荧光光谱法研究牛血清蛋白在尿素体系中的变性

应用荧光光谱技术,对尿素与牛血清蛋白在30℃水溶液中的结合作用及造成牛血清蛋白变性的过程进行了研究,获取了尿素诱导牛血清蛋白变性时相对荧光强度和峰位的变化规律.用Pace等提出的公式分析了相对荧光强度数据,得到了牛血清蛋白变性时的伸展分数fu随溶液pH值和尿素浓度的变化规律.求出了变性平衡常数Ku,伸展吉布斯自由能△G...     

光谱法研究变性剂诱导的猪胃蛋白酶和牛血清蛋白折叠中间态的分布和过渡

利用紫外光谱和荧光光谱分别测定了变性剂诱导的猪胃蛋白酶和牛血清蛋白的残余活性,并根据它们的两个特征展开参数定量描述了猪胃蛋白酶和牛血清蛋白天然态、折叠中间态和完全展开态随变性液中变性剂浓度的分布和过渡。结果表明,在盐酸胍诱导的猪胃蛋白酶"三态"去折叠过程中,当盐酸胍浓度约为1.5mol/L时,折叠中间态的浓度达到最大,约占溶液中总猪胃蛋白酶的12%;在脲诱导的牛血清蛋白"三态"去折叠过程中,当脲浓度约为2.0mol/L时,折叠中间态的浓度达到最大,约占溶液中总牛血清蛋白的41%;而在盐酸胍诱导的牛血清蛋白"四态"去折叠过程中,当盐酸胍浓度约为0.4mol/L、1.2mol/L时,第一和第二折叠中间态浓度分别达到最大,约占溶液中总牛血清蛋白的20%和70%。     

牛血清蛋白在盐酸胍和尿素体系中变性的微量热研究

在30 ℃时用恒温微量热法研究了不同pH值下盐酸胍、尿素诱导牛血清蛋白变性的过程. 并用Privalov提出的简单键合模型对量热数据进行了分析, 计算了表观键合常数K, 简单键合的单个表观键合自由能ΔG和总吉布斯能ΔG(a), 用变性中点的直线外推方法求出了表观变性焓ΔH_d. 实验结果表明, 牛血清蛋白与盐酸胍的键合在碱性条件下更易进行, 牛血清蛋白在盐酸胍溶液中的变性焓ΔH_d在牛血清蛋白的pH＝6.97和7.05时为350 kJ•mol^－1, 在pH＝9.30时为275 kJ•mol^－1, 表明牛血清蛋白在接近中性时较稳定. 而牛血清蛋白与尿素的键合在酸性条件下更易进行, 此变性焓ΔH_d在牛血清蛋白的pH＝6.97时为295 kJ•mol^－1, 在pH＝7.05和9.30时为230 kJ•mol^－1. 此结果说明牛血清蛋白在两种变性剂溶液中的展开程度是不同的.     

盐酸胍诱导的变性卵清溶菌酶分子重折叠过程及其各稳定构象态的分布和过渡

采用变性和非变性电泳、 高效凝胶排阻色谱、 内源荧光发射光谱和荧光相图以及生物活性测定等方法, 研究了盐酸胍诱导的变性卵清溶菌酶分子的重折叠过程及此过程中卵清溶菌酶分子各稳定构象态的分布和过渡. 结果表明, 当复性液中盐酸胍浓度分别约为5.0和2.4 mol/L时, 变性卵清溶菌酶分子的重折叠过程各存在1个稳定折叠中间态, 重折叠过程符合"四态模型". 在卵清溶菌酶分子四态重折叠过程基础上, 结合盐酸胍与卵清溶菌酶分子之间的缔合-解离平衡, 给出了一个定量描述变性剂诱导的蛋白质分子复性过程中蛋白质分子复性率随溶液中变性剂浓度变化的方程. 该方程包含2个特征折叠参数, 一个是蛋白质分子从一个稳定构象态过渡到另一个稳定构象态的热力学过渡平衡常数k; 另一个是在此过程中平均每个蛋白质分子所结合的变性剂分子数目m. 通过这2个特征折叠参数能够定量描述盐酸胍诱导的变性卵清溶菌酶完全去折叠态、 折叠中间态和天然态分子随复性液中盐酸胍浓度变化的分布和过渡情况.     

牛血清IgG热化学变性和热变性的研究

使用差示扫描量热仪(DSC)和荧光光谱法研究了在pH 7.4时牛血清IgG (bIgG)热变性, 热化学变性和等温化学变性过程(变性剂为尿素和盐酸胍), 首次报道了bIgG在热化学变性和等温化学变性过程中的相关热力学参数. DSC和荧光光谱实验结果表明, bIgG的热变性和热化学变性过程都是较复杂的不可逆过程, 这个过程可被看作一个三态变构过程. DSC实验表明在热化学变性过程中bIgG的变性温度和焓变值会随着环境中的变性剂浓度的升高而降低. 使用荧光光谱法对bIgG在尿素或盐酸胍存在下的等温化学变性过程进行了研究, 结果显示bIgG的化学变性过程也是一个较复杂的非二态过程. 实验数据分析表明, 变性剂尿素和盐酸胍与bIgG之间主要是依靠氢键相互作用的, 而热变性过程中bIgG的凝集是由于bIgG热变性时结构改变后暴露出的疏水结构互相作用造成的. 实验结果还表明单纯的热变性只能导致bIgG的不完全变性, 而即使是在高浓度变性剂存在时的bIgG热化学变性, 尿素和盐酸胍分别导致的bIgG热化学变性的去折叠态也是不同的.     

用尺寸排阻色谱法研究蛋白质的热稳定性

用尺寸排阻色谱ＺｏｒｂａｘＧＦ－２５０凝胶过滤柱研究了核糖核酸酶Ａ,马心肌红蛋白和溶菌酶的热稳定性。测定了蛋白质在尺寸排阻系统中的分配系数Ｋｄ并用荧光检测器分析了加热引起的蛋白质内源性荧光的改变,从而计算出了蛋白质的热变性转化点温度,测定了蛋白质天然态、热变性和盐酸胍诱导变性状态下的斯托期斯半径,实验结果进一步证帝了热变性后蛋白质不是呈真正的随机线状,而是保留了部分紧密的结构区域。用本方法研究了Ｐ     

溶菌酶热变性的DSC研究

用差示扫描量热法研究了固体溶菌酶的热变性以及水溶液中不同变性剂与浓度对溶菌酶变性的影响. 结果表明, 溶剂水的存在及变性剂尿素和盐酸胍的加入使溶菌酶的变性温度降低, 变性焓减小; 同时, 在一定的浓度范围内, 溶菌酶的变性温度和变性焓随变性剂浓度的增大而降低. 盐酸胍的变性效果较尿素强, 这是由于盐酸胍与蛋白质分子间除了氢键作用外还存在着静电作用.     

不同介质中大肠杆菌碱性磷酸酶构象变化的研究

利用色氨酸残基作为内源荧光探针的膜模拟剂（各种表面活性剂）和不同变性剂中对大肠杆菌碱性磷酸酶（ＡＰ）的构象变化进行了系统的研究。通过测定在不同变性时间下盐酸胍浓度对荧光强度的影响以及荧光强度随pＨ有规律的变化,进一步证实了该蛋白质变性过程中形成较稳定中间态的结论。     

盐酸胍诱导的淀粉液化芽孢杆菌α-淀粉酶去折叠过程的研究

分别用内源荧光光谱法、荧光相图法、荧光探针法、荧光猝灭法、蛋白质电泳法以及体积排阻色谱法研究了盐酸胍诱导的淀粉液化芽孢杆菌a-淀粉酶的去折叠过程. 内源荧光光谱和荧光相图结果表明, 当变性液中盐酸胍浓度约为1.0 mol/L时, 芽孢杆菌a-淀粉酶的去折叠过程中出现一个部分折叠中间体, 其去折叠过程符合“三态模型”; 荧光探针结果表明, 在溶液中盐酸胍浓度约为1.0 mol/L时, 中间态芽孢杆菌a-淀粉酶分子中存在着能够与探针分子1-苯胺 基-8-萘磺酸(ANS)结合的稳定的疏水区域; 荧光猝灭研究给出了不同程度变性的淀粉液化芽孢杆菌a-淀粉酶中的Trp的分布情况, 结果表明中间态芽孢杆菌a-淀粉酶分子中能够被碘化钾猝灭的位于分子表面的色氨酸残基数目达到最大的8个; 蛋白电泳和体积排阻色谱结果表明, 在盐酸胍诱导的芽孢杆菌a-淀粉酶分子的整个去折叠过程中, 不会以共价键或非共价键形式形成芽孢杆菌a-淀粉酶分子之间的集聚体或集聚体沉淀. 在此基础上, 对盐酸胍诱导的淀粉液化芽孢杆菌a-淀粉酶的去折叠过程进行了描述.     

脲和盐酸胍诱导的牛碳酸酐酶B的去折叠

采用非变性聚丙烯酰胺凝胶电泳、十二烷基硫酸钠-聚丙烯酰胺凝胶电泳、高效凝胶排阻色谱和激光光散射光谱研究了脲和盐酸胍诱导的牛碳酸酐酶B的去折叠.实验结果表明,在脲和盐酸胍诱导的牛碳酸酐酶B的去折叠过程中,溶液中只含有单分子牛碳酸酐酶B和二分子牛碳酸酐酶B集聚体;二分子牛碳酸酐酶B集聚体是通过单分子牛碳酸酐酶B之间的疏水和...     

Denaturation study of bovine serum albumin induced by guanidine chloride or urea by microcalorimetry

The denaturation of bovine serum albumin (BSA) induced by guanidine chloride or urea at different pH values was studied by isothermal microcalorimetry measurements at 30°C. The simple bonding model, which was developed by Privalov, was employed to obtain the apparent bonding constant K, the apparent singular bonding Gibbs bonding energy ΔG and the total Gibbs energy ΔG(a) between the protein and denaturant, from analysis of the calorimetric data. Furthermore, linear extrapolation at the midpoint of transition was employed to determine the apparent denaturation enthalpy ΔH _d. The results showed that for guanidine chloride, the bonding between BSA and guanidine chloride could proceed more easily in an alkaline condition, and the apparent denaturation enthalpy ΔH _d of BSA due to guanidine chloride was 350 kJ·mol⁻¹ at pH 6.97 and 7.05, while it was 275 kJ·mol⁻¹ at pH 9.30, which indicated that BSA was more stabilized in a neutral condition. However, for urea, the bonding between BSA and urea could proceed more easily in an acidic condition, and the apparent denaturation enthalpy ΔHd of BSA due to urea was 295 kJ · mol⁻¹ at pH 6.97, while it was 230 kJ · mol⁻¹ at pH 7.05 and 9.30. The results indicate that the degree of expansion of BSA in the two denaturants is different. __________ Translated from Acta Chimica Sinica, 2008, 66(5) (in Chinese)     

Effect of pH and guanidine hydrochloride on the conformation of 57 kDa rat liver nuclear thyroid hormone binding protein measured by fluorescence.

The denaturation of the 57 kilodalton (kDa) rat liver nuclear thyroid hormone binding protein (NTHB) by pH and guanidine hydrochloride (GdnHCl) has been investigated with the fluorescence method. The acid and alkaline fluorescence quenching suggests that the structure of NTHB is invariant in the relatively narrow pH region of approximately pH 7-9. A cooperative conformational transition occurred in GdnHCl concentrations of 1.5-2.5 m. The apparent free energy of unfolding of NTHB, delta G(appH2O) was evaluated as 6.31 (+/- 0.12) kcal.mol-1 at pH 7.7, 25 degrees C.     

Analysis of binding interaction between pegylated puerarin and bovine serum albumin by spectroscopic methods and dynamic light scattering

The interaction between bovine serum albumin (BSA) and pegylated puerarin (Pur) in aqueous solution was investigated by UV-Vis spectroscopy, fluorescence spectroscopy and circular dichroism spectra (CD), as well as dynamic light scattering (DLS). The fluorescence of BSA was strongly quenched by the binding of pegylated Pur to BSA. The binding constants and the number of binding sites of mPEG(5000)-Pur with BSA were 2.67±0.12 and 1.37±0.05 folds larger after pegylating, which were calculated from the data obtained from fluorescence quenching experiments. The enthalpy change (ΔH) and entropy change (ΔS) were calculated to be 4.09 kJ mol(-1) and 20.01 J mol(-1) K(-1), respectively, according to Van't Hoff equation, indicating that the hydrophobic force plays a main role in the binding interaction between pegylated Pur and BSA. In addition, the negative sign for Gibbs free energy change (ΔG) implies that the interaction process is spontaneous. Moreover, the results of synchronous fluorescence and CD spectra demonstrated that the microenvironment and the secondary conformation of BSA were changed. Comparing with Pur, all our data collected indicated that pegylated Pur interacted with BSA in the same way as that of Pur, but docked into the hydrophobic pocket of BSA with more accessibility and stronger binding force. DLS measurements showed monomethoxy polyethylene glycol (mPEG) have an effect on BSA conformation, and revealed that changes in BSA size might be due to increases in binding constant and the absolute values of ΔG after Pur pegylation.     

氧氟沙星与脲诱导牛血清白蛋白结合的机制研究

摘要 利用荧光光谱和紫外光谱研究了脲（Urea）对牛血清白蛋白（BSA）结构的影响以及氧氟沙星（Oflxacin）与脲诱导的BSA结合的情况。结果显示：Urea诱导BSA变性历经两步、三态过程,且伴随中间态的形成。随着Urea浓度的增大,BSA荧光强度降低并先蓝移（344 nm～336 nm）,后又红移至350 nm。Urea浓度在4.6～5.2 mol/L范围时,Oflx对BSA中间态有强的猝灭作用（KQ=10.46×104 L/mol, Urea 4.8 mol/L）和较大的结合常数（KA=3.8807×105 L/mol, Urea 4.8 mol/L）,但是结合位点数小（n=0.76, Urea 5.0 mol/L）,能量传递效率低（E=0.3002, Urea 4.8 mol/L）。同步荧光光谱显示：Urea诱导BSA去折叠时,Trp-212残基微环境并未发生改变,而Tyr的最大荧光发射峰蓝移,Oflx的加入诱导Trp-212的微环境更具疏水性。Oflx加速了Urea对BSA的失活作用。     

Mechanistic investigation on binding interaction of bioactive imidazole with protein bovine serum albumin--a biophysical study

The interaction between bioactive imidazole derivative (PPP) and bovine serum albumin (BSA) was investigated using fluorescence and UV-vis spectral studies. The experimental results showed that the fluorescence quenching of BSA by imidazole derivative was the result of the formation of BSA-PPP complex and the effective quenching constants (K(SV)) were 2.66×10(4), 2.56×10(4), and 2.10×10(4) at 301, 310 and 318 K, respectively. Static quenching and non-radiative energy transfer were confirmed to the result in the fluorescence quenching. The binding site number n, apparent binding constant K(A) and corresponding thermodynamic parameters (ΔG, ΔH and ΔS) were measured at different temperatures. The process of binding of PPP molecule on BSA was a spontaneous molecular interaction procedure in which entropy increased and Gibbs free energy decreased.     

Interaction of a Rhodococcus sp. trehalose lipid biosurfactant with model proteins: thermodynamic and structural changes

One major application of surfactants is to prevent aggregation during various processes of protein manipulation. In this work, a bacterial trehalose lipid (TL) with biosurfactant activity, secreted by Rhodococcus sp., has been identified and purified. The interactions of this glycolipid with selected model proteins have been studied by using differential scanning calorimetry (DSC), Fourier-transform infrared (FTIR) spectroscopy, isothermal titration calorimetry (ITC), and fluorescence spectroscopy. Bovine serum albumin (BSA) and cytochrome c (Cyt-c) have been chosen because of their quite different secondary structures: BSA contains essentially no β-sheets and an average 66% α-helix, whereas Cyt-c possesses up to 25% β-sheets and up to 45% α-helical structure. Differential scanning calorimetry shows that addition of TL to BSA at concentrations below the critical micelle concentration (cmc) shifts the thermal unfolding temperature to higher values. FTIR indicates that TL does not alter the secondary structure of native BSA, but the presence of TL protects the protein toward thermal denaturation, mainly by avoiding formation of β-aggregates. Studies on the intrinsic Trp fluorescence of BSA show that addition of TL to the native protein results in conformational changes. BSA unfolding upon thermal denaturation in the absence of TL makes the Trp residues less accessible to the quencher, as shown by a decrease in the value of Stern-Volmer dynamic quenching constant, whereas denaturation in the presence of the biosurfactant prevents unfolding, in agreement with FTIR results. In the case of Cyt-c, interaction with TL gives rise to a new thermal denaturation transition, as observed by DSC, at temperatures below that of the native protein, therefore facilitating thermal unfolding. Binding of TL to native BSA and Cyt-c, as determined by ITC, suggests a rather nonspecific interaction of the biosurfactant with both proteins. FTIR indicates that TL slightly modifies the secondary structure of native Cyt-c, but protein denaturation in the presence of TL results in a higher proportion of β-aggregates than in its absence (20% vs 3.9%). The study of Trp fluorescence upon TL addition to Cyt-c results in a completely opposite scenario to that described above for BSA. In this case, addition of TL considerably increases the value of the dynamic quenching constant, both in native and denatured protein; that is, the interaction with the glycolipid induces conformational changes which facilitate the exposure of Trp residues to the quencher. Considering the structures of both proteins, it could be derived that the characteristics of TL interactions, either promoting or avoiding thermal unfolding, are highly dependent on the protein secondary structure. Our results also suggest the rather unspecific nature of these interactions. These might well involve protein hydrophobic domains which, being buried into the protein native structures, become exposed upon thermal unfolding.     

Study on the Interaction between Theasinesin and Bovine Serum Albumin by Fluorescence Method

Theasinesin (TS), a polymer of epigallocatechin gallate, is the main active component of tea polyphenols. Several studies indicate that tea polyphenols have extensive pharmacology activity. However, there is little research about the transportation and metabolism of tea polyphenols in vivo. Serum albumin is a most important protein serving as a depot protein and as a transport protein for many drugs and other bioactive small molecules. This study observed the interaction between TS and bovine serum albumin (BSA) by fluorescence and absorption spectroscopy. The results showed that both static and dynamic quenching occurred in the fluorescence quenching of BSA by TS. The binding sites number is 1.1845 and the binding sites may close to the tyrosine residues. The thermodynamic parameters ΔH°, ΔG°, ΔS° at temperatures 310 K were calculated 1.7 KJ, ?35.4 KJ, and 0.12 KJ. The negative sign of free energy (ΔG°) means that the interaction process is spontaneous. The positive enthalpy (ΔH°) and entropy (ΔS°) values of the interaction of TS and BSA indicate that the binding is mainly entropy-driven and the enthalpy is unfavorable for it, the hydrophobic forces playing a major role in the reaction. A distance of 4.037 nm was found between donor (BSA) and acceptor (TS), obtained according to the F?rster theory of non-radiation energy transfer, which indicates that the energy transfer from BSA to TS occurs with high probability. The results of synchronous fluorescence spectra and UV–vis absorption spectra showed that the peptide strands of BSA molecules extended more and the hydrophobicity decreased with the addition of TS.     

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。