【作者】 方茹；

【导师】 邹国林；

【作者基本信息】 武汉大学 ， 生物化学与分子生物学， 2004， 硕士

【摘要】 采用L-苯丙氨酸为探针,研究了用芳环羟基化法（aromatic hydroxylation）检测·OH的可行性。结果表明:使用液相色谱仪的荧光检测器（FLD）和荧光分光光度计的荧光光谱（FS）两种方法平行检测Cu²⁺-H₂O₂体系中产生的。试验采用的激发波长277nm,发射波长306nm。体系在反应前后的荧光变化,可反映·OH产生量。对FLD与FS所得数据进行了比较分析,结果显示两种方法具有较高一致性。由于FS使用混合体系检测,易对荧光的产生造成干扰,故此法虽然经济简便,但应用范围受到一定限制;而FLD法无上述干扰。同时使用HPLC—FLD检测了7种过渡金属离子参与类Fenton反应产生·OH的能力。实验结果表明Cr³⁺、Fe²⁺、Co²⁺、Cu²⁺、Pb²⁺产生·OH的能力较强,而Ni²⁺和Cd²⁺产生·OH的能力极弱。·OH清除剂脱铁敏（desferrioxamine,DFO）或甘露醇,能明显抑制产物L-Tyr的荧光强度变化,说明·OH的产生受到抑制,结果表明HPLC方法准确可靠。使用分光光度法对上述离子参与类Fenton反应产生·OH的实验进行了验证,得到了基本一致的结果,但芳环羟基化荧光法的结果更稳定,灵敏度更高,故此方法对于·OH检测以及抗·OH机理和应用研究等方面具有较高应用价值。 基于Mn²⁺和Mn³⁺是抑制还是促进·OH产生的争论,利用电子自旋共振法、芳环羟基化法和电泳法研究了Mn²⁺和Mn³⁺参与类Fenton反应的情况。结果表明Mn²⁺和Mn³⁺在离子浓度较高时抑制·OH的产生,而降低Mn²⁺和Mn³⁺的离子浓度,·OH的产生明显受到促进,·OH清除剂能明显抑制Mn²⁺更多还原

【Abstract】 In this study, RP-HPLC with fluorescence detection and Fluorescence spec trum（excitation 277nm and emission 306nm） was used to detect hydroxyl radic al generated by Cu（Ⅱ）-H₂O₂ （Fenton like reaction） system, using a fluorescenc e probe, L-phenylalanine, which could be converted to highly fluorescent L-tyr osine in the presence of -OH. Before and after reaction, the variety of fluores cence intensity reflected the generation of OH. The data of two methods wer e compared. By this, we can conclude that the result of two methods have qu ite coherence. Furthermore, the fluorescence spectrum is a more convenient m ethod. But the fluorescence intensitydetected by fluorescence spectrum reflectedall fluorescent components in the system.Other .OH generation system, such as Fe（Ⅱ）—H₂O₂ or phenanthroline- Cu（Ⅱ） coordinate—H₂O₂, influenced the a ccuracy of the result. So the method applied to detect OH by FS is confined.On the other hand, RP-HPLC with fluorescence detectionwas used to detect hydroxyl radical generated by Fenton like reaction systemparticipatated by seve n 3d-transition metal-ions,using a fluorescence probe,L-phenylalanine,which coul d be converted to highly fluorescent L-tyrosine in the generation of OH. the variety of fluorescence intensity reflected the generation of OH.The experiment al results indicated that ability of Cr（Ⅲ）,Fe（Ⅱ）,Co（Ⅱ）,Cu（Ⅱ）,Pb（ Ⅱ） to produce ? OH is relatively strong , but the ability of Cd（ Ⅱ） and Ni（ Ⅱ） is poor. The intensityof fluorescence in the systems could be inhibited obviously by mannite andde sferrioxamine（DFO）,which proved OH was cleard. We used the UV spectrome ter and gotthe same results. Aromatic hydroxylation was more effective and re liable. So this sensitive method can be applied to study the hydroxyl radical and hydroxyl radicalscavenger.Based on the dispute whether Mn（ II ）and Mn（III） inhibit or accelerate the production of OH, the article studied on the Fenton-like reaction which Mn（II） and Mn（III） participated in with the method of electron spin resonance,, aromatic hydroxylation and Agrose gel electropHoresis. Results indicated that Mn（II） and Mn（III） inhibit the production of -OH when the ionic concentration is higher , but accelerate obviously when it is lower.And the OH producted in the Fenton-like system of Mn（ II ）and Mn（III） was inhibited obviously by scavengerAt the same time the results indicated that Mn（II） inhibited the production of -OH in the Fenton system when the ionic concentration is higher and accelerate when it is lower. This research has made the preliminary explanation to the function mechanisms of Mn（ II） and Mn（III） participating in Fenton-like reaction,and it has provided experiment and theoretical basic about the dispute whether Mn（ II） and Mn（III） inhibit or accelerate the production of OH.更多还原