Thermal stability of bovine serum albumin DSC study

A calorimetric study of thermal denaturation of bovine serum albumin in aqueous solutions has shown essential differences in stability of fatty acid containing and defatted albumin. The first one shows a single endotherm peak in DSC curve near 69°C with enthalpy change about 1000 kJ mol^-1. Defated albumin melts in two different temperature ranges: near 56 and 69°C with enthalpy changes about 300 and 200 kJ mol^-1 respectively. Deconvolution analysis shows that the single endotherm is well approximated as the sum of three independent two-state transitions. Two transitions of bimodal DSC curve for defatted albumin are not of a two-state type. This molecule melts probably as two structurally independent parts. This revised version was published online in July 2006 with corrections to the Cover Date.     

Comparative DSC study of human and bovine serum albumin

The thermal denaturation process of bovine and human both fatty acid containing and fatty acid free albumins in aqueous solution was studied by use of differential scanning calorimetry. Human serum albumins were found to be more stable than their bovine counterparts. Fatty acid free albumins were characterized as generally less stable, more susceptible to aggregation, their unfolding endothermic transition was less cooperative and with the smaller degree of reversibility. Deconvolution analysis with using a non-two-state model with two component transitions showed essential differences in the thermodynamic parameters between all studied albumins, particularly regarding the high-temperature component transition.     

Stability of bovine serum albumin at different pH

Summary The effect of pH on the thermal denaturation of BSA containing fatty acids was studied by use of differential scanning calorimetry (DSC). Thermal scanning of BSA aqueous solutions gave various types of DSC curves depending on the protein concentration and on the pH. The broad bimodal endothermic transition was suggested to be connected with loose protein structure in contradistinction to single peak for compact molecule structure. The propensity toward precipitation at pH conditions ranging from 3.8 to 5 was observed. A scan-rate independent and partly reversible behavior of the thermal heating of BSA was found. Deconvolution of DSC traces in non-two-state model with assumption of two- or three-component transition allowed to study the effect of pH on different parts of BSA molecule.     

Effect of ethanol on the thermal stability of human serum albumin

The effect of ethanol on human serum albumin stability in aqueous solution was studied by use of differential scanning calorimetry. A deconvolution of DSC traces in 2-state model with ΔC _p=0 and ΔC _p≠0 was performed and analysed to obtain information on the interaction of ethanol with different parts of albumin molecule both fatty acid containing and fatty acid free. The differences in ethanol binding affinity for both kinds of albumin were found. At very low concentrations ethanol was observed to be a stabilizer of the folded state of albumin contrary to the higher concentration where its binding to the unfolded protein predominates.     

渗透压法对牛血清蛋白在NaCl水溶液中相互作用的研究

测量了牛血清蛋白不同pH值和不同离子强度Nacl水溶液中的渗透压,计算了渗透第二维里系数,按照Prausnitz提出的分子热学模型计算了蛋白质之间的静电排斥能、色散吸引能和离子排斥体积产生的吸引势能,并对这三种势能与溶液pH值和离子强度的变化关系进行讨论。     

锌离子对缓冲液中槲皮素和牛血清白蛋白相互作用的影响

采用荧光光谱法和紫外光谱法研究了Zn2+离子对槲皮素(Qct)和牛血清白蛋白(BSA)在pH=7.4的Tris-HCl缓冲溶液中相互作用的影响;根据荧光猝灭双倒数图计算了Qct和BSA之间的结合常数和结合位点数.结果表明,Qct和Zn2+离子都可以使BSA的荧光强度发生猝灭;Qct和BSA之间的结合常数为3.17×107L.mol-1.s-1,结合位点数为1.32.定量计算表明,加入Zn2+离子后,Qct与BSA间的结合常数显著降低、结合位点数减小,表明Zn2+离子参与了Qct与BSA的结合过程.     

DSC study of the association of ethanol with human serum albumin

Human serum albumin unfolding in ethanol/water mixtures was studied by use of differential scanning calorimetry. Ethanol-induced changes in DSC curves of defatted and non-defatted albumin were markedly different. In the presence of ethanol, bimodal denaturation transition for fatty acid free albumin was observed while that for albumin containing endogenous fatty acids was single and more sharpen than in aqueous solution. Ethanol was found to decrease the thermal stability of albumin due to the binding to the unfolded state to a higher degree than to the native state, thus favouring unfolding. The binding with different affinities has been suggested depending on ethanol concentration range.     

Counterion-accelerated diffusion of aqueous serum albumin

A modified Harned conductimetric procedure has been used to measure binary diffusion coefficients at 25°C for aqueous sodium and potassium salts of the protein bovine serum albumin (M _n BSA; M=Na⁺ or K⁺; n=2 to 23). The condition of electroneutrality requires that the BSA ions diffuse at the same speed as the relatively mobile sodium or potassium counterions. Consequently, the protein ions diffuse 3 to 8 times more rapidly than the isoionic protein diffuses. An approximate analysis of counterion-accelerated diffusion of high molecular weight solutes is presented which shows that the increase in diffusivity with the number of counterions is a thermodynamic effect brought about by an increase in the free energy gradient of the solutes.     

Binding of naproxen to bovine serum albumin and tryptophan-modified bovine serum albumin

Two classes of binding sites, a single high-affinity site with an association constant of 4·8×10⁶ M⁻¹ and two low-affinity sites with association constant of about 0·05×10⁶ M⁻¹ have been observed in the interaction of Naproxen with bovine serum albumin (BSA). Chemical modification of two tryptophan residues in BSA with 2-hydroxy-5-nitrobenzyl bromide has led to a reduction in the association constant of the high-affinity site by 89% and its number of binding sites by 66% suggesting the involvement of tryptophan residues in the high-affinity site. In contrast, the two low-affinity sites were not affected by the modification. Binding of Naproxen to the low-affinity sites of BSA induces microdisorganisation of the albumin structure leading to conformational changes as evident from fluorescence measurements with 1-anilino-8-naphthalenesulphonic acid as the probe.     

甲苯胺蓝与牛血清白蛋白作用的光谱特性研究

应用荧光光谱法研究了甲苯胺蓝(TB)与牛血清白蛋白(BSA)相互作用的光谱特性.测定了TB与BSA在20℃,30℃和40℃3个温度下的结合常数KA分别为8.94×103,1.75×104,1.74×104L/mol,结合位点数n分别为0.66,0.71和0.93.实验表明:TB与BSA的荧光猝灭主要为静态猝灭;利用Gibbs-Helmholtz方程计算得到热力学参数ΔH为49.416kJ,ΔS为244.876J.mol-1.K-1,探讨了它们的相互作用机理,结果表明TB主要以氢键作用力与BSA相互作用;应用同步荧光技术研究了TB对BSA构象的影响.     

Temperature‐induced conformational transition of bovine serum albumin in neutral aqueous solution by reversed‐phase liquid chromatography

The temperature‐induced conformational transition of bovine serum albumin (BSA) in neutral aqueous solution was studied using intrinsic fluorescence emission spectrum, reversed‐phase liquid chromatography and sodium dodecyl sulfate–polyacrylamide gel electrophoresis, and the conformation transition thermodynamic parameters were determined in the temperature range 12–50 °C. The results showed that, in the temperature range 12–20 °C, BSA only existed in a single conformation state A, while in the temperature range 22–50 °C, it existed in two different conformation states: A and B. The percentage of conformation state A decreased while that of conformation state B increased with the increase in temperatures, and when temperature approached 50 °C conformation state B accounted for approximately 25% of all conformation states of BSA. In the conformational transition of BSA from conformation state A to conformation state B, the positive enthalpy change, entropy change and free energy changes demonstrated that the conformational transition was endothermic, nonspontaneous and mainly entropy‐driven. © 2013 The Authors. Biomedical Chromatography published by John Wiley & Sons, Ltd.     

Hydrophobic interactions of phenoxazine modulators with bovine serum albumin

The interaction of 10-(3′-N-morpholinopropyl)phenoxazine [MPP], 10-(4′-N-morpholinobutyl)phenoxazine [MBP], 10-(3′-N-morpholinopropyl)-2-chlorophenoxazine [MPCP], 10-(3′-N-piperidinopropyl)-2-chlorophenoxazine [PPCP] or 10-(3′-N-morpholinopropyl)-2-trifluoromethylphenoxazine [MPTP] with bovine serum albumin (BSA) has been studied by gel filtration and equilibrium dialysis methods. The binding of these modulators, based on dialysis experiments, has been characterized using the following parameters: percentage of bound drug (Β), the association constant (K ₁), the apparent binding constant (k) and the free energy change (δF‡). The binding of phenoxazine derivatives to serum transporter protein, BSA, is correlated with their octanol-water partition coefficient, log₁₀ P. In addition, effect of the displacing activities of hydroxyzine and acetylsalicylic acid on the binding of phenoxazine derivatives to albumin has been studied. Results of the displacement experiments show that phenoxazine benzene rings and tertiary amines attached to the side chain of the phenoxazine moiety are bound to a hydrophobic area on the albumin molecule.     

Pulsed-electric-field crossflow ultrafiltration of bovine serum albumin

A conventional crossflow ultrafiltration (CUF) apparatus was modified by the inclusion of electrodes which permitted a pulsed electric field to be produced across the ultrafiltration membrane (PEF-UF process). Using this apparatus, a discontinuous electrophoretic velocity was imposed upon the proteins being concentrated, opposing their convective movement toward the CUF membrane. This resulted in a lower concentration of rejected solute protein in the fluid boundary layer adjacent to the high-pressure side of the membrane and, hence, in a lower solute-related filtration resistance than in the case of conventional ultrafiltration (zero electric field). Studies of the PEF-UF process with bovine serum albumin (BSA) in the range of 0.5–5% w/v demonstrated a 25–40% decrease in the solute-related resistance to the permeate flux compared to the case of a zero electric field. Accordingly, higher permeate fluxes and, therefore, higher rates of concentration of the protein solution were obtained than for conventional crossflow ultrafiltration. When the electric field was reimposed following a period of operation under conventional CUF conditions, the permeate flux could be restored to nearly the same higher value observed initially for the PEF-UF process.     

3-乙基苯并噻唑螺萘并噁嗪与牛血清白蛋白的相互作用

采用荧光光谱法和热力学方法研究了3-乙基苯并噻唑螺萘并噁嗪(EBSN)与牛血清白蛋白(BSA)的相互作用.结果表明,在pH为7.46的Tris-HCl及0.01mol·L-1的NaCl介质中,EBSN能强烈猝灭BSA的荧光,猝灭机理为形成复合物的静态猝灭.在298、306和313K时,两者的表观结合常数Kb分别为1.762 0×104,3.396 3×104和6.123 5×104 L·mol-1.与此同时,EBSN与BSA的结合反应是自发的,作用力主要为疏水作用力;BSA和EBSN的工作曲线分别为F0-F=2.439c-8.322和(F0-F)/F=0.061 89c+0.556 6,对BSA和EBSN的检出限分别为0.015mg·L-1和7.61×10-7 mol·L-1.     

PS-TEPA树脂合成及其吸附蛋白质性质

本文以四乙烯五胺与氯乙酰化聚苯乙烯树脂反应合成PS-TEPA树脂。考察了温度、时间、胺用量以及溶剂等合成条件。研究了PS-TEPA树脂吸附牛血清蛋白的条件,包括吸附温度、吸附时间以及蛋白质的用量。结果发现,当以二氯甲烷作分散剂,合成温度为85℃,反应时间为12 h,投料比为32g四乙烯五胺/g树脂条件下,树脂接枝的增重率和氮元素含量最大;当吸附温度为30℃,吸附时间为12 h,牛血清蛋白溶液的浓度为2.0 mg/mL时,PS-TEPA树脂的蛋白吸附量达到80mg/g树脂,吸附回收率为84%。     

丝裂霉素C与牛血清蛋白结合作用的研究

结合光谱法与微量热法研究了水溶液中丝裂霉素C与牛血清白蛋白分子间的结合反应,测定了反应的结合常数K~A,结合位点数n及热力学函数△~rG~m,△~rH~m和△~rS~m,并确定了分子间作用力性质;依据Forster非辐射能量转移机制,确定了授体-受体间的结合距离和能量转移效率;采用同步荧光技术考察了丝裂霉素C对牛血清白蛋白构象的影响。     

5种荧光染料探针与牛血清白蛋白相互作用研究

采用荧光光谱法研究了水溶液中四溴荧光素、四氯四溴荧光素、二碘荧光素、乙基罗丹明B、健那绿B等5种荧光染料探针与牛血清白蛋白(BSA)的相互作用.实验表明,这5种染料探针同牛血清白蛋白结合时,疏水作用力起决定性作用,静电力起次要作用,相比之下牛血清白蛋白(BSA)结合阴离子的能力最强,其次为中性分子,最后为阳离子.通过疏水作用力,5种染料均是以非极性苯基进入BSA疏水性腔体中同色氨酸残基发生作用,弱氢键的形成加强了这种作用力,且使得光谱间能量转移效率明显提高.5种染料探针的极性部位由于极性和空间效应的原因难以进入腔体内部,致使反应均按接近1∶1的方式进行.     

固定化牛血清白蛋白吸附材料的制备及其对胆红素的吸附性能

降低高胆红素血症和重症肝炎患者血液中异常升高的胆红素浓度是血浆交换和血液灌流等疗法的目标之一。本文通过共价键合的方法将牛血清白蛋白(BSA)固定在甲基丙烯酸缩水甘油酯共聚三羟甲基丙烷三甲基丙烯酸酯[poly(GMA-co-TMPTMA)]大孔树脂微球上,制备得到对胆红素具良好吸附性能的固定化BSA吸附材料(BIA),吸附容量达48.7mg/g。由于血清白蛋白对胆红素的强烈相互作用,胆红素溶液中游离BSA的存在会显著降低BIA对胆红素的吸附量。BIA对胆红素的吸附量随吸附温度升高而增加。BIA在-80℃下储存31d后性能仍然稳定,对胆红素的吸附量几乎不变。上述结果表明所制备的BIA为以特异性吸附胆红素为目的的血液净化材料提供了新的选择。     

三价铁对灯盏花素与牛血清白蛋白相互作用的影响

运用荧光光谱、紫外吸收光谱探讨了Fe3+对灯盏花素(BR)与牛血清白蛋白(BSA)相互作用的影响;从Fe3+与BSA的静电作用、配位结合以及Fe3+与BR的配位作用等方面分析了影响BR与BSA相互作用的因素.结果表明,Fe3+不改变BSA与BR的作用机制,但使得二者结合的猝灭常数、结合常数及结合位点数减小.     

孔雀石绿与牛血清白蛋白作用的光谱研究

应用荧光及紫外光谱法研究了孔雀石绿(MG)与牛血清白蛋白(BSA)相互作用的光谱特性。测定了16℃、26℃、36℃三个温度下的结合常数KA(7.066×103、4.638×103、1.338×103)和结合位点数n(1.2、1.1、1.0)。结果表明:MG对BSA内源荧光的猝灭机理主要为静态猝灭;MG主要以范德华力与BSA相互作用;同步荧光技术研究了MG对BSA构象的影响,表明BSA的荧光主要源于色氨酸残基,MG对BSA的构象有影响;利用F ster偶极-偶极非辐射能量转移理论,计算了三个温度下MG与BSA的作用距离(r16℃=3.30,r26℃=3.314,r36℃=3.58nm),表明MG对BSA的荧光猝灭中存在能量转移。