Isoentropic and Isoenthalpic Temperatures of Protein Unfolding in Hydrophobic Interaction Chromatography

The thermal behaviors of five proteins in hydrophobic interaction chromatography （HIC） were investigated in the temperature range from 0 to 50℃. The thermodynamic parameters （△H°,△S°, △Cp°and △G°） of these proteins in the process of retention and unfolding were determined. The existence of enthalpy and entropy convergence with temperature was confirmed. The differences of the isoentropic and isoenthalpic temperatures for protein unfolding in HIC system from the traditional solution were elucidated.

Comparison of the Contributions of Tetrahydrofurfuryl Alcohol and PEG to a-Chymotrypsin Renaturation with High Performance Hydrophobic Interaction Chromatography

The contributions of tetrahydrofurfuryl alcohol (THFA) and polyethylene glycol (PEG) to the renatured efficiency of a-chymotrypsin were investigated and compared with each other. The maximum increments of bioactivity recovery of a-Chy were found to be 25.1% for THFA, 10.4% for PEG, respectively. The experimental results indicated that the denaturant solution containing THFA contributed more to the renaturation of a-Chy in high performance hydrophobic interaction chromatography (HPHIC) than that containing PEG, when the concentration of THFA was 3.2%, the bioactivity recovery of a-Chy is the highest.

高效液相色谱-质谱技术在蛋白质组学中的应用

蛋白质组学研究在生物医学领域发挥了重要作用,然而研究面临的主要难点在于其研究对象的复杂性和多样性。随着质谱技术的快速发展,高效液相色谱-质谱(HPLC-MS)分离分析复杂生物样品已经成为蛋白质组学研究的基础工具。蛋白质组学的研究从肽段分离,延伸到蛋白质和蛋白质复合物的分离,随着分析物的分子质量不断增大,其结构和组成复杂性也持续增加,分子特性也发生改变。面对不同的蛋白质组学研究对象,选择不同的分离模式、分离条件以及固定相参数是进行深度蛋白质组学研究的关键。本文综述了实验室常用的液相色谱分离模式,包括反相色谱(RPLC)、亲水相互作用色谱(HILIC)、疏水相互作用色谱(HIC)、离子交换色谱(IEC)和体积排阻色谱(SEC),以及其不同的组合模式与质谱联用在自下而上(bottom-up)分析、自上而下(top-down)分析、蛋白-蛋白相互作用分析中应用的研究。具体分析了色谱流动相与被分析对象之间的兼容性问题、色谱流动相与质谱兼容性问题,以及多维色谱中不同色谱模式之间流动相的兼容性问题。重点关注存在不兼容问题时研究者所提出的解决方案。此外,本文还评述了HPLC-MS结合样本前处理的方法在外泌体和单细胞蛋白质组学中的应用研究。总之,文章聚焦于近年来HPLC-MS技术在蛋白质组学中的研究进展,旨在为未来蛋白质组学领域的研究提供参考。

HPHIC固定相对脲变α-糜蛋白酶复性的影响

依据计量置换保留理论所得到的参数lgI,来测定不同构象态α-糜蛋白酶(α-Chy)在两种不同高效疏水相互作用色谱(HPHIC)固定相表面的折叠自由能,发现脲变α-Chy在HPHIC固定相表面获取的折叠自由能比溶液中的高很多,不同HPHIC固定相表面为脲变α-Chy提供不同的折叠自由能,且都随变性剂脲浓度的增大而增大;通过对不同HPHIC色谱柱后复性α-Chy的比活测定,还发现脲变α-Chy的复性效率与其从固定相表面的折叠自由能有关,同一构象的α-Chy从固定相表面得到的折叠自由能越高越有利于其折叠成天然蛋白质。

Synthesis of Novel Hydrophobic Media and Purification of Recombinant HBsAg

A novel hydrophobic medium with propyl as functional group and Sepharose 6B as matrix was designed and synthesized. The comparison of the hydrophobic medium synthesized with the commercial products was made by hydrophobic interaction chromatography(HIC) in isolating recombinant hepatitis B surface antigen(r HBsAg). r HBsAg was further purified to the final products by following a downstream procedure . The results indicate that the synthesized hydrophobic medium possesses a stable structure and desired physical and chemical properties. They were used to purify r HBsAg with a high yield and purity. Both the immunity and stability of hepatitis vaccine made by the r HBsAg products have reached the same level as other similar kinds of products.

Resolution enhancement in hydrophobic interaction chromatography via electrostatic interactions

In this work,a new type of hydrophobic stationary phase that provide electrostatic interactions with analytes was developed by bondingβ-phenylethylamine as a functional ligand to silica.This stationary phase can separate proteins with similar hydrophobicity that traditional hydrophobic resins cannot.Hen egg white was separated to examine the selectivity.The results show that the introduced electrostatic interactions are an important factor for the resolution enhancement and the new resin could have important applications in separation and purification of biological macromolecules.

Mechanism of simultaneously refolding and purification of proteins by hydrophobic interaction chromatographic unit and applications

The hydrophobic amino acid residues of a denatured protein molecule tend to react with the particles of the stationary phase of hydrophobic interaction chromatography (STHIC). These hydrophobic interactions prevent the denatured protein molecules from aggregating with each other. The STHIC can provide high enough energy to a denatured protein molecule to make it dehydration and to refold it into its native or various intermediate states. The outcome not only depends on the specific interactions between amino acids, the structure of STHIC, but also depends on the association between the STHIC and mobile phase. The mechanism of protein refolding and the principle of its quality control by HPHIC were also presented. By appropriate selection of the Chromatographie condition, several denatured proteins can be refolded and separated simultaneously in a single Chromatographic run. A specially designed unit, with diameter much larger than its length, was designed and employed for both laboratory and preparative scales. That unit for the simultaneous renaturation and purification of proteins (USRPP) had the following four functions: to completely remove denaturant, to renature proteins, to separate renatured proteins from impurities, and to easily recycle waste denaturant. The efficiencies of refolding and purification of proteins by the USRPP are almost comparable to a usual long Chromatographie column in laboratory. In preparative scale, USRPP can be easily, rapidly, and economically applied requiring a low pressure gradient. As an example, recombinant human interferon-γ is employed to elucidate the application of the preparative USRPP.

Applying flexible molecular docking to simulate protein retention behavior in hydrophobic interaction chromatography

Interaction between proteins and stationary phase in hydrophobic interaction chromatography (HIC) is differentiated into two thermodynamic processes involving direct nonbonding/conformation interac- tion and surface hydrophobic effect of proteins, hence quantitatively giving rise to a binary linear rela- tion between HIC retention time (RT) at concentrated salting liquid and ligand-protein binding free en- ergy. Then, possible binding manners for 27 proteins of known crystal structures with hydrophobic ligands are simulated and analyzed via ICM flexible molecular docking and genetic algorithm, with re- sults greatly consistent with experimental values. By investigation, it is confirmed local hydrophobic effects of proteins and nonbinding/conformation interaction between ligand and protein both notably influence HIC chromatogram retention behaviors, mainly focusing on exposed portions on the protein surface.

Hydrophobic interaction membrane chromatography for bioseparation and responsive polymer ligands involved

Hydrophobic interaction chromatography （HIC） is a rapid growing bioseparation technique, which separates biomolecules, such as therapeutic proteins and antibodys, based on the reversible hydrophobic interaction between immobilized hydrophobic ligands on chromatographic resin spheres and non-polar regions of solute molecule. In this review, the fundamental concepts of HIC and the factors that may affect purification efficiency of HIC is summarized, followed by the comparison of HIC with affinity chromatography and ion-exchange chromatography. Hydrophobic interaction membrane chromatography （HIMC） combines the advantages of HIC and membrane process and has showed great potential in bioseparation. For better understanding of HIMC, this review presents an overview of two main concerns about HIMC, i.e. membrane materials and hydrophobic ligands. Specifically, cellulose fiber-based membrane substrate and environment-responsive ligands are emphasized.

Effect of ligand structure of stationary phase of high performance hydrophobic interaction chromatography on renaturation efficiency of GuHCl-denaturedα-chymotrypsin

The renaturation of the denaturedα-chymotrypsin(α-Chy)with 1.7 mol·L^(-1)guanidine hydrochloride(GuHCI)by three kinds of stationary phase of high performance hydrophobic interaction chromatography(STHIC)with a comparable hydrophobicity but different ligand structures was investigated.The obtained result indicates that the ligand structures of the three STHIC contribute to the renaturation efficiency ofα-Chy in the order of the end ligands PEG-600<phenyl group<tetrahydrofurfuryl alcohol(THFA).

Retention model of protein for mixed-mode interaction mechanism in ion exchange and hydrophobic interaction chromatography

A unified retention equation of proteins was proved to be valid for a mixed-mode interaction mechanism in ion exchange chromatography (IEC) and hydrophobia interaction chro-matography (HIC). The reason to form a 'U' shape retention curve of proteins hi both HIC and IEC was explained and the concentration range of the strongest elution ability for the mobile phase was determined with this equation. The parameters in this equation could be used to characterize the difference for either HIC or IEC adsorbents and the changes in the molecular conformation of proteins. With the parameters in this equation, the contributions of salt and water in the mobile phase to the protein retention in HIC and IEC were discussed, respectively. In addition, the comparison between the unified equation and Melander' s three-parameter equation for mixed-mode interaction chromatography was also investigated and better results were obtained in former equation.

用低交换容量聚苯乙烯型强酸性阳离子交换树脂柱色谱分离中性氨基酸

通过磺化苯乙烯 -二乙烯苯共聚物或商品化的聚苯乙烯型强酸性阳离子交换树脂 ( 0 0 1× 7)的逆磺化反应 ,得到一系列不同交换容量的聚苯乙烯型强酸性阳离子交换树脂 .研究了丙氨酸和缬氨酸及缬氨酸和亮氨酸在这些树脂柱上的色谱分离 .结果表明 ,用两种方法得到的树脂对丙氨酸和缬氨酸的色谱分离性能基本相同 ;同时中性氨基酸与聚苯乙烯型强酸性阳离子交换树脂之间的作用包括离子作用和疏水作用 ,且二者之间存在协同作用 .

一种疏水色谱填料的特性及应用的研究

以交联壳聚糖为基质 ,正戊醛为配基 ,利用改进的方法制备了疏水作用色谱 (HIC)填料 ,并对该色谱填料的吸附行为和应用作了研究。结果表明 ,此类填料对蛋白质的吸附行为符合疏水相互作用理论 ,对α 淀粉酶的纯化活性回收率大于 80 %。

刀额新对虾变应原的分离、鉴定与纯化

目的通过对我国常见的刀额新对虾变应原蛋白进行分离、鉴定与纯化,以提供虾特异性变应原用于食物变态反应疾病的诊断和治疗。方法通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)分离刀额新对虾的蛋白质组分并测定其分子量,收集过敏病人血清,采用免疫印迹(Western-blotting)法鉴定其变应原成分,通过离子交换层析对刀额新对虾变应原进行初步纯化,通过疏水层析和凝胶过滤层析进一步对变应原纯化。结果刀额新对虾有17条蛋白带,其中主要条带有10条,68和36 kD为可与虾过敏性病人血清IgE结合的特异性变应原;通过各种层析方法纯化出刀额新对虾68 kD变应原,纯度为96%。结论对刀额新对虾变应原进行分离、鉴定和纯化,得到高纯度的68 kD刀额新对虾变应原,为临床虾变态反应疾病的诊断和治疗奠定基础。

重组灵芝免疫调节蛋白的纯化及其性质

采用超滤浓缩、强阴离子交换、疏水作用和凝胶色谱等方法,对毕赤酵母表达的rGlip进行分离和纯化,对离子交换色谱中rGlip与固相结合的最佳pH值进行了考察,并对纯化产物的活性进行了鉴定.rGlip在215nm处有强的紫外吸收,经激光解析电离时间飞行质谱鉴定其相对分子量为12722,经反相液相色谱鉴定纯度≥97%.设计rGlip的疏水作用色谱,有效地去除色素.凝血实验结果表明,rGlip可以凝集绵羊血红细胞,但对人血A,B,AB和O型等红细胞无凝集作用,有类似凝集素的生物学活性.

金属螯合亲和色谱中的疏水作用

通过考察盐溶盐和盐析盐浓度对蛋白质在IDA裸柱和金属螯合柱上保留行为的影响,详细研究了金属螯合色谱中的疏水作用,疏水作用的发生、形成的条件以及不同条件下对蛋白质保留值的贡献。实验结果表明,在高浓度和低浓度的盐溶盐以及低浓度盐析盐中,蛋白质在金属螯合柱上的保留主要受静电和配位作用控制,而疏水作用对蛋白质的保留影响很小。对弱亲和性的金属螯合柱以静电作用为主,其大小可用参数Q表征;对强亲和性的IDA-Cu(Ⅱ)柱以配位作用为主。仅在高浓度的盐析盐中,金属螯合柱才呈现较强的疏水作用,支配蛋白质保留。实验证明,金属螯合色谱中疏水作用主要来自固定相间隔臂中的疏水碳链和盐析盐对蛋白质的增疏作用,利用这种疏水作用有可能改善金属螯合色谱分离的选择性。

疏水膜色谱法对生物大分子的快速纯化

首次采用自制的分别级合了辛基、丁基、苯基及聚乙二醇-4000的4种常用疏水基团的纤维素疏水膜色谱柱，以键合了辛基、苯基的SepharoseCL－4B凝胶柱为对照，考察了疏水膜色谱柱对牛血清白蛋白（BSA）的动态吸附容量及流速对吸附容量的影响。疏水膜色谱柱对蛋白及酶具有较好的疏水吸附及纯化作用，但吸附容量比相应的琼脂糖凝胶柱低得多。增大流速及降低蛋白溶液质量浓度对疏水膜色谱柱的吸附容量影响较小，这些性能使膜色谱柱非常适合于大体积低质量浓度蛋白溶液（如基团工程培养液）的分离纯化。牛肝过氧化氢酶（BLC）经苯基膜色谱柱一步纯化，比活力提高11．8倍，蛋白回收率近100％，分离过程仅十几分钟。

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构象变化的表征。蛋白质的Ｚ值随脲浓度的增大而减小。除分子构象变化的因素外，Ｚ值变化与脲参与置换剂分子间的计量置换过程有关。Ｚ值变化的连续性与否直接反映了蛋白质分子构象的连续性变化或突变。

疏水作用柱层析技术纯化HBsAg条件的筛选

在应用流水作用柱层析纯化HBsAg中，对流水介质的功能基类型、交联密度、盐的浓度、操作温度等决定疏水性强弱的因素作了比较研究。结果表明，应用以C4为功能基的Butyl—Sepharose（6～8μmol／ml）和以C3为功能基的Propyl－Sepharose（20～22μmol／ml）疏水介质、8％（NH4）2SO4，18～24℃等条件纯化HBsAg时结果较理想。

舟山眼镜蛇毒细胞毒素的分离纯化及其体外抗肿瘤活性

目的从眼镜蛇毒中分离纯化细胞毒素 F(CTX F)并鉴定其活性。方法应用凝胶过滤、离子交换柱色谱及疏水柱色谱等方法从舟山眼镜蛇毒中分离纯化CTX F ,以SRB法观察CTX F对体外培养癌细胞的杀伤作用。结果眼镜蛇毒粗毒经凝胶过滤获得 4个蛋白峰 ,将CTX所在第Ⅳ峰用阳离子交换柱色谱获A、B、C、D、E、F和G等7个组分 ,其中E、F和G具CTX活性 ,将F组分再经凝胶过滤和疏水色谱进一步纯化得不含磷酯酶A2 (PLA2 )的CTX纯品 ,暂定名为CTX F ,它对多种癌细胞株有杀伤作用。结论应用凝胶过滤、离子交换和疏水色谱等方法可从眼镜蛇毒中获得不含PLA2 的CTX 。