Structure of molecular associates of fluorescent probes in solutions of human serum albumin

Spectral-luminescent characteristics and molecular association processes in solutions of human serum albumin are analyzed at different pH values for three fluorescent probes (eosin, erythrosin, and fluorescein). Common features for all three probes in protein solutions are quenching of the fluorescence, a red shift of the fluorescence maximum, a decrease in the degree of association, and an increase in the angle between dipole moments of dye molecules in dimers. This being so, differences between fluorescein and its halogen derivatives (eosin and erythrosin) are observed in the pH dependences of fluorescence, degree of association, and the angle between dipole moments of probe molecules in dimers. Translated from Zhurnal Prikladnoi Spektroskopii, Vol. 75, No. 6, pp. 782–787, November–December, 2008.     

Raman spectroscopy in investigations of secondary structure of human serum albumin at binding of nanomarkers of fluorescein family

The influence of binding of nanomarkers of fluorescein family to HSA on secondary structure of this protein at different values of pH was investigated by Raman spectroscopy method. The greatest changes in secondary structure of HSA, consisting in decreasing of α-helix sites, at binding of fluorescein to HSA occur at pH 5–6. The greatest changes in secondary structure of HSA, consisting in decreasing of α-helix sites, at binding of eosin or erythrosin to HSA take place at values of pH, smaller 5. The differences in changes in secondary structure of HSA at binding of these three nanomarkers are explained by dependences of binding of nanomarkers to HSA on pH which determined by value of electronegativity of atoms of lateral radicals in structural formulas of nanomarkers and, therefore, by value of pK of their ionized groups.     

Determination of the binding constants of nanomarkers of the fluorescein family to human serum albumin

Using three models, the constant of quenching of fluorescence of nanomarkers of the fluorescein family and the actual constants of its binding to human serum albumin (HSA) at different values of pH are determined. The presence of two mechanisms of binding of nanomarkers of the fluorescein family to HSA and anti-cooperativity are shown. The dependence of the constants of the quenching fluorescence of nanomarkers on pH was found: for fluorescein this was nonlinear, for its halogen derivatives (erythsosine, eosin, and Rose Bengal) it was monotonous and decreased with an increase of pH. It is shown that the electronegativity of the atoms in the structural formulas of nanomarkers of the fluorescein family influences the values of the constants of binding of nanomarkers to HSA.     

Molecular association processes and fluorescent characteristics of nanomarkers of the fluorescein family in solutions of bovine serum albumin

An analysis of the molecular association and fluorescent characteristics of nanomarkers of the fluorescein family, viz., fluorescein, erythrosine, eosine, and Rose Bengal, in BSA solutions was conducted. For all the markers a decreasing degree of molecular association was observed in the BSA solutions as compared with the solutions without protein. In the solutions with BSA, fluorescence quenching and red shifting of the fluorescence spectrum maximum occurred for the solutions with BAS compared with solutions without protein for the markers of the fluorescein family. The dependences of the degree of molecular association on pH differed for fluorescein and its halogen derivatives. The efficiency dependences of nanomarker binding with BSA on pH differed for fluorescein and its halogen derivatives.     

The fluorescence characteristics and the molecular association of the Rose Bengal nanomarker in human serum albumin solutions

The decrease in the degree of molecular association of the Rose Bengal nanomarker in solutions with the addition of human serum albumin (HSA) has been revealed. It has been observed that in solutions with the addition of HSA the fluorescence quenching and the shifting of the fluorescence spectrum peaks of Rose Bengal to the red take place. It has been shown that the dependence of the effective binding constant of binding Rose Bengal to HSA steadily decreases with an increase in the pH value. It has been established that the values of the molecular association degree of Rose Bengal and the values of the effective constant of its binding to HSA depend on the magnitude of the electronegativity of the atoms in its structural formula, as well as on the pK values of its ionizable groups.     

Investigation of the rotational diffusion of the Rose Bengal fluorescent nanomarker in human serum albumin solutions

The polarized fluorescence of the Rose Bengal fluorescent nanomarker in HSA solutions was investigated and parameters of its rotational diffusion were calculated. The increase in the degree of fluorescence polarization, rotational relaxation time, and the effective hydrodynamic radius of Rose Bengal, as well as the decrease of the rotational diffusion coefficient in HSA solutions, were found. The effects of the electronegativity of atoms in the structure of the nanomarker on the parameters of its rotational diffusion were established based on comparison of Rose Bengal with other nanomarkers of the homologous family.     

Denaturation of human serum albumin initiated by cetyltrimethylammonium bromide as monitored via the intrinsic fluorescence of the protein and fluorescence of an eosin probe

An analysis of the intrinsic fluorescence of the protein and the fluorescence of the eosin molecular probe in solutions of the protein was used to study the denaturation of human serum albumin (HSA) under the action of cetyltrimethylammonium bromide (CTAB), a cationic detergent, at various pH values. The denaturation of HSA under the influence of CTAB is a single-stage process over the entire pH range covered, pH (3.5–8.0). The maximum possible loosening of HSA globules in the presence of CTAB is reached at [CTAB] = 4 mmol/l. This detergent effectively denatures HSA at pH values higher than the pI of the protein (4.7).     

HSA-磷钼杂多酸缔合纳米微粒体系荧光猝灭机理研究

在pH 7.40的Tris-HCl缓冲溶液中, 磷钼杂多酸(PMA)呈浅黄色,在可见光区没有明显的吸收峰;人血清白蛋白(HSA) 呈无色,在可见光区也没有明显的吸收峰,但在350 nm处有一荧光峰。当有PMA存在时,HSA与PMA形成缔合纳米微粒, HSA-PMA缔合纳米微粒的粒径约为80 nm。经研究发现HSA对PMA有增色和减色效应,PMA对HSA有荧光猝灭作用;PMA对色氨酸(Trp)和酪氨酸(Tyr)也有一定的荧光猝灭作用,但这两种荧光猝灭机理不同,且没有形成缔合纳米微粒。PMA对色氨酸(Trp)和酪氨酸的荧光猝灭作用主要是由于PMA 在发射波长范围内存在一定的分子吸收,即通常所报道过的能量转移所至。研究结果表明,HSA-PMA缔合纳米微粒和界面的形成是导致该体系的荧光猝灭、共振散射增强及增色和减色效应的根本原因。     

叶酸与人血清白蛋白结合作用的光谱研究

在不同温度下的pH 7.4的Tris-HCl缓冲溶液体系中, 采用荧光光谱、紫外吸收光谱和同步荧光光谱研究了人血清蛋白与叶酸的相互作用。研究表明,这种相互作用使人血清白蛋白发生内源荧光猝灭,属于静态猝灭机制。通过计算得到人血清蛋白与叶酸在17和37 ℃下静态猝灭的猝灭速率常数分别为7.396 6×104和7.2652×104 L·mol-1、结合常数分别为7.50×104和1.98×105 L·mol-1、结合位点数均为1。根据Förster非辐射能量转移机理,求算出给体(HSA)与受体(叶酸)间的作用距离和能量转移效率分别为1.77和0.052 65 nm,并结合热力学参数说明了叶酸分子与人血清白蛋白的作用以疏水作用为主,同时也存在静电引力。利用同步荧光光谱研究了人血清蛋白与叶酸的相互作用中HSA的构象变化,发现色氨酸残基所处环境的疏水性降低,说明叶酸分子进入了人血清白蛋白的疏水腔中。     

Complexation of insecticide chlorantraniliprole with human serum albumin: Biophysical aspects

Chlorantraniliprole is a novel insecticide belonging to the diamide class of selective ryanodine receptor agonists. A biophysical study on the binding interaction of a novel diamide insecticide, chlorantraniliprole, with staple in vivo transporter, human serum albumin (HSA) has been investigated utilizing a combination of steady-state and time-resolved fluorescence, circular dichroism (CD), and molecular modeling methods. The interaction of chlorantraniliprole with HSA gives rise to fluorescence quenching through static mechanism, this corroborates the fluorescence lifetime outcomes that the ground state complex formation and the predominant forces in the HSA-chlorantraniliprole conjugate are van der Waals forces and hydrogen bonds, as derived from thermodynamic analysis. The definite binding site of chlorantraniliprole in HSA has been identified from the denaturation of protein, competitive ligand binding, and molecular modeling, subdomain IIIA (Sudlow's site II) was designated to possess high-affinity binding site for chlorantraniliprole. Moreover, using synchronous fluorescence, CD, and three-dimensional fluorescence we testified some degree of HSA structure unfolding upon chlorantraniliprole binding.     

荧光光谱法结合分子对接研究人血清白蛋白 对齐墩果酸与熊果酸的异构体识别作用

五环三萜化合物齐墩果酸(OA)与熊果酸(UA)为同分异构体,具有相似的理化性质和稍有差异的药理活性。目前人们主要采用各种色谱、质谱类方法实现对OA与UA的异构体识别,未见使用荧光光谱法的报道。提出了一种使用荧光猝灭法实现对OA与UA异构体识别的方法。首先考察了两种常见的血清蛋白-牛血清白蛋白(BSA)与人血清白蛋白(HSA)同OA与UA的作用情况,结果表明OA与UA均可有效地猝灭BSA与HSA的荧光发射。对所得荧光猝灭数据计算可知OA,UA与BSA,HSA作用的双分子猝灭速率常数(K_q)均远大于生物大分子荧光猝灭所观察到的最大散射碰撞速率常数2.0×1010 L·(mol·s)-1,说明猝灭类型均为静态猝灭,即OA与UA均是通过与BSA及HSA形成稳定复合物方式实现荧光猝灭的。应用双对数方程对所得荧光猝灭数据计算可知OA,UA与BSA,HSA所形成的复合物中结合位点数在0.90～1.26之间,说明所形成的复合物为1∶1型。BSA与OA,UA所形成复合物的表观结合常数(K_A)为同一数量级,相差不大,但是HSA与OA,UA所形成复合物的K_A差别很大,HSA-UA复合物的KA比HSA-OA复合物高124.91倍,表明HSA-UA复合物的稳定性更强。同步荧光实验结果显示,OA与UA的加入对于HSA波长差(Δλ)为60 nm同步荧光光谱的影响大于Δλ为15 nm的同步荧光光谱,由此可以说明OA与UA在HSA上的结合位点可能位于Trp残基附近。分子对接模拟计算结果表明OA与UA均对接在HSA结构中一个疏水性空腔中,主客体之间存在强烈的氢键与疏水作用。OA同Arg218,His242,Pro447等残基间存在氢键作用,键长分别为2.95,2.97与3.17 Å,此外还与Lys195,Lys199,Trp214,Arg222,Leu238,Asp451和Tyr452等七个氨基酸残基间存在疏水作用。UA同Trp214,Arg218和Lys444等残基存在氢键作用,键长分别为3.01,2.88与2.65 Å,此外还与Leu198,Gln221,Arg222,Asn295,Val343,Pro447,Cys448,Asp451和Val455等9个氨基酸残基间存在疏水作用。由于UA同HSA作用位点数目多于OA,说明UA与HSA疏水性空腔的空间匹配程度更高。因此,认为HSA-UA与HSA-OA复合物间稳定性差异是HSA实现对OA与UA异构体识别的原因。     

Spectral and fluorescent indication of the acid-base properties of biopolymer solutions

Methods of absorption and fluorescent spectroscopy and fluorescent chronoscopy are used to investigate aqueous fluorescein solutions with pH values increasing from 1.79 to 11.67. For each method, the calibration curves are drawn for the fraction of dianion fluorescein species, quantum fluorescence yield, and amplitude dianion contribution to the fluorescence intensity on the pH values of aqueous solutions. The calibration curves allow the pH values of aqueous fluorescein solutions with gelatin (pH = 6.4 ± 0.1, 6.6 ± 0.2, and 6.6 ± 0.1), starch (pH = 6.3 ± 0.1, 6.3 ± 0.2, and 6.4 ± 0.1), and acid-soluble chitosan additions (pH = 5.4 ± 0.1, 5.6 ± 02, and 5.8 ± 0.1) to be estimated by these three methods. The pH values of biopolymer solutions obtained by the potentiometer method for starch (6.51) and gelatin (6.69) and by the three spectral and fluorescent methods are close; they are significantly underestimated (3.85) for chitosan solutions.     

同步与三维荧光光谱法研究日落黄和人血清蛋白的相互作用

日落黄是一种具有潜在危害性的人工合成色素。选用人血清蛋白为研究对象,利用荧光光谱法分析了日落黄对人血清蛋白荧光的猝灭作用,确定了荧光猝灭反应的猝灭常数Ksv,结果表明日落黄对人血清蛋白的荧光猝灭作用类型为静态猝灭。运用同步荧光光谱法分析日落黄与人血清蛋白结合表明：日落黄与人血清蛋白的结合靠近色氨酸附近,引发色氨酸残基趋向伸展状态,人血清蛋白的结构发生改变。此外,在模拟人体生理条件下选用10种不同的金属离子加入到日落黄和人血清蛋白的反应体系中,运用三维荧光法检测金属离子对体系的影响,结果表明Cu2+,Pb2+,Ni2+和Mn2+对猝灭过程有着促进作用,其中Ni2+的影响最大,增幅达 22.6 %;而Fe2+和Zn2+对猝灭过程有着抑制作用,抑制率达14.12%和14.2%。本研究的实验方法适用于研究食品添加剂的毒性,有助于保护食品安全,维护人体健康。     

Spectroscopic study on interaction between bisphenol A or its degraded solution under microwave irradiation in the presence of activated carbon and human serum albumin

In this study, the interaction between bisphenol A (BPA) or its degraded solution under microwave irradiation after their adsorption on activated carbon (AC/MW) and human serum albumin (HSA) was investigated by UV-vis and fluorescence spectroscopy techniques. The results showed that BPA could bind to HSA molecule, which could cause the stretch of peptide chains. Also, the degraded BPA solution with a few residues could still interact with HSA. Otherwise, the influences of pH and ionic strength on the interaction were estimated. The fluorescence quenching modes of HSA initiated by BPA at three temperatures (298, 310 and 315 K) were all obtained using Stern-Volmer and Lineweaver-Burk equations. The number of binding sites (n), binding constants (K_D) and energy transfer efficiency (E) were all calculated. The thermodynamic parameters (ΔH, ΔG and ΔS) and binding distances (r) were all measured at the three temperatures, respectively. Synchronous fluorescence spectroscopy was also carried out.     

Separate and simultaneous binding effects of aspirin and amlodipine to human serum albumin based on fluorescence spectroscopic and molecular modeling characterizations: A mechanistic insight for determining usage drugs doses

The binding of aspirin (ASA) and amlodipine (AML) to human serum albumin (HSA) in aqueous solution was investigated by multiple techniques such as fluorescence quenching, resonance light scattering (RLS), three-dimensional fluorescence spectroscopy, FT-IR and zeta-potential measurements in an aqueous solution at pH=7.4. For the protein-ligand association reaction, fluorescence measurements can give important clues as to the binding of ligands to proteins, e.g., the binding mechanism, binding mode, binding constants, binding sites, etc. Fluorescence spectroscopy showed that ASA and AML could quench the HSA fluorescence spectra, and this quenching effect became more significant when both ASA and AML coexisted. The results pointed at the interaction between HSA and both drugs as ternary systems decreasing the binding constant and binding stability of the HSA-drug complex as a binary system. Therefore, by reducing the amount of drugs transported to their targets, the free drug concentration of the target would be reduced, lowering the efficacy of the drugs. It was demonstrated that there exists antagonistic behavior between the two drugs when it comes to binding of HSA. Furthermore, the fluorescence results also showed that the quenching mechanism of HSA-drug complexes as binary and ternary systems is a static procedure. The number of binding sites of HSA-ASA, (HSA-AML)ASA, HSA-AML and (HSA-ASA) AML were 1.31, 0.92, 1 and 0.93, respectively. Due to the existence of the antagonistic action between ASA and AML, the binding distance r was reduced. The results of synchronous fluorescence and three-dimensional fluorescence spectra showed that the antagonistic action between ASA and AML would alter the micro-environment around Trp and Tyr residues. Moreover, the simultaneous presence of ASA and AML during binding to HSA should be taken into account in multidrug therapy, as it induces the necessity of a monitoring therapy owing to the possible increase of uncontrolled toxic effects. Molecular dynamic studies showed that the affinity of each of the drugs to HSA was reduced in the presence of significant amounts of the other. In the interaction of HSA with both drugs, the zeta potential of the ternary system is more negative than its binary counterpart. The zeta-potential results suggested induced conformational changes on HSA that confirmed the experimental and theoretical results.     

Hydrophobic Interactions in Donor-Disulphide-Acceptor (DSSA) Probes Looking Beyond Fluorescence Resonance Energy Transfer Theory

Donor –linker –acceptor (DSSA) is a concept in fluorescence chemistry with acceptor being a fluorescent compound (FRET) or quencher. The DSSA probes used to measure thiol levels in vitro and in vivo. The reduction potential of these dyes are in the range of ?0.60 V, much lower than the best thiol reductant reported in literature, the DTT (?0.33 V). DSSA disulphide having an unusually low reduction potential compared to the typical thiol reductants is a puzzle. Secondly, DSSA probes have a cyclized rhodamine ring as acceptor which does not have any spectral overlap with fluorescein, but quenches its absorbance and fluorescence. To understand the structural features of DSSA probes, we have synthesized DSSANa and DSSAOr. The calculated reduction potential of these dyes suggest that DSSA probes have an alternate mechanism from the FRET based quenching, namely hydrophobic interaction or dye to dye quenching. The standard reduction potential change with increasing complexity and steric hindrance of the molecule is small, suggesting that ultra- low Eo’ has no contribution from the disulphide linker and is based on structural interactions between fluorescein and cyclized rhodamine. Our results help to understand the DSSA probe quenching mechanism and provide ways to design fluorescent probes.     

利复星与人血清白蛋白作用的荧光光谱研究

用荧光光谱研究了药物利复星(Levofloxacin)与人血清白蛋白(HSA)的作用和影响,测量发现人血清白蛋白的最大激发峰位于286.70 nm处。在向该溶液滴加Levofloxacin时,原有的343.70 nm发射峰强度明显减弱, 且向长波长稍有移位,并出现了位于503.96 nm的新荧光发射峰(利复星的发射峰), 利复星对HSA荧光有猝灭作用。利复星Levofloxacin的503.96 nm荧光的激发峰则位于300.16和336.16 nm。当向该溶液滴加利复星时,300.16和336.16 nm的激发峰仅向长波长方向稍有移动。利复星对HSA的离解常数K_d=3.65×10-5(mol·L-1)。利复星的结合常数为K_S=2.742×104(L·mol-1)。利复星-HSA体系的猝灭过程不是因为分子扩散和碰撞所引起的动态猝灭,而是分子之间结合形成了化合物所引起的静态猝灭。利复星对HSA的能量转移效率为E=0.372, 利复星和人血清白蛋白的色氨酸残基的结合位置为R=1.933 nm。     

三种肉桂酰胺衍生物的制备及其与人血清白蛋白的结合

基于临床上肉桂酰胺类药物的广泛应用及优异性能, 以间羟基肉桂酸为母体, 分别与不同氨基酸反应, 设计合成了3种未见报道的肉桂酰胺类衍生物, 并用MS、IR、¹H NMR、¹³C NMR进行结构表征.采用分子对接技术和荧光光谱法、同步荧光光谱法、紫外-可见光谱法共同研究了3种衍生物分别和人血清白蛋白(HSA)相结合的机理.AutoDock对接显示, 这3种衍生物结合在HSA亚结构域ⅡA(即site Ⅰ)的疏水腔内, 维系衍生物与HSA的主要作用力为氢键和范德华力, 同时还存在着疏水作用.光谱实验结果表明, 在体外生理条件下, 衍生物都与HSA形成复合物, 对HSA内源荧光产生静态猝灭, 且对其构象产生影响.根据不同温度下的热力学函数, 确定主要作用力均是氢键和范德华力.分子对接与实验获得了一致的结果.     

荧光法研究木犀草素与人血清白蛋白的相互作用

用荧光光谱、同步荧光光谱和紫外吸收光谱方法,研究了木犀草素与人血清白蛋白（HSA）的相互作用。研究表明木犀草素对HSA有较强的荧光猝灭作用,根据不同温度下木犀草素对HSA的荧光猝灭作用,利用Stern-Volmer方程处理实验数据,表明木犀草素对HSA的荧光猝灭作用属于静态猝灭。根据Fōrster非辐射能量转移理论计算了木犀草素与HSA间的结合常数和结合位点数,求得了木犀草素与HSA间的结合距离r。热力学数据表明二者主要靠疏水作用力结合。同时用同步荧光光谱探讨了木犀草素对HSA构象的影响。     

Spectroscopic and Molecular Modeling Studies of the Interaction Between 4′-O-(α-L-Oleandrosyl)daunorubicin and Human Serum Albumin and Its Analytical Application

4′-O-(α-L-Oleandrosyl)daunorubicin (ODNR) is a disaccharide analogue of daunorubicin with potent antitumor activity against leukemia cell line K562 cells and colon cancer cell line SW620 cells. In this paper, the binding interaction of ODNR with human serum albumin (HSA) was investigated under simulative physiological conditions by fluorescence spectroscopy in combination with UV absorption spectroscopy and molecular modeling method. A strong fluorescence quenching reaction of ODNR to HSA was observed and the quenching mechanism was suggested as static quenching according to the Stern-Volmer equation. The binding constants (K) at different temperatures as well as thermodynamic parameters, enthalpy change (ΔH) and entropy change (ΔS), were calculated according to relevant fluorescent data and Van’t Hoff equation. The hydrophobic interaction was a predominant intermolecular force in order to stabilize the complex, which was in agreement with the results of molecular modeling study. In addition, the effects of other ions on the binding constants were also studied. Moreover, the synchronous fluorescence technique was successfully employed to determine the total proteins in serum, urine and saliva samples at room temperature under the optimum conditions with a wide linear range and satisfactory results.