三维荧光光谱法研究蛋白质溶液构象

采用三维荧光光谱法(三维荧光光谱、三维荧光偏振光谱)结合内源荧光探针色氨酸对牛血清白蛋白(BSA)和鸡蛋白蛋白(EA)及其在不同条件下的构象变化进行了研究.结果表明,三维荧光光谱法是一种研究蛋白质溶液构象很有效的分析方法,该方法能够较直观地表明色氨酸残基在蛋白质分子中的微环境及其在不同条件下的构象变化,并得出了一些有价值的结果.     

用硝基苯萃取法在拉曼测试中猝灭荧光

本文报道了一种测试拉曼光谱时把硝基苯掺入蛋白质溶液再加以萃取的预处理方法，该方法能迅速猝灭蛋白质分子的荧光。得到稳定清晰的拉曼图谱，而且不影响对蛋白质分子的构象分析。     

苯胺蓝黑与人血清白蛋白的相互作用及对其构象的影响

利用荧光光谱和同步荧光光谱研究了不同温度下苯胺蓝黑与人血清白蛋白相互作用时的荧光猝灭及构象的变化情况。实验结果表明,苯胺蓝黑与人血清白蛋白之间可以发生相互作用,而且有较强的结合。同步荧光光谱研究了人血清白蛋白与苯胺蓝黑的相互作用中人血清白蛋白构象的变化,结果显示二者结合改变了蛋白质的微环境。热力学参数说明小分子与蛋白质的作用以疏水作用为主。     

噻唑橙类菁染料与生物大分子结合的光谱特性的研究

研究了两个噻唑橙类菁染料TO-1和TO-2与核酸、蛋白质结合的光谱特性.发现染料喹啉环中氮上取代基对染料标记生物大分子的荧光特性有着很大的影响.羧基的引入,使染料TO-2与核酸结合后的荧光性能下降,但同时也提高了它与蛋白质结合的光物理性能.研究表明:在喹啉环氮上引入适当的亲水性取代基,可以使噻唑橙类菁染料的应用范围拓展到蛋白质标记领域.并初步研究了蛋白质浓度的变化对染料TO-2的荧光光谱的影响以及TO-2在不同pH值的蛋白质溶液中的荧光性质.     

荧光成像在生物分析中的应用

荧光显微镜与荧光光谱仪耦合系统可获取显微荧光成像及微区荧光光谱、荧光寿命的测定信息,广泛应用于细胞、组织中蛋白质的结构功能分析,核酸的识别检测,金属离子、自由基的定量测定,以及纳米生物探针的研制等生物分析研究的热点领域。本文引用文献46篇,综述了荧光成像在生物分析中的应用新进展。     

三维荧光光谱结合物理和数学分离研究溶解性有机质组成

采用分子排阻色谱和激发/多波段发射荧光检测器,结合三维荧光光谱和平行因子分析,研究了新、老填埋垃圾渗滤液中溶解性有机质( DOM)的组成。实验结果表明,两种渗滤液来源的DOM均含有类蛋白和类腐殖质物质。在新填埋垃圾渗滤液中,类蛋白物质有4种存在形态,包括大分子蛋白质形态、高/低分子量腐殖质结合态和多肽/氨基酸形态;在老填埋垃圾渗滤液DOM中,类蛋白物质只有两种形态,分别为大分子蛋白质形态和腐殖质结合态。相比于分子排阻色谱,三维荧光光谱结合平行因子分析能够分辨出腐殖质和非腐殖质结合态的类蛋白物质,但不能有效区分蛋白质和以多肽/氨基酸形态存在的类蛋白物质。结果表明,三维荧光光谱结合平行因子分析和分子排阻色谱,可以表征DOM中不同形态分布的类蛋白和类腐殖质物质。     

光谱法研究蛋白质与表面活性剂的相互作用

结合本课题组的工作, 较系统地总结了近年来有关紫外吸收光谱、荧光光谱、圆二色光谱和电子自旋共振光谱技术在蛋白质-表面活性剂混合体系研究中的应用. 大量研究表明, 借助于光谱技术不仅可以研究蛋白质结构与功能的关系, 而且可以探讨蛋白质与表面活性剂的作用机理.     

荧光探针在蛋白质研究中的应用

荧光探针技术是研究蛋白质在水溶液中构象的一种有效方法。利用它可以测定蛋白质分子的疏水微区内两基团的距离以及酶与底物结合过程中蛋白质构象的变化等。本文综述了荧光探针技术在蛋白质研究中的一些进展。     

时间分辨发射光谱的应用研究进展

时间分辨荧光技术是研究超分子组装微环境变化的主要手段之一。荧光体与蛋白质、内膜及胶束等相互作用引起最大荧光强度和量子效率急剧变化反映了激发态反应情况,激发态过程通常用时间分辨发射光谱(TRES)测定。国内在这一领域的研究虽有成果,但与国外相比仍有一定差距。本文对时间分辨荧光光谱的应用研究进展进行了综述,阐述了其历史、原理、检测方法及近几年国内外的应用研究进展情况。     

荧光法研究酚藏花红与牛血清白蛋白的相互作用

用荧光光谱、同步荧光光谱和紫外吸收光谱研究了牛血清白蛋白与酚藏花红的结合反应特征。在不同的pH条件下,酚藏花红与蛋白质的反应导致了蛋白质荧光猝灭及酚藏花红的荧光增强,其荧光猝灭值与酚藏花红的浓度成正比,可用于酚藏花红的分析测定;而酚藏花红荧光增强值与蛋白质浓度成正比,又可用于蛋白质的分析测定。用Stern-Volmer方程和Lineweaver-Burk双倒数函数处理实验数据,得到10℃和20℃时动态猝灭常数和静态猝灭结合常数、反应的热力学参数和结合位点数。根据F彲ster能量转移原理计算出20℃时酚藏花红与牛血清白蛋白的结合距离为r=2.22 nm。     

二维相关荧光光谱技术

从发展历史、计算方程、一般规则和特有性质等方面系统地介绍了近年来在二维相关荧光光谱技术方面的方法探索和应用进展。以不同的外扰方式，如浓度、激发波长、猝灭以及其他如pH等分类，举例阐述了二维荧光相关光谱的可操作性及其应用，并与普通一维荧光光谱比较，说明了二维荧光相关光谱技术的优势。     

Monofluorination and Trifluoromethylation of BODIPY Dyes for Prolonged Single‐Molecule Detection

Electrophilic monofluorination with Selectfluor and nucleophilic trifluoromethylation with the Ruppert–Prakesh reagent of dimethyl‐, tetramethyl‐ and pentamethyl‐substituted boron dipyrromethenes (BODIPY) are investigated. Monofluorinated dyes are synthesized with low yields (<30 %), however trifluoromethyl derivatives are obtained in moderate to high yields (≈40–90 %). All compounds are characterized by steady‐state and time‐resolved fluorescence spectroscopy, the photostability is investigated with fluorescence correlation spectroscopy (FCS) and total internal reflection fluorescence microscopy (TIRF). Monofluorination hardly affects the spectroscopic parameters of the unsubstituted parent compounds, but distinctly enhances the photostability, whereas trifluoromethylation leads to a hypsochromic shift by up to 17 nm in both absorption and emission, slightly enhanced intersystem crossing, and higher photostability. Further development of soft fluorination and trifluoromethylation methods is therefore highly desired.     

中药成分五味子甲素及其金属离子复合物与血清白蛋白相互作用的研究

在模拟人体生理条件下,采用紫外光谱法、荧光光谱法和同步荧光光谱法研究五味子甲素及其金属离子复合物与血清白蛋白(SA)的结合作用。结果表明:五味子甲素与金属离子形成1∶1复合物;五味子甲素对SA的荧光猝灭机制为静态猝灭和非辐射能量转移;五味子甲素与牛血清白蛋白(BSA)和人血清白蛋白(HSA)的结合常数K、结合位点数n、结合距离r分别为3.58×104L·mol-1和6.42×103L·mol-1、0.944和0.993、2.18nm和1.63nm,其作用力以氢键和范德华力为主。同步荧光光谱法的结果表明:结合位点靠近色氨酸,并使色氨酸的疏水性减弱。共存金属离子对五味子甲素与SA的相互作用有一定的影响。     

Fluorescence Spectroscopy of Single Molecules under Ambient Conditions: Methodology and Technology

This review presents an overview of the fluorescence detection and spectroscopy of single molecules (SMS) in liquids and on surfaces under ambient conditions. The various techniques of SMS, such as confocal epifluorescence detection and wide‐field imaging are presented and discussed, together with the different methods of data analysis such as fluorescence correlation spectroscopy and burst‐by‐burst analysis. Selected applications of the various techniques in physics, chemistry, and biology are described.     

Investigation on the photo-induced de-oxygenation process of myoglobin in aqueous solution by use of fluorescence spectroscopy

A photo-induced de-oxygenation process of myoglobin (Mb) in aqueous solution was investigated by use of fluorescence spectroscopy. The spectra are characterized by the fluorescence intensity declining gradually after each scan,and the decay of fluorescence intensity being significant in each scan,which is assigned to the release of oxygen from the opening of the heme-pockets induced by illumination. More illumination will cause more release of oxygen; if the temperature of an Mb solution is increased when it is illuminated,the rate of de-oxygenation will be higher. It was found that ligand-oxygen in Fe-porphyrin could be removed from Mb by nitrogen. This indicates that the interac-tion between oxy-Mb and other different gases can be tested by the method of fluorescence spectros-copy. In addition,fluorescence spectroscopy can be employed to probe the energy transfer between Fe-porphyrin and tryptophan or tyrosine in Mb molecules.     

铱(IV)离子与人血丙种球蛋白的作用研究

在0.1 mol•L^－1醋酸-醋酸钠(pH 5.0)体系中, 采用紫外吸收光谱、荧光光谱及同步荧光光谱法研究了人血丙种球蛋白(gamma seroglobulinum humanum, 简称GSH)与铱(IV)离子的相互作用. 结果表明, Ir(IV)离子使人血丙种球蛋白的构象发生了改变, α-螺旋含量减少, 并且用同步荧光光谱发现Ir(IV)离子与人血丙种球蛋白的作用位点更接近于色氨酸, 从而使色氨酸残基的疏水性略有减小. 荧光光谱结果表明Ir(IV)对人血丙种球蛋白内源荧光(342 nm)产生了较强的荧光猝灭作用, 根据不同温度下Ir(IV)对人血丙种球蛋白的荧光猝灭作用, 证明了这种荧光猝灭为静态猝灭机制, 计算了其结合常数和结合位点数, 从而得出了静电作用力为其主要的作用力.     

Influence of axial coordination on the photophysical property of excited state of zinc porphyrin

The phenomenon that metalloporphyrins are coordinated to axial ligands is very common especially in green-plant and photosynthetic bacteria, where such coordination impacts a signifi-cant effect on both the primary charge separation in the reaction center and energy transfer in an-tenna systems[1]. In addition, it plays a key role in photodynamic therapy and medicine synthesis in a sense that some metalloporphyrins have been assembled by coordination bonds to anti-cancer drugs, enhancing the se…     

Optical saturation in fluorescence correlation spectroscopy under continuous-wave and pulsed excitation.

A detailed theoretical and experimental study of the dependence of fluorescence correlation measurements on optical excitation power due to optical saturation effects is presented. It is shown that the sensitivity of a fluorescence correlation measurement on excitation power becomes increasingly stronger for decreasing excitation power. This makes exact measurements or diffusion coefficients with fluorescence correlation spectroscopy rather difficult. A strong difference of this behavior for continuous-wave and pulsed excitation is found.     

Cr(Ⅵ)与牛血清白蛋白的相互作用

采用荧光光谱、紫外光谱、CD光谱法研究了K2Cr2O7与牛血清白蛋白(BSA)的相互作用。实验结果表明,铬(Ⅵ)使BSA的紫外吸收降低,峰位红移,表明铬(Ⅵ)与BSA发生较强的相互作用;铬(Ⅵ)酸根离子与BSA形成基态复合物导致BSA内源荧光猝灭,猝灭机理主要为静态猝灭。测定了不同温度下该反应的热力学参数,ΔGθ0,ΔHθ和ΔSθ分别为-12.60kJ/mol和56.60J/(mol.k),表明上述作用过程是一个熵增加、自由能降低的自发分子间作用过程,铬(Ⅵ)酸根离子与BSA之间以静电作用力为主;非辐射能量转移机理确定了铬(Ⅵ)与牛血清白蛋白中色氨酸残基之间的距离r=2.85nm;同步荧光和CD光谱研究表明,铬(Ⅵ)使BSA的二级结构发生改变,α-螺旋含量降低,色氨酸残基所处微环境的极性减小。     

Molecular Fluorescence Imaging Spectroscopy for Mapping Low Concentrations of Red Lake Pigments: Van Gogh's Painting The Olive Orchard

Vincent van Gogh used fugitive red lake pigments that have faded in some paintings. Mapping their distribution is key to understanding how his paintings have changed with time. While red lake pigments can be identified from microsamples, in situ identification and mapping remain challenging. This paper explores the ability of molecular fluorescence imaging spectroscopy to identify and, more importantly, map residual non‐degraded red lakes. The high sensitivity of this method enabled identification of the emission spectra of eosin (tetrabromine fluorescein) lake mixed with lead or zinc white at lower concentrations than elemental X‐ray fluorescence (XRF) spectroscopy used on account of bromine. The molecular fluorescence mapping of residual eosin and two carmine red lakes in van Gogh's The Olive Orchard is demonstrated and compared with XRF imaging spectroscopy. The red lakes are consistent with the composition of paint tubes known to have been used by van Gogh.