法。探讨了HRP浓度和pH值对NO-HRP体系的影响,优化了实验条件。在最佳实验条件下,研究了共存物质如NO2-,NO3-,H2O2对NO浓度检测的影响。建立了标准曲线的回归方程:h=8.89×10-2CNO–1.56×10-3,其NO检测的线性浓度范围为0.085-1.3μM,相关系数r=0.9966(n=5)。此方法的相对标准偏差均小于3%,检出限为0.032μM(S/N=3)。该方法成功地应用于检测血清样品中的NO的浓度。
    In this paper, binding of nitric oxide (NO) to horseradish peroxidase (HRP) has been investigated by UV absorption spectrometry, and based on the reaction of HRP with NO, a novel detection method of NO has been developed with second-order derivative spectrometry.Binding of nitric oxide to HRP has been investigated by UV absorption spectrometry in 0.2mol/L anaerobic phosphate buffer solution (pH 7.4). Based on this binding equilibrium, a model equation for evaluating the binding constant of NO to HRP is developed and the binding constant is calculated to be (1.55±0.06)×104L/mol, indicating that HRP can form a stable complex with nitric oxide. The type of inhibition by NO is validated on the basis of studying initial reaction rates of HRP-catalyzed oxidation of guaiacol in the presence of hydrogen peroxide and nitric oxide. The inhibition mechanism is found to follow an apparent non-competitive inhibition by Lineweaver–Burk method. Based on this kinetic mechanism, the binding constant is also calculated to be (5.22±0.06)×104L/mol. The values of the binding constant determined by the two methods are almost identical. This further demonstrated that the non-competitive inhibition mechanism suggested in the study was reasonable. The non-competitive inhibition model is also applicable to studying the effect of NO on other metalloenzymes, which catalyze the two-substrate reaction with the“ping-pong”mechanism.Based on the reaction of HRP with nitric oxide, a novel detection method of NO has been developed with second-order derivative spectrometry in an anaerobic phosphate buffer solution. The effects of pH and HRP concentration on the determination of NO in HRP system were investigated, and the conditions for the measurements were optimized. Some possible coexisting substances, such as nitrite, nitrate and hydrogen peroxide, were tested. The linear regression equation of standard curve was found to be h=8.89×10-2CNO–1.56×10-3 with the relevant coefficient of 0.9966 (n=5) in the NO concentration range of 0.085-1.3μM. The relative standard deviations were less than 3%. Based on the standard deviation of 5 bank measurements and a signal-to-noise ratio of 3, the detection limit for NO was 0.032μM. The proposed method was successfully applied to the determination of nitric oxide levels in serum samples.
        

辣根过氧化物酶与一氧化氮作用机理及其在一氧化氮测定中的应用

中文摘要9-10
ABSTRACT10
1 引言12-39
    1.1 NO 的性质12-13
        1.1.1 NO 的物理性质12-13
        1.1.2 NO 的化学性质13
    1.2 NO 在生物体中的研究13-24
        1.2.1 NO 在生物体中的主要反应13-18
            1.2.1.1 与氧气的反应14-15
            1.2.1.2 与超氧离子的反应15
            1.2.1.3 与金属离子的反应15-16
            1.2.1.4 与硫醇的反应16-17
            1.2.1.5 与自由基的耦合反应17
            1.2.1.6 与生物分子的间接反应17-18
        1.2.2 NO 在动物体中的研究18-22
            1.2.2.1 动物体中NO 的产生途径18-19
            1.2.2.2 NO 在动物体中的作用19-22
        1.2.3 NO 在植物体中的研究22-24
            1.2.3.1 植物中NO 的产生途径22-23
            1.2.3.2 NO 在植物体中的作用23-24
    1.3 溶液中NO 的检测24-33
        1.3.1 液相中NO 的间接测定25-27
            1.3.1.1 Griess 法(重氮化反应法)25
            1.3.1.2 高铁血红蛋白法25-26
            1.3.1.3 化学发光法26
            1.3.1.4 荧光分光光度法26-27
            1.3.1.5 电子自旋共振波谱法(ESR、EPR)27
        1.3.2 液相中NO 的直接测定27-29
            1.3.2.1 电化学还原法28
            1.3.2.2 电化学氧化法28-29
        1.3.3 NO 传感器29-33
            1.3.3.1 NO 电化学传感器(NO 电极)29-31
            1.3.3.2 NO 光化学传感器31-33
    1.4 辣根过氧化物酶33-37
        1.4.1 HRP 的结构34-35
        1.4.2 HRP 的作用机制35-37
            1.4.2.1 作用机制36
            1.4.2.2 芳香族底物的结合位点36-37
            1.4.2.3 国内外关于HRP 与NO 的研究37
    1.5 论文研究内容及意义37-39
2 材料与方法39-44
    2.1 仪器与试剂39-40
    2.2 实验方法40-44
        2.2.1 NO 与HRP 作用机理的研究40-42
            2.2.1.1 NO 对HRP 紫外吸收光谱的影响40-41
            2.2.1.2 不同浓度NO 对HRP 紫外吸收光谱的影响的测定41
            2.2.1.3 H_20_2 对HRP-NO 体系紫外吸收光谱的影响41
            2.2.1.4 愈创木酚与NO、H_20_2 的反应41
            2.2.1.5 动力学测定41-42
        2.2.2 建立二阶导数光谱法检测NO42-44
            2.2.2.1 二阶导数光谱的与一般的紫外光谱的比较42
            2.2.2.2 HRP 浓度对二阶导数光谱中h 值的影响42
            2.2.2.3 pH 值对二阶导数光谱中h 值的影响42
            2.2.2.4 建立标准曲线42-43
            2.2.2.5 干扰因素N0_2~-,N0_3~-,H_20_2 对二阶导数光谱中h 值的影响43
            2.2.2.6 测定血清中NO 的浓度43-44
3 结果与讨论44-58
    3.1 NO 与HRP 作用机理的研究44-53
        3.1.1 紫外光谱分析44-45
        3.1.2 NO 与HRP 的结合常数45-47
        3.1.3 NO 对HRP 活性的竞争抑制机理47-53
    3.2 建立二阶导数光谱法检测NO53-58
        3.2.1 紫外吸收光谱与二阶导数光谱进行光谱分析53-54
        3.2.2 HRP 浓度的影响54-55
        3.2.3 pH 值的影响55
        3.2.4 标准曲线的建立55-56
        3.2.5 干扰试验56-57
        3.2.6 方法的应用57-58
4 结论58-59
    4.1 NO 与HRP 作用机理的研究58
    4.2 建立二阶导数光谱法检测NO58-59
参考文献59-70
致谢70-71
攻读学位期间发表的学术论文目录71
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