Thermodynamic and structural changes associated with the interaction of a dirhamnolipid biosurfactant with bovine serum albumin

The interaction of a dirhamnolipid biosurfactant secreted by Pseudomonas aeruginosa with bovine serum albumin was studied by means of various physical techniques. Binding of the biosurfactant to bovine serum albumin was first characterized by isothermal titration calorimetry, showing that one or two molecules of dirhamnolipid, in the monomer state, bound to one molecule of the protein with high affinity. These results were confirmed by surface tension measurements in the absence and presence of bovine serum albumin. As seen by differential scanning calorimetry, dirhamnolipid shifted the temperature of the thermal unfolding of bovine serum albumin toward higher values, thus increasing the stability of the protein on heating. The impact of dirhamnolipid on the structure of the native protein was low, since most of the secondary structure remained unaffected upon interaction with the biosurfactant, as shown by FTIR spectroscopy. However, 2D correlation infrared spectroscopy indicated that the sequence of temperature-induced structural changes in native bovine serum albumin was modified by the presence of the biosurfactant. The consequences of these results in relation to possible applications of these dirhamnolipid biosurfactants for protein studies are discussed.     

Photochemical modification of 10 kDa polyethersulfone ultrafiltration membranes for reduction of biofouling

Poly(ether sulfone) 10 kDa ultrafiltration membranes were modified by photolysis using ultraviolet light and graft polymerization of hydrophilic monomers onto the membrane surface to create more hydrophilic and lower fouling membrane surfaces. The modified membrane surfaces were characterized by FTIR/ATR and captive bubble contact angle measurements to determine chemical and hydrophilicity changes during modification. The modified membranes were compared with an unmodified poly(ether sulfone) (control) membrane as well as a commercial regenerated cellulose and a low protein adsorbing poly(ether sulfone) membrane using a newly developed standardized filtration protocol with 1 wt% bovine serum albumin. The best performing modified membrane was with N-vinyl-2-pyrrolidinone and showed a 25% increase in hydrophilicity, a 49% decrease in bovine serum albumin fouling, and a 4% increase in bovine serum albumin retention compared to the unmodified poly(ether sulfone) membrane. While the regenerated cellulose membrane had the lowest fouling and the low protein adsorbing membrane had the highest flux of all tested membranes, the N-vinyl-2-pyrrolidinone-modified membranes had the best combination of low fouling and high flux.     

分析测试新成果(160~168)金属吸附剂用于去除血清蛋白及核苷检测

利用多糖与金属离子复合制备了一种高效的蛋白质吸附剂.海藻酸钠和羧甲基纤维素钠是两种富含羟基和羧基的多糖, 具有较强的金属亲和性.将其用钙离子交联后制备成金属-多糖复合材料, 进一步修饰铜离子, 得到蛋白质吸附剂.吸附剂对富含组氨酸的牛血红蛋白的吸附量可以达到33 g/g, 对少量组氨酸的牛血清白蛋白的吸附量也可以达到9.8 g/g.蛋白质吸附剂对人血血清进行两次吸附后, 可以去除其中98%的蛋白, 能够满足人血血清中核苷类物质的直接色谱进样检测.     

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.     

荧光光谱在研究氯霉素与沙拉沙星间拮抗作用中的应用

氯霉素(CHL)和沙拉沙星(SLFX)均能够猝灭牛血清白蛋白(BSA)的荧光. 当两种药物共存时使BSA荧光进一步猝灭, 据此利用荧光光谱法研究了氟喹诺酮类药物SLFX与CHL间相互作用. 结果表明: 两种药物间存在拮抗作用, 使药物与蛋白的结合稳定性增加, 致使能够转运到作用部位产生药理效应的游离型药物含量减少, 造成药效降低; 药物对蛋白荧光的猝灭属于静态猝灭; 药物与蛋白结合位点数约为1. 根据Forster非辐射能量转移理论, 确定了药物与蛋白之间的结合距离r, 药物间拮抗作用的存在使r值降低, 结合距离减小. 同步荧光光谱研究表明, 药物间的拮抗作用对蛋白质构象产生影响, 使蛋白质分子伸展, 疏水性降低.     

Conformational stability and antibody response to the 18kda heat- shock protein formulated into different vehicles

Protein stability is one of the most important obstacles for successful formulation in the development of new-generation vaccines. Here, the 18kDa heat-shock protein (18kDa-hsp) was chemically modified though conjugation with bovine serum albumin or by esterification with N-hydroxysuccinimide ester of palmitic acid. The biologically active conformation of the protein was preserved after chemical modification. The immune responses to the recombinant 18kDa-hsp fromMycobacterium leprae were studied in different presentations: free, copolymerized with bovine serum albumin in aggregates (18kDa-hsp-BSA), and either surface linked to liposomes or entrapped into liposomes. Measuring the antibody production of immunized genetically selected mice has compared the adjuvant effects of liposomes and proteic copolymer. Among the two liposome preparations, the strongest response was obtained with the surface-exposed antigen-liposomes. The copolymer 18kDa-hsp-BSA conferred a high titer of antibody in injected mice, and persisted 70 d after immunization. This approach should prove very useful for designing more effective vaccines by using 18kDa-hsp as carrier protein.     

Ionic liquid-assisted SDS-PAGE to improve human serum protein separation

Ionic liquid (IL)-assisted sodium dodecyl sulfate polyacrylamide gel electrophoresis (ILs-SDS-PAGE) was presented to improve protein separation. ILs were employed during the preparation process of polyacrylamide gel, then the modified gel was used for commercial protein marker, binary bovine serum albumin/lysozyme (BSA/Lyz) and human serum separation. The influence of ionic liquid concentration, cation alkyl chain length, cation and anion types on proteins separation were investigated. The results showed that ILs played a role in improving some protein separation, and ILs-SDS-PAGE provided higher resolution and separation efficiency than ordinary SDS-PAGE for low and middle relative molecular mass proteins in human serum. In addition, the principle of ILs-SDS-PAGE was discussed and the comparison of ILs-SDS-PAGE with ordinary SDS-PAGE and Native PAGE was made.     

Preparation of composite monoliths of quaternized chitosan and diatom earth for protein separation

In this study, composite monoliths with porous structures were prepared using quaternized chitosan and diatom earth for protein separation. Quaternized chitosan (N-[(2-hydroxy-3-trimethylammonium)propyl] chitosan chloride) dissolved in water was mixed with diatom earth and crosslinked with glutaraldehyde under low-temperature conditions to form a cryogel. Interconnected porous monoliths were obtained after removing ice crystals from the cryogel. The monoliths adsorbed bovine serum albumin selectively from the solution mixture of bovine serum albumin and bovine ɤ-globulin, and bovine ɤ-globulin was recovered in the flow-through fraction. The adsorption selectivity was enhanced by changing the solution pH from 6.8 to 5.5. The adsorption of bovine serum albumin by the monolith was replicated at least five times following its washing with a buffer containing 400 mM NaCl and subsequent regeneration with a 10 mM acetate buffer. The composited monolith is a promising adsorbent for the removal of acidic proteins, such as serum albumin contamination in neutral proteins, for example, ɤ-globulins, in bioproduction processes.     

Studies on electrochemical oxidation of azithromycin and its interaction with bovine serum albumin

A novel nanoparticle film modified electrode has been constructed using a glassy carbon electrode (GCE) coated with a carbon nanotube-dihexadecylphosphate (DHP) film. This modified electrode exhibits an enhanced effectiveness for the oxidation of azithromycin. A method is also described for the evaluation of azithromycin-bovine serum albumin (BSA) interaction. The electrochemical behavior of azithromycin as well as its interaction with BSA at this nanoparticle film electrode has been investigated by cyclic voltammetry, linear sweep voltammetry, differential pulse voltammetry and chronocoulometry. The binding number and association constant between azithromycin and bovine serum albumin have been obtained.     

Competitive adsorption of collagen and bovine serum albumin—effect of the surface wettability

The competitive adsorption of collagen and bovine serum albumin (BSA) on surfaces with varied wettability was investigated with imaging ellipsometry, and ellipsometry. Silane modified silicon surfaces were used as substrates. The results showed that surface wettability had an important effect on protein competitive adsorption. With the decrease of surface wettability, the adsorption of collagen from the mixture solution of collagen and BSA decreased, while the adsorption of BSA increased.     

How does bovine serum albumin prevent the formation of kidney stone? A kinetics study

To attain a better understanding of the crystallization of calcium oxalate crystals under the influence of the protein bovine serum albumin, we examined not only the nucleation kinetics but also the structural synergy between the biomineral and the biosubstrate. It follows that during the crystallization process of calcium oxalate crystals bovine serum albumin inhibits the nucleation of calcium oxalate by increasing the kink kinetics barrier. The results of scanning electron microscopy and X-ray diffraction show, however, that bovine serum albumin promotes the formation of calcium oxalate dihydrate. Apart from this, bovine serum albumin facilitates the ordered calcium oxalate crystal assembly by suppressing the supersaturation-driven interfacial structure mismatch. The physics questions behind the mentioned effects have been addressed from the kinetics point of view. This may explain why bovine serum albumin plays an important role in suppressing urine stone formation.     

Surface‐Imprinting Sensor Based on Carbon Nanotubes/Graphene Composite for Determination of Bovine Serum Albumin

A novel molecularly imprinted sensor was firstly prepared based on a carbon nanotubes/graphene composite modified carbon electrode (MIPs/CNT/GP/CE) for the selective determination of bovine serum albumin. The molecularly imprinted sensor was tested by differential pulse voltammetry (DPV) to investigate the relationship between the response current and bovine serum albumin concentration. The results showed that a wide linear range (1.0×10^?4 to 1.0×10^?10 g mL^?1) for the detection of bovine serum albumin with a low detection limit of 6.2×10^?11 g mL^?1 for S/N=3 was obtained. The novel imprinted sensor exhibited high selectivity, sensitivity, and reproducibility, which provided an applicable way for sensor development.     

Study of Protein Adsorption/Adhesion Behaviors on Solid Beads Surface with Different Surface Properties

Three kinds of chitosan (CS) derivatives have been prepared via polyethyleneimine (PEI) or polyethylene glycol (PEG) together with PEI modified chitosan. FTIR, x-ray photoelectron spectroscopy (XPS), and thermal gravimetric analysis (TGA)/differential thermal analysis (DTA) proved the successful linkage, and XPS and zeta potential analysis show that these derivatives beads possess different surface nature. The PEI modified chitosan derivative is made in acid solution (A-PEI-CS) and the one added with additional PEG (PEG–CS-PEI) has showed higher swelling degree than their counterpart derivative prepared at basic condition (B-PEI-CS). Although the nitrogen content of A-PEI-CS is higher than that of B-PEI-CS, due to the better surface hydrophilicity, the B-PEI-CS beads are more capable of adsorbing bovine serum albumin (BSA) protein. Electrostatic attraction facilitates protein adsorption in a narrow acidic pH range while hydrophilicity hinders protein adhering to the beads’ surface at any pH.     

Preparation and characterization of Zonyl-coated nanodiamonds with antifouling properties

The paper reports on covalent linking of a modified amphiphilic polymer, the alkynyl-terminated Zonyl, to azide-terminated nanodiamonds by click chemistry. An analysis of the hybrid particle stability is presented based on size and zeta potential measurements. The antifouling character of the grafted nanodiamonds was investigated using bovine serum albumin as a model protein. The protein adsorption was quantified using a Bradford assay and found to be reduced by 30% in the presence of the Zonyl layer.     

An automated flow-injection serial dynamic dialysis technique for drug-protein binding studies

An automated flow-injection analyzer is interfaced with a dialysis unit to study drug-protein protein binding interactions. The binding of some sulfonamides to bovine serum albumin is studied by means of the automated system and gives results similar to those obtained by other procedures. A usual time for a complete run is 100 min, including calibration. The dialysable sulfonamides are quantified spectrophotometrically by using a modified Bratton-Marshall method. The system is also used for the calculation of dialysis rate constants.     

Thiophilic interaction chromatography of serum albumins

An investigation of the binding of native and recombinant human serum albumin and bovine serum albumin on three thiophilic gels, PyS, 2S, and 3S was performed. In addition to these proteins, we studied serum albumins from several species such as goat, rabbit, guinea pig, rat, hamster, baboon, and pig. Our results reveal that recombinant human serum albumin (rHSA) binds completely to PyS whereas native human serum albumin and bovine serum albumin bind only partially to PyS. The binding affinities of rHSA, human serum albumin and bovine serum albumin to 2S and 3S gels are less than their binding to PyS. Serum albumins from goat, rabbit, guinea pig, rat, hamster, baboon, and pig bind much stronger to 3S gel than human and bovine serum albumins. The binding of pig and hamster serum albumins is stronger than that of rat, goat, baboon, and rabbit.     

Direct conjugation of semiconductor nanoparticles with proteins

Nanocrystalline CdS particles directly conjugated with bovine serum albumin (BSA) protein were prepared by applying the supercritical fluid processing technique, rapid expansion of a supercritical solution into a liquid solvent. The direct conjugation takes advantage of the unique features of the process for nanoparticle formation. The BSA-conjugated CdS nanoparticles in stable aqueous suspension or in the solid state were characterized by using microscopy, X-ray diffraction, and optical spectroscopy methods. The results show that well-dispersed CdS nanoparticles are coated with BSA in a core-shell-like arrangement and that the protein species associated with the nanoparticles remain functional according to the modified Lowry assay. These BSA-conjugated CdS nanoparticles are also strongly luminescent, with the luminescence spectrum contributed to primarily by the exciton emission.     

The electrochemical behavior of N-n-undecy1-N＇-（sodium-p- amino-benzenesulfonate） thiourea and its interaction with bovine serum albumin

In pH 5.5 phosphate buffer solution, N-n-undecyl-N＇-（sodium-p-amino-benzenesulfonate） thiourea （UPT） produced a pair of redox peaks on the bare glassy carbon electrode. At the multi-walled carbon nanotube （MWNT） modified electrode, the electrochemical behavior of UPT enhanced greatly. In the presence of bovine serum albumin （BSA）, the peak currents of UPT decreased linearly due to the formation of a super-molecular complex. This method was successfully applied to the determination of BSA in a bovine serum sample.     

Integration of carboxyl modified magnetic particles and aqueous two-phase extraction for selective separation of proteins

Both of the magnetic particle adsorption and aqueous two-phase extraction (ATPE) were simple, fast and low-cost method for protein separation. Selective proteins adsorption by carboxyl modified magnetic particles was investigated according to protein isoelectric point, solution pH and ionic strength. Aqueous two-phase system of PEG/sulphate exhibited selective separation and extraction for proteins before and after magnetic adsorption. The two combination ways, magnetic adsorption followed by ATPE and ATPE followed by magnetic adsorption, for the separation of proteins mixture of lysozyme, bovine serum albumin, trypsin, cytochrome C and myloglobin were discussed and compared. The way of magnetic adsorption followed by ATPE was also applied to human serum separation.     

Thermo‐sensitive surface molecularly imprinted magnetic microspheres based on bio‐macromolecules and their specific recognition of bovine serum albumin

Bovine serum albumin imprinted magnetic microspheres, with functional monomers of modified chitosan, N‐isopropylacrylamide and sulfobetaine methacrylate, were successfully prepared and characterized in detail. Computational analyses showed that during the preparation process, modified chitosan can effortlessly form multiple non‐covalent bonds with protein molecules. Temperature‐sensitive N‐isopropylacrylamide improves the elution efficiency by abating the mass transfer resistance. Meanwhile, the zwitterionic sulfobetaine methacrylate strongly interacts with H₂O molecules, remarkably reducing the non‐specific adsorption. The specific bovine serum albumin adsorption performances of the prepared imprinted material were then determined. The adsorption amount reached 86.87 mg/g and the imprinting factor was 6.49. These excellent specific adsorption properties are attributed to the synergetic effects of the different monomers. The fabricated imprinted material not only exhibits great prospects as a biosensor or separation material for protein molecules, but also provides a collaborative strategy for preparing multi‐functional imprinted materials.