Self‐diffusion in a hyaluronic acid–albumin–water system as studied by NMR

Hyaluronic acid (HA) is an anionic biopolymer that is present in many tissues and can be involved in cancerous neoformations. HA can form complexes with proteins (particularly, serum albumin) in the body. However, HA structures and processes involving HA have not been extensively studied by NMR because the molecule's rigid structure makes these studies problematic. In the current work, self‐diffusion of HA and bovine serum albumin (BSA), and water in solutions was measured by ¹H pulsed field gradient NMR (PFG NMR) with a focus on the HA‐BSA‐D₂O systems at various concentrations of BSA and HA. It was shown that in the presence of even a small amount of HA, the self‐diffusion coefficient (SDC) of BSA decreases. To explain this fact, three hypotheses were proposed and analyzed. The first one was based on the effect of slowing down of water mobility in the presence of HA. The second hypothesis suggested an effect of mechanical collisions of BSA with HA molecules. The third hypothesized that BSA and HA molecules form a complex where BSA molecules reduced in mobility. It was shown that the third mechanism is the most likely. The state of the BSA molecules in the BSA‐HA‐D₂O system corresponds to a ‘fast exchange’ condition from the NMR point of view: BSA molecules reside in the ‘free’ and ‘bound’ (with HA) states for much shorter time than the diffusion time of the PFG NMR experiment, 7 ms. The fractions of ‘bound’ BSA molecules in the BSA‐HA complex were estimated. Copyright © 2012 John Wiley & Sons, Ltd.     

Temperature‐responsive “tadpole‐shaped” protein–polymer hybrids and their self‐assembly behavior

The temperature‐responsive poly (N, N‐diethylacrylamide) (pDEAAm) with narrower molecular weight distribution was prepared by the atom transfer radical polymerization and characterized by ¹HNMR and gel permeation chromatography. The temperature‐responsive “tadpole‐shaped” BSA–pDEAAm hybrids were fabricated via a free Cys‐34 residue of bovine serum albumin (BSA) site specifically binding to the end group disulfide bonds of pDEAAm and characterized by native‐polyacrylamide gel electrophoresis (Native‐PAGE) and matrix‐assisted laser desorption/ionization time of flight mass spectrometry. Their temperature‐responsive behaviors were measured by ultraviolet‐visible spectra (UV‐Vis). The lower critical solution temperature (LCST) of the pDEAAm was identified as 28°C, and the LCST of BSA–pDEAAm hybrids was identified as 31°C. The morphologies of BSA–pDEAAm hybrids self‐assembled in the aqueous solutions with two different temperatures at 25 °C and 40°C were investigated by transmission electron microscopy. Below the LCST of BSA–pDEAAm hybrids, the separate spherical nanoparticles were observed. In contrast, bundles and clusters were observed above the LCST of BSA–pDEAAm hybrids. The results suggested that the self‐assembly morphology of BSA–pDEAAm hybrids depended upon the pDEAAm block in BSA–pDEAAm hybrids, and the morphology transitions were effected by the LCST of BSA–pDEAAm hybrids. It would be expected to be used in biomedicine and materials science. Copyright © 2016 John Wiley & Sons, Ltd.     

Interaction Between <Emphasis Type="Italic">Trans</Emphasis>-resveratrol and Serum Albumin in Aqueous Solution

The interaction between trans-resveratrol (TR) with bovine serum albumin (BSA) in aqueous solution was investigated by means of fluorescence, synchronous fluorescence and infrared spectroscopy. The fluorescence of BSA can be quenched remarkably by TR in aqueous solution. A notable red-shift of the maximum emission of BSA from 340 to 353 nm together with appearance of an isoemissive point at 395 nm were observed. The results indicate that TR binds to BSA, forming a TR–BSA complex. The TR–BSA binding distance was determined to be less than 7 nm, suggesting that energy transfer from BSA to TR may occur. The interaction process is spontaneous. Based on the obtained thermodynamic parameters, electrostatic forces may play a major role in this process. Both synchronous fluorescence and FT-IR spectra confirmed the interaction, and indicate the conformational changes of BSA.     

A new specific metal ion chelated‐poly(N‐vinylimidazole) gel sorbents for albumin adsorption‐desorption

Poly(N‐vinylimidazole) (PVIm) hydrogels were prepared by γ‐irradiating binary mixtures of N‐vinylimidazole‐water in a ⁶⁰Co‐γ source having 4.5 kGy/h dose rate. These affinity gels having different swelling ratio of Cu(II)‐chelated, Co(II)‐chelated and plain PVIm in acetate buffer were used in the albumin adsorption studies. Bovine serum albumin (BSA) adsorption on these gels from aqueous solutions containing different amounts of BSA at different pH adjusted with acetate and phosphate buffer was investigated in batch reactors. The adsorption capacities of BSA on/in the gels were decreased dramatically by increasing the ionic strength (I) adjusting with NaCl. BSA adsorption capacities of the metal ion‐chelated gels were higher than the plain PVIm gel even if the swelling ratio of the metal ion‐chelated gels was very low comparing to the PVIm gel. The rigidity of the metal ion‐chelated gel is very high and it can be used for the column applications. More than 95% of BSA were desorbed in 3 h in the desorption medium containing KSCN for PVIm gel and EDTA for metal ion‐chelated gels. These results indicate that PVIm and metal ion‐chelated PVIm gels are very efficient to remove BSA and the different metal ion‐chelated PVIm gels show different affinity for BSA or biomolecules.     

Protein‐Directed Synthesis of Mn‐Doped ZnS Quantum Dots: A Dual‐Channel Biosensor for Two Proteins

Proteins typically have nanoscale dimensions and multiple binding sites with inorganic ions, which facilitates the templated synthesis of nanoparticles to yield nanoparticle–protein hybrids with tailored functionality, water solubility, and tunable frameworks with well‐defined structure. In this work, we report a protein‐templated synthesis of Mn‐doped ZnS quantum dots (QDs) by exploring bovine serum albumin (BSA) as the template. The obtained Mn‐doped ZnS QDs give phosphorescence emission centered at 590 nm, with a decay time of about 1.9 ms. A dual‐channel sensing system for two different proteins was developed through integration of the optical responses (phosphorescence emission and resonant light scattering (RLS)) of Mn‐doped ZnS QDs and recognition of them by surface BSA phosphorescent sensing of trypsin and RLS sensing of lysozyme. Trypsin can digest BSA and remove BSA from the surface of Mn‐doped ZnS QDs, thus quenching the phosphorescence of QDs, whereas lysozyme can assemble with BSA to lead to aggregation of QDs and enhanced RLS intensity. The detection limits for trypsin and lysozyme were 40 and 3 nM , respectively. The selectivity of the respective channel for trypsin and lysozyme was evaluated with a series of other proteins. Unlike other protein sensors based on nanobioconjugates, the proposed dual‐channel sensor employs only one type of QDs but can detect two different proteins. Further, we found the RLS of QDs can also be useful for studying the BSA–lysozyme binding stoichiometry, which has not been reported in the literature. These successful biosensor applications clearly demonstrate that BSA not only serves as a template for growth of Mn‐doped ZnS QDs, but also impacts the QDs for selective recognition of analyte proteins.     

Separation of tryptic peptides of native and glycated BSA using open‐tubular CEC with salophene–lanthanide–Zn2+ complex as stationary phase

Open‐tubular CEC (OT‐CEC) with a new stationary phase, salophene–lanthanide–Zn²⁺ complex, has been applied to the separation of tryptic peptides of native BSA and BSA glycated by glucose and ribose. Glycation of proteins (non‐enzymatic modification by sugars) significantly affects their properties and it is of great importance from a physiological point of view. Separation of tryptic peptides of glycated BSA by CZE was poor because of their strong adsorption to the bare fused silica capillary. An improved separation of tryptic peptides of both native and glycated BSA was achieved by OT‐CEC in the fused silica capillary non‐covalently coated with salophene–lanthanide–Zn²⁺ complex, which suppressed the adsorption of peptides to the capillary and via specific interactions with some (glyco)peptides enhanced selectivity of the separation. Significant differences have been found in OT‐CEC analyses of tryptic hydrolysates of native and glycated BSA. In OT‐CEC‐UV profile of tryptic peptides of native BSA, 44 peaks could be resolved, whereas a reduced number of 38 peaks were observed in the profile of tryptic peptides of glucose‐glycated BSA and only 30 peaks were found in the case of ribose‐glycated BSA. The developed OT‐CEC can be potentially used for monitoring of protein glycation.     

Spectroscopic Studies on the Binding of Kaempferol‐3,7‐α‐L‐rhamnopyranoside to Bovine Serum Albumin

The binding of kaempferol‐3,7‐α‐L‐rhamnopyranoside (KRR) with bovine serum albumin (BSA) was investigated by different spectroscopic methods under simulative physiological conditions. Analysis of ?uorescence quenching data of BSA by KRR at different temperatures using Stern‐Volmer methods revealed the formation of a ground state KRR‐BSA complex with moderate binding constant of the order 10⁴ L·mol^?1. The existence of some metal ions could weaken the binding of KRR on BSA. The changes in the van't Hoff enthalpy (ΔH⁰) and entropy (ΔS⁰) of the interaction were estimated to be ?26.53 kJ·mol^?1 and 3.33 J·mol^?1·K^?1 and both hydrophobic and electrostatic forces contributed to stabilizing the BSA‐KRR complex. According to the F?ster theory of non‐radiation energy transfer, the distance r between the donor (BSA) and the acceptor (KRR) was obtained (r=2.83 nm). Site marker competitive experiments showed that KRR could bind to Site I of BSA. In addition, synchronous fluorescence, UV‐Vis absorption and circular dichroism (CD) results indicated that the KRR binding could cause conformational changes of BSA.     

Amperometric Immunosensor for the Determination of α‐1‐Fetoprotein Based on Core‐Shell‐Shell Prussian Blue‐BSA‐Nanogold Functionalized Interface

In the present work, a newly functional nanoparticle has been prepared to immobilize the protein for the detection of α‐1‐fetoprotein (AFP). Prussian blue (PB) nanoparticle was initially synthesized under ultrasonic condition, then bovine serum albumin (BSA) was used to coat the PB nanoparticle to improve the stability of the PB nanoparticle as well as functionalize the surface of PB nanoparticle, and then gold colloids were loaded on the BSA‐coated PB nanoparticle to construct a core‐shell‐shell nanostructure via the conjunction of thiolate linkages or alkylamines of the BSA. Finally, a convenient, effective and sensitivity amperometric immunosensor for the detection of α‐1‐fetoprotein (AFP) was constructed by the employment of these functional core‐shell‐shell microspheres. The preparation of the nanoparticle (Au‐BSA‐PB NPs) was characterized by transmission electron microscopy (TEM), and the assembly of the biosensor was characterized with cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The dynamic range of the resulted immunosensor for the detection of AFP is from 0.02 ng/mL to 200.0 ng/mL with a detection limit of 0.006 ng/mL (S/N=3). Moreover, this biosensor displays good selectivity, stability and reproducibility.     

Development of a Novel Covalent Folate‐Albumin‐Photosensitizer Conjugate

There is considerable interest in the development of novel and more efficient delivery systems for improving the efficacy of photodynamic therapy (PDT). The authors in this highlighted issue describe the synthesis and the photobiological characterizations of two photosensitizer (PS) conjugates based on β‐carboline derivatives covalently conjugated to folic acid (FA) coupled to bovine serum albumin (BSA) as a carrier system specifically targeting cancer cells overexpressing FA receptor alpha (FRα). Accordingly, only the FA–BSA–β‐carboline conjugates are internalized specifically in FRα‐positive cells and are proved to be phototoxic. On the other hand, albumin–β‐carboline conjugates without FA or β‐carboline derivatives alone are not internalized and nontoxic. This conjugate is among the first to produce a conjugate composed of a PS and FA molecules that are directly conjugated to BSA. In addition, the in vitro studies are the first evidence that directly conjugated FA‐BSA can be used as carriers to selectively enhance cytotoxicity by PDT relative to unmodified PS or nontargeted BSA‐PS. This strategy is a positive step forward for the covalent design and construction of a photodynamic nanomedicine for FR‐positive tumors.     

Preparation and characterization of a polystyrene/bovine serum albumin nanoparticle‐coated capillary for chiral separation using open‐tubular capillary electrochromatography

Polystyrene (PS) nanoparticles coated by BSA, hereafter denoted as PS/BSA, were prepared and chemically immobilized for the first time onto a capillary inner wall for open‐tubular CEC (OTCEC). EOF and scanning electron micrography were used to characterize the prepared nanoparticle‐coated capillaries. To investigate the performance of the prepared columns in OTCEC, chiral separation of d ,l ‐tryptophan (dl ‐Trp) was performed in monolayer BSA‐modified capillary and PS/BSA nanoparticle‐coated columns. The results indicated that the nanoparticle‐modified column afforded a higher resolution compared with the monolayer type. Rapid enantioseparation of dl ‐Trp (within 3 min) was achieved with the PS/BSA‐immobilized column using an electroosmotic pump‐assisted CEC. Enantiomer separations of other compounds like dl ‐tyrosine and warfarin were also achieved with the column. Besides, run‐to‐run and column‐to‐column repeatabilities of the PS/BSA‐coated column in the chiral separation were systematically introduced.     

Mixed‐monolayer of N‐hydroxysuccinimide‐terminated cross‐linker and short alkanethiol to improve the efficiency of biomolecule binding for biosensing

The goal of this study was to use a novel surface chemistry for modifying gold surfaces to decrease the steric hindrance, minimize the nonspecific bindings while providing directed immobilization of proteins for advancing the transducer property and to provide a biosensing platform for surface plasmon resonance (SPR) applications. Mixed self‐assembled monolayers (mSAMs) were prepared using 3,3′‐Dithiodipropionic acid di (N‐hydroxysuccinimide ester) (DSP) and 6‐mercapto‐1‐hexanol (MCH) and the selected model proteins bovine serum albumin (BSA) and lysozyme were tested for binding efficiency. First, binding of these two proteins at constant concentration to different DSP:MCH mSAMs were compared to deduce the best molar ratio for forming mSAM using a continuous flow system coupled to SPR. Coincidently the maximum protein binding DSP:MCH mSAM were the same for both proteins. The change in Response Unit (∆RU) signal due to protein binding between DSP SAM and maximum protein binding DSP:MCH mSAM for lysozyme binding was more in comparison to BSA binding. Second, the effect of BSA and lysozyme concentration on binding efficiency to maximum protein binding DSP:MCH mSAM were compared and discussed. Lysozyme and BSA were shown to reach saturations on the same monolayer at concentrations of 5.7x10⁻⁵ and 8.96x10⁻⁶ [M] respectively, hence the molar ratio for limit concentrations is 6:1. The DSP SAM, MCH SAM, and DSP:MCH mSAMs where maximum and minimum protein binding occurs were also characterized with XPS and Attenuated total reflectance‐Fourier transform infrared (ATR‐FTIR) spectroscopy. Blank gold surface, maximum protein binding DSP:MCH mSAM and BSA immobilized DSP:MCH mSAM on gold surface were also investigated utilizing tapping mode AFM.     

叔丁醇-水混合溶剂的混合状态对牛血清白蛋白构象的影响

应用动态光散射法测定了叔丁醇(TBA)-水混合溶剂中牛血清白蛋白(BSA)流体动力学半径, 并通过分析BSA的荧光光谱和紫外-可见光吸收光谱, 研究BSA在TBA-水混合溶剂中的构象变化. 同时, 通过分析TBA-水二元体系和BSA-TBA-水三元体系静态散射光的变化, 探讨TBA-水溶剂体系的混合状态及其对BSA在水溶液中构象变化的影响. 结果表明, TBA-水溶剂体系的混合状态与BSA的构象变化密切相关, 低浓度的混合溶剂中, 水分子在TBA周围形成疏水水化结构, 与蛋白质疏水基团的选择性结合, 破坏了蛋白质的稳定结构, 但是, 少量TBA的加入削弱了蛋白质疏水基团间的疏水相互作用, 有利于蛋白质形成更紧密的构象; 高浓度的混合溶剂中, TBA分子相互聚集形成胶束, 削弱了对蛋白质的变性作用.     

Calcium Phosphate Nanocarriers Dual‐Loaded with Bovine Serum Albumin and Ibuprofen: Facile Synthesis,Sequential Drug Loading and Sustained Drug Release

A simple and green strategy is reported for the preparation, drug loading, and release properties of a drug delivery system consisting of calcium phosphate (CP) nanocarriers dual‐loaded with bovine serum albumin (BSA) and hydrophobic drug ibuprofen (IBU). The sequential loading of BSA and IBU in calcium phosphate nanocarriers and in vitro simultaneous release of BSA and IBU are realized and investigated. In this method, BSA, which is used as a model protein drug, is encapsulated in situ in calcium phosphate nanocarriers. Subsequently, the typical hydrophobic drug IBU is loaded in the BSA/CP drug delivery system, forming the IBU/BSA/CP dual drug delivery system. The experiments reveal that the preloaded BSA not only reduces the cytotoxicity of calcium phosphate nanocarriers but also significantly improves the IBU drug loading capacity in calcium phosphate nanocarriers and greatly extends the duration of drug release. Thus, the as‐prepared IBU/BSA/CP dual drug delivery system is promising for drug delivery applications.     

BSA‐imprinted synthetic receptor for reversible template recognition

A novel approach to the manufacturing of protein‐responsive imprints on a home‐made chitosan substrate was established together with m‐aminophenylboronic acid (APBA) as a functional monomer. The produced polymers were characterized using both (1) equilibrium adsorption assays and (2) high performance liquid chromatography analysis. Results confirmed that the synthesized BSA‐MIP (molecularly imprinted polymer) has a high affinity towards its template compared to the determined control proteins. The produced BSA‐MIP featured largely in its good adsorption reversibility, especially in competitive binding assays, which is of great biological significance in separations. Non‐specific binding was reduced to almost zero in a BSA/BHb competitive binding event. An excellent HPLC profile of template recognition was found for BSA‐MIP, even under harsh mobile phase conditions. In the present work, the adopted trapped‐template‐release method permits recovery of bound BSA [1]. The strategy of making an artificial protein‐receptor with high adsorption affinity and reversibility is promising in on‐line isolation of target protein from complicated biological environments.     

羟丙基-β-环糊精对1-羟基芘与牛血清白蛋白相互作用的影响

在模拟生理条件下, 利用荧光光谱、 圆二色光谱和紫外-可见吸收光谱探究了羟丙基-β-环糊精(HPCD)对1-羟基芘(1-OHPyr)与牛血清白蛋白(BSA)相互作用的影响及其相关机制. 结果表明, 291 K下HPCD可使1-OHPyr与BSA的结合常数降低为1.45×10⁵ L/mol; 导致1-OHPyr-BSA体系中BSA的α-螺旋含量恢复, 色氨酸(TRP)残基周围微环境的极性变化减弱; 致使1-OHPyr及BSA的荧光寿命均延长, 且二者的结合距离增大. 初步研究结果表明, HPCD与1-OHPyr的包络作用是HPCD影响BSA与1-OHPyr结合的主要原因.     

基于壳聚糖衍生物的蛋白质药物载体材料的制备及性能

在离子液体均相体系中合成了一种新型两亲性窄分子量分布的低聚壳聚糖衍生物月桂基-琥珀酰化壳聚糖(LSCOS). 以LSCOS为载体材料, 以牛血清蛋白(BSA)为模板蛋白, 以戊二醛为交联剂, 用油包水(W/O)乳化交联法制备了包载BSA的BSA/LSCOS缓释载药微球. 通过扫描电子显微镜(SEM)、 透射电子显微镜(TEM)及紫外-可见光谱(UV-Vis)研究了BSA/LSCOS比率和戊二醛/LSCOS比率对微球的形貌结构、 包埋率、 载药率和体外药物释放特性的影响. 结果表明, 在离子液体中合成的LSCOS包覆了BSA, 形成的微球粒径约为1 μm, 微球表面随BSA用量的增加变得光滑, 随戊二醛用量的增加变得粗糙. BSA的累积释放率与BSA包载量成正比, 与交联剂添加量成反比, 因此, 可通过控制蛋白质药物的添加比率和交联剂用量来控制蛋白质药物体外释放率.     

Adsorption of bovine serum albumin to acrylamide–itaconic acid hydrogels

In this study, acrylamide–itaconic acid hydrogels containing different amounts of itaconic acid prepared by irradiating with γ radiation are discussed. They have been used in experiments of swelling, diffusion and bovine serum albumin (BSA) adsorption. Maximum and minimum swellings were observed with water (1520%) and BSA (890%), respectively. Diffusion of water, NaCl and BSA within hydrogels were found to be non-Fickian in character. In the experiments of BSA adsorption, type III adsorption was found. The hydrogel prepared with 60 mg itaconic acid and irradiated at 2.00 kGy was found to be the best adsorption system for BSA. The adsorption capacity of acrylamide–itaconic acid hydrogel was found to exceed that of acrylamide hydrogel by more than 80–100%.     

Study of nobiletin binding to bovine serum albumin by capillary electrophoresis–frontal analysis and circular dichroism

A very recent epidemiological study provided strong support for nobiletin (NOB) as a potential candidate chemopreventive agent against cancer. From the pharmacology point of view, drug–protein interactions are determining factors in therapeutic, pharmacodynamic and toxicological drug properties. In this work, for the first time, detection of NOB at near‐physiological conditions was accomplished by means of capillary electrophoresis–frontal analysis (CE‐FA), and then the binding constants of NOB with bovine serum albumin (BSA) at the same conditions were determined. Complexation of NOB–BSA led to a decrease of the height for free NOB with increasing concentration of BSA. These results revealed the presence of a single class of binding site on BSA, and provided the binding constant of 10³/m , showing the strong affinity of NOB for BSA. Furthermore, circular dichroism spectra showed that, when the molar ratio of NOB to BSA was up to 2:1, NOB did not affect the overall protein conformation significantly and the protein thus retained a native‐like structure. These results may provide important information for preclinical studies of nobiletin in pharmaceutical research. Copyright © 2010 John Wiley & Sons, Ltd.     

BSA‐Polysaccharide Interactions at Negatively Charged Electrode Surface. Effects of Current Density.

Constant current chronopotentiometric stripping (CPS) peak H due to catalytic hydrogen evolution reaction on Hg‐containing electrodes appeared useful in the analysis of protein complexes with single‐stranded and double‐stranded DNA as well as with peptides. In dependence on stripping current (I_str), structural transition of the protein alone or in complexes can be followed as a result of the protein exposure to electric field effects. For the first time we show here that the CPS analysis can be used for the study of the interaction of BSA with a polysaccharide namely sodium alginate (SA). BSA‐SA complex formation was accompanied by the shift of the structural transition of BSA to lower ‐I_str intensities. Another polysaccharide dextran did not alter I_str‐dependent structural transition of BSA. BSA‐SA complex can be disturbed by an electric field effect or high ionic strength confirming the electrostatic nature of BSA‐SA interaction.