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.     

金属硫蛋白异构体及亚型异构体的液相色谱分离与质谱鉴别

建立了金属硫蛋白(MT)异构体及亚型异构体的色谱分离与质谱鉴别方法。将金属硫蛋白混合物通过弱阴离子DEAE Sephadex A-25离子交换柱,结合离线电感耦合等离子体质谱(ICP-MS)对锌诱导金属硫蛋白的两个异构体MT-1和MT-2进行分离和检测;利用Sephadex G-25凝胶排阻色谱柱对得到的两个金属硫蛋白异构体进行脱盐;探索脱盐后的金属硫蛋白异构体在不同色谱条件下的C18反相色谱柱上的保留行为,进而实现各个亚型异构体的分离;通过在线电喷雾质谱检测实现了对金属硫蛋白各个亚型异构体的鉴别。结果表明,通过优化色谱条件,由离子交换色谱及凝胶排阻色谱得到的金属硫蛋白各亚型异构体在酸性条件下均得到了良好的分离,质谱检测结果与前人的文献报道结果一致。该方法可使金属硫蛋白各异构体均达到最佳的分离效果。     

克拉维酸钾及主要原料中大分子蛋白杂质的测定EI北大核心CSCD

建立了凝胶排阻色谱对克拉维酸钾及其原料克拉维酸叔辛胺中的残余蛋白进行检测的方法。采用TSK gel G3000 SWXL凝胶色谱柱(5μm,7.8×300 mm),流动相为磷酸盐缓冲溶液,流速为0.8 m L/min,紫外检测波长为220 nm。对照品牛血清白蛋白(BSA)在0.249~49.9μg/m L内线性关系良好(R2=0.9997),平均回收率为101.2%,RSD为0.7%,检测限为0.05μg/m L。方法为同类药物中的大分子蛋白检测提供了有效手段。     

高效凝胶排阻色谱法测定牛血清白蛋白相对分子质量及其分布

建立了一种高效凝胶排阻色谱快速测定牛血清白蛋白的相对分子质量及其分布的方法。使用凝胶色谱仪,TSKgel G3000SWXL型(300 mm×7.8 mm)高效凝胶排阻色谱柱,以0.05 mol/L磷酸盐缓冲溶液(pH 6.8)–0.3mol/L氯化钠溶液为流动相,流速为1 mL/min,DAD检测器,检测波长为220 nm,选择300~330 nm作为参比波长,信号采集时间为15 min。测得质控样牛血清白蛋白相对分子质量标准物质峰值相对分子质量的均值为7.160×104Da,与参考值的相对误差为7.76%,组间相对标准偏差为0.49%。采用该法对市售牛血清白蛋白样品的相对分子质量分布进行测试,组内相对标准偏差为0.004%~0.014%,组间相对标准偏差为1.89%。该法可以满足一般实验室对牛血清白蛋白样品的分子量分布测定的要求。     

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.     

水溶性凝胶渗透色谱法测定碱木素分子量

应用水溶性凝胶渗透色谱法测定了碱木素的分子量,与脂溶性凝胶渗透色谱法的测定结果基本一致。色谱柱为Ｕｌｔｒａｈｙｄｒｏｇｅｌ凝胶色谱柱,流动相为氢氧化钠溶液（ｐＨ１２）。系统考察了不同流动相对碱木素在凝胶柱上吸附的影响,结果发现,增大流动相离子强度有利于减少碱木素的吸附,但流动相的酸度对于碱木素吸附具有更重要的影响,ｐＨ值高时,碱木素吸附减弱。实验同时表明,高分子量的碱木素更易在凝胶柱上发生吸附。     

丁基胺丙基硅胶毛细管整体柱的制备及其电色谱性能研究

采用溶胶-凝胶技术制备了丁基胺丙基硅胶毛细管整体柱,此整体固定相表面同时含有能产生阳极的电渗流的仲胺官能团和产生疏水作用的正丁基和丙基官能团。对所制备的整体柱电色谱性能进行了详细的表征和分析。考察了流动相pH值对电渗流的影响;对烷基苯同系物、有机酸酸性化合物和苯胺类碱性化合物保留行为进行了研究,并对其可能的保留机理进行了探讨。实验结果表明,对于中性化合物的保留机理主要基于反相作用;而对于酸性化合物的保留行为则是基于混合模式作用机理,即除了电泳作用外,还包括阴离子交换和疏水作用。碱性化合物在丁基胺丙基硅胶毛细管整体柱上的峰形较好,没有明显的峰拖尾现象。     

杜氏盐藻多糖的高效体积排阻色谱的保留特性及其分析方法的研究

通过对从杜氏盐藻中提取出的不同多糖级分在高效体积排阻色谱柱(Waters Ultrahydragel Linear,7.8 mm i.d.×300 mm,2根串联)上的保留特性的考察及其分离分析条件的优化,建立了高效体积排阻色谱分析盐藻多糖平均相对分子质量及其分布的方法。结果表明:流动相中盐的种类及其浓度、pH值对3种酸性多糖级分(特别是硫酸化多糖级分PD4a)的保留行为有显著影响;在柱温为45 ℃,流速为0.9 mL/min条件下,使用0.1 mol/L的NaAc水溶液作流动相基本上能消除非特异性吸附作用及分子间缔合等因素的干扰,使各多糖级分基本以非缔合状态按立体排除机制保留和分离。在优化的色谱条件下,测得的盐藻多糖5个级分的重均相对分子质量(Mw)分别为1548000,33000,67000,424000,10000;测得的硫酸化多糖级分PD4a的Mw和峰面积的相对标准偏差分别为1.7%和 0.88%(n=5)。     

烷基酚聚氧乙烯醚在不同液相色谱分离模式中的分离研究

考察了烷基酚聚氧乙烯醚在反相色谱、正相键合色谱、硅胶吸附色谱、体积排阻色谱4种不同液相色谱分离模式中的分离效果,分别采用Kromasil C_(18)(250 mm× 4.6 mm,5 μm)、Agilent ZORBAX NH2(250 mm× 4.6 mm,5 μm)、Waters Spherisorb S3W(150 mm×2.0 mm,3 μm)和Shodex MSpak GF-310 2D(150 mm×2.0 mm,5 μm)色谱柱,以225 nm为紫外检测波长,对不同液相色谱分离模式的流动相组成、梯度洗脱条件、柱温、流速等进行了优化,并对烷基酚聚氧乙烯醚在不同液相色谱分离模式中的保留机理进行了初步探讨.结果表明,正相键合色谱实现了烷基酚聚氧乙烯醚的最佳分离;硅胶吸附色谱和体积排阻色谱的分离效果较正相键合色谱稍差.     

小肽在反相色谱中的保留行为

]本文系统地考察了反相色谱中小肽的保留行为,包括氨基酸疏水性对小肽保留值的贡献,流动相有机溶剂浓度、pH值和无机盐浓度及柱温对小肽（主要是二肽、三肽和二肽异构体）的保留值和选择性的影响。     

Fractionation of apple procyanidins by size-exclusion chromatography.

Oligomeric constituents of apple procyanidins were fractionated by size-exclusion chromatography using a TSKgel Toyopearl HW-40F column. The best separation was obtained using a mobile phase of acetone-8 M urea (6:4; adjusted to pH 2) at a flow-rate of 1.0 ml/min. In this chromatographic system, the use of 8 M urea in the mobile phase resulted in a molecular sieve effect without any surface affinity interaction between the gel beads and the procyanidin molecules. Each fraction obtained was examined by reversed-phase high-performance liquid chromatography and time-of-flight mass spectrometry. The order of elution of the procyanidins from the column was coincident with their degree of polymerization.     

Interaction of urea with amino acids: implications for urea-induced protein denaturation

The molecular mechanism of urea-induced protein denaturation is not yet fully understood. Mainly two opposing mechanisms are controversially discussed, according to which either hydrophobic, or polar interactions are the dominant driving force. To resolve this question, we have investigated the interactions between urea and all 20 amino acids by comprehensive molecular dynamics simulations of 22 tripeptides. Calculation of atomic contact frequencies between the amino acids and solvent molecules revealed a clear profile of solvation preferences by either water or urea. Almost all amino acids showed preference for contacts with urea molecules, whereas charged and polar amino acids were found to have slight preferences for contact with water molecules. Particularly strong preference for contacts to urea were seen for aromatic and apolar side-chains, as well as for the protein backbone of all amino acids. Further, protein-urea hydrogen bonds were found to be significantly weaker than protein-water or water-water hydrogen bonds. Our results suggest that hydrophobic interactions are the dominant driving force, while hydrogen bonds between urea and the protein backbone contribute markedly to the overall energetics by avoiding unfavorable unsatisfied hydrogen bond sites on the backbone. In summary, we suggest a combined mechanism that unifies the two current and seemingly opposing views.     

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).     

邻苯二甲醛-尿素柱前衍生高效液相色谱法快速检测枸杞中牛磺酸

干枸杞经粉碎、匀浆、离心后,通过阳离子交换柱脱去样品中其它氨基酸,再通过Ｚｏｒｂａｘ－Ｃ８柱进行柱前衍生分离。衍生剂：Ａ．４％ＯＰＡ甲醇溶液;Ｂ．尿素∶磷酸钠盐缓冲液（ｐＨ６．８）＝１∶３（Ｗ／Ｖ）。流动相：甲醇∶０．０１ｍｏｌ／Ｌ乙酸钠溶液（ｐＨ６．８）＝３５∶６５（Ｖ／Ｖ）。紫外检测波长３３０ｎｍ。牛磺酸浓度在０．１～１．０ｍｍｏｌ／Ｌ范围内可被定量测定。回收率可达１００．３１％±１．９８％,变异系数（ＣＶ）为１．９４％。     

Uptake of hydrophobic amino acids and sodium chloride by a strong cation exchanger: Application to desalting and separation by preparative liquid chromatography

Summary The uptake of sodium chloride, leucine and phenylalanine by a strong cation-exchange resin in the sodium form have been compared. Sodium chloride was excluded from the resin, as indicated by its parabolic upwards curved isotherm, and the corresponding desorption edge was sharpened (constant pattern). Isotherms for both amino acids constructed from a single elution experiment displayed slightly downwards curvature; the corresponding desorption edges spread in contrast to the breakthrough ones. The affinities of both amino acids for the resin were higher (from 15 to 40 %) in the presence of 1 M sodium chloride, indicating that hydrophobic interactions played a significant role in the mechanism of sorption. As expected from the isotherms, a mixture of 1 M sodium chloride, leucine and phenylalanine was separated with satisfactory recovery of both amino acids for a column loading of 10 % of the bed volume. Real mother liquors have also been separated under the same conditions.     

体积排阻色谱法研究变性溶菌酶分子复性过程中集聚体的形成

在体积排阻色谱柱上研究了还原剂存在时脲和盐酸胍变性的3种溶菌酶溶液的复性和分离过程。当变性液中原始溶菌酶浓度大于10 g/L时,变性溶菌酶在体积排阻色谱柱上除了复性为与未变性溶菌酶出峰时间相同的复性态溶菌酶分子外,还形成了溶菌酶折叠中间体的二分子集聚体。这个结果得到了用稀释法复性时溶菌酶的蛋白电泳检测结果的支持。与稀释法复性相比较,用体积排阻色谱法复性时所形成的折叠中间体二分子集聚体的量要远远低于用稀释法所形成的集聚体的量。     

离子色谱法测定卷烟主流烟气中的硫化氢

建立了测定卷烟主流烟气中硫化氢含量的离子色谱法.采用玻璃纤维滤片捕集卷烟烟气粒相物,并用0.5%(体积分数)乙二胺-50 mmol/L氢氧化钠-250 mmol/L乙酸钠溶液萃取粒相物,吸收液吸收气相物中的硫化氢,合并粒相萃取液与气相吸收液,经离子色谱柱分离,以1.5 mol/L氢氧化钠-1 mol/L乙酸钠-2%(体积分数)乙二胺(40∶50∶10,体积比)为流动相,安培检测器检测并施加-100 mv的检测电位.运用方法对12种市售卷烟样品进行了测定,结果表明:方法的线性范围为0.1~5.0μg/mL,检出限为1.03μg/mL,定量限为3.41μg/mL,回收率为102.3%~107.2%,相对标准偏差小于5%.方法处理简单、准确度高,可以用于卷烟烟气中硫化氢含量的测定.     

Separation of cationic analytes by nonionic micellar electrokinetic chromatography using polyoxyethylene lauryl ether surfactants with different polyoxyethylene length

Although nonionic micellar electrokinetic chromatography is used for the separation of charged compounds that are not easily separated by capillary zone electrophoresis, the effect of the hydrophilic moiety of the nonionic surfactant has not been studied well. In this study, the separation of ultraviolet‐absorbing amino acids was studied in electrokinetic chromatography using neutral polyoxyethylene lauryl ether surfactants (Adekatol) in the separation solution. The effect of the polyethylene moiety (the number of repeating units was from 6.5 to 50) of the hydrophobic test amino acids (methionine, tryptophan, and tysorine) was studied using a 10 cm effective length capillary. The separation mechanism was based on hydrophobic as well as hydrogen bonding interactions at the micellar surface, which was made of the polyoxyethylene moiety. The length of the polyoxyethylene moiety of the surfactants was not important in nonionic micellar electrokinetic chromatography mode.     

Hydrophobic properties of maize zein

The proteins of the zein complex from maize endosperm have been studied with the aid of hydrophobic chromatography. Their best separation was achieved on a column with TSK gel HW-65f. By comparing the results of fractionation by hydrophobic chromatography with those of electrophoresis, it was found that electrophoresis in PAAG under denaturing conditions separates the zein protein into groups according to their surface hydrophobicity. The most hydrophobic is the high-molecular-mass group of zein polypeptides.V. R. Vil'yams Kazakh Scientific-Research Agricultural Institute, Alma-Ata Province. Translated from Khimiya Prirodnykh Soedinenii, No. 1, pp. 105–108, January–February, 1991.     

Some factors affecting separation and detection of amino acids by high-performance anion-exchange chromatography with integrated pulsed amperometric detection

Some factors influencing the separation and detection of amino acids by high-performance anion-exchange chromatography with integrated pulsed amperometric detection were investigated. These factors include eluent concentration, column temperature, and detection waveform. The selectivity changes in weakly retained amino acids are slight with changing sodium hydroxide eluent concentration. When sodium acetate eluent concentration is changed, the selectivity variations between strongly retained amino acids containing two carboxyl groups and containing only one carboxyl group are obviously different. Significant but slight selectivity changes in weakly retained amino acids can be achieved through changing the column temperature. Sodium hydroxide and sodium acetate eluent concentration affect the detection of amino acids. Detection sensitivity of amino acids can be improved by increasing the concentration of sodium hydroxide and sodium acetate in a certain concentration range. The detections of amino acids at two different detection waveforms were compared. The hydroxyl amino acids can be selectively detected by choosing a modified detection waveform. The optimized gradient elution condition and column temperature for analyzing 19 amino acids were obtained. The time for the gradient elution program was 60 min. The column temperature was 35 degrees C. Under the optimized conditions, detection limits for 19 amino acids were 0.15-4.52 pmol. The calibration graphs of peak area for all the analytes were linear for about three orders of magnitude. The RSDs (n=5) of peak area were 0.6-5.6%. The determination of trace amino acid impurities in valine product is shown as an application example.