Isothermal Titration Microcalorimetric Studies of the Effect of Temperature on Hydrophobic Interaction between Proteins and Hydrophobic Adsorbents

This study attempted to comprehend how temperature affects hydrophobic interaction between proteins and hydrophobic adsorbents. By equilibrium batch analysis, we measured the adsorption isotherm to evaluate the protein-adsorbent affinity, while isothermal titration calorimetry was used to measure the adsorption enthalpy. In addition, the affinity and enthalpy differences between two proteins, alpha-chymotrypsinogen A and trypsinogen, with two adsorbents, butyl-Sepharose and octyl-Sepharose gel, under varying temperatures were studied with respect to the exposed hydrophobic segments of the protein and ligand hydrophobicity. The enthalpies obtained in this investigation can be used to more thoroughly understand the hydrophobic interaction between proteins and adsorbents. First, the adsorption isotherm experiments reveal that the adsorption quantity of the proteins with the Sepharose gels increases with temperature. For a microcalorimetric measurement, as temperature is increased from 298 to 310 K, the DeltaH value of alpha-chymotrypsinogen A with butyl-Sepharose increases, while the DeltaH value of trypsinogen is reduced. This is likely due to the fact that alpha-chymotrypsinogen A has a higher area of exposed hydrophobic segments than trypsinogen does. This observation also implies that as temperature increases, the interaction mechanism of alpha-chymotrypsinogen A with butyl-Sepharose changes from an adsorption-dominated process to a partitioning process. In addition, for octyl-Sepharose, the DeltaH value of alpha-chymotrypsinogen A is positive and decreases with temperature increment. However, the DeltaH value of trypsinogen was positive and increased with temperature. Therefore, we conclude that as temperature increases, the interaction mechanism of the proteins for octyl-Sepharose is a partitioning-dominated process. Copyright 2000 Academic Press.     

Interaction Forces between Hydrophobic and Hydrophilic Self-Assembled Monolayers

An investigation is presented of the interaction of charged self-assembled monolayers (SAMs) with a monoprotic ionizable acid functional group (-COOH) and uncharged SAMs with a methyl terminated functional group (-CH(3)). The strength of the interactions are determined using an atomic force microscope. For all electrolyte conditions investigated the interactions are not well described by a summation of van der Waals attractions and electrostatic repulsions in a manner suggesting that van der Waals attractions are screened. The repulsions are accurately described as corresponding to two surfaces of different charge interacting with surface charges that are independent of separation (i.e., the constant charge model). A small adhesion force was observed under all conditions and its magnitude increased with NaCl concentration. Copyright 2000 Academic Press.     

明胶与AgBr晶体颗粒相互作用的研究

It was found that IR absorption band at 1383 cm - 1 was stronger for AgBr crystal particles . This band was not observed but there was an amide I band at 1630 cm - 1 or 1640 cm - 1 for gelatin-AgBr emulsion after ensymolysis. This variation meant that gelatin was adsorbed on the AgBr crystal particles. The IR analysis was performed for gelatin with added Br- to show the possible interaction between gelatin and AgBr particles. The frequence of NH3+ absorption band for gelatins with added Br- was lower than that for gelatins without added Br-. The frequence shift of NH3+ band around 3300 cm-1 in the spectrum of gelatins indicated that gelatin could be adsorbed by AgBr with NH3+ and Br- in addition to chemical interaction between Ag+ and N atom or/and between Ag+ and S atom. Moreover,there was probably a kind of force between the hydrophobic parts of AgBr crystal surface and the hydrophobic side-chain groups of gelatin. It may be the van der Walls force. It had influence on IR spectrum of emulsion from 1000 cm-1 to 1200 cm-1 . Because there was a difference in photographic characters or crystal particle size between fish gelatin emulsion and bovine gelatin emulsion,their interactions were compared. In general,IR study suggested that gelatin was adsorbed on AgBr crystal particles . This adsorption could be caused by coulombic and van der Walls forces apart from the chemical affinity between gelatin and AgBr crystal particles.     

Polymer Polydispersity Effect on Depletion Interaction between Colloidal Particles

The effect of polymer polydispersity on the polymer‐induced interaction between colloidal particles due to non‐adsorbing ideal chains is investigated. An analytical theory is developed for the polymer‐segment density between two plates and in the space surrounding two spheres by extending a recently proposed superposition approximation to include polymer polydispersity. Monte Carlo computer simulations were made to test the validity of the analytical theory. The polymer densities predicted by the superposition approximation are in reasonable agreement with simulation results for the polydisperse case. The simulations show that depletion leads to a size fractionation of the polymers. It is shown that size polydispersity has a small effect on the interaction between two parallel plates but a more significant effect on the interaction between two spheres. The range of the potential increases and the contact potential drops with increasing polydispersity.

Polymer‐segment density as a function of y for three values of x, as indicated, in the space surrounding two colloidal spheres with radius R = R_g0 and h = 0.48R_g0. Symbols are the MC results: polydisperse polymer (○; z = 1) and monodisperse polymer (•) samples. Curves are the predictions of the product‐function approximation for monodisperse polymer (solid lines) and polydisperse polymer (z = 1, dashed lines).     

Studies on Interaction between Gatifloxacin and Bovine Serum Albumin by Spectroscopy

The interaction of gatifloxacin （HGA） with bovine serum albumin （BSA） at 15 and 37 ℃ has been investigated by fluorescence quenching spectroscopy in aqueous solution. The bimolecular quenching rate constant was determined by Stem-Volmer curves and the values were Kq=9.28× 10^12 L·mol^-1·s^-1 （15 ℃） and Kq=8.51 ×10^12 L·mol^-1·s^-1 （37 ~C）. The results showed that the fluorescence quenching mechanism of BSA by HGA was a static quenching procedure. The thermodynamic parameters indicated that electrostatic forces played major role in the interaction of BSA with HGA. Studies on the relationship between the concentration of HGA and the fluorescence intensity of BSA showed that BSA and HGA bound at the molar ratio 1 ： 1 and the equilibrium constant K0 was 6.80 ×10^4 L·mol^-1. The binding distances between BSA and HGA and the energy transfer efficiency were obtained based on the Ftrster＇s theory.     

A Molecular Torsion Balance Study: A Nearby Anionic Group Exerts Little Influence on Hydrophobic Interactions between Nonpolar Surfaces

Polar groups have a solvent ordering effect on water and therefore may affect hydrophobic binding energies for nearby lipophilic surfaces. This would mean that determinations of excess surface free energy association energies require consideration of nearby polar functional groups. This paper reports results of a study to measure this possible effect. It was concluded from the models used here that an anionic polar group nearby a hydrophobic surface has little or no effect on the magnitude of hydrophobic association.     

用疏水色谱对还原型胍变性牛胰岛素的折叠特性研究

用疏水相互色谱(HPHIC)对还原胍变性牛胰岛素在疏水界面上的折叠与复性进行了研究.结果表明,采用普通流动相时,对还原胍变胰岛素的复性效果较差,而采用氧化型流动相可使其复性效率提高到66%,并用反相色谱(RPLC)、紫外吸收光谱、荧光光谱及MALDI-TOF对其复性效果进行了验证.同时与体积排阻色谱(SEC)和稀释法对还原胍变胰岛素的复性结果进行了比较.结果表明,SEC根本无法使还原胍变胰岛素复性,而稀释法的复性效率仅有2%.这进一步表明HPHIC是变性蛋白复性的有效工具,变性蛋白在疏水界面折叠过程中,蛋白质与固定相之间的疏水相互作用对蛋白折叠起着关键性的作用,是蛋白折叠的主要驱动力.     

红外分光光度法测定工作场所空气中矿物油雾浓度

以玻璃纤维滤膜采集工作场所空气中矿物油雾,用四氯化碳浸提,采用红外分光光度法测定矿物油雾含量.对采样方法、玻璃纤维滤膜处理方法进行了探讨.矿物油雾浓度在0.0～252.0μg/mL范围内与吸光度呈良好的线性关系,相关系数r=0.9995,加标回收率为98.7％～102.3％,方法检出限为2.0 μg/mL,测定结果的相对标准偏差为0.80％～ 1.39％(n=6).该方法适用于工作场所空气中矿物油雾浓度的测定.     

流变学法研究表面活性剂与HPAM的相互作用

流变学法研究表面活性剂与HPAM的相互作用;聚合物;表面活性剂;相互作用;流变学方法     

氟喹诺酮类药物与牛血清白蛋白作用的研究

用荧光猝灭法研究了ＢＳＡ与ＮＦＬＸ及ＯＦＬＸ之间的相互作用，根据Ｆｏｒｓｔｅｒ的能量转移理论，确定了能量给体－受体间的距离，根据荧光猝灭谐绘制了猝灭曲线，确定其猝灭过程是静态猝灭，用Ｌｉｎｅｗｅａｖｅｒ－Ｂｕｒｋ双倒数曲线测定ＮＦＬＸ－ＢＳＡ和ＯＦＬＸ－ＢＳＡ络合物的离解常数，并详细研究了１０种阳离子，４种阴离子与药物对ＢＳＡ竞争结合的影响，推测了药物与蛋白的主要作用力。     

光谱法研究吗啡与核酸的相互作用

采用紫外光谱、荧光光谱和圆二色光谱等手段对吗啡与小牛胸腺脱氧核糖核酸(ctDNA)、酵母核糖核酸(yRNA)的作用方式进行了研究,发现核酸对吗啡有较强的荧光猝灭作用,且核酸的紫外吸收光谱和吗啡的荧光发射光谱有一定程度的重叠,据此求得了其结合反应的结合常数、结合位点数和基本热力学参数等,证实了吗啡通过沟渠方式与核酸作用。     

药物小分子白杨素与人血清白蛋白结合作用的光谱法研究

应用荧光光谱法、共振瑞利散射光谱法和同步荧光法,研究了生理条件下白杨素与人血清白蛋白(HSA)相互作用.研究表明,白杨素与HSA发生作用并形成了新的基态化合物,静态猝灭是导致HSA内源荧光猝灭的主要原因;求得不同温度 (17℃、26℃和35℃) 下白杨素与HSA作用的结合常数分别为2.373×106、1.680×106和1.346×106 L·mol-1;由求得的热力学参数,确定了白杨素与HSA间的结合反应主要由静电引力驱动.根据Frster非辐射能量转移理论,计算出白杨素在蛋白质中的结合位置与214位色氨酸残基间的距离为3.52 nm;同步荧光光谱表明,白杨素使得HSA的二级结构发生了变化.     

Separation of Proteins on Polar Bonded Phases by Hydrophobic Interaction Chromatography

Abstract

Hydrophobic interaction chromatography (HIC) with a polar bonded phase (“Acetamide”) developped for size exclusion chromatography (SEC) is described. Retention of proteins depends on the surface area of the stationary phase, the pH and ionic strength of the eluent. For efficient separation the pore diameter should be 25 nm or more. The surface area should be large to achieve retention even at low ionic strength. Separation is only possible with a gradient from high to low ionic strength. Gradient volumes of 10 empty column volumes with column lengths above 15 cm are recommended. Selectivity can be optimized via pH adjustment. The advantage of this column packing is its applicability for two different separation modes: SEC and HIC.     

荧光光谱法研究大豆甙元与牛血清白蛋白的相互作用

采用荧光光谱技术研究了大豆甙元与牛血清白蛋白(BSA)的相互作用.研究结果表明:290 K、303 K、310K、315 K温度下大豆甙元对BSA的猝灭速率常数Ksv随着温度升高逐渐降低,且均大于最大动态猝灭速率常数2×1010 L·mol -1·s-1,表明大豆甙元对BSA的荧光猝灭属静态猝灭过程.根据F(o)rst...     

荧光法研究奥沙利铂与牛血清白蛋白的相互作用

在Tris缓冲溶液(pH7.0)体系中,用荧光光谱及同步荧光光谱技术研究水溶液中奥沙利铂与牛血清白蛋白的相互作用。结果表明,奥沙利铂对牛血清白蛋白内源荧光(345nm)产生较强的荧光猝灭作用,根据不同温度下奥沙利铂对牛血清白蛋白的荧光猝灭作用,证明其为静态猝灭机制,运用位点模型计算出298、308K时结合常数KA(分别为4.22×104、3.95×104L.mol-1)和结合位点数n(分别为1.02、1.01),根据热力学参数确定其作用力以静电作用为主;运用Fster偶极-偶极非辐射能量转移原理,测定了奥沙利铂与牛血清白蛋白的结合距离r(5.67nm);用同步荧光技术考察了奥沙利铂对牛血清白蛋白构像的影响。     

荧光光谱法研究原花青素与牛血清白蛋白的相互作用

在pH=7.40的Tris-HCl缓冲体系中,采用荧光光谱技术研究了原花青素与牛血清白蛋白(BSA)的相互作用.根据295 K、303 K、310 K、315 K温度下的猝灭常数,表明原花青素对BSA的荧光猝灭为静态猝灭过程,由热力学参数焓变(△rHm)和熵变(△rSm)均大于零,推断出原花青素与BSA之间主要靠疏水作...     

土贝母皂苷II与人血清白蛋白相互作用机制的光谱研究

人血清白蛋白多种结合位点的存在使其成为许多药物可能的结合靶点. 土贝母皂苷具有广泛的生理和药理活性, 它与蛋白质相互作用机制的研究对于深入了解其药理药效具有重要的意义. 采用荧光光谱法研究了土贝母皂苷II (TBMSⅡ)与人血清白蛋白(HSA)之间的相互作用, 根据Stern-Volmer荧光淬灭方程计算得293, 298, 303, 308 K时TBMSⅡ与HSA相互作用的结合常数分别为1.002×10⁵, 0.701×10⁵, 0.514×10⁵, 0.411×10⁵ L•mol^－1. 由实验计算出热力学参数焓变ΔH为－44.829 kJ•mol^－1, 熵变ΔS为－57.497 J•mol^－1•K^－1, 表明分子间的氢键及疏水作用是TBMSⅡ-HSA复合物的主要作用力, 结合位点位于HSA的亚结构ⅡA, 这与分子模拟方法的结果相一致. 依据能量转移原理求得TBMSⅡ与HSA间的距离为4.95 nm|三维、同步荧光光谱及圆二色谱的结果表明TBMSⅡ的加入使HSA构象发生变化, α-螺旋结构有所下降.     

白藜芦醇与CopC的相互作用研究

白藜芦醇是一种天然的植物抗毒素,存在于花生、葡萄和多种浆果之中。CopC是一种由β-折叠片组成的铜伴侣蛋白,含有102个氨基酸,Cu~+结合在C端,Cu~(2+)结合在N端,其作为氧化还原开关参与铜的调控,它与小分子的作用影响其氧化还原开关功能。本研究主要利用红外光谱(FTIR)、圆二色谱(CD)、荧光寿命、三维荧光光谱、荧光光谱和分子对接等方法研究白藜芦醇与CopC的相互作用。通过FTIR、CD、化学变性和荧光寿命测定表明,白藜芦醇的结合使得CopC的稳定性降低,可能源于CopC的β-折叠片含量减少,自由卷曲含量增加。此外,三维荧光光谱和荧光光谱表明,白藜芦醇与CopC结合后,其荧光光谱出现蓝移,表明白藜芦醇可能结合在CopC的疏水区域;并且,可得白藜芦醇与CopC的结合比为1∶1,结合常数为(6.76±0.17)×10~5 L·mol~(-1),疏水作用力是维持两者之间形成复合物的主要作用力。通过F9rster能量转移和分子对接模拟确定CopC中白藜芦醇和色氨酸之间的平均结合距离,模拟的结果和实验结果具有较好的一致性。本文结果将为阐明CopC的铜调控机理提供帮助。     

改进荧光光谱法研究药物与血清白蛋白的相互作用

为了解决蛋白与药物分子的荧光光谱相互作用分析存在的缺点,采用小分子为荧光检测对象,以血清白蛋白(SA)为猝灭剂,研究了模拟生理条件(pH=7.4)下伯氨喹、十二烷基苯磺酸钠和西酞普兰与SA的相互作用。测得伯氨喹、十二烷基苯磺酸钠和西酞普兰与SA的结合常数Kb分别为8.16×104L/mol、4.14×106L/mol和6.08×107L/mol。这种改进荧光光谱法能更准确和全面地表达出SA与药物的相互作用信息。通过紫外吸收光谱相互作用分析法对改进方法进行了验证。并推测出药物与蛋白之间存在一种"点对面"的作用方式。     

荧光法研究偏钒酸钠与牛血清白蛋白的相互作用

本文用荧光光谱和紫外可见吸收光谱研究了在模拟人体生理条件下，偏钒酸钠与牛血清白蛋白(BSA)结合反应的特征，研究了紫外灯(253.7 nm)照射对偏钒酸钠与BSA结合的影响。紫外吸收光谱显示，加入偏钒酸钠后，牛血清白蛋白的紫外吸收降低，表明偏钒酸钠与BSA形成了缔合物。荧光猝灭光谱显示偏钒酸钠对牛血清白蛋白有较强的荧光猝灭作用，荧光猝灭机理符合静态机制。缔合物的稳定常数分别为：K_s=0.357×10⁴(25 ℃)，K_{s相似文献}