真核表达与原核表达的德国小蠊变应原Bla g 2蛋白结构的光谱学研究

在基因工程技术中,采用原核表达系统获得的重组蛋白是以包涵体的形式存在,在细胞内聚集成无生物活性的不溶性颗粒,而采用真核表达系统收获的蛋白已经是经过正确的体内折叠的活性蛋白。文章分别用真核与原核表达系统表达重组变应原,通过对不同来源目的蛋白的荧光光谱和CD谱的比较和分析,对真核系统与原核系统表达出的Bla g 2蛋白的高级构象进行研究,阐明了Bla g 2在溶液中的二级结构,并推断出其三级结构组成类型。     

椰子花粉过敏原profilin蛋白体外重折叠过程的光谱学研究

在基因工程技术中,采用原核表达系统获得的重组蛋白通常都是以包涵体的形式存在.利用圆二色性光谱、荧光光谱和同步荧光光谱对尿素诱导的重组椰子化粉泛过敏原profilin蛋白包涵体的复性过程进行了系统的光谱学研究,得到了profilin蛋白复性过程的光谱学特征;结合生物信息学方法,通过对profilin蛋白的二级结构和三级结构的预测,进一步分析了复性过程中profilin蛋白构象变化的特点,初步建市了一种新的检测重组变应原蛋白复性程度的光谱学实验方法,为重组蛋白包涵体复性机理的深入研究进行了有益的探索.     

固相金属亲和层析纯化全长人PPARγ重组蛋白

建立全长人PPARγ重组蛋白的固相金属亲和层析纯化方法.利用E.coli BL21 (DE3)细胞外源性表达出全长人PPARγ重组蛋白,采用固相金属亲和层析法纯化目标蛋白.通过考察咪唑浓度对纯化的影响,确定在8 mol/L尿素下,用含50 mmol/L咪唑的缓冲液冲洗,200mmol/L咪唑缓冲液洗脱,可获得纯度为95％的目标蛋白,透析复性后经放射配体受体饱和结合实验测定全长人PPARγ重组蛋白的解离常数Kd为106士6nmol/L.结果表明本文所建立的方法能够成功纯化出高纯度的目标蛋白,经复性后,可获得用于结构和功能研究的全长人PPARγ重组蛋白.     

荧光光谱法研究盐酸胍浓度不同时变性胰蛋白酶的构象变化

蛋白变性过程中间体的存在是蛋白变性及复性动力学研究中不可缺少的证据。以胰蛋白酶为模型蛋白 ,用荧光光谱法系统地研究了在不同浓度变性剂盐酸胍存在时胰蛋白酶构象的变化 ,并与活性数据进行了对比。发现胰蛋白酶荧光光谱发射波长随变性剂盐酸胍浓度增大而逐渐增大 ,并且当盐酸胍浓度达到 2mol·L- 1 时胰蛋白酶的最大发射波长达到最大值 ,其后随盐酸胍浓度的增大最大发射波长反而逐渐减小 ,当盐酸胍浓度大于 3mol·L- 1 呈现不变的趋势。也就是说 ,在低浓度变性剂环境下 ,胰蛋白酶存在着一个与天然态和完全变性态的分子构象都不同的中间体状态 ,这个中间体状态的荧光发射波长最大 ,荧光发射强度也最大 ,而以此状态为复性起点 ,最终得到的复性产率也最低。对此原因从分子结构的基础上进行了探讨。     

尺寸排阻色谱柱上尿素梯度复性全长人PPAR-γ

建立尺寸排阻色谱柱上尿素梯度复性全长人PPAR-γ重组蛋白的方法。利用尺寸排阻色谱柱上尿素梯度除去变性剂直接复性变性蛋白。添加尿素和精氨酸抑制聚集以提高复性产率,加入Zn2+促进变性蛋白形成正确折叠。复性后,重组蛋白的尺寸排阻色谱保留体积增大;紫外光谱最大吸收波长从234.0nm变为280.8nm;基质辅助激光解析-电离飞行时间质谱分析重组蛋白的完整性,测得分子量与理论值一致;放射配体受体结合饱和实验测得解离常数(Kd)为89±4nmol/L;复性产率为52.7%。结果表明本文所建立的方法能成功用于复性变性全长人PPAR-γ,获得可用于结构和功能研究的具有生物学活性的全长人PPAR-γ蛋白。     

不同生境条件下藻蓝蛋白活体荧光光谱特性研究

蓝藻生物量检测技术发展是应对目前频繁发生的水华事件的重要环节。藻蓝蛋白作为蓝藻的特异性蛋白,在一定程度上比叶绿素更能准确反应自然水体中的蓝藻生物量,因而成为蓝藻生物量检测技术的重要指标。本文利用三维荧光光谱技术,以不同光照、不同生长期的铜绿微囊藻、鱼腥藻活体为研究对象,比较了单点法和包络法两种光谱解析方法的可靠性,探究了不同生境条件下的藻蓝蛋白活体荧光光谱特性。结果表明：(1)荧光光谱强度随生长期延长而增大;(2)采用包络法解析藻蓝蛋白特征荧光光谱的方法比单点法更为可靠;(3)在不同生境条件下,铜绿微囊藻藻蓝蛋白活体荧光激发波长基本保持614 nm、发射波长基本保持654 nm不变,鱼腥藻藻蓝蛋白活体荧光激发波长随生长期在610和620 nm之间波动减小,发射波长随生长期在650和660 nm之间波动增大。这种波动与藻种样品颗粒度大小和光谱扫描模式有关。该研究结果为发展蓝藻生物量活体荧光监测技术发展提供了实验基础。     

二硫键的氧化还原状态对Trx融合赤霉酸诱导富含半胱氨酸蛋白内源荧光及变性过程影响

在采用亲和层析、SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)对原核表达的赤霉酸诱导的富含半胱氨酸蛋白(Trx-GcGASA)进行纯化、鉴定的基础上,运用稳态荧光光谱手段研究了二硫苏糖醇(DTT)、氧化型谷胱甘肽(GSSG)、过氧化氢、盐酸胍(GdnHCl)对Trx-GcGASA内源荧光及变性过程的影响,发现(1)在中性缓冲体系中融合蛋白的内源荧光以305 nm的酪氨酸的荧光发射为主;(2)伴随着二硫键还原,融合蛋白中色氨酸和酪氨酸的相对荧光强度比值从0.7变化至1.8倍左右;(3)经过0.5 mmol.L-1GSSG、5 mmol.L-1过氧化氢处理后,酪氨酸和色氨酸的荧光强度下降约12~21%;(4)无论是否采用1 mmol.L-1DTT处理,6 mol.L-1盐酸胍均不能诱导融合蛋白彻底变性;(5)二硫键的存在与否影响了盐酸胍诱导的变性过程。通过两态模型拟合获得Trx-GcGASA变性过程Gibbs自由能变化ΔG约为3.7 kJ.mol-1。相关工作不仅为深入研究融合伴侣Trx对GcGASA变性热力学、动力学及复性过程影响奠定了基础;同时,也为通过光谱手段获取GcGASA的结构信息提供了基础的数据。     

高压及热处理对氮碳薄膜激光诱导荧光效率的影响

 应用激光激发荧光光谱实验对经热处理后及高压条件下的氮碳薄膜荧光光谱进行了测量分析。实验显示,热处理效应和高压效应均导致薄膜荧光效率降低,前者表现为不可恢复,后者为可恢复即卸压后荧光效率的恢复,表明导致荧光光谱效率降低的微观机制不同,为氮碳薄膜荧光模型提供了新的实验证据。     

高压及热处理对氮碳薄膜激光诱导荧光效应的影响

应用激光激发荧光光谱实验对经热处理后及高压条件下的氮碳薄膜荧光光谱进行了测量分析。实验显示,热处理效应和高压效应均导致薄膜荧光疚降有者表现为不可恢复,后者为可恢复即卸压后荧光效率的恢复,表明导致荧光光谱效率低的微观机制不同,为氮碳薄膜荧光模型提供了新的实验证据。     

基于光谱技术分析不同还原糖对Ara h2糖基化反应的影响

糖基化反应能诱导食品中蛋白质的结构发生改变;Ara h2是花生中的主要蛋白组分之一,可以作为一种模式蛋白研究花生蛋白糖基化产物的结构变化。不同还原糖对Ara h2糖基化反应的影响目前未见相关报道。以花生蛋白Ara h2为研究对象,通过SDS-PAGE、内源荧光、同步荧光、紫外、圆二色谱、红外等光谱技术研究Ara h2糖基化前后分子量、二级、三级结构以及官能团的变化,分析六种还原糖(核糖、木糖、半乳糖、葡萄糖、果糖、乳糖)对花生蛋白Ara h2糖基化产物结构的影响,阐明经不同还原糖修饰后花生蛋白Ara h2的结构变化。SDS-PAGE电泳表明木糖和核糖修饰的花生蛋白Ara h2电泳条带明显上移,糖基化程度最大;紫外光谱分析表明糖基化反应会改变花生蛋白Ara h2的吸收峰强度。五碳糖修饰的花生蛋白Ara h2具有最强的吸收强度,其中五碳糖中木糖的吸收峰强度最大;内源荧光、同步荧光和三维光谱实验结果表明,糖基化修饰会使花生蛋白Ara h2的荧光强度降低,且五碳糖修饰的Ara h2荧光强度最低。分析认为由于糖基化修饰使花生蛋白Ara h2的结构展开,导致芳香族氨基酸暴露在水环境中,从而引起荧光...     

C型肉毒毒蛋白的拉曼光谱研究

Ｃ型肉毒毒蛋白的拉曼光谱研究伍铁桥，刘新民，黄天荃，鲍培谛，杨志荣，刘世贵（四川大学成都６１００６４）ＳｔｕｄｉｅｓｏｎＴｈｅＲａｍａｎＳｐｅｃｔｒｏｓｃｏｐｙｏｆＢｏｔｕｌｉｎＴｙｐｅＣ￥ＷｕＴｉｅｑｉａｏ；ＬｉｕＸｉｎｍｉｎ；ＨｕａｎｇＴｉａｎｑ...     

Fluorescence Origin of 6,P-toluidinyl-naphthalene-2-sulfonate (TNS) Bound to Proteins

6,P-toluidinylnaphthalene-2-sulfonate (TNS) is a highly fluorescent molecule when dissolved in a low polarity medium or when bound to proteins. The aim of the present work is to explain origin of this fluorescence, to find out how the medium (solvent, protein matrix) affects fluorescence observables such as lifetimes and spectra and finally to put into evidence possible relation that exists between these observables and fluorophore structure. To achieve our goal we performed studies on TNS dissolved in ethanol, at high concentrations in water (aggregated form) and bound to proteins. Our experiments allowed us to find out that TNS in the three environments has different structures. Presence of three lifetimes observed in proteins and in water instead of one lifetime found in ethanol can be assigned to the high contact between TNS molecules. Our results are discussed in terms of solvent polarity and interaction within fluorophore molecules bound to proteins.     

基于紫外光诱导血浆的三维同步荧光光谱共振能量转移及能量再吸收的分析

利用三维同步荧光光谱技术研究了不同浓度的血浆溶液在紫外光波段的同步荧光光谱.实验结果表明：血浆蛋白的主要的激发峰主要有三个,分别在257 nm、274 nm和280 nm附近,同步荧光光谱峰值大小随着血浆的浓度的变化而有所不同.通过改变Δλ值而获得的同步荧光光谱,表明血浆存在三个同步荧光峰,其Δλ值分别为90 nm、72 nm和44 nm,根据实验数据计算,表明血浆的各个内源荧光团之间存在着荧光共振能量转移以及二次光吸收现象.     

吲哚基二聚体菁类探针同步荧光测定蛋白质的研究

基于蛋白质对双嵌吲哚染料具有良好的荧光增强作用,以新型水溶性吲哚基同型二聚体探针I,建立了一种灵敏的蛋白质同步荧光分析体系。实验考察了吲哚探针的荧光特征、吲哚探针浓度、缓冲体系pH、盐浓度等参数对体系荧光的影响。在酸性条件下,蛋白质分子与探针I发生结合作用,同步荧光明显增强并向长波方向发生红移,且同步荧光强度与蛋白质浓度成良好的线性关系。在最优条件下,牛血清白蛋白BSA的线性响应范围5.00×10-7～2.50×10-5 g·mL-1,检测限(3σ/K)为3 ×10-8 g·mL-1;测定了血清蛋白BSA的合成样品,不同浓度BSA样品回收率为98.6%～103.0%,相对标准偏差1.1%～1.9%;与蛋白质紫外标准测定法比较,测定偏差为0.4%～3.9％。     

Study of interaction between protein and main active components in Citrus aurantium L. by optical spectroscopy

The interaction between flavonoids and proteins was investigated by fluorescence and absorption spectroscopy. The binding parameters of drugs with proteins were obtained according to the corrected fluorescence data by an improved calculation method. The ΔH, ΔS and ΔG obtained indicate that the van der Waals or hydrogen bond, electrostatic force and hydrophobic forces all play a role in the interaction of drugs with proteins. Based on Förster's theory, the binding average distance r between the protein and drug was evaluated and found to be less than 3 nm. The interaction of drug-metal ion complexes and proteins was also investigated.     

Studies on the interaction of gliclazide with bovine serum albumin by fluorescence and synchronous fluorescence spectroscopy

The interaction between gliclazide and bovine serum albumin was investigated by fluorescence and synchronous fluorescence spectroscopy. From the experimental results, it was found that the quenching mechanism was static. The results of the synchronous fluorescence obtained indicated that the binding of gliclazide with bovine serum albumin could affect conformation in bovine serum albumin. In addition, the binding constants (K_a), binding sites (n), thermodynamic parameters, binding forces, Hill’s coefficient, and binding rate of gliclazide to protein calculated from two methods using the same equation were consistent at three different temperatures (298, 310, 318 K). This indicated that as a useful supplement to the conventional method, synchronous fluorescence spectroscopy could be used to study the mechanism of drugs and proteins. The conclusion was verified by UV/vis method.     

A Simple Model-Free Method for Direct Assessment of Fluorescent Ligand Binding by Linear Spectral Summation

Fluorescent tagged ligands are commonly used to determine binding to proteins. However, bound and free ligand concentrations are not directly determined. Instead the response in a fluorescent ligand titration experiment is considered to be proportional to the extent of binding and, therefore, the maximum value of binding is scaled to the total protein concentration. Here, a simple model-free method is presented to be performed in two steps. In the first step, normalized bound and free spectra of the ligand are determined. In the second step, these spectra are used to fit composite spectra as the sum of individual components or linear spectral summation. Using linear spectral summation, free and bound 1-Anilinonaphthalene-8-Sulfonic Acid (ANS) fluorescent ligand concentrations are directly calculated to determine ANS binding to tear lipocalin (TL), an archetypical ligand binding protein. Error analysis shows that the parameters that determine bound and free ligand concentrations were recovered with high certainty. The linear spectral summation method is feasible when fluorescence intensity is accompanied by a spectral shift upon protein binding. Computer simulations of the experiments of ANS binding to TL indicate that the method is feasible when the fluorescence spectral shift between bound and free forms of the ligand is just 8 nm. Ligands tagged with environmentally sensitive fluorescent dyes, e.g., dansyl chromophore, are particularly suitable for this method.

Formation of soybean protein isolate-hawthorn flavonoids non-covalent complexes: Linking the physicochemical properties and emulsifying properties

In recent years, more and more attention had been paid to the combination of proteins and flavonoids, and several flavonoids had been reported to improve the physicochemical and emulsifying properties of proteins. This study investigated the effects of ultrasonic treatment (450 W for 10 min, 20 min, and 30 min) on the physicochemical properties, antioxidant activity, and emulsifying properties of soy protein isolate (SPI) -hawthorn flavonoids (HF) non-covalent complexes. The results showed that the addition of HF to SPI and 20 min of ultrasound could reduce α-helix and random coil, increase β-sheet and β-turn, and enhance fluorescence quenching. In addition, it decreased the particle size, zeta potential, surface hydrophobicity, and turbidity to 88.43 or 95.27 nm, −28.80 mV, 1250.42, and 0.23, respectively. The protein solubility, free sulfhydryl group, antioxidant activity, emulsifying activity index, and emulsifying stability index all increased to 73.93%, 15.07 μmol/g, 71.00 or 41.91%, 9.81 m²/g, and 67.71%, respectively. Moreover, high-density small and low-flocculation droplets were formed. Therefore, the combined ultrasound treatment and addition of HF to SPI is a more effective method for protein modification compared to ultrasound treatment alone. It provides a theoretical basis for protein processing and application in the future.