Insights into the energetics and mechanism underlying the interaction of tetraethylammonium bromide with proteins

Calorimetry has been employed to investigate the quantitative energetic aspects and mechanism underlying protein–tetraethylammonium bromide (TEAB) interactions. Differential scanning calorimetry and UV–Visible spectroscopy have been used to study the thermal unfolding of three proteins of different structure and function (bovine serum albumin, α-lactalbumin, and bovine pancreatic ribonuclease A). The mode of interaction has been studied by using isothermal titration calorimetry, which demonstrates the absence of appreciable specific binding of TEAB to the protein. This suggests the involvement of solvent mediated effects and, possibly weak non-specific binding. The thermal unfolding transitions were found to be calorimetrically reversible for α-lactalbumin and bovine pancreatic ribonuclease A and partially reversible in the case of bovine serum albumin. The results indicate protein destabilization promoted by the TEAB interaction. The preferential interaction parameters of TEAB with α-lactalbumin and ribonuclease A confirm that an increased interaction of the hydrophobic groups of the TEAB with that of the protein upon denaturation is responsible for the reduced thermal stability of the protein. The decrease in the thermal stability of proteins in the presence of TEAB is well supported by a red shift in the intrinsic fluorescence of these proteins leading to conformational change thereby shifting the native ? denatured equilibrium towards right. The forces responsible for the thermal denaturation of the proteins of different structure and function in the presence of TEAB are discussed.     

Thermodynamics of the interaction of a dirhamnolipid biosurfactant secreted by Pseudomonas aeruginosa with phospholipid membranes

Rhamnolipids are bacterial biosurfactants produced by Pseudomonas spp. These compounds have been shown to present several interesting biological activities, restricting the growth of Bacillus subtilis and showing zoosporicidal activity on zoosporic phytopathogens. It has been suggested that the interaction with the membrane could ultimately be responsible for these actions. Therefore, it is of great interest to gain insight into the molecular mechanism of the interaction of purified rhamnolipids with the various phospholipid components of biological membranes. In this work, the critical micelle concentration (cmc) of a purified dirhamnolipid produced by Pseudomonas aeruginosa has been determined both by isothermal titration calorimetry and surface tension measurements. The partition coefficients from water to membranes of different compositions, as well as the corresponding thermodynamic parameters, have been determined by isothermal titration calorimetry measurements. The results indicate that dirhamnolipid membrane partitioning is an entropically driven process. The presence of cholesterol in the membrane decreases the partition of dirhamnolipid. On the other hand, phosphatidylethanolamine stimulates dirhamnolipid binding, whereas lysophosphatidylcholine opposes binding, suggesting that the biosurfactant behaves as an inverted-cone-shaped molecule. The values obtained for the cmc and the partition constant are considered in relation to the surfactant potency of dirhamnolipids.     

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.     

Quantitative aspects of recognition of the antibiotic drug oxytetracycline by bovine serum albumin: Calorimetric and spectroscopic studies

A quantitative understanding of the mode of interaction of drugs with target proteins provides a guide for the synthesis of new drug molecules. The binding of the antibiotic drug oxytetracycline with serum albumin has been studied by a combination of isothermal titration calorimetry (ITC), differential scanning calorimetry (DSC), steady-state and time-resolved fluorescence spectroscopy, and circular dichroism spectroscopy. The values of the binding constant (K), enthalpy change (ΔH), entropy (ΔS), and stoichiometry of binding have been determined along with the associated conformational changes in the protein. Oxytetracycline binds to bovine serum albumin with a 1:1 stoichiometry and with a weakly temperature dependent association constant of 1.8 · 10⁴ at T = 298.15 K. The effect of ionic strength, tetrabutylammonium bromide, and sucrose on the thermodynamic parameters obtained from ITC and DSC measurements indicate involvement of predominantly ionic and hydrophobic interactions with a minor hydrogen bonding contribution in the drug-protein complexation. The DSC results on the binding of oxytetracycline with bovine serum albumin in the absence and presence of these additives provide quantitative information on the effect of drugs on the stability of bovine serum albumin, and suggest preferential complexation of one of the domains of the protein. The results further indicate that the drug occupies binding site II on bovine serum albumin.     

The interaction of cationic and anionic porphyrins with the bovine serum albumin in borate buffer

The interaction of water-soluble porphyrins with the bovine serum albumin in borate buffer at pH 8.6 has been studied. The localization of porphyrins in the protein globule has been determined. It was established that the native conformation of albumin upon binding with the porphyrins is preserved, however, the anionic porphyrins are exhibit wedging effect on the albumin domains. The binding constants were obtained from fluorescence spectroscopy data.     

咪唑型离子液体与牛血清蛋白的缔合特性

通过紫外-可见光谱、荧光光谱、同步荧光光谱、圆二色谱、衰减全反射红外光谱、负染-透射电镜、等温滴定微量热等实验方法系统地探讨了咪唑型离子液体与牛血清蛋白(BSA)的缔合特性.结果发现,离子液体[Bmim]Cl的加入使得BSA的紫外吸收强度增加,同时也会导致其荧光猝灭,并且这种猝灭是静态猝灭.同步荧光的研究结果表明,[Bmim]Cl分子可与蛋白质中接近色氨酸残基的区域发生相互作用,使蛋白质的构象和内部的疏水结构发生改变;负染色法透射电镜直观地显示了加入离子液体后形成的蛋白质-离子液体复合物结构逐渐变大;圆二色谱和衰减全反射红外光谱表明:在离子液体与BSA缔合过程中,离子液体的加入使得BSA二级结构中的α-螺旋和β-折叠的含量降低,从而引起蛋白质二级结构的变化;表面张力法和等温滴定微量热法进一步证实上述缔合作用为静电作用和疏水作用共同作用的结果,但离子液体的烷基链与BSA疏水内腔之间的疏水作用是离子液体与BSA缔合的主要驱动力.     

甲基紫与牛血清白蛋白作用的荧光光谱研究

在pH 7.40的Tris-HC1缓冲溶液体系中,采用荧光光谱法和同步荧光光谱法研究了甲基紫(MV)与牛血清白蛋白(BSA)相互作用.结果表明MV与BSA相互作用两者存在一个结合位点,结合常数(KA)为7.628×103 L/mol,MV与BSA主要发生疏水作用,反应是一个吸热、熵增的自发过程.     

光谱法研究喜树碱与牛血清白蛋白的相互作用

采用多种光谱技术对喜树碱和牛血清白蛋白的相互作用进行了研究.结果表明喜树碱和牛血清白蛋白可形成基态复合物,引起牛血清白蛋白内源荧光猝灭.通过计算获得了二者在不同温度下的结合常数及结合位点数.根据喜树碱和牛血清白蛋白结合的热力学参数,确定了二者之间主要为疏水作用力.根据F(o)rster非辐射能量转移理论确定了喜树碱和牛血清白蛋白的作用距离.同步荧光光谱显示喜树碱主要与蛋白中色氨酸残基发生相互作用,改变其周围的局部构象.红外光谱提示喜树碱可引起蛋白的构象发生改变,α-螺旋二级结构减少.     

Competitive adsorption of sodium dodecyl sulfate and polyethylene oxide at the air/water interface

Geoinspired synthetic chrysotile, which represents an ideal asbestos reference standard, has been utilized to investigate homomolecular exchange of bovine serum albumin (BSA), the major plasma protein, between the adsorbed and dissolved state at the interface between asbestos fibers and biological medium. FTIR spectroscopy has been used to quantify BSA structural modifications due to surface adhesion on chrysotile fibers as a function of the surface coating extent. Circular dichroism spectroscopy has been used to investigate the adsorption/desorption equilibrium through analysis of the BSA structural perturbations after protein desorption from chrysotile surface. Data results show clearly that in the solid state BSA modifications are driven by surface interaction with the substrate, following a bimodal adsorption evidenced by two different binding constants. On the other hand, BSA desorbed in solution is able to rearrange, in the lack of substrate, although keeping irreversible modifications with respect to the native species. The lack of regaining its native structure certainly affects albumin interaction with biological environment. The present investigation on the stoichiometric synthetic geoinspired chrysotile nanocrystals is the first approach toward a deeper attempt to use standard synthetic chrysotile reference samples in mimicking the behavior of asbestos fibers and allows to better understand their interaction with a biological environment.     

Interaction of polyethyleneimine-functionalized ZnO nanoparticles with bovine serum albumin

In biological fluids, nanoparticles are always surrounded by proteins. As the protein is adsorbed on the surface, the extent of adsorption and the effect on the protein conformation and stability are dependent on the chemical nature, shape, and size of the nanoparticle (NP). We have carried out a detailed investigation on the interaction of bovine serum albumin (BSA) with polyethyleneimine-functionalized ZnO nanoparticles (ZnO-PEI). ZnO-PEI was synthesized using a wet chemical method with a core size of ~3-7 nm (from transmission electron microscopy). The interaction of BSA with ZnO-PEI was examined using a combination of calorimetric, spectroscopic, and computational techniques. The binding was studied by ITC (isothermal titration calorimetry), and the result revealed that the complexation is enthalpy-driven, indicating the possible involvement of electrostatic interaction. To investigate the nature of the interaction and the location of the binding site, a detailed domain-wise surface electrostatic potential calculation was performed using adaptive Poisson-Boltzmann software (APBS). The result shows that the protein surface can bind the nanoparticle. On binding ZnO-PEI, the protein gets destabilized to some extent, as displayed by CD (circular dichroism) and FTIR (Fourier transform infrared) spectroscopy. Chemical and thermal denaturation of BSA, when carried out in the presence of ZnO-PEI, also indicated a small perturbation in the protein structure. A comparison of the enthalpy and entropy components of binding with those derived for the interaction of BSA with ZnO nanoparticles explains the effect of hydrophilic cationic species attached on the NP surface. The effect of the NP surface modification on the structure and stability of BSA would find useful applications in nanobiotechnology.     

Protein denaturation kinetic processes of a simple and a complex reaction mechanism analyzed by an iso-conversional method

The protein denaturation kinetic processes of a simple and a complex reaction mechanism represented by bovine serum albumin and hen egg-white lysozyme were analyzed by an iso-conversional method using differential scanning calorimetry. After differential scanning calorimetry using the iso-conversional method, the results were found to pose distinct contrasts between the two proteins. Bovine serum albumin showed an increasing peak temperature of the transition as the scan rate and protein concentration increased, whereas hen egg-white lysozyme exhibited almost constant peak temperature. The differential scanning calorimetry transition of bovine serum albumin was calorimetrically irreversible, while one part of hen egg-white lysozyme denaturation process was irreversible during which aggregation occurred and the other part was reversible. The iso-conversional method indicated that the value of bovine serum albumin apparent activation energy hardly varied with the degree of conversion, which showed that the denaturation kinetic process should conform to single reaction model. Using the master plots method, the most possible kinetic model for bovine serum albumin denaturation might be described by F _n kinetic model. On the contrary, the hen egg-white lysozyme value of apparent activation energy decreased with the increase of degree of conversion. It was not a process involving the two standard reversible states, and can be described by the simple Lumry–Eyring model. The iso-conversional method provides new opportunities in exploring a simple and a complex reaction mechanism of protein denaturation.     

三七总皂甙对牛血清白蛋白溶液构象的影响

应用衰减全反射傅立叶变换红外光谱结合荧光光谱和紫外光谱研究了中药三七 的有效成分三七总皂甙与牛血清白蛋白（BSA）的相互作用，采用对蛋白质红外光 谱酰氨Ⅰ带和酰氨Ⅲ带进行曲线拟合的方法，定量分析了不同浓度三七总皂甙对 BSA二级结构的影响，发现随着三七总皂甙浓度的增加，蛋白分子结构逐渐发生了 由螺旋向折叠的转化。a-螺旋结构减少了3%，β-折叠结构增加了约5%，其它二级 结构没有明显的变化，红外差谱和荧光光谱的结果为药物与蛋白质的作用引起牛血 清白蛋白溶液构象的变化提供了佐证，紫外光谱反映了单体皂甙与蛋白质的结合常 数的差异。     

Spectroscopic studies on the interaction of bovine (BSA) and human (HSA) serum albumins with ionic surfactants

Bovine (BSA) and human (HSA) serum albumins are frequently used in biophysical and biochemical studies since they have a similar folding, a well known primary structure, and they have been associated with the binding of many different categories of small molecules. One important difference of BSA and HSA is the fact that bovine albumin has two tryptophan residues while human albumin has a unique tryptophan. In this work results are presented for the interaction of BSA and HSA with several ionic surfactants, namely, anionic sodium dodecyl sulfate (SDS), cationic cethyltrimethylammonium chloride (CTAC) and zwitterionic N-hexadecyl-N,N-dimethyl-3-ammonium-1-propanesulfonate (HPS), as monitored by fluorescence spectroscopy of intrinsic tryptophans and circular dichroism spectroscopy. On the interaction of all three surfactants with BSA, at low concentrations, a quenching of fluorescence takes place and Stern-Volmer analysis allowed to estimate their 'effective' association constants to the protein: for SDS, CTAC and HPS at pH 7.0 these constants are, respectively, (1.4+/-0.1) x 10(5) M(-1), (8.9+/-0.1) x 10(3) M(-1) and (1.4+/-0.1) x 10(4) M(-1). A blue shift of maximum emission is observed from 345 to 330 nm upon surfactant binding. Analysis of fluorescence emission spectra allowed to separate three species in solution which were associated to native protein, a surfactant protein complex and partially denatured protein. The binding at low surfactant concentrations follows a Hill plot model displaying positive cooperativity and a number of surfactant binding sites very close to the number of cationic or anionic residues present in the protein. Circular dichroism data corroborated the partial loss of secondary structure upon surfactant addition showing the high stability of serum albumin. The interaction of the surfactants with HSA showed an enhancement of fluorescence at low concentrations, opposite to the effect on BSA, consistent with the existence of a unique buried tryptophan residue in this protein with considerable static quenching in the native state. The effects of surfactants at low concentrations were very similar to those of myristic acid suggesting a non specific binding through hydrophobic interaction modulated by eletrostatic interactions. The changes in the vicinity of the tryptophan residues are discussed based on the recently published crystallographic structure of HSA myristate complex (S. Curry et al., Nat. Struct. Biol. 5 (1998) 827).     

氨基硫脲芳基铱抗癌物与牛血清蛋白相互作用机制

在生理条件下,利用紫外-可见光谱和荧光光谱分别研究氨基硫脲芳基铱抗癌物与牛血清蛋白的相互作用。确定作用机制,讨论结合力类型,并研究共存离子对结合常数的影响。实验结果表明,间甲氧基苯甲醛4-苯基-3-氨基硫脲芳基铱(TSC-Ir-6)配合物对牛血清白蛋白(BSA)的内源性荧光有猝灭作用,其猝灭类型为静态猝灭;通过计算二者相互作用时的热力学参数,其结果表明TSC-Ir-6与BSA的结合是一个自发过程(ΔG<0),且体系ΔH<0,ΔS<0,其相互作用力类型为氢键和范德华力,结合位点约为1。共存离子的存在使TSC-Ir-6与BSA之间的结合常数明显增大,结合力更强,提高了其在血浆中的滞留时间,可能得到更好的治疗效果。     

Study on the binding of puerarin to bovine serum albumin by isothermal titration calorimetry and spectroscopic approaches

The interaction of a flavonoid molecule (puerarin) with bovine serum albumin (BSA) was characterized by isothermal titration calorimetry (ITC), optical spectroscopic technique, and molecular modeling method under physiological conditions. The binding parameters for the reaction were calculated according to ITC experiments at different temperatures. The thermodynamic parameters, negative enthalpy changes (ΔH), and positive entropy (ΔS) indicated that the binding processes were entropically driven. The alterations of protein secondary structure in the presence of puerarin in aqueous solution were estimated by the evidences from FT-IR and CD spectroscopy with reductions of α-helices. On the basis of fluorescence resonance energy transfer (FRET) between excited tryptophan in BSA and BSA bound puerarin, the critical transfer distance and mean distance between tryptophan in BSA and puerarin were estimated.     

水杨酸金属配合物与牛血清白蛋白的相互作用

本文采用荧光法研究了水杨酸金属配合物与牛血清白蛋白的相互作用。观察到水杨酸金属配合物对牛血清白蛋白荧光产生猝灭现象,猝灭方式为静态猝灭。计算了结合常数和结合位点数。并且用圆二色谱法研究水杨酸金属配合物对牛血清白蛋白二级结构的影响,发现水杨酸金属配合物的存在明显改变牛血清白蛋白的构象。     

Interaction of bovine serum albumin with chrysotile: spectroscopic and morphological studies

The biodurability of chrysotile fibers, which is related to their cytotoxicity and mutagenic responses, is strongly affected by the surface chemical adsorption of biological molecules. Natural chrysotile is a heterogeneous material in both structure and composition. The availability of synthetic stoichiometric chrysotile of constant structure and uniform morphology has allowed us to investigate its interaction with bovine serum albumin (BSA). By using transmission electron microscopy (TEM) and atomic force microscopy (AFM), we have obtained the first morphological evidence of albumin adsorption onto chrysotile nanocrystals. FTIR spectroscopy was used to quantify modifications of BSA secondary structure that were induced by the surface interaction. The protein transition to beta-turns allows a stronger interaction between the protein hydrophilic side-chains and the charged asbestos surface, which is consistent with hydrogen bonds involving the superficial OH groups. Synthetic stoichiometric chrysotile nanocrystals were shown to be an ideal reference standard with which to study the interaction of asbestos fibers with biological systems, in order to elucidate the chemical mechanisms of asbestos toxicity.     

新型长侧链双羧基功能单体N-(p-乙烯基苄基)-N,N-二[2-(3-羧基丙酰氧基)乙基]胺的合成及其对牛血清白蛋白印迹识别的应用

设计合成了一种新型功能单体N-(p-乙烯基苄基)-N,N-二[2-(3-羧基丙酰氧基)乙基]胺. 采用质子核磁共振、红外光谱及元素分析对单体分子的结构进行了表征, 利用荧光猝灭法和同步荧光法研究了单体与牛血清白蛋白的结合机理, 结果表明在pH 7.4离子强度为0.5 mol•L^－1条件下, 单体与牛血清白蛋白中的色氨酸残基形成稳定的复合物, 其结合比为2∶1, 表观结合常数K_A＝2.239×10¹¹ L²•mol^－2. 以该单体为功能单体, 牛血清白蛋白为模板分子, N,N'-亚甲基双丙烯酰胺为交联剂和多孔聚偏二氟乙烯膜为支持膜, 在水介质中制备了一个分子印迹聚合物复合膜. 渗透实验表明, 这个印迹复合膜对模板分子牛血清白蛋白的渗透量要远高于对照的人血清白蛋白和卵蛋白, 通过与非分子印迹膜对照也说明了此分子印迹复合膜对模板分子高的渗透选择性.     

Study on the Interaction of Matrine with Bovine Serum Albumin

The interaction of matrine (MAT) with bovine serum albumin (BSA) was studied via applying isothermal titration calorimetry, fluorescence and circular dichroism spectra. Important thermodynamic parameters were obtained based on the assumption that there were several classes of binding sites on the biomacromolecules and the supposition that the binding of the drug with the protein could be represented by the Langmuir absorption model. Analysis of the thermodynamic data revealed that there were two classes of binding sites on the biomacromolecules for the ligand molecules. This result was confirmed by the spectroscopic results.     

Calorimetric and structural investigation of the interaction of lysozyme and bovine serum albumin with poly(ethylene oxide) and its copolymers

This work reports investigations aiming at verifying the occurrence of specific interactions between lysozyme or bovine serum albumin (BSA) and poly(ethylene oxide) and its copolymers with poly(propylene oxide). Thermal stability of these proteins, followed by means of high sensitivity DSC, was found to be mostly unaffected by the presence of these polymers. Chromatographic experiments (reverse-phase HPLC and size exclusion chromatrography) did not reveal any sign of specific interaction for these mixtures, either. Isothermal titration calorimetry revealed an increase in enthalpy for the mixtures, represented by a positive enthalpy of transfer for these proteins from buffer to polymer solutions. Moreover, SAXS analyses confirmed that at ambient temperatures these polymers do not affect lysozyme structure. In summary, no evidence is found to support earlier suggestions that some kind of complex could be formed between these proteins and poly(ethylene oxide) or its copolymers, but the present results suggest the occurrence of entropically driven hydrophobic effects.