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反相高效液相色谱与质谱联用分析合成七肽的消旋产物 总被引:2,自引:0,他引:2
采用反相高效液相色谱(RP-HPLC)与质谱(MS)联用技术对固相法化学合成七肽(H2N-PFNSLAI-COOH,M r 760.9)时出现的消旋产物进行了分析。根据弱疏水性合成七肽粗品特性,提出了充分利用吸附和分配双重保留机制的“早期吸附”概念,以此优化色谱条件得到4个峰形良好的色谱峰;质谱分析表明:4谱峰具有相同的m/z761.5[M H] 值,对每一消旋产物进行在线多肽测序,并利用串联质谱(MSn)谱图叠加的方法,得到b轴和y轴两套片段信息,成功确定了合成七肽的4个消旋产物。 相似文献
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高效液相色谱分离纯化血管紧张素转换酶活性抑制肽 总被引:6,自引:0,他引:6
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 相似文献
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反相高效液相色谱法分离制备蜂毒肽类似物 总被引:1,自引:0,他引:1
应用高效制备液相色谱法,对5种合成的蜂毒肽类似物分离制备。当用极性较强的洗脱液做流动相时,主峰的保留时间短,且主峰和前后的杂质峰不能很好的分开;随着洗脱液极性减弱至某一值时,主峰和杂质峰的保留时间均向后延长,且时间间隔加大,可以成功地对多肽样品进行分离制备。有多肽分子中各氨基酸保留常数加和值的方法可以预测不同多肽的保留时间,为选择分离纯化多肽所需的流动相提供了参考作用。经半制备分离纯化后的产物用分析型RP-HPLC测定达到了很高的纯度,氨基酸分析结果表明得到了所需的肽段,可以用于下一步的研究工作。 相似文献
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分子印迹技术在天然产物有效成分分离纯化中的应用 总被引:6,自引:0,他引:6
天然产物体系复杂,大分子和小分子、生命和非生命物质共存,多存在结构相近的异构体,且有效成分含量低,采用一般的分离方法富集难度较大。分子印迹技术是制备高选择性分离介质的有效技术手段,分子印迹聚合物(MIP)的选择性强,分离操作简单,在各种分离纯化中展现了良好的应用前景。本文对近年来分子印迹技术在天然产物活性组分(如生物碱、甾体、多元酚、黄酮等)的分离纯化中的应用进行了综述,并介绍了本课题组利用MIP从天然产物中分离纯化莽草酸等活性成分的研究进展。 相似文献
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变性蛋白表面的疏水氨基酸残基有与疏水色谱固定相(STHIC)颗粒相互作用的倾向, 两者之间的疏水相互作用能够抑制变性蛋白分子间的相互聚集. 同时疏水色谱固定相还能在分子水平上给变性蛋白分子提供足够高的能量, 使其瞬时脱水并折叠成其天然构象或不同的折叠中间体. 变性蛋白在疏水界面上的折叠不仅取决于其氨基酸之间的特异性相互作用及疏水色谱固定相的结构, 而且还取决于固定相和流动相之间的协同作用. 同时, 还提出了高效疏水相互色谱(HPHIC)进行蛋白折叠的机理及其进行蛋白折叠时能实现质量控制的原理. 在适当的色谱条件下, HPHIC 可使几种变性蛋白一步实现复性及同时纯化. 此外, 还设计制造出了直径比柱长大得多的实验室型和制备型“变性蛋白复性及同时纯化装置, USRPP”, 该“装置”具有完全除去变性剂、使蛋白质复性, 与杂蛋白分离及易于回收变性剂的“一石四鸟”功能. 该“装置”对变性蛋白的复性和纯化效率与通常使用的长柱相当. 在制备规模情况下, 该“装置”可以在低压梯度条件下简便、快速、而经济地应用于重组蛋白药物的制备. 文中以重组人干扰素-γ为例, 说明了制备型“装置”在其复性及同时纯化生产工艺中的应用. 相似文献
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本文报道的内容为胰泌素基因的化学合成和克隆。胰泌素为一由2了个氨基酸组成的肠胃道多肽激素,该多肽合成基因的顺序由计算机辅助设计,共216个核苷酸碱基;合成基因选用酵母细胞偏爱的密码子,并在其中设置了一些便于今后用于基因改造、表达调控和产物分离所需的位点,排除了可能影响酶促连接反应的各种重复顺序;用化学法(固相磷酸三酯法和固相亚磷酸三酯法)合成了12个基因片段,长度为12寡聚核苷酸至24寡聚核苷酸,聚丙烯酰胺凝胶电泳分离纯化合成产物;利用T_4DNA连接酶组建完整的胰泌素基因,克隆于pWR 13质粒中,构建了一个含胰泌素基因的新质粒pWS 1。 相似文献
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应用芴甲氧羰基(Fmoc)固相化学合成了海南捕鸟蛛毒素-Ⅲ,并优化了其最佳氧化复性条件。最佳的氧化复性条件为pH 7.5的重蒸水溶液体系,样品质量浓度为0.1 g/L,还原型谷胱甘肽和氧化型谷胱甘肽的浓度分别为 1.0 mmol/L和0.1 mmol/L、L-Arg浓度为1.0 mol/L。复性产物经质谱测定其相对分子质量为3607.68;与天然毒素等量混合后用高效液相色谱分析得到单一峰;膈神经-膈肌标本生理实验结果表明,合成的毒素具有与天然毒素相同的生物学活性,从而可确定二者在结构与功能上具有一致性。 相似文献
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Flt3配体(FL)是一类具有促进早期造血功能的细胞因子,在促进造血细胞生长发育及造血动员方面具有重要的临床应用价值。为了用基因工程方法获得大量用于临床和研究的重组人FL(rhFL)蛋白质,本文对在大肠杆菌(E. coli)中表达得到的Flt3配体的包涵体进行回收、洗涤,溶解于8 mol/L脲后在高效疏水相互作用色谱(HPHIC)柱上进行rhFL包涵体的复性与同时纯化,并对其保留特征和复性规律进行了研究。结果表明,在连续进样、变性蛋白质质量浓度为8.51 g/L、固定相选用端基为PEG800、流动相添加4 mol/L脲、1.8 mmol/L 还原型谷胱甘肽(GSH)和0.3 mmol/L氧化型谷胱甘肽(GSSH)、pH 7.0的优化条件下,复性与同时纯化rhFL包涵体的质量回收率为36.9%,纯度达94.5%以上。本文仅用一步HPHIC法成功地复性与同时纯化了rhFL蛋白质,为获得高活性的rhFL产品奠定了一定的工作基础。 相似文献
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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 chromatographic 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 相似文献
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Based on three-state renaturation process of denatured proteins, an equation describing the effect of denaturant concentration on renaturation yield of denatured proteins was presented. By this equation, two parameters n(m1 -m2) and Ka can be obtained. The former indicates the difference in the number of denaturant molecules between the renaturation process of n number of refolding intermediates from refolding intermediate state to native state and their aggregate process from refolding intermediate state to aggregate state, the latter denotes the apparent aggregate equilibrium constant for protein molecules aggregated from native state to aggregate state, and from them, the characteristics of the renaturation process of denatured proteins in denaturant solution can be identified. This equation was tested by the renaturation processes of denatured egg white lysozyme in guanidine hydrochloride and urea solutions, with the results to show that when guanidine hydrochloride and urea concentrations were separately higher than 1.25 and 3.00 mol/L or separately lower than 1.00 and 3.00 mol/L, the refolding intermediates of egg white lysozymes were more easily aggregated to aggregate state or more easily renatured to native state, respectively. Under different initial total egg white lysozyme concentrations in urea solution, the refolding egg white lysozyme intermediates could be deduced to have a tendency to form a bimolecular intermediate aggregate, and this inference was further confirmed by their nonreducing SDS-PAGE and size exclusion chromatography. 相似文献
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YanWANG BoLinGONG XinDuGENG 《中国化学快报》2003,14(8):828-831
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. 相似文献
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The denatured state of a miniprotein BBA1 is studied under the native condition with the AMBER/Poisson-Boltzmann energy model and with the self-guided enhanced sampling technique. Forty independent trajectories are collected to sample the highly diversified denatured structures. Our simulation data show that the denatured BBA1 contains high percentage of native helix and native turn, but low percentage of native hairpin. Conditional population analysis indicates that the native helix formation and the native hairpin formation are not cooperative in the denatured state. Side-chain analysis shows that the native hydrophobic contacts are more preferred than the non-native hydrophobic contacts in the denatured BBA1. In contrast, the salt-bridge contacts are more or less nonspecific even if their populations are higher than those of hydrophobic contacts. Analysis of the trajectories shows that the native helix mostly initiates near the N terminus and propagates to the C terminus, and mostly forms from 3(10)-helix/turn to alpha helix. The same analysis shows that the native turn is important but not necessary in its formation in the denatured BBA1. In addition, the formations of the two strands in the native hairpin are rather asymmetric, demonstrating the likely influence of the protein environment. Energetic analysis shows that the native helix formation is largely driven by electrostatic interactions in denatured BBA1. Further, the native helix formation is associated with the breakup of non-native salt-bridge contacts and the accumulation of native salt-bridge contacts. However, the native hydrophobic contacts only show a small increase upon the native helix formation while the non-native hydrophobic contacts stay essentially the same, different from the evolution of hydrophobic contacts observed in an isolated helix folding. 相似文献
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Some new information on the conformation of xanthan in aqueous solutions is given. A single helical chain conformation characterizes xanthan in the native state. When heated over the critical temperature for conformational change ( helixŕ coil), the xanthan is denatured and renatured when it is cooled down in a localy double helical structure. A galactomannan was also characterized and its persistence length was obtained (Lp ≈︁ 90A°). Then mixtures of the galactomannan and xanthan were investigated to propose a mechanism for the specific gelation. From the results of microcalorimetry and circular dichroism, it is concluded that a complex is formed between one disordered xanthan chain and one galactomannan chain and that an ordered conformation is stabilized at temperatures lower than 25°C when the galactomannan has a M/G ratio of ≈︁ 3. This temperature corresponds to the sol-gel transition. This is the first time that a structure of the crosslink points is demonstrated. 相似文献
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In our previous work we characterized the Ca binding properties of BLA by analysis of Ca-induced renaturation in the presence
of urea. In this study we focused on the renaturing capacity of CaCl2 in the absence of urea and analysed the apparent thermodynamic and kinetic properties of renatured BLA by DSC, CD and dynamic
light scattering (DLS) measurements. Ca-free protein did not return fully to the native state even after extensive dialysis
against high concentrations of calcium. Thus Ca removal provokes changes in protein structure that can not be reversed completely
even by addition of an excess of calcium. However, activity studies performed simultaneously with the DSC experiments showed
a significant return of enzymatic activity despite the failure of complete return of native stability. This conclusion is
in excellent agreement with findings of Machius et al. who suggested on the basis of their X-ray studies that it is not possible to remove CaI and CaII without introducing significant
structural changes. It is important to note that all unfolding reactions of metal-free, partially renatured and native protein
were found to be irreversible. Therefore the DSC transition curves were fitted using irreversible transition models. It turned
out that the apparent heat capacity profiles of partially renatured BLA could be well represented by superposition of two
irreversible processes each following the two-state irreversible model. 相似文献
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Many prokaryotic and eukaryotic proteins are modified by post‐translational conjugation to short‐chain poly[(R)‐3‐hydroxybutyrate] (cPHB). The relative lability of ester bonds raises the concern that the cPHB may be substantially degraded by chemical hydrolysis during protein purification, thus increasing the difficulty of its detection and measurement. Here, we compare rates of acid‐ and base‐catalyzed hydrolysis of cPHB conjugated to native and denatured proteins at room temperature. E. coli cytoplasmic proteins, native or denatured by addition of guanidium hydrochloride, were treated with aqueous solutions of H2SO4 or NaOH at concentrations ranging from 0.1–2.0n . The loss of cPHB was measured as a function of time by a chemical assay. We find that cPHB conjugated to native proteins is surprisingly resistant to both acid‐ and base‐catalyzed hydrolysis, whereas cPHB conjugated to denatured proteins is proficiently degraded at rates proportional to acid or base concentration. The results suggest that cPHB occupies a highly protective environment within native proteins. 相似文献