光诱导的肌红蛋白去氧及人血清白蛋白的光谱学研究

发布时间：2018-03-20 06:11

本文选题：肌红蛋白　切入点：人血清白蛋白　出处：《大连大学》2007年硕士论文　论文类型：学位论文

【摘要】： 本论文首次用我们建立的荧光光谱法研究了光诱导下Mb(WT)和Mb(D44K)的去氧过程,比较了它们在光诱导的去氧等方面的不同;又用紫外、荧光和圆二色光谱学方法研究了秋水仙碱与人血清白蛋白之间的相互作用。结果发现: 1.荧光光谱法在研究光诱导的Mb去氧方面,比之拉曼、紫外等光谱法具有灵敏、方便、峰单一容易识别不被其它峰干扰等优点。含氧Mb在光照下发生去氧作用,光照量越大,去氧量越大;光照下的温度越高,去氧越快;N2可以把Mb活性中心铁卟啉中的配位氧脱下来,因而可用荧光光谱法作含氧Mb与CO、CO2、H2、Cl2、NO、CS2等各种气体相互作用的去氧研究。此外,荧光光谱法还可用作Mb中色氨酸和酪氨酸残基与铁卟啉之间的传能研究。 2. Mb(D44K)的去氧速率比Mb(WT)慢,更加难于去氧。430nm激发时,Mb(D44K)在597.9nm和628.8nm处出现两个荧光发射峰,不同于Mb(WT)仅在597nm处出现一个荧光发射峰。经研究证明628.8nm处荧光峰是Mb3+-H2O型中的H2O峰。光照也使此峰的荧光强度下降,但比去氧的速率慢。进一步研究发现,597nm处Mb(D44K)的荧光效率比Mb(WT)的荧光效率低。传能实验表明Mb表面44位氨基酸由Asp突变为Lys后,不影响Mb(D44K)中色氨酸和酪氨酸残基传递给铁卟啉的荧光效率,但使Mb(D44K)中色氨酸和酪氨酸残基的荧光效率变高。430nm是研究Mb(WT)和Mb(D44K)光照去氧的最佳激发波长。 3.秋水仙碱使人血清白蛋白的紫外吸收增强,特征荧光峰猝灭,并且随温度升高猝灭常数Ksv降低。求算了不同温度下秋水仙碱与人血清白蛋白相互作用的平衡常数与结合位点数。根据Van′t Hoff方程计算出?H = -11.66 KJ/mol,?S= 51.507J/mol·K,得出二者之间的作用力主要是静电作用力。圆二色光谱测得加入秋水仙碱后,人血清白蛋白的α-螺旋降低,二级结构改变。表明秋水仙碱对人血清白蛋白的荧光猝灭机制属于形成配合物所引起的静态猝灭。
[Abstract]:In this paper, we first studied the deoxygenation process of MbCWTand MbD44K) by using the fluorescence spectrometric method established by us for the first time, and compared their differences in photoinduced deoxygenation, and used UV, UV, UV, UV, UV, UV, UV, UV, UV, UV, UV, UV. The interaction between colchicine and human serum albumin was studied by fluorescence and circular dichroism. 1. The fluorescence spectrum method is more sensitive and convenient than Raman and ultraviolet spectroscopy in studying the deoxygenation of Mb induced by light, and it is easy to identify the single peak without interference by other peaks. The higher the amount of illumination is, the more the oxygen Mb is deoxidized under light. The higher the amount of deoxidization and the higher the temperature under light, the faster the deoxygenation can remove the coordination oxygen from the iron porphyrin in the active center of Mb. Therefore, the interaction of oxygen containing Mb with various gases, such as COO2CO2H2Cl2Cl2NOCS2 and so on, can be studied by fluorescence spectrometry. Fluorescence spectroscopy can also be used to study the energy transfer between tryptophan and tyrosine residues in Mb and iron porphyrin. 2. The deoxygenation rate of MbD44K) is slower than that of MbCWT), and it is more difficult to deoxidize D44K at 430nm. Two fluorescence emission peaks appear at 597.9 nm and 628.8 nm, respectively. The fluorescence peak at 628.8 nm is the H 2O peak in Mb3 H 2O type, and the fluorescence intensity of this peak is also decreased by illumination. But the rate of deoxygenation was slower than that of deoxygenation. The fluorescence efficiency of MbD44K at 597 nm was lower than that of MbCWT. the energy transfer experiment showed that 44 amino acids on Mb surface mutated from Asp to Lys. The fluorescence efficiency of tryptophan and tyrosine residues transferred to iron porphyrin was not affected, but the fluorescence efficiency of tryptophan and tyrosine residues in MbD44K) was increased by .430 nm. 3.Colchicine enhanced the ultraviolet absorption of human serum albumin and quenched the characteristic fluorescence peak. The equilibrium constants and binding sites of colchicine interacting with human serum albumin at different temperatures were calculated. The equilibrium constants and binding sites of colchicine and human serum albumin were calculated by Van't Hoff equation. H = -11.66 KJ / mol? S = 51.507 J / mol 路K, the main force between them was electrostatic force. The 伪 -helix of human serum albumin was decreased after colchicine was added to the circular dichroism spectrum. The change of secondary structure indicates that the fluorescence quenching mechanism of colchicine on human serum albumin belongs to the static quenching caused by the formation of complex.
【学位授予单位】：大连大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R341

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