【作者】 徐芹；

【导师】 张志超；

【作者基本信息】 大连理工大学 ， 生物化工， 2007， 硕士

【摘要】 利用¹³C核磁共振波谱及圆二色光谱，研究了L-半胱氨酸（Cys）和还原型谷胱甘肽（GSH）与Hg²⁺的结合模式以及它们之间的区别。¹³C核磁共振波谱和圆二色光谱的结果同时显示Hg²⁺∶Cys（GSH）在1∶2到2∶3之间时，Cys和GSH中的巯基都继续参与结合。而且，在这两个比例之间GSH中除了巯基参与结合外，谷氨酸残基也参与了结合。奇怪的是，尽管在与Hg²⁺结合时GSH比Cys的结合位点多，Hg-Cys却比Hg-GSH具有更强的稳定性。另外，圆二色光谱的结果显示，对于Hg-Cys和Hg-GSH，汞-巯基金属簇都是在1∶5到1∶2 Hg²⁺∶Cys（GSH）之间形成的，其主要的存在形式均为1∶2 Hg²⁺∶Cys（GSH）。将原创性的汞离子荧光分子探针EPNP进一步发展应用到细胞生物学、动物学和神经毒理学领域。结合倒置荧光显微镜，在新生小鼠神经元细胞和神经组织内，均首次实现了小于μM水平上，高选择性、可视化地研究其内汞离子的蓄积和分布。研究中，我们找到了截止到目前的神经系统汞中毒的最小计量和最短的染毒时间，分别为0.1μg／g体重、3天；无论是对细胞的观察还是对组织的研究，实验结果均明确表明在我们所涉及的注射剂量和注射时间范围内汞首先进入的是大脑海马的神经元，在脊髓神经元细胞中始终没有发现汞离子的存在；研究结果还发现，新生小鼠同成年鼠体内汞离子的储存和运输可能存在较大差异，究其原因很有可能是新生小鼠的血脑屏障发育不成熟，不能像成年鼠那样有效地抵御有毒Hg²⁺的入侵。更多还原

【Abstract】 The binding modes of Hg²⁺ by Glutathione （GSH） and L-cysteine （Cys） have been studied and the difference between them was investigated by means of carbon-13 magnetic resonance and Circular dichroism spectra. The ¹³C NMR and CD spectra all implied that the binding sites at the molar ratio of 1:2 to 2:3 Hg²⁺:Cys （GSH）—the sulfhydryl groups of Cys and GSH were still bound to Hg²⁺; in this span, not only the sulfhydryl group binds to Hg²⁺, but also the glutamyl end was involved in the binding of GSH to Hg²⁺. Surprisingly, the binding capacity of Hg²⁺ with Cys was powerful than that of GSH although more binding sites were available for GSH. In addition, the CD spectral envelope illustrated that the only one metal cluster was formed at Hg:GSH and Hg:Cys molar ratios of 1:5-1:2, and the 1:2 complex was the main species both for GSH-Hg²⁺ and Cys-Hg²⁺.The original Hg²⁺ fluorescence sensor EPNP, is developed to apply on cell biology, zoology and neurotoxicology. By fluorescent microscope, the study of mercuric ion in neurons and nerval tissue is high selectively and visibly achieved for the first time. So far, we have found the lowest injected dose and the shortest time that could be seen in the neurons and nerval tissue, that is 0.1μg/g body weight and three days. By contrast, no Hg²⁺ was observed in spinal cord neurons and spinal tissue sections. The differences of the mercuric ion between the neonatal mice and the adult mice may be resulted by the immature blood-brain barrier of the neotatal mice, which are different from those of the adult, render the brain of neonatal highly susceptible to insults from exposure to toxic metals.更多还原