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).     

The Photochemical Study of HSA and BSA with Resonance Light-Scattering and Fluorescence Spectra

In recent years, people have paid close attention to the physiological harms induced byultraviolet (UV) irradiation. The serum albumin, which constitutes 60% of blood plasma,has very important physiological functions. Therefore, to study their photochemicalreaction is of great significance. The metal ions, little molecules and medicines etcinteracting with HSA or BSA have been reported ','*"', but it has not been repoFted aboutusing RLS to study the photochemical reaction of HSA or BSA.…     

Fluorescence Spectroscopic Studies on the Interaction of Oleanolic Acid and its Triterpenoid Saponins Derivatives with Two Serum Albumins

The interaction of oleanolic acid (OA) and its glycosylated derivatives (LL-2 and LL-4) with human and bovine serum albumins were investigated using the methods of fluorescence spectroscopy. The spectroscopic analysis of the fluorescence quenching that occurs when OA and its derivatives interact with serum albumin indicates that these quenching constants are inversely correlated with temperature and the quenching process involves static interactions. The binding affinity of OA and OA-derived compounds to bovine serum albumin (BSA) and human serum albumin (HSA) follow the trend LL-4 > LL-2 > OA, suggesting that glycosylation of OA can facilitate its binding to serum albumins. Additionally, the binding affinity of these compounds to HSA is stronger than it is to BSA. The calculated thermodynamic parameters suggest that hydrophobic interactions dominate these interaction processes. We also found that only a single type of binding site exists for OA and its derivatives to HSA and BSA. Synchronous fluorescence results indicate that the binding of OA, LL-2 and LL-4 to BSA and HSA can lead to the conformational changes around the tryptophan residues of the two serum albumins. These results provided valuable clues to the pharmacokinetics and the pharmacologic activities of OA and its types of triterpenoid saponins derivatives.     

三维同步偏振荧光光谱研究蛋白质溶液变性时氨基酸残基的发光行为

将偏振技术、同步技术与三维技术结合起来的三维同步偏振荧光光谱(TDSPS)能分辨蛋白质溶液中的色氨酸(Trp)和酪氨酸(Tyr)残基,具有同步光谱分辨率高、三维技术信息丰富的优点．本文用TDSPS表征牛血清白蛋白(BSA)、人血清白蛋白(HSA)受各种因素影响(酸效应、碱效应、盐效应、猝灭剂等)时Trp,Tyr残基荧光光谱的变化,用于区分HSA和BSA．     

Quenching of tryptophan fluorescence in various proteins by a series of small nickel complexes

A series of twelve anionic, cationic, and neutral nickel(II) complexes have been synthesized and characterized. The interaction of these complexes with bovine serum albumin (BSA), human serum albumin (HSA), lysozyme (Lyso), and tryptophan (Trp) has been studied using steady-state fluorescence spectroscopy. Dynamic and static quenching constants have been calculated, and the role played in quenching by the ligand and complex charge investigated. The nickel complexes showed selectivity towards the different proteins based on the environment surrounding the Trp residue(s). Only small neutral complexes with hydrophobic ligands effectively quenched protein fluorescence via static quenching, with association constants ranging from 10(2) M(-1) (free Trp) to 10(10) M(-1) (lysozyme), indicating a spontaneous and thermodynamically favorable interaction. The number of binding sites, on average, was determined to be one in BSA, HSA and free Trp, and two in lysozyme.     

Fluorescence spectroscopic studies on binding of a flavonoid antioxidant quercetin to serum albumins

Binding of quercetin to human serum albumin (HSA) was studied and the binding constant measured by following the red-shifted absorption spectrum of quercetin in the presence of HSA and the quenching of the intrinsic protein fluorescence in the presence of different concentrations of quercetin. Fluorescence lifetime measurements of HSA showed decrease in the average lifetimes indicating binding at a location, near the tryptophan moiety, and the possibility of fluorescence energy transfer between excited tryptophan and quercetin. Critical transfer distance (R _o ) was determined, from which the mean distance between tryptophan-214 in HSA and quercetin was calculated. The above studies were also carried out with bovine serum albumin (BSA).     

Spectroscopic Investigation of the Interactions of Cryptotanshinone and Icariin with Two Serum Albumins

The interactions of two drugs, cryptotanshinone (CTS) and icariin, with bovine serum albumin (BSA) and human serum albumin (HSA) have been investigated using multiple spectroscopic techniques under imitated physiological conditions. CTS and icariin can quench the fluorescence intensity of BSA/HSA by a static quenching mechanism with complex formation. The binding constants of CTS–BSA, CTS–HSA, icariin–BSA and icariin–HSA complexes were observed to be 1.67 × 10⁴, 4.04 × 10⁴, 4.52 × 10⁵ and 4.20 × 10⁵ L·mol^?1, respectively at 298.15 K. The displacement experiments suggested icariin/CTS are primarily bound to tryptophan residues of the proteins within site I and site II. The thermodynamic parameters calculated on the basis of the temperature dependence of the binding constants revealed that the binding of CTS–BSA/HSA mainly depends on van der Waals interaction and hydrogen bonds, and yet the binding of icariin–HSA/BSA strongly relies on the hydrophobic interactions. The binding distances between BSA/HSA and CTS/icariin were evaluated by the Föster non-radiative energy transfer theory. The results of synchronous fluorescence, 3D fluorescence, FT-IR and CD spectra indicates that the conformations of proteins were altered with the addition of CTS or icariin. In addition, the effects of some common ions on the binding constants of CTS/icariin to proteins are also discussed.     

Spectroscopic studies on the interaction between riboflavin and albumins

The interactions between riboflavin (RF) and human and bovine serum albumin (HSA and BSA) were studied by using absorption and fluorescence spectroscopic methods. Intrinsic fluorescence emission spectra of serum albumin in the presence of RF show that the endogenous photosensitizer acts as a quencher. The decrease of fluorescence intensity at about 350 nm is attributed to changes in the environment of the protein fluorophores caused by the ligand. The quenching mechanisms of albumins by RF were discussed. The binding constants and binding site number were obtained at various temperatures. The distance between albumins and RF in the complexes suggests that the primary binding site for RF is close to tryptophan residue (Trp214) of HSA and Trp212 of BSA. The hydration process of albumins has also been discussed.     

分子内电荷转移荧光探针1-酮-2-（对二甲氨基苯亚甲基）四氢萘与人血清白蛋白作用

采用荧光及紫外光谱研究了1-酮-2-（对二甲氨基苯亚甲基）-四氢萘（KDTN）与人血清白蛋白（HSA）相互作用的光谱特性。 结果表明,静态猝灭和非辐射能量转移是导致KDTN对HSA荧光猝灭的主要原因。 测得17、27和37 ℃ 3个温度下的结合常数K_A分别为1.633×10⁸、0.7998×10⁸和0.347×10⁸ L/mol,结合位点数n分别为1.7、1.6和1.7;据Forster偶极 偶极非辐射能量转移理论,计算得到KDTN与HSA在3个温度下的作用距离r分别为2.64、2.59和2.64 nm;能量转移效率E分别为0.5100、0.4797和0.4210。 热力学参数表明,二者主要以范德华力或氢键结合;用同步荧光技术研究了KDTN对HSA构象的影响,结果表明,KDTN的加入对HSA构象影响不大。     

芘与人血清白蛋白和牛血清白蛋白结合位点微环境极性的差异

利用芘(Pyr)的微环境极性探针性质, 采用稳态荧光光谱、 荧光共振能量转移技术结合分子对接法, 对比分析了Pyr分别与人血清白蛋白(HSA)和牛血清白蛋白(BSA)作用机制的差异. 结果表明, HSA和BSA中Pyr的I₁/I₃平均值分别为1.36和0.92; Pyr与HSA和BSA的结合常数分别为1.86×10⁷和1.71×10⁵ L/mol; Pyr与HSA和BSA中色氨酸残基表观距离分别为2.37和2.34 nm. Pyr在HSA和BSA中不同的结合位点位于ⅠB子域和ⅠA子域, 其结合位点周围氨基酸残基的极性是影响Pyr I₁/I₃值的主要原因之一. 实验证实Pyr与HSA和BSA结合作用位点处的微环境极性存在差异.     

光谱法测定伊曲康唑与牛血清和人血清白蛋白相互作用

用荧光光谱和紫外吸收光谱法, 在pH=7.4±0.1的0.1 mol·L-1磷酸缓冲溶液中, 研究了伊曲康唑与牛血清白蛋白(BSA)和人血清白蛋白(HSA)的相互作用. 实验结果表明, 伊曲康唑与牛血清白蛋白和人血清白蛋白作用的猝灭常数均随着温度的升高而降低, 伊曲康唑可以有规律地使血清白蛋白内源荧光猝灭, 其猝灭机理可认为是伊曲康唑与白蛋白形成复合物的静态猝灭. 获得了在不同温度下, 伊曲康唑与血清白蛋白作用的结合常数以及△G、△H和△S等热力学参数. 根据所得结果可推断伊曲康唑与白蛋白的作用力主要为疏水作用力, 同时, 利用荧光共振能量转移理论(FRET)计算得出了伊曲康唑与白蛋白结合位置的距离d. 而且, 利用同步荧光光谱和紫外光谱揭示了该反应中蛋白的结构和其微环境的变化.     

Affinity of two novel five-coordinated anticancer Pt(II) complexes to human and bovine serum albumins: a spectroscopic approach

The interactions of two organoplatinum complexes, [Pt(C^N)Cl(dppa)], 1, and [Pt(C^N)Cl(dppm)], 2 (C^N = N(1), C(2')-chelated, deprotonated 2-phenylpyridine, dppa = bis(diphenylphosphino)amine, dppm = bis(diphenylphosphino)methane), as antitumor agents, with bovine serum albumin (BSA) and human serum albumin (HSA) have been studied by fluorescence and UV-vis absorption spectroscopic techniques at pH 7.40. The quenching constants and binding parameters (binding constants and number of binding sites) were determined by fluorescence quenching method. The obtained results revealed that there is a strong binding interaction between the ligands and proteins. The calculated thermodynamic parameters (ΔG, ΔH, and ΔS) confirmed that the binding reaction is mainly entropy-driven, and hydrophobic forces played a major role in the reaction. The displacement experiment shows that these Pt complexes can bind to the subdomain IIA (site I) of albumin. Moreover, synchronous fluorescence spectroscopy studies revealed some changes in the local polarity around the tryptophan residues. Finally, the distance, r, between donor (serum albumin) and acceptor (Pt complexes) was obtained according to F?rster theory of nonradiation energy transfer.     

Mechanism of interaction of vincristine sulphate and rifampicin with bovine serum albumin: A spectroscopic study

The mechanism of interaction of vincristine sulphate (VS) and rifampicin (RF) with bovine serum albumin (BSA) has been studied by quenching of BSA fluorescence by RF/VS. The Stern-Volmer plot indicates the presence of a static component in the quenching mechanism. Results also show that both the tryptophan residues of BSA are accessible to VS and RF. The high magnitude of rate constant of quenching indicates that the process of energy transfer occurs by intermolecular interaction and VS/RF-binding site is in close proximity to the tryptophan residues of BSA. Binding studies in the presence of a hydrophobic probe, 8-anilino-1-naphthalene-sulphonic acid sodium salt (ANS) indicate that the VS and RF compete with ANS for hydrophobic sites on the surface of BSA. Small decreases in critical micellar concentrations (CMC) of anionic surfactants in presence of VS/ RF show that the ionic character of VS/RF also contributes to binding. The temperature dependence of the association constant is used to estimate the values of the thermodynamic parameters involved in the interaction of VS/RF with BSA and the results indicate that hydrophobic forces play a significant role in the binding. Circular dichroism studies reveal that the change in helicity of BSA are due to binding of VS/RF to BSA.     

Complexation of serum albumins and triton X-100: Quenching of tryptophan fluorescence and analysis of the rotational diffusion of complexes

The polarized and nonpolarized fluorescence of bovine serum albumin and human serum albumin in Triton X-100 solutions is studied at different pH values. Analysis of the constants of fluorescence quenching for BSA and HSA after adding Triton X-100 and the hydrodynamic radii of BSA/HSA–detergent complexes show that the most effective complexation between both serum albumins and Triton X-100 occurs at pH 5.0, which lies near the isoelectric points of the proteins. Complexation between albumin and Triton X-100 affects the fluorescence of the Trp-214 residing in the hydrophobic pockets of both BSA and HSA.     

用荧光猝灭和荧光加强两种理论研究喹诺酮类新药与白蛋白的作用

应用荧光加强和荧光猝灭两种理论公式, 对四种喹诺酮类药物与人血清和牛血清白蛋白的作用作进行了对比研究, 对药物与白蛋白的结合特点和通常的表征量(解离常数、 猝灭常数、 猝灭效率、 能量转移效率、 给体 受体作用距离等)进行了深入地分析; 在白蛋白与药物结合类型上, 四种药物对HSA和BSA的猝灭实验结果表明, 这种由给体-受体结合引起的猝灭作用类型不是由生物大分子血清白蛋白单方面决定的, 而是由血清白蛋白与药物、 即给体与受体两者的分子结构和相互匹配共同决定的.     

Interactions between protein and porphyrin-containing cyclodextrin supramolecular system: a fluorescent sensing approach for albumin

The interactions of meso-tetraphenylporphyrin (TPP), meso-tetraphenylporphyrin cobalt(ii) (CoTPP) and protein in the presence of a cyclodextrin derivative, heptakis(2,6-di-O-n-octyl)-beta-cyclodextrin (Oc-beta-CD), have been investigated. In the presence of Oc-beta-CD, significant increase of TPP fluorescence was realized, but the increased fluorescence was quenched by CoTPP. To further investigate the fluorescence-quenched system and explore its potential application in bioanalysis, a strategy has been devised to restore the quenching fluorescence of TPP upon interacting with protein. The restoration of TPP fluorescence in the present system is fast and accomplished upon interaction with bovine serum albumin (BSA) or human serum albumin (HSA). On the basis of the spectroscopic measurement and excited state fluorescence lifetime, the mechanism of TPP fluorescence quenching is attributed to formation of a ground-state complex of TPP and CoTPP, and the fluorescence restoration is attributed to the binding of CoTPP with the protein molecule which destroys the aggregate, releasing the free base porphyrin. With optimized conditions, the calibration equations are linear from 0.80 to 75.4 microg mL(-1) BSA and from 3.20 to 93.2 microg mL(-1) HSA. The corresponding detection limits are 0.32 microg mL(-1) for BSA and 1.06 microg mL(-1) for HSA, respectively. The method was used for the direct assay of HSA content in human serum. The result is comparable to that obtained by another method. The recovery from BSA in synthetic sample is also satisfactory.     

Spectroscopic studies on the mode of interaction of an anticancer drug with bovine serum albumin

The mechanism of interaction of vinblastin sulphate (VBS) with bovine serum albumin (BSA) has been reported. Association constant for VBS-BSA binding was found to be 3.146+/-0.06 x 10(4) M(-1). Stern-Volmer analysis of fluorescence quenching data showed that the fraction of fluorophore (protein) accessible to the quencher (drug) was close to unity indicating thereby that both tryptophan residues of BSA are involved in drug-protein interaction. The rate constant for quenching, greater than 10(10) M(-1) S(-1), indicated that the drug-binding site is in close proximity to tryptophan residues of BSA. Binding studies in the presence of an hydrophobic probe, 8-anilino-1-naphthalein-sulphonic acid, sodium salt (ANS) indicated that there is hydrophobic interaction between VBS and probe and they do not share common sites in BSA. Thermodynamic parameters obtained from data at different temperatures showed that the binding of VBS to BSA involves predominant hydrophobic forces. The effects of some additives and paracetamol on binding of VBS-BSA have also been investigated. The CD spectrum of BSA in presence of VBS shows that the binding of VBS leads to change in the helicity of BSA.     

PHOSPHORESCENCE LIFETIME STUDIES OF INTERACTIONS BETWEEN SERUM ALBUMINS AND SODIUM DODECYL SULFATE

Binding of sodium dodecyl sulfate (SDS) to bovine serum albumin (BSA) and human serum albumin (HSA) in aqueous solutions at room temperature induces significant changes in the phosphorescence lifetime of tryptophan (Trp) residues. A steep rise of the phosphorescence lifetime from 1.9 ms to 10.0 ms for BSA and from 1.9 ms to 5.5 ms for HSA is observed when the total SDS concentration increased from 0.0 mM to 0.22 mM at 1 mg/mL protein concentration. As the total SDS concentrationis further inccreased to 2.2 mM, a slower increase in the phosphorescence lifetime is observed, from 10.0 ms to 19.5 ms for BSA and from 5.5 ms to 7.2 ms for HSA. It appears that the phosphorescence lifetime modifications are mainly due to an increase of protein matrix rigidity around Trp residues. The observed differences (between HSA and BSA) allow us to distinguish the contribution of the two Trp residues to the BSA phosphorescence.     

Interaction Between Fluorinated Amphiphilic Copolymer P(HFMA)-g-P(SPEG) and BSA

A novel fluorinated amphiphilic copolymer P(HFMA)-g-P(SPEG) was synthesized. The interactions between P(HFMA)-g-P(SPEG) and bovine serum albumin (BSA) were studied by synchronous fluorescence and intrinsic fluorescence spectroscopy. It was concluded through synchronous fluorescence that P(HFMA)-g-P(SPEG) mainly bound to tryptophan residues of BSA. Intrinsic fluorescence results revealed that BSA and P(HFMA)-g-P(SPEG) had strong interactions. The mechanism of quenching belonged to dynamic quenching and the main sort of binding force was hydrophobic force. The hydrophobic interaction between P(HFMA)-g-P(SPEG) and BSA was conformed by micropolarity and TEM photographs.     

Mechanism of interaction of hypoglycemic agents glimepiride and glipizide with human serum albumin

The mechanism of interaction of hypoglycemic drugs, glimepiride and glipizide with human serum albumin (HSA) has been studied using fluorescence spectroscopy. The results are discussed in terms of the binding parameters, thermodynamics of the binding process, nature of forces involved in the interaction, identification of drug binding site on serum albumin and the fluorescence quenching mechanism involved. The association constants were of the order of 10⁵ and glipizide was found to have much higher affinity for HSA than glimepiride at all temperatures. Thermodynamic parameters for the binding suggested that hydrophobic interactions are primarily involved in the binding of these drugs to HSA. However, glimepiride and glipizide appear to cause temperature-dependent conformational changes in the albumin molecule and, therefore, the nature of interaction varied with temperature. Glimepiride and glipizide bind to both site I and site II on HSA, but the primary interaction occurs at site II. The binding region in site II is different for the two drugs. Stern-Volmer analysis of quenching data indicated that tryptophan residues of HSA are not fully accessible to the drugs and a predominantly dynamic quenching mechanism is involved in the binding. Results can provide useful insight into prediction of competitive displacement of these drugs by other co-administered drugs and excipients, resulting in serious fluctuations of the blood glucose levels in diabetic patients.