Z及logI对离子交换色谱中脲变溶菌酶分子构象变化的表征

以溶菌酶(Lys)为目标蛋白,用计量置换保留理论(SDT-R)中的参数Z和logI对弱阳离子交换色谱(WCX)中“准天然态”和脲变还原与非还原的两种变性状态的Lys的分子构象变化进行了表征。发现在流动相中含有脲时,蛋白的保留仍服从SDT理论,可以准确测定在特定脲浓度条件下Lys的Z及logI值。结果表明,3种分子构象状态的Lys的Z值均随脲浓度改变呈现不连续变化;“准天然态”Lys在同一脲浓度条件下的Z值比变性状态的大,logI比变性状态的小,而非还原变性态和“准天然态”Lys的Z和logI值比较接近。还     

参数Z对疏水色谱中胍变蛋白质分子构象变化的表征

用计量置换参数Ｚ对疏水色谱流动相中存在盐酸胍时蛋白质分子构象变化进行了表征。除溶菌酶外，其余４种蛋白质的Ｚ值随盐酸胍浓度的增大先增大，而后减小。将盐酸胍和脲对蛋白质Ｚ值的影响进行了比较后发现，在疏水色谱中Ｚ值作为蛋白质分子构象变化表征的一个重要的结论是随变性剂浓度的增大，埋藏在蛋白质分子内部的疏水性氨基酸残基暴露到分子表面的程度逐渐增大，造成了Ｚ值随蛋白质分子构象变化程度的增大而减小。蛋白质的Ｚ值随盐酸胍及脲浓度变化的不同特点，反映了两者对蛋白质变性机理的不同。     

参数Z对疏水色谱中胍变蛋白质分子构象亦化的表征

用计量置换参数Ｚ对疏水色谱流动相中存在盐酸胍时蛋白质分子构象变化进行了表征。除溶菌酶外，其余４种蛋白质的Ｚ值随盐酸胍浓度的增大先增大，而后减小。蛋白质的Ｚ值随盐酸及脲浓度的增大，埋藏在蛋白质分子内     

液相色谱中一个新的表征参数Z Ⅳ.反相液相色谱中甲酸浓度与生物大分子的构象变化

反相高效液相色谱中蛋白质分子的Z值可以作为平衡状态下蛋白分子构象变化的表征,它与蛋白分子起始状态构象无关。在异丙醇-甲酸-水体系中蛋白的Z值会因流动相中甲酸浓度的增大而减小。除蛋白分子构象变化因素外,蛋白Z值的变化也与甲酸参与蛋白与置换剂分子间的计量置换过程有关。在蛋白分子未完全失去分子立体结构时,其Z值与分子量的立方根成正比。当甲酸浓度足够大时,例如大于40％(V/V)时,蛋白分子完全失去三维结构。     

用参数Z表征疏水色谱中脲浓度与蛋白质分子的构象变化

研究了５种标准蛋白在流动相中含有不同脲浓度条件下的疏水色谱保留行为。当脲浓度不变时，蛋白质的保留仍然服从计量置换保留模型，并可测定在该特定脲浓度条件下蛋白质的Ｚ值。计量置换参数Ｚ可作为疏水色谱中生物大分子的构象变化的表征。     

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。     

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.     

高效弱阳离子交换色谱法对脲还原变性溶菌酶的折叠研究

用高效弱阳离子交换色谱(HPWCX)对脲还原变性溶菌酶(Lys)进行了复性研究. 在流动相中脲浓度固定为4.0 mol•L^－1和选用对天然态蛋白有稳定作用的硫酸铵为盐或置换剂时, 在蛋白浓度为15.0～50.0 mg•mL^－1时, HPWCX法比稀释法活性回收率高. 为了提高Lys的质量及活性回收率对所用色谱条件进行了优化研究, 当蛋白起始浓度为20.0 mg•mL^－1时, Lys的质量回收率和活性收率分别为97.8%和95.4%. 表明此种方法简便且有可能对其他还原变性蛋白的复性具有通用性.     

反相液相色谱中温度对蛋白分子构象变化的新表征

液相色谱中溶质计量置换保留模型的Ｚ值可用来对反相高效液相色谱中热变蛋白的分子构象进行表片。发现在异丙醇－甲酸４４％（Ｖ／Ｖ）－水体系为流动相对完全变性蛋白的Ｚ值随温度升高而降低。且Ｚ值与绝对温度例数（１／Ｔ）成正比，因此蛋白分子构象变化可用Ｚ对１／Ｔ作图所得两条直线的斜率差值来表征，差值愈大，蛋白分子构象变化愈甚。该两直线的交点即为蛋白分子构象的突变温度。与通常采用的１ｇＫ＇对１／Ｔ的作图相比，用     

液相色谱中一个新的表征参数Z：Ⅳ.反相液相色谱中甲酸浓度与生物大…

反相高效液相色谱中蛋白质分子的Ｚ值可以为平衡状态下蛋白分子构象变化的表征，它与蛋白分子起始状态构象无关，在异丙醇－甲醇－水体系中蛋白的Ｘ值会因流动相中甲酸浓度的增大而增小，除蛋白分子构象变化因素外，蛋白Ｚ值的变化也与甲酸参与蛋白与置换剂分子间的讲师置换过程有关，在蛋白分子未完全失去分子立体结构时，其Ｚ值与分子的立方根成正比，当甲酸浓度足够大量，例如大于４０％（Ｖ／Ｖ）时，蛋白分子完全失去三维结构。     

Refolding of Denatured／Reduced Lysozyme Using Weak－Cation Exchange Chromatography

Oxidative refolding of the denatured/reduced lysozyme was investigated by using weak-cation exchange chromatography (WCX). The stationary phase of WCX binds to the reduced lysozyme and prevented it from forming intermolecular aggregates. At the same time urea and ammonium sulfate were added to the mobile phase to increase the elution strength for lysozyme. Ammonium sulfate can more stabilize the native protein than a common eluting agent,sodium chloride. Refolding of lysozyme by using this WCX is successfully. It was simply carried out to obtain a completely and correctly refolding of the denatured lysozyme at high concentration of 20.0 mg/mL.     

氨基己酸为配基的新型弱阳离子交换/疏水双功能色谱固定相对蛋白质的分离纯化

以硅胶为基质、氨基己酸为配基制备了一种新型弱阳离子交换/疏水（WCX/HIC）双功能混合模式色谱固定相。该固定相配基具有一定的疏水性且含有羧基,在高盐浓度下表现为HIC的性质,可作为HIC固定相使用;在低盐浓度条件下表现为离子交换的性质,可作为WCX固定相使用。分别考察了该介质在WCX和HIC两种模式下对标准蛋白质的分离性能,并与商品柱进行比较。结果表明,所合成的WCX/HIC双功能固定相在WCX和HIC两种模式下对蛋白质均有较高的分离度和选择性,且分离能力与商品柱相当,两种模式下标准蛋白质的质量和活性回收率均大于93%,表明该柱具有“一柱二用”的功能,适于生物大分子的分离纯化。基于此双功能色谱柱构建的在线单柱二维液相色谱（2DLC-1C）可在60 min内实现8种蛋白质的快速分离。在70 min内完成了对蛋清中溶菌酶的二维纯化,纯度可达到98.3%。该技术中一根色谱柱可当作两根色谱柱使用,对蛋白质组学研究和重组蛋白药物的生产具有重要的应用价值。     

Studies on the refolding of the reduced-denatured insulin with size exclusion chromatography

The refolding of the reduced-denatured insulin from bovine pancreas was investigated with the size exclusion chromatography (SEC). It was shown that the reduced-denatured insulin originally denatured with 7.0 mol·L-1 guanidine hydrochloride (GuHCI) or 8.0 mol·L-1 urea could not be refolded with a non-oxidized mobile phase. Although the oxidized and reduced glutathione (GSSG and GSH) were employed in the oxidized mobile phase, the reduced-denatured insulin still could not be renatured. However, in the presence of 2.0 mol·L-1 urea in the oxidized mobile phase employed, the reduced-denatured insulin can be refolded with SEC, and the aggregation of denatured insulin can be diminished by urea. In addition, the disul-fide exchange of reduced-denatured insulin also can be accelerated with GSSG/GSH in the oxidized mobile phase. The three disulfide bridges of insulin were formed correctly and the reduced-unfolded insulin can be renatured completely. The results were further tested with re-versed-phase liquid chromatography (RPLC) and hydrophobic interaction chromatography (HIC).     

Synthesis of Monodisperse Poly（glycidylmethacrylate-co-ethylene dimethacrylate） Beads and Their Application in Separation of Biopolymers

Introduction Since 19541 the polymeric separation media has attracted much attention due to their chemical stability over the entire pH range. The rigid, highly cross-linked styrene copolymers were first used for chromatography by Moore.2 The macroporous copolymers currently available are not only chemically stable but also more resistant to mechanical forces prevailing in a column and therefore are comparable to the traditional packings based on silica gel. Most polymer separation media are …     

Microcalorimetric determination of displacement adsorption enthalpies of protein refolding on a moderately hydrophobic surface at 308 K

Both microcalorimetric determination of displacement adsorption enthalpies ΔH and measurement of adsorbed amounts of guanidine – denatured lysozyme (Lys) refolding on the surface of hydrophobic interaction chromatography (HIC) packings at 308 K were carried out and compared with that at 298 K. Study shows that both temperature and concentration of guanidine hydrochloride (GuHCl) affect the molecular mechanism of hydrophobic interaction of protein with adsorbent based on the analysis of dividing ΔH values into three kinds of enthalpy fractions. The adsorption in higher concentrations of GuHCl (>1.3 mol L^–1) at 308 K is an enthalpy-driving process, and the adsorption under other GuHCl concentrations is an entropy-driving process. The fact that the Lys denatured by 1.8 mol L^–1 GuHCl forms a relatively stable intermediate state under the studied conditions will not be changed by temperature.     

反相/亲水色谱法分析糖苷类化合物

根据皂苷、黄酮苷等糖苷类化合物的结构特点,采用亲水色谱模式分析该类化合物,以弥补反相色谱模式分离结构类似糖苷类化合物选择性的不足。首先选择14个糖苷类化合物,比较了反相色谱柱(XAqua C18)和亲水色谱柱(Click XIon)的分离效果,评价了反相/亲水色谱的正交性,并构建了反相/亲水二维体系用于西洋参样品的分离。结果表明,糖苷类化合物在反相及亲水色谱柱上均有很好的保留,但两者具有不同的分离选择性,14种物质在反相和亲水柱的出峰顺序有较大差异,在反相色谱中不易分离的人参皂苷Rg1和Re在亲水色谱柱可获得很好的分离,反相/亲水模式分离糖苷化合物具有很好的正交性。以构建的RPLC/HILIC二维色谱体系分离西洋参样品,有效地提高了分离能力及峰容量,有利于后续更多极性及微量化合物的制备、结构表征与活性研究,且该方法操作简便、流动相兼容性好,可作为糖苷分离分析、制备的有效手段,也可以为其他中药复杂体系的分析提供参考。     

Separation of statistical poly[(N-vinyl pyrrolidone)-co-(vinyl acetate)]s by reversed-phase gradient liquid chromatography

Although size exclusion chromatography (SEC) has been used successfully to determine the molecular weight distribution (MWD) of statistical poly[(N-vinyl pyrrolidone)-co-(vinyl acetate)]s [PVPVAs], SEC cannot separate the copolymers according to their chemical composition. In this article, the separation of commercial PVPVAs with varying chemical compositions is reported, by aqueous reversed-phase gradient liquid chromatography (RPLC) using polystyrene-divinylbenzene-based wide pore columns. RPLC-SEC cross-fractionation indicates the presence of molar mass dependant effects during RPLC separation due to broad MWD for the copolymer studied; therefore the width of the RPLC peak could not be associated entirely with chemical composition distribution of the copolymer. Coupling of RPLC with online FTIR spectroscopy reveals the increase of VA content with increasing THF gradient, an indication of interaction mechanism between VA repeating units and the stationary phase for water soluble PVPVAs. Separation of water insoluble PVPVAs and PVAs by the RPLC are possibly based on both interaction and precipitation/redissolution mechanisms.     

Synthesis of non-porous poly(glycidylmethacrylate-co-ethylenedimethacrylate) beads and their application in separation of biopolymers

The monodisperse, 5.0 μm non-porous poly(glycidylmethacrylate-co-ethylenedimethacrylate) (P_GMA/EDMA) beads were prepared by a single-step swelling and polymerization method. The seed particles prepared by dispersion polymerization exhibited good absorption of the monomer phase. Based on this media, a weak cation exchange (WCX) stationary phase for high performance liquid chromatography (HPLC) was synthesized by a new chemical modification method. The prepared resin has advantages of biopolymer separation, high column efficiency, low column backpressure, high protein mass recovery and good resolution for proteins. The measured bioactivity recovery for lysozyme was 97 ± 5%. The dynamic protein loading capacity of the synthesized WCX packings was 20.5 mg/g. Four proteins were completely separated in 3.0 min using the synthesized WCX stationary phase. The experimental results show that the obtained WCX resin has very weak hydrophobicity. The WCX resin was also used for the rapid separation and purification of lysozyme from egg white in 3.0 min with only one step. The purity and specific bioactivity of the purified lysozyme was found more than 95% and 70.264 IU/mg, respectively.