Interaction of Potassium Mono and Di Phosphates with Bovine Serum Albumin Studied by Fluorescence Quenching Method

The interactions between potassium mono and di phosphates and bovine serum albumin (BSA) were studied using fluorescence spectroscopy (FS) and ultraviolet spectroscopy (UV). The experimental results showed that the potassium mono and di phosphates could insert into the BSA and quench the inner fluorescence of BSA by forming the potassium mono phosphate—BSA and pottassium di phosphate—BSA complexes. It was found that the static quenching was the main reason leading to the fluorescence quenching. It was conformed by XRD and SEM techniques.     

Interaction of 1-cyanoethyl-5-chlorouracil with human and bovine serum albumins

The interactions of human (HSA) and bovine (BSA) serum albumins with 1-cyanoethyl-5-chlorouracil (CECU) were investigated by fluorescence spectroscopy, UV absorption spectroscopy, and molecular modeling methods under the simulated physiological conditions. The results of fluorescence measurements indicate that CECU has a strong ability to quench the intrinsic fluorescence of both HSA and BSA through a static quenching procedure. The binding constants (K) at different temperatures and thermodynamic parameters, enthalpy change (ΔH), and entropy change (ΔS) were calculated according to fluorescence data. The results show that hydrophobic interaction is a predominant intermolecular force for stabilizing the complex, which is in agreement with the results of molecular modeling study. The effect of some normal ions on the binding constants is also discussed. Published in Zhurnal Prikladnoi Spektroskopii, Vol. 75, No. 5, pp. 737–745, September–October, 2008.     

Biophysical Studies on the Interactions of a Classic Mitochondrial Uncoupler with Bovine Serum Albumin by Spectroscopic,Isothermal Titration Calorimetric and Molecular Modeling Methods

The interaction between a classic uncoupler (2,4-dinitrophenol, DNP) and bovine serum albumin (BSA) was investigated by fluorescence spectroscopy under the physiological conditions. The fluorescence quenching constants were calculated by the Stern-Volmer equation, and based upon the temperature dependence of quenching constants, it was proved that DNP caused a static quenching of the intrinsic fluorescence of BSA. Owing to the static quenching mechanism, different associative binding constants at various temperatures were determined and thus the thermodynamic parameters, namely enthalpy (ΔH = −21.12 kJ mol⁻¹) and entropy changes (ΔS = 23.51 J mol⁻¹ K⁻¹) could be calculated based on the binding constants. Moreover, the enthalpy and entropy changes are consistent with the “Enthalpy-Entropy Compensation” equation obtained from our previous work. The negative enthalpy and positive entropy indicated that the electrostatic interactions played a major role in DNP-BSA binding process. Site marker competitive displacement experiments were carried out by using fluorescence and isothermal titration calorimetry (ITC) methods. These results showed that DNP bound with high affinity to Sudlow’s site I (subdomain IIA) of BSA. The distance (r = 3.78 nm) between donor (BSA) and acceptor (DNP) was obtained according to the mechanism of fluorescence resonance energy transfer (FRET). Furthermore, the results of synchronous fluorescence and circular dichroism (CD) spectroscopic studies indicated that the microenvironment and the secondary conformation of BSA were altered. The above results were supported by theoretical molecular modeling methods.     

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.     

Spectroscopic Identification of Interactions of Pb2+ with Bovine Serum Albumin

The effect of Pb²⁺ targeted to bovine serum albumin (BSA) in vitro was investigated by fluorescence, synchronous fluorescence, UV absorption and circular dichroism (CD) spectrophotometry. The characteristic fluorescence of BSA was quenched, which indicated that Pb²⁺ changed the skeleton of BSA and caused the gradual exposure of aromatic amino acid residues (Trp, Tyr, Phe) in the internal hydrophobic region of BSA. When the concentration of Pb²⁺ was higher than 1 × 10⁻⁴ mol/L, the BSA was completely denatured. The excess lead ion interacted with the aromatic amino acid residues of BSA exposed to the solution, which decreased the fluorescence of BSA further. According to the experiment results, we found that a lead-BSA complex was formed following static quenching and the binding site was calculated approximately equal to 1. This work reflected the interaction mechanism of BSA and Pb²⁺ from the perspective of spectroscopy.     

Studies on Interaction of CdTe Quantum Dots with Bovine Serum Albumin Using Fluorescence Correlation Spectroscopy

Luminescent quantum dots (QDs) have widely used in some biological and biomedical fields due to their unique and fascinating optical properties, meanwhile the interaction of QDs with biomolecules recently attract increasing attention. In this paper, we employed fluorescence correlation spectroscopy (FCS) to investigate the nonspecific interaction between CdTe QDs and bovine serum albumin (BSA) as a model, and evaluate their stoichiometric ratio and association constant. Our results documented that BSA was able to bind to CdTe QDs and form the QD–BSA complex by a 1:1 stoichiometric ratio. The association constant evaluated is 1.06 ± 0.14 × 10⁷ M⁻¹ in 0.01 M phosphate buffer (pH = 7.4). Furthermore, we found that QD–BSA complex dissociated with increase of ion strength, and we speculated that the interaction of CdTe QDs with BSA was mainly attributed to electrostatic attraction. Our preliminary results demonstrate that fluorescence correlation spectroscopy is an effective tool for investigation of the interaction between quantum dots (or nanoparticles) and biomolecules.     

烟碱与牛血清白蛋白相互作用的光谱研究

在0.1 mol·L-1的磷酸氢二钠-柠檬酸体系中,采用荧光光谱、紫外吸收光谱研究了牛血清白蛋白(BSA)与烟碱的相互作用。荧光滴定表明这种相互作用使BSA的内源荧光猝灭,尼古丁和BSA形成1∶1稳定复合物。不同温度和酸度下的猝灭作用证实其静态猝灭行为和疏水作用机制。紫外吸收光谱和同步荧光光谱表明,相互作用引起BSA构象变化,而同步荧光光谱提示结合位点更接近于色氨酸。     

Interaction of BSA with proflavin: A spectroscopic approach

The interaction of bovine serum albumin (BSA) with proflavin was investigated by spectroscopic tools like absorption and fluorescence spectroscopy as well as laser flash photolysis. Absorption spectroscopy proved the formation of ground-state BSA-proflavin complex. Proflavin was found to quench the intrinsic fluorescence of BSA via static quenching. High value of quenching constant suggested that energy transfer occurred from BSA to proflavin. Distance between the fluorophore in the protein and the ligand (proflavin) was evaluated. Binding constant and number of binding site were determined for proflavin-BSA interaction both in phosphate buffer (pH∼6.8) and in sodium dodecylsulphate media. The values of the thermodynamic parameters suggested that the key interacting forces are van der Waal's interaction and hydrogen bonding. Laser flash photolysis study reconfirmed the formation of complex between BSA and proflavin.     

甲钴胺与牛血清白蛋白相互作用的光谱特性

应用荧光光谱法、紫外吸收光谱法及共振光散射法,研究了甲钴胺 (Mecobalamin) 与牛血清白蛋白 (BSA) 之间的相互作用。在pH=7.40的三羟甲基胺基甲烷-盐酸 (Tris-HCl) 缓冲溶液中,随着甲钴胺浓度的增加,BSA的荧光强度、共振散射光强度逐渐减弱。通过计算不同温度(293,303,310 K)下的猝灭常数 (K_sv=5.40×10⁴,6.90×10⁴,8.00×10⁴ L/mol) 及扫描紫外吸收光谱,确定了甲钴胺对牛血清白蛋白的猝灭机理为动态猝灭。测定了该反应的表观结合常数 (K_A=1.68×10⁴,4.34×10⁴,7.90×10⁴ L/mol)和结合位点数 (n≈1)。利用热力学参数 (ΔH>0、ΔG<0和ΔS>0) 确定了分子间的作用力性质,作用力主要是疏水作用力,作用过程是自发的。同时应用同步荧光技术研究了甲钴胺对BSA构象的影响。结果表明,甲钴胺没有引起BSA构象的变化。     

伊文思蓝与牛血清白蛋白相互作用的研究

利用荧光光谱和紫外吸收光谱研究了伊文思蓝（Evans blue,EB）与牛血清白蛋白（Bovine serum albumin,BSA）的相互作用。伊文思蓝与牛血清白蛋白作用,使牛血清白蛋白的荧光发生猝灭,利用Stern-Volmer方程和荧光寿命的测定,确定了伊文思蓝对牛血清白蛋白的荧光猝灭为静态猝灭和非辐射能量转移。实验测得伊文思蓝与牛血清白蛋白的结合常数KBSA-EB为1.122×106L·mol-1,结合点数n为0.994;根据Foerster非辐射能量转移理论,得到伊文思蓝与牛血清白蛋白之间的能量转移效率E为0.276,作用距离r为3.14nm。同时,利用同步荧光光谱研究了牛血清白蛋白的构象变化。     

Synthesis of biomacromolecule-stabilized silver nanoparticles and their surface-enhanced Raman scattering properties

In this work, water soluble silver nanoparticles stabilized by biomacromolecule, were produced through using an aqueous solution of silver nitrate with Bovine Serum Albumin (BSA) under different reducing agents (such as sodium borohydride, hydrazine, N,N-dimethyl formamide) at the room temperature, where BSA provided the main function to form monodispersed silver nanoparticles. UV–vis spectroscopy, Fluorescence spectra, TEM and HR-TEM are used to characterize the BSA-capped silver nanoparticles under different condition. The results show that the formed silver nanoparticles have different size and morphology under the three different reducing agents. Moreover, the fluorescence intensity of BSA was drastically quenched in presence of Ag nanoparticles from the results of fluorescence spectra. Furthermore, the surface-enhanced Raman scattering effects of the formed silver nanoparticles were also displayed and we made a comparison under three different reducing agents.     

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.     

巴比妥钠与牛血清白蛋白结合反应的热力学研究

采用荧光光谱研究了药物巴比妥钠(BBTS)和牛血清白蛋白(BSA)的相互作用机理,运用热力学方法确定了巴比妥钠和牛血清白蛋白主要是以静电力相互作用,该过程是一个熵增加、Gibbs自由能降低的自发超分子作用过程,根据Stern-Volmer方程和Lineweaver-Burk双倒数曲线方程求出了其结合常数在高温时比在低温时小,得到巴比妥钠和牛血清白蛋白的结合比为1∶1,证实了巴比妥钠和牛血清白蛋白之间的作用是生成复合物的静态猝灭过程,并且采用同步荧光技术考察了巴比妥钠对牛血清白蛋白氨基酸残基微环境的影响。     

应用荧光猝灭法研究尼莫地平与牛血清白蛋白的相互作用

应用荧光光谱(FS)和紫外光谱(UV)研究了尼莫地平与牛血清白蛋白(BSA)之间的相互作用。尼莫地平与BSA的结合常数K_A为5.01×104(26 ℃)和4.46×104(36 ℃),尼莫地平在BSA上的结合位点数为1.08±0.01。根据Frster非辐射能量转移理论,求出了尼莫地平与BSA之间的结合距离为3.14 nm(26 ℃)和3.10 nm(36 ℃)。实验表明静态猝灭和非辐射能量转移是 导致尼莫地平对BSA荧光猝灭的两大原因。通过计算热力学参数,可知该药物与牛血清白蛋白的相互作用是一个吉布斯自由能降低的自发过程,且二者之间的作用力以静电相互作用为主。     

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。     

多光谱法与分子对接法研究盐酸四环素与牛血清白蛋白的相互作用

盐酸四环素属于抗生素类, 目前有关盐酸四环素和牛血清白蛋白二级结构的影响及作用机理报道较少。在模拟生理条件下,采用荧光光谱法、三维荧光光谱法、紫外-可见光谱法、圆二色谱法和傅里叶红外光谱法以及分子对接模拟法,研究了盐酸四环素与牛血清白蛋白(BSA)之间的相互作用。荧光光谱表明,盐酸四环素能有效猝灭BSA的内源荧光,猝灭机制属静态猝灭,通过Stern-Volmer方程计算结合常数Ka为2.813×105 L·mol-1(298 K)。根据Vant’s Hoff方程确定结合过程中的热力学参数ΔS=-151.1 J·mol-1·K-1、ΔH=-76.09 kJ·mol-1_, 两者之间作用为氢键和范德华力。同步荧光光谱、紫外光谱、三维荧光光谱、红外光谱、圆二色谱结果证明盐酸四环素能够改变BSA的二级结构和微环境。根据Föster’s非辐射能量转移理论,盐酸四环素与BSA结合距离为0.49 nm。希尔系数(n_H)值小于1,表明盐酸四环素与BSA结合后存在药物间协同作用。圆二色谱(CD)定量测定了盐酸四环素与BSA作用前后的二级结构含量：α-螺旋含量增加了9.16%(1:1)。分子对接模拟表明盐酸四环素通过氢键、疏水作用和范德华力等多种作用力结合在BSA的site Ⅰ(亚域ⅡA)。本研究有助于了解盐酸四环素与BSA的作用机制,也有助于理解盐酸四环素对蛋白质在储运过程中功能的影响。     

贝诺酯与牛血清白蛋白位点选择性结合的分子光谱研究

采用荧光光谱、紫外可见光谱、同步荧光光谱及三维荧光光谱等分子光谱方法,研究了生理条件下贝诺酯(BEN)与牛血清白蛋白(BSA)的相互作用。结果表明,BEN对BSA的内源荧光有显著的猝灭作用,猝灭机理为动态猝灭,二者之间的作用力类型以疏水作用为主,BEN与BSA发生反应后,使BSA的疏水环境极性增强,疏水性减弱,荧光强度降低。测得的表观结合常数和结合位点数分别是1 050 L·mol^-1和0.88,同时测得了焓变(ΔH)、熵变(ΔS)和自由能变(ΔG)等热力学参数。同步荧光和三维荧光光谱的结果表明,BEN使BSA的构象发生改变。利用荧光特异性位点探针DA和DP,通过竞争结合实验,监测BEN与BSA的结合位点,测得了位点Ⅰ和位点Ⅱ的表观结合常数分别为4 300 L·mol^-1和21 200 L·mol^-1,表明BEN与BSA优先在位点Ⅱ结合。     

柠檬酸钠与牛血清白蛋白相互作用的荧光光谱研究

利用荧光光谱技术研究了柠檬酸钠和牛血清白蛋白(BSA)之间的相互作用。柠檬酸钠对BSA荧光的猝灭机制属于形成复合物的静态猝灭过程。在20.0、32.0℃和36.5℃下柠檬酸钠与BSA之间的结合常数Ka分别为2.51×104、1.07×104、9.1×103L.mol-1,结合位点数近似为1。二者结合反应的主要作用力类型为氢键或范德华力。当pH值为7.4时二者的结合作用最强,同步荧光光谱显示两者的结合位点更接近于色氨酸。同时也研究了金属离子对二者作用的影响。     

基于低聚壳聚糖的希夫碱化合物与BSA的相互作用

窄分子量分布低聚壳聚糖CS_<i>n(n表示壳聚糖的聚合度,n=6, 8, 11)和对二甲氨基苯甲醛(DMABA)通过缩合反应得到了新型的基于壳聚糖的希夫碱化合物DMABA-CS_n。利用荧光光谱法,同步荧光光谱法,圆二色谱法(CD)和等温滴定微量热法(ITC)研究了DMABA-CS_n与牛血清白蛋白 (BSA)之间的相互作用。通过荧光光谱法探讨了DMABA-CS_n对BSA的荧光猝灭机制。结果表明,DMABA-CS_<i>n(n=6, 8, 11) 均能使BSA的荧光猝灭,猝灭机制是形成DMABA-CS_n/BSA复合物的静态猝灭。利用同步荧光光谱法和圆二色谱法考察了DMABA-CS_n对BSA构象的影响。研究结果表明,BSA的构象在DMABA-CS_n的溶液微环境中发生了变化。另外,ITC热力学测定结果(ΔH<0, ΔS<0, ΔG<0)表明,BSA与DMABA-CS_n的作用过程是自发进行的放热过程,二者之间的作用力类型主要是氢键和疏水作用。同时,研究结果也说明在一定的分子量范围内,随着CS_n聚合度的增加,DMABA-CS_n更容易与BSA结合。研究结果为DMABA-CS_<i>n(n=6, 8, 11)作为潜在药物的药理作用机制研究提供了一定的理论参考。     

Effect of temperature and pH on interaction between bovine serum albumin and cetylpyridinium bromide: Fluorescence spectroscopic approach

The fluorescence spectroscopic technique has been efficiently employed to investigate the interaction between bovine serum albumin (BSA) and cetylpyridinium bromide (CPB) under different pH and temperature conditions. The binding constant, number of binding sites, thermodynamic parameters such as ΔG, ΔH, ΔS, and nature of binding forces between BSA and CPB were obtained by measuring the steady state fluorescence quenching of BSA by CPB. The experimental results showed that the fluorescence quenching of BSA by CPB was a result of the formation of CPB-BSA complex. The static quenching was confirmed from the Stern-Volmer quenching constant at different temperatures. The effect of CPB on the conformation of BSA was analyzed using synchronous and three-dimensional fluorescence spectroscopy. pH dependence complex formation between BSA-CPB is due to the interaction between cationic side chain of CPB and the net charge developed on BSA. The distance ‘r’ between BSA and CPB was obtained according to the fluorescence resonance energy transfer.