新型单胺氧化酶荧光探针的研究

发布时间：2017-12-27 21:50

本文关键词：新型单胺氧化酶荧光探针的研究　出处：《浙江工业大学》2009年硕士论文　论文类型：学位论文

【摘要】： 单胺氧化酶(MAO)是一种膜结合线粒体酶,在多巴胺氧化代谢中起重要作用。根据其对底物或抑制剂结合的特异性、细胞分布、免疫特异性等特性分为A和B两种形式。它与人体的很多精神性疾病以及健康有着密切的关系,譬如帕金森症、阿尔之海默症、精神分裂症和攻击性行为及癌症等。因此,MAO作为新药物的靶标越来越受到广泛的关注和研究。目前有多种方法检测单胺氧化酶的活性:分光光度法、放射性法、高效液相法以及荧光探针法等。分光光度法、放射性法、高效液相法等都不适合高通量的筛选;而荧光探针具有很高的灵敏度和专一性,并且样品不需要复杂的提纯,因此荧光探针法被广泛应用于生物医学和生物学。本论文首先根据单胺氧化酶的反应机理成功设计和合成了一个探针:4-甲基-7-(3-氨基丙基)-香豆素。该探针并无荧光,但经与单胺氧化酶作用,3-氨基丙基转化为醛,接着发生消除反应后,生成了具有强烈荧光性能的4-甲基-7-羟基香豆素。因此通过检测荧光的变化就可检测MAO的活性。接着利用从人胎盘和牛肝中提取的MAOA和MAOB粗酶,系统的研究了探针的检测条件:2h,温度为37℃,硼酸缓冲液pH=8.4,最后对他们的动力学常数进行了测定。结果为MAOA:K_m=16.04±0.05μmol/L,V_max=0.72±0.01nmol/min/mg;MAOB:K_m=12.25±0.05μmol/L,V_max,=2.57±0.02nmol/min/mg。最后,本论文对探针进行了简单的应用,包括酶提取和人血清的检测。总之,本论文所设计的探针是非常高效和灵敏的,同时它对MAOA和MAOB有选择性。
[Abstract]:Monoamine oxidase (MAO) is a membrane binding mitochondrial enzyme that plays an important role in the oxidative metabolism of dopamine. It is divided into two forms, A and B, according to the specificity, cell distribution, and immunological specificity of the combination of substrates or inhibitors. It is closely related to many mental disorders and health of human body, such as Parkinson's disease, Alzheimer's disease, schizophrenia and aggressive behavior and cancer. Therefore, as the target of new drugs, MAO has attracted more and more attention and research. There are many methods to detect the activity of monoamine oxidase, such as spectrophotometry, radioactivity, high performance liquid phase and fluorescence probe. Spectrophotometry, radioactivity and high performance liquid chromatography are not suitable for high-throughput screening. However, fluorescent probes are highly sensitive and specific, and samples do not need complex purification. Therefore, fluorescence probe method is widely applied in biomedicine and biology.
【学位授予单位】：浙江工业大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R341

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