A method of chemiluminescence coupled with ultrafiltration for investigating the interaction between ibuprofen and human serum albumin

In acidic media, ibuprofen substantially enhanced the weak chemiluminescence (CL) produced by sodium sulfite and potassium permanganate. The increased signals were linearly correlated with ibuprofen concentrations ranging from 1.2 × 10^‐3 to 4.8 μM, with a detection limit of 4.8 × 10^‐4 μM. Two ultrafiltration (UF) membranes were used to construct a unit for trapping 0.15 and 0.75 μM human serum albumin (HSA) and coupled online with the CL system. At low HSA concentrations, the numbers of bound molecules per binding site were calculated to be 0.9 for Sudlow site I and 6.2 for Sudlow site II. The association constants on these binding sites were 5.9 × 10⁵ and 3.4 × 10⁴ M^‐1, respectively. Our CL–UF protocol presents a rapid and sensitive method for studies on drug–protein interaction. Copyright © 2012 John Wiley & Sons, Ltd.     

Chromatographic analysis of allosteric effects between ibuprofen and benzodiazepines on human serum albumin

The effects of (R)- and (S)-ibuprofen on the binding of benzodiazepines to human serum albumin (HSA) were examined by biointeraction chromatography. The displacement of benzodiazepines from HSA by (R)- and (S)-ibuprofen was found to involve negative allosteric interactions (or possible direct competition) for most (R)-benzodiazepines. However, (S)-benzodiazepines gave positive or negative allosteric effects and direct competition when displaced by (R)- or (S)-ibuprofen. Association equilibrium constants and coupling constants measured for these effects indicated that they involved two classes of ibuprofen binding regions (i.e., low- and high-affinity sites). Based on these results, a model was proposed to explain the binding of benzodiazepines to HSA and their interactions with ibuprofen. This model gave good agreement with previous reports examining the binding of benzodiazepines to HSA.     

Structural studies of bovine,equine, and leporine serum albumin complexes with naproxen

Serum albumin, a protein naturally abundant in blood plasma, shows remarkable ligand binding properties of numerous endogenous and exogenous compounds. Most of serum albumin binding sites are able to interact with more than one class of ligands. Determining the protein‐ligand interactions among mammalian serum albumins is essential for understanding the complexity of this transporter. We present three crystal structures of serum albumins in complexes with naproxen (NPS): bovine (BSA‐NPS), equine (ESA‐NPS), and leporine (LSA‐NPS) determined to 2.58 Å (C2), 2.42 Å (P6₁), and 2.73 Å (P2₁2₁2₁) resolutions, respectively. A comparison of the structurally investigated complexes with the analogous complex of human serum albumin (HSA‐NPS) revealed surprising differences in the number and distribution of naproxen binding sites. Bovine and leporine serum albumins possess three NPS binding sites, but ESA has only two. All three complexes of albumins studied here have two common naproxen locations, but BSA and LSA differ in the third NPS binding site. None of these binding sites coincides with the naproxen location in the HSA‐NPS complex, which was obtained in the presence of other ligands besides naproxen. Even small differences in sequences of serum albumins from various species, especially in the area of the binding pockets, influence the affinity and the binding mode of naproxen to this transport protein. Proteins 2014; 82:2199–2208. © 2014 Wiley Periodicals, Inc.     

Bovine serum albumin‐confined silver nanoclusters as fluorometric probe for detection of biothiols

Fluorescent bovine serum albumin‐confined silver nanoclusters (BSA–AgNCs) were demonstrated to be a novel and environmentally friendly probe for the rapid detection of biothiols such as cysteine (Cys), homocysteine (Hcy) and glutathione (GSH). The sensing was ascribed to the strong affinity between the mercapto group of the biothiols and the silver nanoclusters. The fluorescence intensity of BSA–AgNCs was quenched efficiently on increasing the concentration of biothiol, corresponding with a red‐shift in emission wavelength. However, the fluorescence of the silver nanoclusters was almost unchanged in the presence of other α‐amino acids at 10‐fold higher concentrations. By virtue of this specific response, a new, simple and rapid fluorescent method for detecting biothiols has been developed. The linear ranges for Cys, Hcy and GSH were 2.0 × 10^‐6 to 9.0 × 10^‐5 M (R² = 0.994), 2.0 × 10^‐6 to 1.2 × 10^‐4 M (R² = 0.996) and 1.0 × 10^‐5 to 8.0 × 10^‐5 M (R² = 0.980), respectively. The detection limits were 8.1 × 10^‐7 M for Cys, 1.0 × 10^‐6 M for Hcy and 1.1 × 10^‐6 M for GSH. Our proposed method was successfully applied to the determination of thiols in human plasma and the recovery was 94.83–105.24%. It is potentially applicable to protein‐stabilized silver nanoclusters in a chemical or biochemical sensing system. Copyright © 2014 John Wiley & Sons, Ltd.     

Variable responsiveness of rat tracheal epithelial cells to bovine serum albumin in serum-free culture

Summary The colony-forming efficiency of rat tracheal epithelial (RTE) cells was determined in serum-free media containing different types of commercially available bovine serum albumin (BSA): crude fraction V, essentially globulin-free, essentially fatty-acid-free, and essentially globulin- and fatty-acid-free BSA. RTE cells exhibited a concentration-dependent increase in colony-forming efficiency in response to crude fraction V BSA. Similar results were obtained using essentially globulin-free BSA. However, deletion of cholera toxin from the medium resulted in a decrease in the colony-forming efficiency for cells plated in high concentrations (>2 mg/ml) of globulin-free, but not one type of fraction V, BSA. Essentially fatty-acid-free or essentially fatty-acid- and globulin-free BSA stimulated RTE cell colony formation at low concentrations (less than 2.5 to 5 mg BSA/ml) but resulted in concentration-dependent decreases in colony-forming efficiency at higher concentrations. The response of cells to these BSAs was not dependent on cholera toxin. Finally, commerically available fraction V BSA prepared by heat shock, dialysis, charcoal treatment, and deionization was stimulatory at low concentrations but inhibitory at high concentrations. These data suggest that impure preparations of BSA can, under different conditions, stimulate or inhibit cell proliferation and that the expression, of these activities is affected by the method of BSA preparation, the concentration of BSA used, and, in some cases, by the presence or absence of cholera toxin. Research conducted with support from the Office of Health and Environmental Research, U.S. Department of Energy, Washington, DC, under contract no. DE-AC04-76EV01013 in facilities fully acredited by the American Association for Laboratory Animal Care.     

Effect of prototypic drugs ibuprofen and warfarin on global chaotropic unfolding of human serum heme-albumin: a fast-field-cycling 1H-NMR relaxometric study

Human serum albumin (HSA) is the most prominent protein in plasma, but it is also found in tissues and secretions throughout the body. The three-domain design of HSA provides a variety of binding sites for many ligands, including heme and drugs. HSA has been used as a model multidomain protein to investigate how interdomain interactions affect the global folding/unfolding process. Here, we report on the reversible chemical denaturation of heme-HSA involving three different conformational states (F, N, and B, occurring at pH 4.0, 7.0, and 9.0, respectively) and on the effect of prototypic drugs ibuprofen and warfarin on thermodynamics of the reversible unfolding process. Chaotropic unfolding of heme-HSA in the F, N, and B conformations is governed by different thermodynamic regimes, with the B form showing an entropic stabilization of the structure that compensates an enthalpic destabilization, and the F form easily unfolding under entropic control. Warfarin and ibuprofen binding stabilizes heme-HSA in both N and B states.     

Studies on binding interactions between clenbuterol hydrochloride and two serum albumins by multispectroscopic approaches in vitro

In this study, binding properties of clenbuterol hydrochloride (CL) with human serum albumin (HSA) and bovine serum albumin (BSA) were examined using constant protein concentrations and various CL contents under physiological conditions. The binding parameters were confirmed using fluorescence quenching spectroscopy at various temperatures. The experimental results confirmed that the quenching mechanisms of CL and HSA/BSA were both static quenching processes. The thermodynamic parameters, namely, enthalpy change (ΔH) and entropy change (ΔS), were calculated according to the van't Hoff equation, which suggested that the electrostatic interactions were the predominant intermolecular forces in stabilizing the CL–HSA complex, and hydrogen bonds and van der Waals force were the predominant intermolecular forces in stabilizing the CL–BSA complex. Furthermore, the conformational changes of HSA/BSA in the presence of CL were determined using the data obtained from three‐dimensional fluorescence spectroscopy, ultraviolet‐visible absorption spectroscopy and circular dichroism spectroscopy. Copyright © 2013 John Wiley & Sons, Ltd.     

Comparative study of the interactions between bisphenol-A and its endocrine disrupting analogues with bovine serum albumin using multi-spectroscopic and molecular docking studies

Interaction studies of bisphenol analogues; biphenol-A (BPA), bisphenol-B (BPB), and bisphenol-F (BPF) with bovine serum albumin (BSA) were performed using multi-spectroscopic and molecular docking studies at the protein level. The mechanism of binding of bisphenols with BSA was dynamic in nature. SDS refolding experiments demonstrated no stabilization of BSA structure denatured by BPB, however, BSA denatured by BPA and BPF was found to get stabilized. Also, CD spectra and molecular docking studies revealed that BPB bound more strongly and induced more conformational changes in BSA in comparison to BPA. Hence, this study throws light on the replacement of BPA by its analogues and whether the replacement is associated with a possible risk, raising a doubt that perhaps BPB is not a good substitute of BPA.     

血清蛋白与4,5-二溴荧光素相互作用及其分析应用的研究

在 0 .10mol/mL的醋酸溶液中 ,4,5 二溴荧光素能与血清蛋白形成稳定的复合物 ,最大吸收波长 482nm ,与试剂比较 ,红移了 12nm。据此建立了测定血清蛋白的方法 ,用于BSA和HSA的测定 ,分别在 2～ 14mg·L-1有线性关系 ,表观摩尔吸光系数分别为 3.12× 10 5L·mol-1·cm-1和 3.2 7× 10 5L·mol-1·cm-1。应用该法测定了人血清样品总蛋白含量 ,结果令人满意。     

Study on the synthesis of sulfonamide derivatives and their interaction with bovine serum albumin

Three sulfonamide derivatives (SAD) were first synthesized from p‐hydroxybenzoic acid and sulfonamides (sulfadimidine, sulfamethoxazole and sulfachloropyridazine sodium) and were characterized by elemental analysis, ¹H NMR and MS. The interaction between bovine serum albumin (BSA) and SAD was studied using UV/vis absorption spectroscopy, fluorescence spectroscopy, time‐resolved fluorescence spectroscopy and circular dichroism spectra under imitated physiological conditions. The experimental results indicated that SAD effectively quenched the intrinsic fluorescence of BSA via a static quenching process. The thermodynamic parameters showed that hydrogen bonding and van der Waal's forces were the predominant intermolecular forces between BSA and two SADs [4‐((4‐(N‐(4,6‐dimethylpyrimidin‐2‐yl)sulfamoyl)phenyl)carbamoyl)phenyl acetate and 4‐((4‐(N‐(5‐methylisoxazol‐3‐yl)sulfamoyl)phenyl)carbamoyl)phenyl acetate], but hydrophobic forces played a major role in the binding process of BSA and 4‐((4‐(N‐(6‐chloropyridazin‐3‐yl)sulfamoyl)phenyl) carbamoyl)phenyl acetate. In addition, the effect of SAD on the conformation of BSA was investigated using synchronous fluorescence spectroscopy and circular dichroism spectra. Molecular modeling results showed that SAD was situated in subdomain IIA of BSA. Copyright © 2014 John Wiley & Sons, Ltd.     

羧甲基化壳聚糖季铵盐与牛血清白蛋白的相互作用研究

应用荧光光谱研究了羧甲基化壳聚糖季铵盐(CMCQA)与牛血清白蛋白(BSA)的相互作用.研究表明:CMCQA对BSA内源性荧光猝灭机制属于CMCQA和BSA形成复合物所引起的静态猝灭.在室温下,二者的结合常数为2.45×104 L/mol,结合位点数为1.04.二者主要靠静电引力相互作用.     

The investigation of the interaction between orientin and bovine serum albumin by spectroscopic analysis

In this paper, the interaction between orientin and bovine serum albumin (BSA) was examined using fluorescence and absorbance spectroscopy. The analysis of the quenching mechanism was done using Stern–Volmer plots which exhibit upward (positive) deviation. A linear response to orientin was shown in the concentration range between 3 and 50 μM. The experimental results showed the presence of a static quenching process between orientin and BSA. The thermodynamic parameters ΔH, ΔS and ΔG were also calculated and suggested that the hydrophobic and electrostatic interactions played an important role in the interaction between orientin and BSA. Furthermore, the distances between BSA and orientin were determined according to Förster non‐radiation energy transfer theory. In addition, the results of the synchronous fluorescence obtained indicated that the binding of orientin with BSA could affect conformation in BSA. Copyright © 2013 John Wiley & Sons, Ltd.     

In vitro study on binding interaction of quinapril with bovine serum albumin (BSA) using multi-spectroscopic and molecular docking methods

The binding interaction between quinapril (QNPL) and bovine serum albumin (BSA) in vitro has been investigated using UV absorption spectroscopy, steady-state fluorescence spectroscopic, synchronous fluorescence spectroscopy, 3D fluorescence spectroscopy, Fourier transform infrared spectroscopy, circular dichroism, and molecular docking methods for obtaining the binding information of QNPL with BSA. The experimental results confirm that the quenching mechanism of the intrinsic fluorescence of BSA induced by QNPL is static quenching based on the decrease in the quenching constants of BSA in the presence of QNPL with the increase in temperature and the quenching rates of BSA larger than 10¹⁰ L mol^?1 s^?1, indicating forming QNPL–BSA complex through the intermolecular binding interaction. The binding constant for the QNPL–BSA complex is in the order of 10⁵ M^?1, indicating there is stronger binding interaction of QNPL with BSA. The analysis of thermodynamic parameters together with molecular docking study reveal that the main binding forces in the binding process of QNPL with BSA are van der Waal’s forces and hydrogen bonding interaction. And, the binding interaction of BSA with QNPL is an enthalpy-driven process. Based on Förster resonance energy transfer, the binding distance between QNPL and BSA is calculated to be 2.76 nm. The results of the competitive binding experiments and molecular docking confirm that QNPL binds to sub-domain IIA (site I) of BSA. It is confirmed there is a slight change in the conformation of BSA after binding QNPL, but BSA still retains its secondary structure α-helicity.     

Comparative studies on the interactions of dihydroartemisinin and artemisinin with bovine serum albumin using spectroscopic methods

The interactions of dihydroartemisinin (DHA) and artemisinin (ART) with bovine serum albumin (BSA) have been investigated using fluorescence, UV/vis absorption and Fourier transform infrared (FTIR) spectra under simulated physiological conditions. The binding characteristics of DHA/ART and BSA were determined by fluorescence emission and resonance light scattering (RLS) spectra. The quenching mechanism between BSA and DHA/ART is static. The binding constants and binding sites of DHA/ART–BSA systems were calculated at different temperatures (293, 298, 304 and 310 K). According to Förster non‐radiative energy transfer theory, the binding distance of BSA to DHA/ART was calculated to be 1.54/1.65 nm. The effect of DHA/ART on the secondary structure of BSA was analyzed using UV/vis absorption, FTIR, synchronous fluorescence and 3D fluorescence spectra. In addition, the effects of common ions on the binding constants of BSA–DHA and BSA–ART systems were also discussed. Copyright © 2014 John Wiley & Sons, Ltd.     

Conditions for reproducible detection of calmodulin and S100 beta in immunoblots

The conditions used in some immunoblot procedures can fail to detect calmodulin, S100 proteins, and other proteins with similar physical properties. We describe here some of the basis of this difficulty, and provide an immunoblot protocol that allows the rapid and reproducible detection of calmodulin and S100 beta in crude biological samples. These proteins are rapidly transferred from sodium dodecyl sulfate-polyacrylamide gels to membrane matrices, and retention on the matrix is enhanced by a glutaraldehyde fixation step. Either nitrocellulose or a positively charged membrane filter (ZetaProbe) can be used as the immobilizing matrix. By combining microslab gel electrophoresis, 30 min electrophoretic transfer, and glutaraldehyde fixation of nitrocellulose paper, an immunoblot analysis can be done in an 8-hr day.     

Determination of ibuprofen in pharmaceutical formulations using time-resolved terbium-sensitized luminescence.

A sensitive and specific luminescence method for the determination of ibuprofen (IB) in pharmaceutical formulations in aqueous solution is described. The method is based on the luminescence sensitization of terbium (Tb(3+)) by formation of ternary complex with IB in the presence of tri-n-octylphosphine oxide (TOPO) and Tween-20 as surfactant. The luminescence signal for Tb-IB-TOPO is monitored at lambda(ex) = 229 nm and lambda(em) = 545 nm. Optimum conditions for the formation of the complex in aqueous system, were 16 mmol/L TRIS buffer, pH 5.7, TOPO 200 micromol/L and 15 micromol/L of Tb(3+), which allows for the determination of 9.7 x 10(-7) - 9.7 x 10(-6) mol/L IB with a detection limit of 1.2 x 10(-7) mol/L. The relative standard deviations of the method were <1.4%, indicating excellent reproducibility. The proposed method was successfully applied for the assays of IB in pharmaceutical formulations with average recoveries of 100.3-102.5%.     

光谱及分子模拟法对HSA和BSA与酪氨酸激酶抑制剂Imatinib相互作用的比较分析

研究一种酪氨酸激酶抑制剂（tyrosine kinase inhibitor, TKI）伊马替尼（imatinib, IMA）与人血清清蛋白（HSA）及牛血清清蛋白（BSA）的相互作用,比较分析HSA和BSA与IMA相互作用机制的差异. 模拟生理条件下,计算机模拟技术结合荧光光谱和紫外光谱法,研究IMA与蛋白质的作用机制. 分子模建IMA与血清清蛋白的结合模型,表明伊马替尼与蛋白质的相互作用力为疏水作用力,兼有氢键作用. 光谱结果表明,IMA与HSA和BSA的相互作用表现为静态结合过程,结合强度较强,IMA与HSA和BSA分子的结合距离r值较小,说明发生了能量转移现象. IMA对HSA和BSA的结构域微区构象产生影响,使结合位域的疏水性发生改变. 荧光相图技术解析出IMA与HSA和BSA反应构象型态的变迁为“二态”模型. HSA与IMA相互作用的热力学参数表明,IMA与HSA之间是以疏水作用为主的分子间作用,而IMA与BSA之间的作用力为氢键和范德华力,兼有少量的疏水作用力. 光谱实验与计算机模拟结果基本一致,可为研究IMA与HSA和BSA相互作用本质提供一定参考.