流动相组成、浓度和pH对蛋白质在金属螯合柱上的保留特性的影响

 系统研究了流动相中盐的性质和浓度、溶液 pH以及竞争配体对蛋白质在金属螯合色谱中保留值的影响。导出了描述蛋白质在金属螯合色谱中保留特征的数学表达式 ,提出用洗脱强度指数ε表征盐溶液的洗脱能力。根据不同色谱条件下蛋白质的保留特性 ,发现蛋白质在金属螯合色谱中的保留是配位、静电和疏水的协同作用。对与蛋白质强结合的金属螯合柱 ,以配位作用为主 ,静电作用为辅 ;对弱结合的金属柱 ,以静电作用为主 ,配位作用为辅。在流动相中加入高浓度非成络盐 ,可增强蛋白质和固定相间的疏水作用。     

金属螯合亲和色谱中固定金属与蛋白质的作用

在不同PHNaCl的磷酸缓冲体系，比较了牛血清蛋白（BSA）、核糖核酸酶（RNase)、细色素C（Cyt-C)和溶菌酶（Lys)在IDA裸柱和一些金属螯合柱上的保留特性，考察了固定金属对蛋白质保留行为的影响，指出蛋白质在强结合IDA－Cu柱上的保留主要受固定金属和蛋白质间配位作用支配，在弱亲和的IDA－Ni,IDA-Co和IDA－Zn柱上的保留主要受静电作用控制，配位作用为辅，讨论了金属螯合亲和色谱中影响蛋白质和金属配位的主要因素，金属离子的电荷和半径，配位原子对中心离子外层d轨道的影响，以及蛋白质表面配位的组氨酸数目，离解常数和取向，影响金属螯合配体和蛋白质静电作用的主要因素为溶液的PH和蛋白质的等电点pI.     

盐酸胍对疏水色谱中蛋白质保留行为的影响

研究了变性剂酸胍对几种蛋白质在高效疏水色谱中保留行为的影响，用液相色谱中溶质的计量置换保留模型和测流动相表面张力及蛋白质紫外吸收光谱方法研究的结果表明：在低浓度范围内，盐酸胍主要以疏水作用影响蛋白质的保留；而在高浓度区域内，盐酸胍的存在则显著影响蛋白质分子的构象，使其保留行为发生变化。     

采用柔性分子对接技术模拟蛋白质在疏水作用色谱上的保留行为

将蛋白质与疏水作用色谱(HIC)固定相相互作用分为直接非键/构象作用和蛋白质表面疏水效应两个热力学过程, 从而定量给出了处于浓盐析盐水溶液中HIC保留时间与配基/蛋白质结合自由能之间的二元线性关系. 通过ICM柔性分子对接策略及遗传算法(GA)对27个已知晶体结构的蛋白质与疏水配基的可能结合方式进行模拟和分析, 所得结果与实验观测情况吻合良好. 研究表明, 蛋白质局部疏水效应以及配基与蛋白质的非键/构象作用皆对HIC色谱保留行为影响显著, 且作用区域多集中于蛋白质表面突出部位.     

一种金属螯合连续棒色谱柱的制备及色谱性能

金属螯合色谱（IMAC）在生物大分子的分离和纯化中有广泛的应用,通过改变IMAC的洗脱条件和被螯合的金属离子种类,生物大分子可获得选择性分离。目前,IMAC通常以有机和无机微球作固定相,由于柱死体积的存在,使色谱柱空间利用率低,且降低了蛋白质分离的柱效。近年报道的连续棒色谱柱是由直径1μm左右的微粒堆积而成的整体,消除了色谱柱的死体积,该类色谱柱用于蛋白的反相、疏水、离子交换和亲和色谱分离均可获得高的分离效率。然而,至今尚未见到对金属螯合连续棒色谱柱制备及应用的研究报道。对蛋白的IMAC分离机理研究中,研究流动相pH对分离的影响是主要手段之一,但通常研究的pH值的5.0-9.0的窄范围内。本文提出和制备了以交联聚甲基丙烯酸缩水甘油酯连续棒为基质的金属螯合色谱柱,并研究了pH在2.0-11.0的较宽范围内对蛋白保留的影响。     

蛋白质在合成阳离子交换剂上的色谱特性研究

用国产材料按间接法合成了螯合型弱阳离子交换剂,详细研究了合成填料的色谱性能,并与商品柱的分离效能进行了比较;在宽温度范围内研究了蛋白质在弱阳离子交换系统中的色谱热力学,测定了蛋白质在色谱过程变性时的热力学参数 (△H0和△S0) 和补偿温度β,提出用标准熵变△S0判断蛋白质的构象变化和用△H0与△S0的补偿关系鉴定蛋白质各变体在色谱系统保留机理的同一性。考察了螯合型弱阳离子交换剂与金属离子的作用,研究了蛋白质在金属螯合色谱中的保留机理。     

固定金属离子亲和色谱--蛋白质分离的方法、原理、特性和应用

介绍了固定金属离子亲和色谱法(IMAC)的方法原理、金属螯合柱的制备、固定金属离子与蛋白质的相互作用以及影响这些作用的因素、不同色谱条件下各种作用力对蛋白质保留值的贡献、蛋白质的洗脱原理和IMAC在蛋白质分离纯化中的应用,论述了IMAC的特点、不足、克服的方法和今后应解决的问题。     

蛋白质在固定Zn2+金属螯合亲和色谱中的变性热力学

在15~85℃宽温度范围,研究了蛋白质在固定Zn2 金属螯合色谱系统中的热行为和变性热力学。实验结果表明,蛋白质在色谱过程都有一个固定的热转变温度:核糖核酸酶(RNase)、α-胰凝乳蛋白酶原A(α-Chy)的热转变温度约为55℃,细胞色素C(Cyt-C)和溶菌酶(Lys)约为65℃;,热转变温度的出现标志蛋白质构象发生变化;利用Van′tHoff作图测定了蛋白质在色谱系统热变性时的标准焓变ΔH°和标准熵变ΔS°,提出用标准熵变ΔS°和自由能变ΔG°判断蛋白质构象变化;利用ΔH°-ΔS°的线性关系估算了蛋白质热变性时的补偿温度,鉴定了蛋白质各变体在金属螯合色谱中保留机理的同一性,RNase、Cyt-C、Lys和α-Chy的补偿温度分别为55℃、65.8℃、65.2℃和54.8℃;根据蛋白质热变性时的补偿温度和构象变化熵变Δ(ΔS°)的大小,讨论了蛋白质在阳离子交换色谱和固定Zn的金属螯合色谱体系中的热稳定性。实验证明,在IDA裸柱引入Zn2 后蛋白质在色谱系统中的热稳定性减小,平均补偿温度从65.3℃降低到59.7℃,而构象变化熵变的绝对值大幅度升高。     

谷胱甘肽键合柱对蛋白的选择性研究

通过将谷胱甘肽键合到硅胶表面合成了同时具有弱阳离子交换(WCX)、疏水(HIC)和氢键作用的多功能色谱填料, 该固定相在HIC和WCX模式下对蛋白都有很好的分离效果. 实验通过计量置换保留模型对蛋白在谷胱甘肽键合柱上的色谱保留行为及机理进行了研究, 结果发现, 在流动相盐浓度较低时蛋白根据自身等电点高低通过静电作用力得以分离, 而在高盐浓度下疏水和氢键作用力共同决定蛋白的保留. 这种多作用力保留模式可有效提高色谱柱的选择性, 尤其为蛋白质、多肽及氨基酸的高效分离提供新的解决思路.     

使用疏水作用色谱研究蛋白质的构象变化

研究了高效疏水作用液相色谱中(HIC)色谱条件改变对蛋白质构象的影响。发现固定相配体的疏水性、温度及流动相中盐的阴离子、阳离子和pH值都影响蛋白质的构象。     

Effects of Immobilized Metal Ion on Retention Behaviors of Proteins in Metal Chelate Affinity Chromatography

The chromatographic behaviors of proteins on iminodiacetic acid (IDA) column with and without immobilized metal ion were examined in detail. Comparing the effects of pI, solution pH, and salt concentration on retention of proteins in cation‐exchange chromatography (CEC) and metal chelate affinity chromatography (MCAC), the retention mechanism of proteins was investigated in MCAC. By aid of observing the retention characteristics of proteins on naked IDA and metal chelate columns in high concentration salt‐out salt solution, the hydrophobic interaction in MCAC and the influence of metal ion on it were proved. In terms of the comparison of the thermodynamics of proteins in CEC and MCAC, the thermostability, the conformational change entropy Δ(ΔS⁰) and enthalpy Δ(ΔH⁰), compensation temperature β, the driving force and caloritic effect of proteins in MCAC were discussed. The identity of retention mechanism at protein thermal denaturation in CEC and MCAC was demonstrated by using the compensation relationship between ΔH⁰ and ΔS⁰.     

Retention Behavior of Proteins on Glutamic Acid-Boned Silica Stationary Phase

A glutamic acid-bonded silica (Glu-silica) stationary phase with cation-exchange properties was synthesized using l-glutamic acid as ligand and silica gel as matrix. The effects of solution pH value, salt concentration and metal ion on the retention of proteins were examined. The standard protein mixture was separated with a prepared chromatographic column and an iminodiacetic acid column, and compared. The influence of the binding capacity of an immobilized metal ion on the complexation of metal chelate column was studied. The results indicate that the obtained column displays cation-exchange characteristic and better separation ability for proteins. As fixing metal ion on the Glu-silica column, retention of proteins on the column is a cooperative interaction of metal chelate and cation-exchange. The stationary phase shows the typical metal chelate properties with the increase of the sorption capacity of immobilized metal ion.     

Chromatographic behaviors of proteins on cation-exchange column

A weak cation-exchanger (XIDACE-WCX) has been synthesized by the indirect method. The chromatographic characteristics of the synthesized packing was studied in detail. The standard protein mixture and lysozyme from egg white were separated with the prepared chromatographic column. The chromatographic thermodynamics of proteins was studied in a wide temperature range. Thermodynamic parameters standard enthalpy change (deltaH0) and standard entropy change (deltaS0) and compensation temperature (beta) at protein denaturation were determined in the chromatographic system. By using obtained deltaS0, the conformational change of proteins was judged in the chromatographic process.The linear relationship between deltaH0 and deltaS0 can be used to identify the identity of the protein retention mechanism in the weak cation-exchange chromatography. The interaction between weak cation-exchanger and metal ions was investigated. Several metal chelate columns were prepared. The effects of introducing metal ion into the naked column on protein retention and the retention mechanism of proteins in the metal chalet affinity chromatography were discussed.     

Analysis of pH-dependent protein interactions with gel filtration medium.

A prepacked Superose 12 HR 10/30 column was used to study the effects of elution ionic strength and pH on the chromatographic behaviour of a strong hydrophobic Clostridium thermocellum endoglucanase (1) and two weak hydrophobic proteins, Clostridium thermocellum endoglucanase C and egg white lysozyme. Ion-exclusion or ion-exchange interactions between weakly hydrophobic proteins and the gel matrix were observed at low ionic strength, depending on whether the pH of the elution buffer was higher or lower than the pI values of the proteins. These interactions were due to the presence of negatively charged groups on the surface of Superose and could be eliminated at any pH by adding electrolyte at a concentration determined by its chemical identity. The optimum results were observed with sodium sulphate at a concentration of 100 mM. The chromatographic behaviour of strong hydrophobic endoglucanase (1) on a Superose column as a function of pH was much more complex because of two interplaying effects, electrostatic and hydrophobic. Ideal size-exclusion chromatography could be achieved only in a narrow range of the conditions: first, the mobile phase must contain a weak salting-out electrolyte such as NaCl, and second, the mobile phase pH must be high enough that hydrophobic interactions between the solute and support are balanced by their electrostatic repulsion. At pH greater than pI, the retardation of endoglucanase (1) gradually increased with decreasing pH as a result of lowering of repulsive electrostatic interactions whether or not the buffer ionic strength was high. At pH less than pI a drastic increase in the capacity factor k' was observed owing to the additivity of hydrophobic and ion-exchange effects.(ABSTRACT TRUNCATED AT 250 WORDS)     

Peculiar effects of alkali thiocyanates on the activity coefficients of aromatic hydrocarbons in water

Among twenty-two 1:1 electrolytes examined, LiSCN, CsSCN, KSCN and CsI have a considerable effect on the aqueous solubilities of a series of nonelectrolytes. LiSCN shows a salting-in effect for all the nonelectrolytes examined including 1-octanol, benzene, naphthalene and biphenyl while CsSCN and CsI show a salting-out effect for 1-octanol but a salting-in effect for all the aromatic hydrocarbons. The effect of NaSCN and KSCN on the solubilities of naphthalene and biphenyl results in an anomalous concentration depencence, i.e., both salting-in and salting-out effects occur depending upon the electrolyte concentration. It is suggested that the anomalous salt effects may partly be explained through perturbation of preexisting ion-ion structural hydration interaction upon introduction of the solute molecules.     

Salting-in effect of ionic liquids on poly(N-vinylimidazole) hydrogels

This work attempts to study the interaction of two ionic liquids (1-ethyl-3-methylimidazolium tosylate and 1-hexyl-3-methylimidazolium chloride) with chemically crosslinked poly(N-vinylimidazole) in aqueous media, and to compare it with that of NaCl, a typical salting-out electrolyte. The three salts show a salting-in effect whose intensity was measured on the basis of the decrease of the polymer–solvent interaction parameter and the increase of swelling with increasing ionic strength. It was thus found that the salting-in effect is the same for the two salts with the same anion (chloride), while the intensity of the salting-in effect exerted by the tosylate ionic liquid is larger. The coefficient of selective sorption, which expresses the salt excess inside the swollen gel with respect to the external solution was determined by comparing the initial and equilibrium compositions of the immersion bath. These results are discussed in terms of the hydrophobic character of the ions involved.