疏水表面对牛血清清蛋白二级结构的诱导

本文结合圆二色谱,椭圆偏振术及放射性示踪技术研究了疏水表面对牛血清清蛋白二级结构的诱导作用,实验结果表明：处于不同阶段的蛋白质分子由于受到不同程度的诱导作用而呈现不同的二级结构特征。初始阶段吸附的ＢＳＡ分子丧失有序结构,形成以无规卷曲为主的二级结构。中间阶段吸附的ＢＳＡ呈现出以β结构为主的二级结构。而后期吸附的ＢＳＡ分子保存了大部分α螺旋结构。作者认为疏水表面对ＢＳＡ二级结构的诱导与表面的疏水作用     

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.     

牛血清白蛋白对Cu+与δ-MnO2间吸附平衡性质的影响

研究了溶液中牛血清白蛋白(BSA)的存在对Cu2 与δ-MnO2间吸附平衡性质的影响,旨在增进人们对Cu2 环境化学行为的认识.结果表明:BSA存在下,Cu2 在δ-MnO2表面的吸附率-pH曲线呈典型的"S"形,加入BSA使Cu2 的吸附突跃向高pH方向移动,影响程度与BSA的加入方式有关,其次序为Ⅳ(先加BSA后加Cu2 )＞Ⅲ(Cu2 和BSA同时加)≈Ⅱ((先加Cu2 后加BSA);BSA存在下,Cu2 的等温吸附线遵循Langmuir等温吸附方程,Cu2 的饱和吸附量随BSA浓度的增大而减小,并随BSA的加入方式而改变,其次序为Ⅰ(不加BSA)＞Ⅱ≈Ⅲ＞Ⅳ;Cu2 在δ-MnO2上的饱和吸附量随温度的升高而增大.在pH=3.0条件下,Cu2 在δ-MnO2上的吸附是完全可逆的,但pH=5.0的条件下,吸附是不可逆的,BSA对Cu2 的吸附可逆性没有影响.     

光谱和微量热法分析柑橘苷与BSA分子间作用及构建其理论模型

光谱和微量热法分析柑橘苷(naringin,NAR)与牛血清白蛋白（bovine serum albumin, BSA）分子间作用,构建NAR与BSA分子间作用的理论模型。采用紫外-荧光光谱法解析Fōrster方程求得NAR与BSA分子间作用及分子间作用的临界距离r,等温滴定微量热技术测定NAR与BSA分子间作用的积分量热曲线,获得Δ H并通过Gibbs-Helmholtz方程获取Δ S和Δ G。基于光谱和微量热辅助分析,构建NAR与BSA分子间作用的理论模型。结果表明,光谱法测出NAR与BSA发生分子间作用,NAR与BSA分子间作用的临界距离为2.06 nm,表明NAR与BSA分子间作用为短程分子间作用。微量热法成功测定出NAR与BSA分子间的热力学参数Δ H<0,Δ S>0,Δ G<0,说明NAR与BSA分子间作用是自发进行的放热相互作用。依据Ross理论分析NAR与BSA分子间作用力主要是疏水作用力和静电作用力。模型构建结果说明,NAR与BSA分子间作用主要发生在BSA的domain IIA区域,NAR与BSA分子间作用力主要是静电作用力,兼有范德华作用力和氢键。实验与理论模型构建结果基本一致。本研究工作可为深入了解蛋白质与大分子化合物间的作用以及研究微观药理学机制提供有益的参考。     

表面活性剂增敏--牛血清白蛋白荧光猝灭法测定槐花中芦丁含量

目的:应用牛血清白蛋白荧光猝灭法建立一种测定槐花中芦丁含量的新方法。方法:牛血清白蛋白(BSA)具有很强的内源荧光性,而芦丁溶液本身不产生荧光。当芦丁与BSA结合后,会导致其荧光强度下降,表面活性剂吐温-80(T-80)对体系有促进荧光猝灭作用。BSA在λex=338nm处的荧光猝灭程度与芦丁的量在一定浓度范围内呈良好的线性关系,据此建立测定槐花中芦丁含量的新方法。结果:该结合物的最大发射波长为λmax=338nm,与芦丁摩尔浓度在6×10-7~3.0×10-5mol.L-1范围内线性关系良好。其线性回归方程为ΔF=136.36CRu(×10-5mol.L-1)-0.5454,相关系数r=0.9976,检出限为1.58×10-7mol.L-1,RSD为2.8%~4.3%,加标回收率为97.6%~101.2%。结论:本方法操作简便、快速,用于实际样本的测定,结果满意。     

酸性品红与牛血清白蛋白变色反应机理的研究

利用光谱探针技术研究酸性品红(fuchsine ac id,FA)与牛血清白蛋白(bovine serum album in,BSA)的变色反应机理,考察了不同实验条件对FA-BSA复合物吸收光谱的影响。实验结果表明,FA与BSA分子相互作用产生变色反应的机理主要是由FA与BSA间的疏水相互作用引起,而静电作用则是形成FA-BSA桃红色复合物的必要条件。     

表面活性剂增敏——牛血清白蛋白荧光猝灭法测定槐花中芦丁含量

目的：应用牛血清白蛋白荧光猝灭法建立一种测定槐花中芦丁含量的新方法。方法：牛血清白蛋白（BSA）具有很强的内源荧光性,而芦丁溶液本身不产生荧光。当芦丁与BSA结合后,会导致其荧光强度下降,表面活性剂吐温-80（T-80）对体系有促进荧光猝灭作用。BSA在λex=338nm处的荧光猝灭程度与芦丁的量在一定浓度范围内呈良好的线性关系,据此建立测定槐花中芦丁含量的新方法。结果：该结合物的最大发射波长为λmax=338nm,与芦丁摩尔浓度在6×10-7-3.0×10-5mol.L-1范围内线性关系良好。其线性回归方程为ΔF=136.36CRu（×10-5mol.L-1）-0.5454,相关系数r=0.9976,检出限为1.58×10-7mol.L-1,RSD为2.8%-4.3%,加标回收率为97.6%~101.2%。结论：本方法操作简便、快速,用于实际样本的测定,结果满意。     

从反胶束溶液中反萃牛血清白蛋白的研究

由近红外光谱证实,十六烷基三甲基溴化铵(CTAB)/正己醇-正辛烷反胶束溶液是牛血清白蛋白(BSA)增溶于非极性有机溶剂的较理想中介。研究了反萃液酸度、离子强度和种类等参数对BSA反萃率的影响,过低的溶液酸度导致BSA变性。适宜的反萃液为:1.0～2.0mol/L KBr,pH4.3～4.9。蛋白质的紫外光谱表明,BSA分子在料液,反胶束溶液和反萃液中的构象大致相同。此外,探讨了反胶束溶液循环使用的效率问题。最后,通过采用适当的相比,成功地实现了蛋白质的回收和浓缩。     

牛血清白蛋白对Cu^2＋与δ-MnO2间吸附平衡性质的影响

研究了溶液中牛血清白蛋白（BSA）的存在对Cu^2＋与δ-MnO2间吸附平衡性质的影响,旨在增进人们对Cu^2＋环境化学行为的认识。结果表明：BSA存在下,Cu^2＋在δ-MnO2表面的吸附率-pH曲线呈典型的“S”形,加入BSA使Cu^2＋的吸附突跃向高pH方向移动,影响程度与BSA的加入方式有关,其次序为Ⅳ（先加BSA后加Cu^2＋）〉Ⅲ（Cu^2＋和BSA同时加）≈Ⅱ（（先加Cu^2＋后加BSA）;BSA存在下,Cu^2＋的等温吸附线遵循Langmuir等温吸附方程,Cu^2＋的饱和吸附量随BSA浓度的增大而减小,并随BSA的加入方式而改变,其次序为I（不加BSA）〉Ⅱ≈Ⅲ〉Ⅳ;Cu^2＋在δ-MnO2上的饱和吸附量随温度的升高而增大。在pH=3．0条件下,Cu^2＋在δ-MnO2上的吸附是完全可逆的,但pH=5．0的条件下,吸附是不可逆的,BSA对Cu^2＋的吸附可逆性没有影响。     

考马斯亮蓝与牛血清白蛋白相互作用机理的研究

利用光谱探针技术研究在酸性溶液中考马斯亮蓝G-250(Coomassie brilliant blue G-250,CBBG)与牛血清白蛋白(bovine serum albumin,BSA)相互作用机理,考察了不同实验条件对CBBG-BSA复合物吸收光谱的影响。实验结果表明:CBBG与BSA相互作用产生光谱蓝移主要是由CBBG与BSA间的疏水相互作用引起,而静电作用则是形成CBBG-BSA蓝色复合物的必要条件。同时,CBBG聚集体的聚集程度是影响CBBG-BSA蓝色复合物形成的重要因素。     

Total internal reflection fluorescence study of energy transfer in surface-adsorbed and dissolved bovine serum albumin

A simple adaptation of a commercial spectrofluorometer allows selective excitation of fluorescent biomolecules adsorbed to a solid surface while they are in equilibrium with a bulk solution. As a demonstration of this technique, we have detected a change in the effective singlet-singlet energy transfer in fluorescence-labeled bovine serum albumin (BSA) upon adsorption to a fused silica surface. The technique combines total internal reflection fluorescence excitation of surface-adsorbed BSA with a fluorescence spectroscopic examination of energy transfer between two different fluorophores that are covalently bound to amino groups in each BSA molecule. Two donor--acceptor pairs were used, 4-chloro-7-nitro-2,1,3-benzoxadiazole-rhodamine and dansyl-eosin. For studies of surface-adsorbed BSA, we constructed a device in which the excitation light of a standard fluorescence spectrometer totally internally reflects from a surface at which adsorbed BSA is in equilibrium with the bulk solution. A shallow evanescent wave is created, which excites fluorescence from only those BSA molecules in close proximity to the surface. Spectral examination shows significantly less effective singlet-singlet energy transfer from the donor to the acceptor in surface-adsorbed BSA relative to that in native bulk-dissolved BSA. Under appropriate and reasonable assumptions, the energy transfer change between native and adsorbed states of fluorescent BSA can be interpreted as a conformational change of BSA upon adsorption.     

Analysis of protein adsorption characteristics to nano-pore silica particles by using confocal laser scanning microscopy

The effect of average pore size of nano-pore silica particles on protein adsorption characteristics was determined experimentally by the dissociation constant and the adsorption capacity determined from the Langmuir equation. As the average pore size was increased from 2.2 to 45 nm, the BSA adsorption capacity increased from 16.8 to 84.3 mg/g-silica so as the equilibrium constant (from 2.6 to 9.4 mg/ml). Using confocal microscopy with fluorescence labeling, we could visualize the protein adsorption in situ and determine the minimum pore size required for efficient intraparticle adsorption. The confocal microscopy analysis revealed that BSA was adsorbed mainly on the surface of the particles with a smaller pore size, but diffused further into the interstitial surface when it was sufficiently large. It was concluded that for BSA whose Stoke's diameter is ca. 3.55 nm the minimum pore size of about 45 nm or larger was required for a sufficient adsorption capacity.     

Protein adsorption at solid-liquid interfaces: Part IV--Effects of different solid-liquid systems and various neutral salts.

Adsorption isotherms of BSA at the solid-water interfaces have been studied as a function of protein concentration, ionic strength of the medium, pH and temperature using silica, barium sulphate, carbon, alumina, chromium, ion-exchange resins and sephadex as solid interfaces. In most cases, isotherms for adsorption of BSA attained the state of adsorption saturation. In the presence of barium sulphate, carbon and alumina, two types in the isotherms are observed. Adsorption of BSA is affected by change in pH, ionic strength and temperature of the medium. In the presence of metallic chromium, adsorbed BSA molecules are either denatured or negatively adsorbed at the metallic interface. Due to the presence of pores in ion-exchange resins, adsorption of BSA is followed by preferential hydration on resin surfaces in some cases. Sometimes two steps of isotherms are also observed during adsorption of BSA on the solid resins in chloride form. Adsorption of BSA, beta-lactoglobulin, gelatin, myosin and lysozyme is negative on Sephadex surface due to the excess adsorption of water by Sephadex. The negative adsorption is significantly affected in the presence of CaCl2, KSCN, LiCl, Na2SO4, NaI, KCl and urea. The values of absolute amounts of water and protein, simultaneously adsorbed on the surface of different solids, have been evaluated in some cases on critical thermodynamic analysis. The standard free energies (delta G0) of excess positive and negative adsorption of the protein per square meter at the state of monolayer saturation have been calculated using proposed universal scale of thermodynamics. The free energy of adsorption with reference to this state is shown to be strictly comparable to each other. The magnitude of standard free energy of transfer (delta G0B) of one mole of protein or a protein mixture at any type of physiochemical condition and at any type of surface is observed to be 38.5 kJ/mole.     

Influence of polymer structure on adsorption behavior at solid–liquid interface by Monte Carlo simulation

Monte Carlo simulations for the adsorption of polymers including random copolymer, homopolymer, diblock copolymer and two kinds of triblock copolymers, respectively, in nonselective solvent at solid–liquid interface have been performed on a simple lattice model. The effect of polymer structure on adsorption properties was examined. In simulations, all polymeric molecules are modeled as self-avoiding linear chains composed of two segments A and B while A is attractive to the surface and B is non-attractive. It was found that for all polymers, the size distribution of various configurations is determined by the linked sequence of segments and the interaction energy between segment and surface. The results of simulation show that the adsorbed amount always increases with increasing bulk concentration but the adsorption layer thickness is mostly dependent on the adsorption energy at a fixed fraction of segments A. On the other hand, diblock copolymer has always the highest surface coverage and adsorbed amount, while random copolymers and homopolymers give generally the smallest surface coverage and adsorbed amount. It is shown that the sequence of polymer chains, i.e. molecular structure, is the most important factor in affecting adsorption properties at the same composition and interaction between segment and surface. The results also show that the adsorption behavior of random copolymers is remarkably different from that of block copolymers, but acting like homopolymer.     

Modification of cellulose films by adsorption of CMC and chitosan for controlled attachment of biomolecules

The adsorption of human immunoglobulin G (hIgG) and bovine serum albumin (BSA) on cellulose supports were investigated. The dynamics and extent of related adsorption processes were monitored by surface plasmon resonance (SPR) and quartz crystal microbalance with dissipation monitoring (QCM-D). Amine groups were installed on the cellulose substrate by adsorption of chitosan from aqueous solution, which allowed for hIgG to physisorb from acid media and produced a functionalized substrate with high surface density (10 mg/m(2)). hIgG adsorption from neutral and alkaline conditions was found to yield lower adsorbed amounts. The installation of the carboxyl groups on cellulose substrate via carboxymethylated cellulose (CMC) adsorption from aqueous solution enhanced the physisorption of hIgG at acidic (adsorbed amount of 5.6 mg/m(2)) and neutral conditions. hIgG adsorption from alkaline conditions reduced the surface density. BSA was used to examine protein attachment on cellulose after modification with chitosan or carboxymethyl cellulose. At the isoelectric point of BSA (pI 5), both of the surface modifications enhanced the adsorption of this protein when compared to that on unmodified cellulose (a 2-fold increase from 1.7 to 3.5 mg/m(2)). At pH 4, the electrostatic interactions favored the adsorption of BSA on the CMC-modified cellulose, revealing the affinity of the system and the possibility of tailoring biomolecule binding by choice of the surface modifier and pH of the medium.     

界面上配基种类对BSA吸附行为的影响

利用双偏振极化干涉测量仪(DPI)研究了界面上配基种类对BSA吸附行为的影响。采用3-氨基丙基三乙氧基硅烷(APTES)、3-(甲氨基)丙基三甲氧基硅烷(MAPTMS)和N,N-二乙基-3-氨基丙基三甲氧基硅烷(DAPTMS)对DPI芯片进行了修饰,利用X射线光电子能谱比较了芯片上不同配基的密度,采用原子力显微镜(AFM)和DPI对界面上BSA吸附行为进行了研究。结果表明APTES修饰界面上BSA呈饼状,高配基密度易导致BSA多位点吸附。相同偶联密度条件下BSA在DAPTMS修饰芯片的吸附量高于MAPTMS修饰芯片,但吸附层厚度一致,表明DAPTMS表面上BSA存在聚集现象;AFM扫描结果与DPI分析结果一致,证明了配基密度和种类不仅影响界面上蛋白质吸附量,而且影响蛋白质吸附密度和表面聚集行为。     

PM-IRRAS investigation of the interaction of serum albumin and fibrinogen with a biomedical-grade stainless steel 316LVM surface

Polarization modulation infrared reflection absorption spectroscopy (PM-IRRAS) was applied to investigate the interaction of bovine serum albumin (BSA) and fibrinogen with a biomedical-grade 316LVM stainless steel surface, in terms of the adsorption thermodynamics and adsorption-induced secondary structure changes of the proteins. Highly negative apparent Gibbs energy of adsorption values revealed a spontaneous adsorption of both proteins onto the surface, accompanied by significant changes in their secondary structure. It was determined that, at saturated surface coverages, lateral interactions between the adsorbed BSA molecules induced rather extensive secondary structure changes. Fibrinogen's two coiled coils appeared to undergo negligible secondary structure changes upon adsorption of the protein, while large structural rearrangements of the protein's globular domains occurred upon adsorption. The secondary structure of adsorbed fibrinogen was not influenced by lateral interactions between the adsorbed fibrinogen molecules. PM-IRRAS was deemed to be viable for investigating protein adsorption and for obtaining information on adsorption-induced changes in their secondary structures.     

Effect of Air Plasma Processing on the Adsorption Behaviour of Bovine Serum Albumin on Spin-Coated PMMA Surfaces

This paper reports the adsorption of Bovine Serum Albumin （BSA） onto Dielectric Barrier Discharge （DBD） processed Poly（methyl methacrylate） （PMMA） surfaces by a Quartz Crystal Microbalance with Dissipation monitoring （QCM-D） technique. The purpose is to study the influence of DBD processing on the nature and scale of BSA adsorption on PMMA surface in vitro. It was observed that DBD processing improves the surface wettability of PMMA film, a fact attributable to the changes in surface chemistry and topography. Exposure of the PMMA to Phosphate Buffed Saline （PBS） solution in the QCM-D system resulted in surface adsorption which reaches an equilibrium after about 30 minutes for pristine PMMA, and 90 minutes for processed PMMA surface. Subsequent injection of BSA in PBS indicated that the protein is immediately adsorbed onto the PMMA surface. It was revealed that adsorption behaviour of BSA on pristine PMMA differs from that on processed PMMA surface. A slower adsorption kinetics was observed for pristine PMMA surface, whilst a quick adsorption kinetics for processed PMMA. Moreover, the dissipation shift of protein adsorption suggested that BSA forms a more rigid structure on pristine PMMA surface that on processed surface. These data suggest that changes in wettability and attendant chemical properties and surface texture of the PMMA surface may play a significant role in BSA adsorption process.     

Adsorption of β-Lactoglobulin onto the Surface of Stainless Steel Particles

The adsorption of β-Lactoglobulin (β-Lg), one of the main constituents of fouling deposits in milk processing, onto the surface of stainless steel particles was studied under various conditions. The adsorption isotherm of β-Lg at 25°C was of the Langmuir type, and the plateau suggested that the surface was covered by a monolayer of β-Lg. The amount of β-Lg adsorbed steeply increased above 65°C. At 75°C, it increased almost linearly with the protein concentration in the bulk solution. Heating and chemical modification of the SH-group caused a much smaller amount of β-Lg to be adsorbed at 75°C. These findings indicate that the thermal aggregation of denatured β-Lg at the surface is important in the adsorption. More β-Lg was adsorbed at pH 4 than at pH 6.85. This suggests that the electrostatic interaction between β-Lg and the surface contributes to the adsorption behavior.     

Reduction of irreversible protein adsorption on solid surfaces by protein engineering for increased stability

The influence of protein stability on the adsorption and desorption behavior to surfaces with fundamentally different properties (negatively charged, positively charged, hydrophilic, and hydrophobic) was examined by surface plasmon resonance measurements. Three engineered variants of human carbonic anhydrase II were used that have unchanged surface properties but large differences in stability. The orientation and conformational state of the adsorbed protein could be elucidated by taking all of the following properties of the protein variants into account: stability, unfolding, adsorption, and desorption behavior. Regardless of the nature of the surface, there were correlation between (i) the protein stability and kinetics of adsorption, with an increased amplitude of the first kinetic phase of adsorption with increasing stability; (ii) the protein stability and the extent of maximally adsorbed protein to the actual surface, with an increased amount of adsorbed protein with increasing stability; (iii) the protein stability and the amount of protein desorbed upon washing with buffer, with an increased elutability of the adsorbed protein with increased stability. All of the above correlations could be explained by the rate of denaturation and the conformational state of the adsorbed protein. In conclusion, protein engineering for increased stability can be used as a strategy to decrease irreversible adsorption on surfaces at a liquid-solid interface.