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.     

热诱导白蛋白与壳聚糖在溶液中的自组装

80℃热诱导牛血清白蛋白(BSA)变性,BSA疏水残基暴露在分子表面,并进一步凝胶化.采用紫外-可见光光度仪(UV-Vis)和动态激光光散射仪(DLS)考察了热处理时间、溶液p H值、壳聚糖(CS)与BSA配比等因素对CS与BSA自组装行为的影响.加热时间介于15~20 min,BSA与CS发生自组装;BSA/CS混合体系p H大于BSA等电点(p I=4.7)时,BSA与壳聚糖静电作用增强,随着p H增大,出现沉淀.BSA浓度过高,变性BSA之间聚集作用增强,导致体系出现沉淀.研究结果显示,CS阻碍了BSA自身凝胶化进程,并与BSA发生自组装.在p H=5.4的溶液中制备了BSA-CS自组装体.采用透射电镜(TEM)观察了自组装体的形态,采用DLS对其粒径及其分布进行了测试.自组装体是以BSA聚集体为核、CS分子链为壳的核壳纳米结构.制备的自组装体室温贮存40天后,其粒径和分布无明显变化.     

铅笔芯电极表面多层自组装膜制备及其对牛血清白蛋白的测定

采用电沉积技术将金沉积在铅笔芯电极(PGE)上,借助Au-S作用,将L-半胱氨酸(L-Cys)组装于金表面,利用吖啶橙(AO)与L-半胱氨酸之间的静电作用,将吖啶橙间接组装于金表面,构建成三层自组装膜电极AO/L-Cys/Au/PGE。将该复合膜电极连接在电化学工作站的工作电极和辅助电极端之间,与参比电极浸入溶液中,构建了一个新的检测回路。采用电化学阻抗谱技术和循环伏安法对多层自组装膜的组装过程进行表征。利用吖啶橙与牛血清白蛋白(BSA)间的相互作用,采用零流电位法,通过对比加入不同浓度BSA后零流电位值的变化(ΔEzcp)对BSA进行检测。结果显示,在1.0×10-9～1.0×10-5mol/L范围内,ΔEzcp与BSA浓度的对数值呈良好线性,相关系数(r)为0.997 0,检出限为1.41×10-11mol/L。该方法选择性和重复性好,有望应用于其它蛋白的测定。     

DMSO对鸡蛋白溶菌酶溶液变性的影响

利用差示扫描量热(DSC)和温度调制差示扫描量热(MDSC)研究了鸡蛋白溶菌酶在纯水及二甲基亚砜(DMSO)/水混合溶剂中的热变性过程, 探讨了酶的浓度、扫描速率和共溶剂的含量对热变性行为的影响规律. 在纯水溶液中, 溶菌酶的变性焓(△Hm)随酶浓度的增大而增大. 而在DMSO/水混合溶剂中, 变性温度(Tm)随DMSO体积分数的增大向低温方向移动, 变性峰变低变宽; 当DMSO体积分数达到70%后, 热变性曲线变成了一条光滑的直线. 另外, 在纯水溶液中溶菌酶的MDSC图除了出现DSC中可观察到的主吸热峰(I)外, 在峰(I)的前面还出现一个小而对称的吸热峰(II), 并且当体系中有DMSO存在时也未能观察到此峰. 当溶菌酶浓度增大时, Tm(II)移向低温, △Hm(II)减小, Tm(I)与Tm(II)之间的距离变长. 吸热峰(II)的出现被认为是由于水溶液中溶菌酶二聚体的可逆离解造成的.     

等转化率法分析溶菌酶在添加剂影响下的热变性行为

利用差示扫描量热法(DSC)研究了溶菌酶在不同浓度的添加剂(蔗糖、葡萄糖、果糖、甘露醇)和不同浓度的磷酸盐缓冲液影响下的热变性过程,并用等转化率法对该过程进行了分析.变性温度Tm随扫描速率和添加剂浓度的增加而提高,随磷酸盐缓冲液浓度的增加而降低.磷酸盐加速了溶菌酶的变性过程,降低了溶菌酶的热稳定性,而添加剂则增强了溶菌酶的热稳定性.在添加剂存在的条件下,溶菌酶变性过程部分为聚集不可逆过程,部分则可逆.等转化率法表明溶菌酶在所有条件下,其表观活化能在不同的转化率下并未保持不变,而是随转化率增大而减小,说明了一个简单的反应机理并不能用来描述溶菌酶的变性过程,其过程并不是标准两态可逆过程,而是一个涉及多种蛋白质状态的复杂过程.     

聚乙烯醇与牛血清白蛋白的相互作用及对其构象的影响

在生理条件下, 使用凝胶过滤色谱、荧光光谱法、差示扫描量热分析和傅里叶变换红外光谱法(FTIR)研究了牛血清白蛋白(BSA)与聚乙烯醇(PVA)的相互作用. 实验结果表明, PVA与BSA结合形成复合物, 在其相互作用过程中, BSA色氨酸的发射荧光部分被猝灭, 但是, 相互作用并没有明显改变色氨酸的微环境; 差示扫描量热分析结果提示, BSA与PVA之间的相互作用可能破坏了PVA或BSA的分子内作用力; 用红外光谱法结合可增强分辨率的傅里叶去卷积技术和高斯曲线拟合技术共同用于对BSA与PVA复合物冻干粉中BSA酰胺Ⅰ带的定量分析, 发现冻干粉BSA分子中与分子间相互作用相关的β-折叠组分含量明显减少, 但是, 可用于衡量冻干状态蛋白质结构完整性的α-螺旋组分含量没有降低. 对冷冻干燥后样品中的可溶性BSA分析结果提示, PVA可以保护BSA冷冻干燥过程中的稳定性.     

氧氟沙星与脲诱牛血清白蛋白结合的机制研究

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(Oflx)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、三态且伴随中问态形成的过程中,随着Urea浓度的增大,BSA荧光强度降低并先蓝移(344～336 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)=10.46X 104L/mol,Urea 4.8 mol/L)和较大的结合常数(KA=3.8807X105L/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,色氨酸残基(Trp-212)微境并未发生改变,而酪氨酸残基(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.     

含盐酸胍β-乳球蛋白A冷、热变性的DSC法研究

运用差示扫描量热法研究了牛β-乳球蛋白A(β-Lg A)在含盐酸肌(GuHCl)的溶液中的冷、热变性过程.实验表明:在pH3以下,热变性后β-Lg A分子结合的质子数几乎不变;GuHCl的存在降低热变性过程活化能.在含2.50及3.06mol/LGuHCl的溶液中,观察到β-LgA既可冷变性又可热变性.冷变性及其复性过程是可再现的.一般地说,热变性过程不可再现.和热变性比较,冷变性过程除变性焓有相反符号外,它的活化能也更低,而且冷变性后伸展的多肽链结合的GuHCl分子数增加得更多.冷变性焓的绝对值大于热变性焓值,表明与盐酸肌的结合是放热性的,是氢键性质的.在热变性过程中单体分子间的协同性较强.用Ooi模型分析,按重原子数平均来说.β-LgA单体分子构象变化对总变性焓的贡献在球蛋白中最低.     

L-半胱氨酸在乙酰二茂铁修饰碳糊电极上的电催化氧化及其电化学动力学性质研究

研究了L-半胱氨酸(L-cysteine,L-Cys)在乙酰二茂铁(acetylferrocene,Afc)修饰碳糊电极(Afc/CPE)上的电催化行为.研究结果表明,Afc/CPE对L-Cys的电化学氧化具有良好的催化作用.用循环伏安法(CV)、计时电流法(CA)测定了L-Cys在Afc/CPE上的电极过程动力学参数.测得Afc分散于液体石蜡中表观扩散系数Dapp=9.49×10-9 cm-2·s-1,电荷传递系数α=0.59,电催化氧化反应速率常数k=(3.76±0.10)×103(mol·L-1)-1·s-1.催化氧化峰电流与L-Cys在浓度8.0×10-6～1.5×10-3mol·L-1范围内呈良好的线性关系,线性回归方程为Ipa(μA)=3.139 c(mmol·L-1) 4.068,r=0.999 7,检出限为2.5 μmol·L-1.该结果可用于对L-Cys的电化学定量测定.     

用差示扫描量热计对低水含量蛋白质热变性的研究——“热变性前新峰”的证实

本文继发现水合牛血清白蛋白存在低温区吸热峰之后,迸一步测定了11个厂家生产的7种高纯低水含量蛋白质的DSC加热扫描曲线.证实在热变性峰之前的低温区无一例外都有一个明显可辨的小吸热峰,我们将它命名为“热变性前新峰”(Pre-thermodenaturation new peak).结果表明“热变性前新峰”的出现是低水含量蛋白质的共性.对新峰规律和机制的研究将有助于人们深入理解蛋白质的变性过程、肽链折叠途径、分子的缔合、解缔合及其溶剂效应。     

Fluorescence Quenching Study on the Interaction of Some Schiff Base Complexes with Bovine Serum Albumin

The interaction of Schiff base ligand A and its three metal complexes [A‐Fe(II), A‐Cu(II), and A‐Zn(II)] with bovine serum albumin (BSA) was investigated using a tryptophan fluorescence quenching method. The Schiff base ligand A and its three metal complexes all showed quenching of BSA fluorescence in a Tris‐HCl buffer. Quenching constants were determined for quenching BSA by the Schiff base ligand A and its metal complexes in a Tris‐HCl buffer (pH=7.4) at different temperatures. The experimental results show that the dynamic quenching constant (K_SV) was increased with increasing temperature, whereas the association constant (K) was decreased with the increase of temperature. The thermodynamic parameters ΔH, ΔG and ΔS at different temperatures were calculated. The ionic strength of the Tris‐HCl buffer had a great influence on the wavelength of maximum emission of BSA. Under low ionic strength, the emission spectra of BSA influenced by A‐Zn(II) had a small blue shift. Compared to A‐Zn(II), the emission spectra of BSA in the presence of the Schiff base ligand A and A‐Cu(II) had no significant λ_em shift. At high ionic strength, the emission spectra of BSA upon addition of the Schiff base A, A‐Fe(II), and A‐Zn(II) all had a red shift, but the emission spectra of BSA had λ_em shift neither at low ionic strength, nor at high ionic strength in the presence of A‐Cu(II). Furthermore, the temperature did not affect the λ_em shift of BSA emission spectra.     

Spectroscopic studies on the interaction between nicotinamide and bovine serum albumin

The interaction of nicotinamide (NA) and bovine serum albumin (BSA) was studied by fluorescence and absorption spectroscopy at different temperatures. The results revealed that NA caused the fluorescence quenching of BSA through a static quenching procedure. The binding constants K(A), and the number of binding sites n, corresponding thermodynamic parameters DeltaG, DeltaH, DeltaS between NA and BSA at different temperatures were calculated. The primary binding pattern between NA and BSA was interpreted as hydrophobic interaction. In addition, the effect of NA on the conformation of BSA was analyzed using synchronous fluorescence spectroscopy. The binding average distance, r between the donor (BSA) and acceptor (NA) was determined based on the F?rster's theory and it was found to be 3.1 nm.     

Study of the interaction between bovine serum albumin and ZnS quantum dots with spectroscopic techniques

The interaction between bovine serum albumin (BSA) and ZnS quantum dots (QDs) was studied by fluorescence, UV-vis spectroscopic techniques. The results showed that the fluorescence of BSA was strongly quenched by ZnS QDs and the quenching mechanism was discussed to be a static quenching procedure, which was proved by quenching rate constant K(q.) The recorded UV-vis data and the fluorescence data quenching by the QDs demonstrated that the interaction between them leads to the formation of QDs-BSA complex. Furthermore, the temperature effects on the structural and spectroscopic properties of individual QDs and protein and their bioconjugates (QDs-BSA) were also researched. It was found that, compared to the monotonically decrease of the individual QDs fluorescence intensity, the temperature dependence of the QDs-BSA emission had a much more complex behavior, highly sensitive to the conformational changes of the protein.     

The investigation of the interaction between NCP-EDA and bovine serum albumin by spectroscopic approaches

The fluorescence and ultraviolet spectroscopies were explored to study the interaction between N-confused porphyrins-edaravone diad (NCP-EDA) and bovine serum albumin (BSA) under simulative physiological condition at different temperatures. The experimental results show that the fluorescence quenching mechanism between NCP-EDA and BSA is a combined quenching (dynamic and static quenching). The binding constants, binding sites and the corresponding thermodynamic parameters (ΔG, ΔH, and ΔS) of the interaction system were calculated at different temperatures. According to F?rster non-radiation energy transfer theory, the binding distance between NCP-EDA and BSA was calculated to be 3.63 nm. In addition, the effect of NCP-EDA on the conformation of BSA was analyzed using synchronous fluorescence spectroscopy.     

大黄酸铜(Ⅱ)配合物与血清白蛋白的相互作用

采用荧光光谱法和紫外光谱法研究了大黄酸铜配合物与牛血清白蛋白之间的相互作用.大黄酸铜配合物能显著猝灭牛血清白蛋白的内源荧光并以静态猝灭为主;计算了298 K和309 K温度下结合常数、结合位点,根据热力学参数判断大黄酸铜配合物与牛血清白蛋白之间具有较强的疏水作用力;依据F?rster的偶极-偶极非辐射能量转移理论,计算出大黄酸铜在蛋白质中结合位置与色氨酸残基间的距离为3.21 nm, 表明大黄酸铜的部分片段能够插入蛋白质分子内部;用同步荧光光谱和圆二色光谱技术探讨了大黄酸铜对牛血清白蛋白构象的影响.     

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。     

Effect of pH on the interaction of vitamin B12 with bovine serum albumin by spectroscopic approaches

The interaction mechanism between vitamin B12 (B12, cyanocobalamin) and bovine serum albumin (BSA) has been investigated by fluorescence, synchronous fluorescence, ultraviolet-vis (UV) absorbance, and three-dimensional fluorescence. The intrinsic fluorescence of BSA was strongly quenched by the addition of B12 in different pH buffer solutions (pH 2.5, 3.5, 5.0, 7.4, and 9.0) and spectroscopic observations are mainly rationalized in terms of a static quenching process at lower concentration of B12 (C(B12)/C(BSA)<5) and a combined quenching process at higher concentration of B12 (C(B12)/C(BSA)>5). The structural characteristics of B12 and BSA were probed, and their binding affinities were determined under different pH conditions. The results indicated that the binding abilities of B12 to BSA in the acidic and basic pH regions (pH 2.5, 3.5, 5.0, and 9.0) were lower than that at simulating physiological condition (pH 7.4). In addition, the efficiency of energy transfer from tryptophan fluorescence to B12 was found to depend on the binding distance r between the donor and acceptor calculated using F?rster's theory. The effect of B12 on the conformation of BSA was analyzed using UV, synchronous fluorescence and three-dimensional fluorescence under different pH conditions. These results showed that the binding of B12 to BSA causes apparent change in the secondary and tertiary structures of BSA.     

Spectroscopic studies on binding of 1-phenyl-3-(coumarin-6-yl)sulfonylurea to bovine serum albumin

The interaction of 1-phenyl-3-(coumarin-6-yl)sulfonylurea (SU22) with bovine serum albumin (BSA) has been investigated by fluorescence quenching spectroscopy combined with UV-absorption, circular dichroism (CD), Fourier transform infrared (FT-IR) spectroscopy techniques under simulative physiological conditions for the first time. Fluorescence data and UV-absorption spectra revealed that the quenching mechanism of fluorescence of BSA by SU22 was a static quenching process and the number of binding sites was about 0.8858; the thermodynamic parameters (DeltaG=-29.23 kJ mol(-1), DeltaH=-47.48 kJ mol(-1), and DeltaS=-61.24 J mol(-1)K(-1)) explained that hydrogen bond and Van der Waals interaction were the main binding force stabilizing the complex. The binding average distance between SU22 and BSA was obtained (3.20 nm) on the basis of the F?rster's theory. In addition, The CD spectra and FT-IR spectra have proved that BSA secondary structure changed in the presence of SU22 in aqueous solution.     

N-烷基-N,N-二(2-羟乙基)-N-甲基溴化铵与牛血清白蛋白的相互作用

用荧光光谱法在298K研究了Tris-HCl缓冲溶液(pH=7.1)中系列N-烷基-N,N-二(2-羟乙基)-N-甲基溴化铵(烷基链长为C12到C16)与牛血清白蛋白(BSA)的结合作用,考察了表面活性剂结构、BSA浓度对结合作用的影响,分别用Stern-Volmer方程、虚拟结合常数模型探讨了表面活性剂在浓度较低区域与BSA的作用机制.结果表明:三种季铵盐表面活性剂均对BSA内源荧光有猝灭作用,并导致其最大发射波长蓝移;表面活性剂的烷基链越长,Stern-Volmer猝灭常数和虚拟结合常数越大,表面活性剂与BSA的结合作用也越强.     

Characterization of the interaction between farrerol and bovine serum albumin by fluorescence and circular dichroism

The binding of farrerol to bovine serum albumin (BSA) in aqueous solution was investigated by fluorescence quenching spectra, synchronous fluorescence spectra, circular dichroism (CD) and the three-dimensional (3D) fluorescence spectra at pH 7.40. The results of fluorescence titration indicated that farrerol could quench the intrinsic fluorescence of BSA in a static quenching way. The cause of showing upward curvy patterns in Stern-Volmer plots was analyzed. The binding sites number n and binding constant K using fluorescence quenching equation at 310 K were calculated. The binding distance and the energy transfer efficiency between farrerol and BSA were also obtained according to the theory of F?rster's non-radiation energy transfer. The effect of some metal ions on the binding constant of farrerol with BSA was also studied. The effect of farrerol on the conformation of BSA was analyzed using CD, synchronous fluorescence spectra and three-dimensional (3D) fluorescence spectra under experimental conditions. Furthermore, the fluorescence displacement experiments indicated that farrerol could bind to the site I of BSA.