血清白蛋白的构象研究Ⅳ．pH诱导HSA构象变化的光谱研究

用紫外吸收二阶导数谱辅以荧光光谱、紫外吸收光谱研究了ｐＨ＝３．０～１１．０范围内ＨＳＡ中芳香氨基酸残基微环境的变化，以此作为推断构象变化的探针。     

杀鼠剂溴鼠灵与牛血清白蛋白相互作用的光谱法研究

利用紫外吸收光谱,荧光光谱和同步荧光技术研究了牛血清白蛋白和杀鼠剂溴鼠灵的相互作用.结果表明溴鼠灵对牛血清白蛋白的内源荧光有较强的猝灭作用,两者形成了新的复合物,属于静态荧光猝灭,并且伴随着分子内的非辐射能量转移.通过双倒数及双对数曲线计算了不同温度下的猝灭速率常数Ksv,结合位点数n,结合常数KA,并根据相对应的热力学参数判断二者之间主要为疏水作用力.依据F(o)rster非辐射能量转移理论求出了溴鼠灵和蛋白质问的结合距离r,确定了溴鼠灵在蛋白质上的结合位置.在20和30℃时r分别为2.84和2.87 nm.同步荧光光谱显示,与溴鼠灵作用后BSA分子的二级结构发生了改变.初步探讨了二者的结合模式与作用机制:溴鼠灵分子通过静电引力靠近蛋白质的疏水腔,并以疏水作用力与疏水腔中的氨基酸残基发生相互作用,导致色氨酸残基微环境极性变化.其结果不但阻止了酪氨酸残基与色氨酸残基间的能量转移,而且使色氨酸残基与溴鼠灵分子间产生非辐射能最转移,从而猝灭BSA的内源荧光.     

光谱法研究变性剂对苦荞麦蛋白质构象的影响

采用紫外差示光谱和荧光光谱研究了不同浓度的乙醇溶液和盐酸胍溶液对苦荞麦蛋白质(TBWSP31)构象变化的影响.研究表明,TBWSP31的色氨酸残基和酪氨酸残基有的位于分子内部,有的暴露于分子表面.乙醇溶液使TBWSP31变性时,仅仅是分子的外层结构发生了变化,分子内部疏水核的变化较小,高浓度的盐酸胍使位于TBWSP31...     

光谱法研究葛根素与牛血清白蛋白的相互作用

采用紫外、同步荧光和圆二色光谱法研究了葛根素（PUE）与牛血清白蛋白（BSA）的相互作用.紫外光谱表明,BSA在230nm和278nm处的吸收峰,随着葛根素浓度的增加而减小.同步荧光光谱表明,葛根素引起BSA中色氨酸残基所处微环境的疏水性降低.圆二色光谱表明,BSA在208nm和222nm处的负峰随着葛根素浓度的增加而增强,BSA中α-螺旋含量也随之增加.这表明葛根素与BSA的相互作用,可使蛋白质分子的疏水作用增强,导致BSA的肽链结构收缩,蛋白质的构象发生变化.     

TrpH—TryOH肽链中电子的逆向转移

本文较详细的研究了色氨酰酪氨酸二肽（ＴｒｐＨ－ＴｙｒＯＨ）中的电子转移，电子该电子转移的速率，甚至电子转移的方向都强烈的依赖于溶液的ｐＨ。而且，该肽中的酪氨酸残基瞬态粒子浓度与色氨酸残基瞬态粒子浓度之比也强烈的依赖于ｐＨ。根据这种依赖性，求得了二肽中的ｐＫａ（ＴｒｐＨ＾＋）和ｐＫａ（ＴｙｒＯＨ），其值分别为６．２和１１．３。     

竹红菌甲素与肌红蛋白和血红蛋白相互作用的同步荧光光谱研究

利用同步荧光光谱方法研究了竹红菌甲素(HA)对肌红蛋白和血红蛋白构象的影响.结果表明,HA能够使肌红蛋白和血红蛋白的构象发生改变,导致蛋白质分子中色氨酸和酪氨酸残基所处微环境由原来的疏水环境不同程度地向亲水环境转变.     

新的植物毒素蒜头果蛋白的荧光光谱研究

蒜头果蛋白(Malanin)是从我国稀有植物蒜头果中分离纯化出的一种具有高细胞毒性的蛋白质.用荧光光谱法研究在温度、酸度,有机溶剂、表面活性剂、变性剂及荧光猝灭剂等不同条件对蒜头果蛋白溶液构象的变化.实验表明,Malanin在天然状态下荧光发射峰位于340 nm处·色氨酸(Trp)残基较大程度位于Malanin分子的疏水区.十二烷基硫酸钠、异硫氰酸胍、丙烯酰胺和碘化钾的加入均可使Malanin的分子构象发生变化,导致分子内Trp残基的荧光猝灭.异硫氰酸胍的加入使Trp残基的荧光发射峰位明显红移,表明位于Malanin分子较疏水环境内的Trp残基相对外露.     

光谱法结合分子动力学模拟研究臭氧对肌红蛋白结构的作用机制

臭氧(O₃)是一种具有强氧化性作用的杀菌消毒剂，因其安全无害等特点已被广泛用于肉制品生产加工的减菌处理，但O₃减菌处理对红肉色泽具有较强的负面作用，且其作用机制尚缺乏研究。针对肌红蛋白（Mb）存在状态是决定红色肉色泽关键因素的基础，通过紫外-可见吸收光谱法、荧光光谱法和圆二色光谱法（CD）研究O₃作用下Mb的光谱特性变化，结合蛋白质氧化特征指标分析和分子动力学模拟技术探究O₃对Mb分子的作用效果与机制。光谱研究结果表明，O₃处理可使Mb的紫外-可见光谱图在412 nm左右处的铁卟啉环特征峰及540和580 nm附近的氧合肌红蛋白（OMb）特征峰的强度减弱，其中铁卟啉环特征峰发生蓝移；利用固定激发波长280 nm下测定Mb内源性荧光和同步荧光光谱表明O₃会降低Mb的荧光强度，增大铁卟啉基团贡献的荧光峰强度和造成酪氨酸残基荧光光谱特征峰的蓝移；O₃作用使Mb三维荧光光谱特征峰强度的下降及光散射强度的增加。以上变化推断出O₃会促进Mb的氧化，造成其氨基酸残基疏水基团裸露，使Mb所处微环境及其蛋白构象改变；CD分析表明O₃与肌红蛋白接触时间越久，蛋白质二级结构变化越明显，造成α-螺旋的含量下降，无规则卷曲增加。辅以检测不同强度O₃处理Mb的含量及性质的变化，可知O₃处理使OMb含量下降，高铁肌红蛋白（MMb）含量增加，同时O₃处理Mb的羰基含量增加和巯基含量下降，这也进一步证实O₃作用促进了Mb的氧化，此外，O₃处理Mb表面疏水性的增强，说明O₃造成Mb体系微环境的极性变化。分子动力学模拟结果显示O₃会提高Mb肽链的RMSD值，影响Mb肽链的稳定性，减弱铁卟啉环与Mb肽链的相互作用；RMSF结果表明Mb活性口袋附近氨基酸残基的变化较大；蛋白质二级结构分析与光谱学试验研究结果一致，Mb的α-螺旋的含量下降，无规则卷曲增加。总而言之，O₃可作用于Mb的氨基酸残基，导致蛋白质二级结构和疏水性改变，并发生蛋白氧化及铁卟啉环暴露，进而引起红色肉色泽发生改变。该研究可为生鲜红肉护色技术制定等提供一定理论依据。     

Cr(Ⅵ)与细胞色素c相互作用的研究

运用荧光光谱、圆二色(CD)谱法研究了K2Cr2O7与细胞色素c的相互作用。结果表明,Cr(Ⅵ)酸根离子与细胞色素c相互作用引起细胞色素c的α-螺旋含量降低,使酪氨酸残基所处微环境的极性增加,色氨酸残基的疏水性增加。Cr(Ⅵ)与细胞色素c的作用位点更接近于色氨酸残基,引起色氨酸残基的微环境变化的程度要大于酪氨酸残基。     

同步荧光光谱技术研究胶原基表面活性剂溶液中分子的聚集行为

基于胶原基表面活性剂(collagen-based surfactant,CBS)中酪氨酸(Tyr)和苯丙氨酸(Phe)的荧光特性,应用恒波长差(Δλ)为15nm的同步荧光光谱技术研究CBS浓度、溶液pH值、NaCl浓度和温度对其在水溶液中分子的聚集行为的影响,并以温度为外扰,利用二维同步荧光相关分析研究CBS分子中Tyr残基和Phe残基随温度变化的响应顺序。结果表明,CBS分子在261和282nm处出现了分别归属于Phe和Tyr的特征吸收峰。随着CBS浓度的升高,CBS分子中Phe残基和Tyr残基数量逐渐增多使CBS分子聚集程度增加,并导致荧光强度增强;CBS溶液pH值(pH 5.0)在等电点附近时,由于CBS分子的疏水作用和氢键形成能力加强,导致CBS分子聚集程度增强;CBS溶液中NaCl浓度的升高,则使CBS分子间排斥力减弱,从而导致CBS分子的聚集;然而温度升高,CBS由聚集状态逐渐变为单分子状态,因猝灭、变性以及氢键形成能力降低,荧光强度逐渐降低。以温度为外扰的二维同步荧光相关光谱分析可知:低温下(10~40℃),CBS聚集体随温度升高逐渐松散,位于聚集体内部的Phe残基比Tyr残基优先响应;而在45~70℃时,CBS由单分子状态逐步水解为无规卷曲构造,Tyr残基的间距变大,氢键形成能力大大降低,Tyr残基比Phe残基优先响应。     

尼古丁与BSA相互作用的光谱研究

用紫外-可见光谱和荧光光谱研究了尼古丁与牛血清白蛋白(bovine serum albumin,BSA)的相互作用。荧光研究表明,尼古丁浓度的增加引起BSA 345 nm处荧光有规律地猝灭。Stern-Volmer方程分析pH5.0,pH 7.4和pH 11.0体系的荧光猝灭机理发现,pH 5.0体系属动态猝灭,而pH 7.4和pH 11.0体系为静态猝灭。Lineweaver-Burk双倒数方程计算pH 7.4和pH 11.0体系在温度为20和37℃条件下尼古丁和BSA的结合常数k分别为:k20℃=140.15 L.mol-1,k37℃=131.83 L.mol-1(pH 7.4)和k20℃=141.76 L.mol-1,k37℃=27.79 L.mol-1(pH 11.0),表明结合常数在pH 7.4条件下受温度的影响要比pH 11.0条件下小,推测是由于不同pH下尼古丁存在的不同形态所致。紫外-可见光谱研究表明,pH 7.4条件下尼古丁浓度的增加引BSA在210 nm处吸收峰吸收强度减小且红移,说明BSA二级结构发生变化,即螺旋结构变松散;紫外二阶导数光谱和同步荧光光谱(Δλ=λem-λex=15 nm和Δλ=λem-λex=60 nm)分析尼古丁对BSA芳香性氨基酸(Trp,Tyr和Phe)残基微环境的变化,结果表明高浓度的尼古丁使所有这些芳香性氨基酸残基微环境由疏水环境转变为亲水环境。     

鸡肝脏谷胱甘肽转移酶的荧光光谱分析

谷胱甘肽转移酶是生物体内一种重要的解毒酶,排除外源和内源毒素,维护肌体的正常功能.文章从荧光角度研究了谷胱甘肽转移酶光谱性质,得到谷胱甘肽转移酶的三维荧光谱图,确定了色氨酸、酪氨酸的荧光峰,以及肽链骨架荧光峰.根据其峰位的红移或蓝移,以及在不同pH条件下氨基酸荧光峰强度、位置变化,推断谷胱甘肽转移酶构象变化以及色氨酸在酶分子中的位置.     

Study of the interaction between tosufloxacin tosylate and bovine serum albumin by multi-spectroscopic methods

The interaction of tosufloxacin tosylate (TSFX) and bovine serum albumin (BSA) was studied by fluorescence spectroscopy, UV–vis spectroscopy and FT-IR spectroscopy. The results indicated that the intrinsic fluorescence of BSA was quenched by TSFX through a static quenching mechanism, and the effective binding constants (K_a) were obtained by means of the modified Stern–Volmer equation. Thermodynamic parameters showed that electrostatic interaction was mostly responsible for the binding of TSFX to BSA. The binding distance (r) between TSFX and Trp-212 was determined to be 3.90 nm according to Föster non-radiative energy transfer theory. BSA had a single class of binding site at Sudlow' site I in subdomain IIA for TSFX. The effects of TSFX on the conformation of BSA were analyzed by synchronous fluorescence spectra and three-dimensional fluorescence spectra, and the results exhibited that the hydrophobicity of tryptophan microenvironment was decreased. In FT-IR spectra, Fourier self-deconvolution, secondary derivative and the curve-fitting process were carried out to obtain the components of BSA secondary structure at 298 K and 310 K. The full basic data indicated that the presence of TSFX resulted in α-helix and β-sheet changing into β-turn and random, which displayed that TSFX induced the unfolding of the polypeptides of BSA.     

Structural and functional changes in ultrasonicated bovine serum albumin solutions

Effects of high-intensity ultrasonication on functional and structural properties of aqueous bovine serum albumin (BSA) solutions were investigated. The functional properties of BSA were altered by ultrasonication. Surface activity of BSA increased. Minimal changes were observed in the global structure of BSA but surface charge increased particularly at basic pH values (e.g. pH>9). While dynamic light scattering measurements indicated that the particle size increased up to 3.4 times after 90 min of sonication, no significant increase in the oligomeric state of BSA using blue native PAGE was observed. The amount of free sulfhydryl groups in BSA after 90 min of sonication decreased. The increased particle size and decreased number of free sylfhydryl groups may be attributed to formation of protein aggregates. Surface hydrophobicity increased and circular dichroism spectroscopy and FTIR analysis indicated changes in the secondary structure of BSA. We hypothesize that mechanical, thermal and chemical effects of ultrasonication resulted in structural changes in BSA that altered the functional properties of the macromolecule which may be attributed to the formation of an ultrasonically induced state that differs from a thermally, mechanically or solvent induced state.     

光谱探针猩红S与牛血清白蛋白结合的发光光谱

利用荧光光谱和紫外吸收光谱,详细研究了不同温度下猩红S(PS)与牛血清白蛋白(BSA)的结合反应,发现PS对BSA的内源性荧光具有较强的猝灭作用,其猝灭机理属于静态猝灭,由此求得PS与BSA间的结合常数、结合位点数及热力学参数等.结果表明:PS与BSA之间形成了1:1稳定复合物,它们之间的作用力主要是静电引力.根据Fo...     

多光谱法和分子对接模拟法研究黄腐植酸和牛血清白蛋白的相互作用

在模拟生理环境中,使用荧光光谱法、紫外光谱法、圆二色谱法、同步荧光光谱法、三维荧光光谱法与分子对接模拟法研究黄腐植酸和牛血清白蛋白(BSA)之间相互作用。在荧光光谱法研究中,经Stern-Volmer方程计算得到298,303和308 K温度下的动态荧光猝灭速率常数K_q和猝灭常数,证明BSA与黄腐殖酸(FA)相互作用的猝灭过程为静态猝灭;同时根据计算得出的结合位点数n都在1附近,FA与BSA体系相互作用比为1∶1;利用静态猝灭双对数方程计算三个温度下的热力学参数,焓变ΔH<0,熵变ΔS＜0,得出结论,FA与BSA之间的主要作用力为氢键和范德华力;ΔG＜0,说明作用过程为自发过程。采用Förster’s偶极-偶极非辐射能量转移理论,计算出结合距离r=6.340 nm,表明BSA与FA之间存在非辐射能量转移。分子对接模拟结果表明FA与BSA残基的结合作用力具有氢键和范德华力,同时二者之间还存在疏水作用力,多种力共同作用使FA与BSA能够稳定结合。通过对FA与BSA相互作用的紫外-可见吸收光谱分析,发现BSA最大吸收峰发生了较为明显的红移,表明FA使BSA的二级结构发生改变。通过研究FA与BSA相互作用的同步荧光光谱,得到FA使BSA中的色氨酸（Trp）残基周围的微环境极性增强,疏水性减弱,亲水性增强,使BSA的蛋白质构象发生了一定程度的改变。通过研究FA与BSA相互作用的三维荧光光谱,峰1（peak 1）与峰2（peak 2）的最大发射波长峰都发生了红移,证明FA与BSA发生了相互作用,FA使BSA周围环境的极性增大,疏水性减小,亲水性增加,BSA蛋白质构象发生变化。最后采用圆二色谱法进行分析,利用软件计算得出该实验相互作用体系下α-螺旋（α-Helix）减少2.3％、β-折叠（β-sheet）增加7.7％、β-转角（β-Turn）增加0.6％和无规则结构（Random coil）含量减少1.2％,β-折叠（β-sheet）含量增加最为明显, 强有力地说明了FA使BSA结构发生了改变。     

Fluorescence Study on the Interaction of Bovine Serum Albumin with P-Aminoazobenzene

In this paper, the interaction between p-aminoazobenzene (PAAB) and BSA was investigated mainly by fluorescence quenching spectra, circular dichroism (CD) and three-dimensional fluorescence spectra under simulative physiological conditions. It was proved that the fluorescence quenching of BSA by PAAB was mainly a result of the formation of a PAAB-BSA complex. The modified Stern-Volmer quenching constant K _a and the corresponding thermodynamic parameters ΔH, ΔG and ΔS at different temperatures were calculated. The results indicated that van der Waals interactions and hydrogen bonds were the predominant intermolecular forces in stabilizing the complex. The distance r?=?4.33 nm between the donor (BSA) and acceptor (PAAB) was obtained according to Förster’s non-radioactive energy transfer theory. The synchronous fluorescence, CD and three-dimensional fluorescence spectral results showed that the hydrophobicity of amino acid residues increased and the losing of α-helix content (from 63.57 to 51.83%) in the presence of PAAB. These revealed that the microenvironment and conformation of BSA were changed in the binding reaction.     

Study of Interaction of Silver Nanoparticles with Bovine Serum Albumin Using Fluorescence Spectroscopy

The interaction between silver nanoparticles (SNPs) and Bovine Serum Albumin (BSA) was investigated at physiological pH in an aqueous solution using fluorescence spectroscopy. The analysis of fluorescence spectrum and fluorescence intensity indicates that SNPs have a strong ability to quench the intrinsic fluorescence of BSA by both static and dynamic quenching mechanisms. Resonance light scattering (RLS) spectra indicated the formation of a complex between BSA and SNP. The number of binding sites ‘n’ and binding constants ‘K’ were determined at different temperatures based on fluorescence quenching. The thermodynamic parameters namely ∆H^°, ∆G^°, ∆S^° were calculated at different temperatures and the results indicate that hydrophobic forces are predominant in the SNP-BSA complex. Negative ∆G^° values imply that the binding process is spontaneous. Synchronous fluorescence spectra showed a blue shift which is indicative of increasing hydrophobicity.     

Denaturation of bovine serum albumin initiated by sodium dodecyl sulfate as monitored via the intrinsic fluorescence of the protein

The tryptophan fluorescence of bovine serum albumin (BSA) is used to study the denaturation transitions in BSA under the influence of sodium dodecyl sulfate (SDS) at various pH values. The stepwise quenching of BSA tryptophan fluorescence and the gradual increase in the degree of anisotropy of BSA tryptophan fluorescence with increasing SDS concentration in solutions indicate the stepwise nature of denaturation: the first stage is a loosening of protein globules, whereas the second is a complete unfolding of the protein amino acid chain. At pH > pI of BSA, the denaturation BSA proceeds through both stages. At pH > pI of BSA, the denaturation BSA runs poorly and stops after the first stage. A more efficient BSA denaturation under the action of SDS occurs at pH < pI of BSA, with the efficiency of BSA denaturation under the influence of SDS decreasing with increasing pH.     

铽离子发光探针研究pH诱导的植物钙调素构象变化

利用脱钙钙调素(apo-CaM),Tb.CaM和Ca.CaM系统的荧光光谱及酪氨酸-铽离子(Tyr-Tb3 )敏化荧光光谱,研究了pH诱导钙调素构象的改变.随着pH值的降低,apo-CaM的荧光强度下降并出现蓝移,Ca.CaM系统的荧光强度较Tb.CaM下降显著,Tyr-Tb3 敏化荧光光谱也有相当程度的降低.对荧光强度的变化与钙调素分子结构和构象的关系和pH诱导钙调素构象改变的机理作了详细的解释,H 可通过与Ca2 和Tb3 产生竞争结合,影响金属离子与钙调素的结合作用,还可以通过正电相斥或与肽链上带负电荷原子相结合而使CaM分子的极性增强,改变CaM表面的疏水性,降低CaM的活性.文章对pH诱导钙调素构象改变在胞外钙调素信号转导机制中的重要意义进行了阐述.