高效液相色谱分离纯化血管紧张素转换酶活性抑制肽

采用分步高效液相色谱法首次从菠菜核酮糖双磷酸羧化酶(英文缩写为Rubisco)的胃蛋白酶-胰酶复合酶水解产物中分离纯化了血管紧张素转换酶(ACE)活性抑制肽。水解产物经ODS柱分离得到活性组分,这些活性组分再依次经过氨基柱(PhA) 氰基柱(CN)和硝基苯乙基柱(NPE)4种色谱柱分离后,得到4种高度纯化的具有抑制ACE活性的多肽。经蛋白质序列自动分析仪测定,4种多肽的结构分别为MRWRD,MRW,IAYKPAG和LRIPVA。采用固相多肽合成法分别合成了这4种肽,并采用测定ACE活性抑制率的方法评价其     

高效液相色谱中的手性两相协同作用

在高效液相色谱中,以纤维素三苯基甲酸酯、纤维素三苯基氨基甲酸酯为手性固定相,以正己烷-异丙醇(体积比为9∶1)为流动相,柱温25 ℃,分别以β-环糊精（β-CD）、2,6-二甲基-β-环糊精(DM-β-CD)、2,3,6-三甲基-β-环糊精(TM-β-CD)为流动相添加剂,分离了DL-色氨酸和DL-苯丙氨酸两种手性化合物,考察是否存在手性固定相和手性添加剂的协同作用。实验结果表明,在流动相中添加β-CD或DM-β-CD至饱和,协同作用不明显;在流动相中添加少量TM-β-CD(即浓度小于0.60 mmol/     

Single Step Purification of a Synthetic Peptide Fragment of Neurofilaments by Preparative High Performance Liquid Chromatography

Abstract

It was recently shown that fragment Lys-Ser-Pro-Val (1,2) is the major site in vivo neurofilament phosforilation. A suitable method for the preparation of this peptide was therefore studied paying particular attention to the purification step. Even thought solubility of this peptide in methyl t-butil ether does not allow the standard purification. It is possible achieve a good separation in a single step by HPLC, after a simple extraction with acidic water.     

Preparative Purification of Peptides by Countercurrent Chromatography on the Ito Coil Planet Centrifuges

Abstract

For the purification of up to 1000 mg of synthetic peptides by countercurrent chromatography, the coil planet centrifuges have proven useful in the research laboratory. Besides the earlier described horizontal flow-through coil planet centrifuge which chromatographs substances in all solvent systems at room temperature, the multi-layer coil planet centrifuge affords more rapid chromatography. However, separations using n-butanol, especially suited for peptides, have to be conducted at elevated temperatures. A new machine that has characteristics of both of the foregoing instruments is the compact horizontal flow-through coil planet centrifuge which promises rapid chromatography with full retention of the stationary phase.     

Neuroendocrine Peptides - Analysis by Reversed Phase High Performance Liquid Chromatography

Abstract

A high performance liquid chromatographic (HPLC) method is described for determining the biologically active neuroendocrine peptides thyrotropin releasing hormone (TRH), leucine (leu) and methionine (met) enkephalin, angiotensin II, delta sleep inducing peptide (DSIP), luteinizing hormone releasing hormone (LHRH), substance P and growth hormone release inhibiting factor (somatostatin). The selection of mobile phases was limited to these systems that do not exhibit strong absorbtion at 215 nm and 254 nm. Under isocratic conditions at room temperatures with the appropriate selection of mobile phase it was possible by reversed phase chromatography to resolve all of the peptides investigated. We can resolve with the systems employed peptides differing by only one amino acid in chain length as well as peptides differing by only one amino acid in the chain sequence. The method is rapid, does not require derivitization, can be used with aqueous matrixes and is sensitive in the nanomolar range.

Our research has shown that most synthetic peptides lack purity and that all of the peptides except LHRH lack stability when stored in aqueous sterile solution at 8°C for four weeks. The implications of this latter finding are under investigation.     

多肽和蛋白质的反相高效液相色谱研究--色谱行为的多样性

以Ｃ８柱为固定相,研究了微管结合蛋白２中两段重要肽段（肽１,肽２）和猪胰岛素在反相色谱上的保留行为。当以甲醇溶液为流动相时,猪胰岛素保留因子的对数值（ｌｎＫ’）随流动相有机溶剂体积分数（ψ）的变化呈现很好的线性关系。当以乙腈为流动相测定肽１的ｌｎＫ’随ψ变化关系时,所得结果显示ｉｎＫ’与ψ呈现良好的二次曲线关系。以乙腈为流动相测定肽１和肽２和Ｖａｎ’ｔ Ｈｏｆｆ曲线（ｌｎｋ’与１／Ｔ的关系图）时,     

应用反相高效液相色谱法分析纯化化学合成肽

ＤＮＡ结合蛋白的化学合成、结构与功能研究有助于阐明基因调控过程的分子机制。主要介绍反相高效液相色谱法（ＲＰ－ＨＰＬＣ）在化学合成ＤＮＡ结合肽分析纯化中的应用。     

Liquid Adsorption Chromatography on Preparative Scale of Protected Synthetic Peptides

Abstract

This paper reports the purification of synthetic protected peptides on a preparative scale by means of adsorption chromatography on silica gel 60 columns.

The protected peptides described were precursors of a free asymmetrical cystine peptide corresponding to the insulin sequence A^18–21-B^19–26 and were obtained in analytically pure form. Selection of solvent systems for isocratic or stepwise elution depended largely on R_F data obtained from thin-layer chromatograms which were used for monitoring and optimizing synthetic reactions.     

Rapid Purification of Synthetic Bombesin by Countercurrent Chromatography on the Multi-Layer Coil Planet Centrifuge

Abstract

A temperature-controlled multi-layer coil planet centrifuge rapidly yields highly efficient preparative separations of polar compounds. Capability of the method was demonstrated on a one-step purification of crude synthetic bombesin with a two-phase solvent system composed of n-butanol/dichloroacetic acid/water (100:1:100). Under an elevated temperature at 45°C to 50°C, the bombesin peak was eluted within two hours. Reversed phase HPLC analysis of the bombesin fractions showed over 98% purity. The method may be applicable to many other peptides and polar compounds.     

High Performance Liquid Chromatography and Proteolytic Enzyme Characterization of Peptides in Tooth Pulp Extracts

Abstract

Metabolic profiles are obtained for peptides contained in tooth pulp extracts. To determine which high performance liquid chromatographic peaks are due to peptides, a series of proteolytic enzymes (chymotrypsin, trypsin, and carboxypeptidase A) are utilized. Results from treatment of extracts with immobilized enzymes demonstrate that virtually all peaks in this reverse phase system are due to peptides. This current study is a necessary component in a larger research program focusing on quantification of enkephalin-and endorphin-related peptides in biologic extracts including brain and tooth pulp tissue.     

Analysis of Group Retention Contributions for Peptides Separated by Reversed Phase High Performance Liquid Chromatography

Abstract

Within the framework provided by solvophobic theory, selectivities for unprotected peptides separated on fully porous, microparticulate, chemically bonded alkylsilicas can be ascribed to differences between the effective hydrophobic contact areas of the solutes. Furthermore, this theoretical treatment predicts that retention behaviour differences can be evaluated from topological parameters which accomodate the influence of amino acid side chain and end group contributions in the retention process. With data obtained for 57 peptides, including a variety of peptide hormones, eluted under the same conditions from a μBondapak C₁₈ column, these predictions have been rigorously tested using two methods of numerical analysis. The results provide further evidence that the hydrophobic group retention contributions of the amino acid residues in small peptides have an essentially additive effect on peptide retention with alkylsilicas. Divergences in retention behaviour are interpreted in terms of specific silanophilic and solvation interactions.     

蛋白质制备色谱的研究

研究了蛋白质纯化制备的二种运行模式,其一,在色谱柱超载下纯化制备蛋白质模式,并成功地用离子交换色谱纯化制备了大豆中的胰蛋白酶,用反相液相色谱纯化了细胞色素Ｃ;其二,用溶质－溶质顶替色谱纯化制备了核糖核酸酶－Ａ,并对顶替色谱过程中诸参数进行了讨论和选择。     

多联吡啶衍生物的RP-HPLC分析

多联吡啶在分子元件、生物模拟、药理病理研究、分析检测等领域中应用前景[1 ] 广阔。我们曾合成了新型多联吡啶衍生物— 6,6″ -二甲基 - 4′-苯基 - 2 ,2′,6′,2″ -三联吡啶 (ＴＰＹ) [2 ] 并研究了它与过渡金属离子形成络合物的性能 ,但其产率低 ,限制了它的实际应用。本文探讨合成ＴＰＹ缩合反应中各物质的色谱行为及各物质ＲＰ -ＨＰＬＣ分离的适宜条件。1　实验部分1 1　仪器与药品Ｗａｔｅｒｓ 60 0高效液相色谱仪 (美国Ｗａｔｅｒｓ公司 ) ,2 4 87紫外 -可见分光光度检测器 ,Ｍ32数据采集和处理系统 ,Ｒｈｅｏｄｙｎｅ 772 5ｉ手…     

高效液相色谱整体柱技术的进展

高效液相色谱整体柱（又名连续床）具有制备相对简单、原料易得以及聚合组分在一定范围内可调节的优点,是近年来得到迅速发展的新型色谱柱。本文综述了目前高效液相色谱（HPLC）制备整体柱的典型高聚物体系、制备各种整体柱时反应条件的影响,并简要介绍了它的表征方法和应用。     

分子印迹液相色谱整体柱

分子印迹是合成预定选择性固定相的新兴技术,整体柱是新型的色谱固定相技术。将分子印迹聚合物与整体柱技术相结合,可以有效提高液相色谱的分离效率,有助于推动整个分离科学的发展,意义重大,是当今分析化学的研究热点。本文就分子印迹液相色谱整体柱的制备合成、色谱分离条件以及物理化学特性评价方法等方面的研究进展进行了较系统的综述,并对该技术目前存在的问题和发展前景进行了探讨。     

用反相高效液相色谱法纯化白细胞介素－2条件初探

用反相高效液相色谱法纯化白细胞介素－２条件初探胡明（沈阳军区军事医学研究所沈阳１１００３１）关键词反相高效液相色谱法，白细胞介素－２１前言高效液相色谱法（ＨＰＬＣ）是一项分辨率很高的液相色谱技术，具有速度快，灵敏度高、样品可回收等优点。除被广泛地应用...     

反相高效液相色谱分离纯化天然除虫菊酯

天然除虫菊酯含有6种有效成分,但结构相似,分离困难。本研究以除虫菊酯精油为原料,通过优化反相高效液相色谱分离条件,分离纯化了除虫菊酯6种有效成分,纯度均达到99%。然后用气相色谱-质谱定性分析确证6种成分。本研究所建立的天然除虫菊酯6种有效成分的反相高效液相色谱分离纯化方法,为天然除虫菊酯除虫菊酯残留检测提供了准确的判断标准,为阐明天然除虫菊酯6种单一有效成分的杀虫机理研究奠定了基础,也为除虫菊酯6种单一有效成分纯品的生产提供了重要的方法参考。     

The Purification of Xanthene Dyes by Reverse Phase High Performance Liquid Chromatography

Abstract

A reliable method for the separation of fluorescein dyes from their impurities was developed using high performance liquid chromatography and involved a μBondapak C₁₈ reverse phase column and mixtures of methanol and ammonium acetate buffer. This technique was used to verify the purity of commercial products as well as to aid in the development of an empirical theory related to retention of halogenated fluorescein dyes by reverse phase columns.     

高效亲和液相色谱法纯化基因重组人白细胞介素-2

1 引言高效亲和液相色谱(HPAC)是将经典亲和色谱的高特异性同高效液相色谱的快速、高效相结合产生的一种蛋白质的分离技术.它使经典亲和色谱原来需要几个小时才能完成的纯化工作缩短为十几分钟或更短,而且纯化倍数高,分离效果好.因此,HPAC在近年基因重组蛋白质的纯化方面得到了广泛的应用.基因重组人白细胞介素-2(rhIL-2)是生物体内具有重要生物学功能的细胞因子,目前已成功地进行了基因表达.本文以自制的rhIL-2单克隆抗体(McAb)的HPAC填料纯化了大肠杆菌(E.coli)表达的rhIL-2.     

Purification of Human Platelet Monoamine Oxidase B by High Performance Liquid Chromatography

Abstract

Human platelet monoamine oxidase (MAO B), a membrane bound enzyme was purified to homogeneity by DEAE-Sephacel column chromatography, chromatofocusing, and high performance liquid chromatography (HPLC). The crucial purification step was HPLC on a anion exchange column (SynChropak AX 300). The HPLC column was eluted initially with potassium phosphate buffer (100 mM, pH 7.4) for 10 min at a flow rate of 1.0 ml/min, followed by a gradient (0–1%) of octyl-β-D-glucopyranoside (octylglucoside) in the same buffer for 10 min, and finally with buffered octylglucoside (1%) for 40 min. The elution of pargyline-bound or active MAO was established by determining either radioactivity in each fraction when MAO B had previously been covalently labeled with [³H]-pargyline [³H(G)] or catalytic activity using [¹⁴C-methylene]-benzylamine as substrate. [³H]-pargyline-bound and active MAO B eluted from the column at approximately 34 min. The extent of homogeneity and the subunit M_r (approximately 59,000) of MAO B were determined by sodium-dodecyl sulfate polyacrylamide gel electrophoresis followed by silver staining for proteins.