溶液中变性剂浓度对蛋白溶菌酶复性的影响

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.     

非还原脲变性蛋白溶菌酶稀释复性过程中集聚现象的研究

用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳、阴极聚丙烯酰胺凝胶电泳和高效凝胶排阻色谱法, 研究了非还原脲变性蛋白溶菌酶在稀释复性过程中的集聚现象. 实验发现, 在整个稀释复性过程中, 没有蛋白溶菌酶集聚体沉淀产生. 当最终复性液中蛋白溶菌酶浓度小于4.0 mg/mL时, 复性过程中不会形成蛋白溶菌酶分子集聚体; 当最终复性液中蛋白溶菌酶浓度介于4.0～8.0 mg/mL时, 复性过程中会形成由非共价相互作用所引起的蛋白溶菌酶二分子和三分子集聚体; 而当最终复性液中蛋白溶菌酶浓度大于8.0 mg/mL时, 复性过程中除了会形成二分子和三分子蛋白溶菌酶集聚体外, 还会形成四分子蛋白溶菌酶集聚体. 在此基础上, 结合文献, 对非还原脲变性蛋白溶菌酶的稀释复性过程进行了描述.     

用蛋白电泳和高效凝胶排阻色谱法分析还原脲变性蛋白溶菌酶稀释复性过程的集聚体

本文利用蛋白电泳和高效凝胶排阻层析法分析了还原脲变性蛋白溶菌酶稀释复性过程中的集聚体。当用复性液稀释复性还原脲变性蛋白溶菌酶时,会迅速产生可观量的沉淀。沉淀和上清液的不连续十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)和高效凝胶排阻层析分析结果表明,还原脲变性蛋白溶菌酶在稀释复性过程中除了能够复性成天然态蛋白溶菌酶分子外,还会形成可溶的蛋白溶菌酶分子二聚体和三聚体,二聚体和三聚体主要是靠分子间二硫键的错配连接而成的;可溶的蛋白溶菌酶分子二聚体之间通过非共价键相互作用而形成集聚体沉淀,而可溶的三聚体溶菌酶分子则仍处于复性液上清液中。     

盐酸胍诱导的变性卵清溶菌酶分子重折叠过程及其各稳定构象态的分布和过渡

采用变性和非变性电泳、 高效凝胶排阻色谱、 内源荧光发射光谱和荧光相图以及生物活性测定等方法, 研究了盐酸胍诱导的变性卵清溶菌酶分子的重折叠过程及此过程中卵清溶菌酶分子各稳定构象态的分布和过渡. 结果表明, 当复性液中盐酸胍浓度分别约为5.0和2.4 mol/L时, 变性卵清溶菌酶分子的重折叠过程各存在1个稳定折叠中间态, 重折叠过程符合"四态模型". 在卵清溶菌酶分子四态重折叠过程基础上, 结合盐酸胍与卵清溶菌酶分子之间的缔合-解离平衡, 给出了一个定量描述变性剂诱导的蛋白质分子复性过程中蛋白质分子复性率随溶液中变性剂浓度变化的方程. 该方程包含2个特征折叠参数, 一个是蛋白质分子从一个稳定构象态过渡到另一个稳定构象态的热力学过渡平衡常数k; 另一个是在此过程中平均每个蛋白质分子所结合的变性剂分子数目m. 通过这2个特征折叠参数能够定量描述盐酸胍诱导的变性卵清溶菌酶完全去折叠态、 折叠中间态和天然态分子随复性液中盐酸胍浓度变化的分布和过渡情况.     

Refolding of detergent‐denatured lysozyme using β‐cyclodextrin‐assisted ion exchange chromatography

Chromatography‐based protein refolding is widely used. Detergent is increasingly used for protein solubilization from inclusion bodies. Therefore, it is necessary to develop a refolding method for detergent‐denatured/solubilized proteins based on liquid chromatography. In the present work, sarkosyl‐denatured/dithiothreitol‐reduced lysozyme was used as a model, and a refolding method based on ion exchange chromatography, assisted by β‐cyclodextrin, was developed for refolding detergent‐denatured proteins. Many factors affecting the refolding, such as concentration of urea, concentration of β‐cyclodextrin, pH and flow rate of mobile phases, were investigated to optimize the refolding conditions for sarkosyl‐denatured lysozymes. The results showed that the sarkosyl‐denatured lysozyme could be successfully refolded using β‐cyclodextrin‐assisted ion exchange chromatography. Copyright © 2012 John Wiley & Sons, Ltd.     

Detection of Chitinolytic Enzymes in Ipomoea Batatas Leaf Extract by Activity Staining after Gel Electrophoresis

A non‐denatured SDS‐PAGE followed by in‐gel activity staining using embedded glycol chitin as a substrate was used to identify the proteins with chitinolyitc activities from sweet potato leaf extract. At least two chitinase activity zones can be clearly identified on the gel at positions with estimated molecular weights of 54.1～55.6 kDa and 39.6 kDa. Furthermore, our data also indicate that the activity of the larger one can withstand the standard SDS‐PAGE sample preparation. Both of these chitinases, however, are different from that of the previously identified chitinase in sweet potato leaves, which has a molecular weight of 16 kDa. By using an embedded substrate, our method has superior sensitivity in detecting chitinases with higher molecular weights. It is a simple, affordable way and may aid in the future discovery of new chitinases.     

Effect of Temperature on Self‐Interaction of Human‐Like Collagen

Effects of temperature on self‐interaction of human‐like collagen (HLC) were investigated by hydrophobic interaction chromatography, calorimetric measurement, and sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE) analysis. Results show that three types of interaction roles may exist between HLC molecules at 3–50°C, which were divided into three narrower temperature ranges. In temperature range from 3–22°C, hydrogen bonding plays a key role in the formation of a gelatinous aggregate. In the range of 22–38°C, hydrophobic bonds accompanied by hydrogen bonds are involved in the formation compact aggregates. When temperature is above 38°C the hydrophobic effect formed in the HLC monomer results in the loss of its ability to self‐interact.     

Refolding and purification of rhNTA protein by chromatography

RhNTA protein is a new thrombolytic agent which has potential medicinal and commercial value. Protein refolding is a bottleneck for large‐scale production of valuable proteins expressed as inclusion bodies in Escherichia coli. The denatured rhNTA protein was refolded by an improved size‐exclusion chromatography refolding process achieved by combining an increasing arginine gradient and a decreasing urea gradient (two gradients) with a size‐exclusion chromatography refolding system. The refolding of denatured rhNTA protein showed that this method could significantly increase the activity recovery of protein at high protein concentration. The activity recovery of 37% was obtained from the initial rhNTA protein concentration up to 20 mg/mL. After refolding by two‐gradient size‐exclusion chromatography refolding processes, the refolded rhNTA was purified by ion‐exchange and affinity chromatography. The purified rhNTA protein showed one band in SDS‐PAGE and the specific activity of purified rhNTA protein was 110,000 U/mg. Copyright © 2008 John Wiley & Sons, Ltd.     

Polyacrylamide Gel Electrophoresis of Insulin

Abstract

Four kinds of polyacrylamide gel electrophoresis (PAGE) were applied to insulin and peroxynitrite‐treated insulin. The Native‐PAGE had a better resolution than sodium dodecyl sulfate (SDS)‐PAGE, SDS‐urea‐PAGE, and even Tricine‐SDS‐PAGE. Reduction and nonreduction of insulin and peroxynitrite‐treated insulin in Native‐PAGE showed that four tyrosine residues in insulin molecular could be nitrated by peroxynitrite and that alkylation with iodoacetamide was better than no alkylation and alkylation with iodoacetic acid, which would introduce negative charges to the peptides. The method of Native‐PAGE was suitable to analysis of insulin and its analogs, even other peptides of low molecular weight.     

Purification,Characterization and Physico‐Chemical Properties of Three Galactose‐Specific Lectins from Pumpkin (Cucurbita Maxima) Seed Kernels

Three galactose‐specific lectins have been isolated and purified from the extract of pumpkin seed kernels by gel filtration on Sephadex G‐75 with 100% ammonium sulfate saturated crude extract, followed by ion exchange chromatography on DEAE‐cellulose and affinity chromatography on Sepharose 4B. All three lectins were found to be homogeneous as judged by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS‐PAGE). The molecular weights of lectins, PSL‐1, PSL‐2 and PSL‐3, as estimated by gel filtration on Sephadex G‐75 were 40,000, 42,000 and 46,000, and by SDS‐PAGE about 39,500, 41,000 and 45,000, respectively. The lectins, PSL‐1, PSL‐2 & PSL‐3 were dimer in nature and the molecular weights of their subunits were about 25,500 and 14,000; 26,000 and 15,000; and 30,500 and 15,000, respectively. The lectins are glycoproteins with a neutral sugar content of 3‐5%. The lectins agglutinated rat red blood cells and the hemagglutination was inhibited specifically by galactose and galactose‐containing saccharides. The lectins exhibited a strong cytotoxic effect in a brine shrimp lethality bioassay.     

脲变性牛碳酸酐酶B的稀释复性过程及其集聚作用研究

采用非变性聚丙烯酰胺凝胶电泳、十二烷基硫酸钠-聚丙烯酰胺凝胶电泳、高效凝胶排阻色谱以及激光光散射光谱研究了脲变性牛碳酸酐酶B的稀释复性过程及其集聚作用。在脲变性牛碳酸酐酶B的稀释复性过程中,当最终复性液中脲浓度大于2.0mol/L时,牛碳酸酐酶B在复性液中以单分子和二分子集聚体形式存在;当最终复性液中脲浓度小于2.0mol/L大于1.0mol/L时,牛碳酸酐酶B在复性液中以单分子、二分子集聚体和少量多分子集聚体形式存在;而当最终复性液中脲浓度小于等于1.0mol/L时,脲变性牛碳酸酐酶B复性时会形成均匀透明的上清和不透明的沉淀,牛碳酸酐酶B在上清和沉淀中达到动态解离平衡,且在两相中都以单分子、二分子集聚体和少量多分子集聚体形式存在。溶液中二分子和多分子牛碳酸酐酶B集聚体是通过牛碳酸酐酶B分子之间的疏水和静电相互作用力而形成的,当溶液中这些成分达到一定浓度并且溶液中脲的浓度小于某一个值时,它们之间会通过非共价形式形成沉淀。     

Controlled pore glass chromatography of protein-sodium dodecyl sulphate complexes.

The inclusion of urea has been found to eliminate adsorption of protein-sodium dodecyl sulphate (SDS) complexes to controlled pore glass. Using buffer containing 6 M urea, 0.5% SDS and glass with pore diameter 12.3 nm, it is possible to determine protein molecular weights in the range 3500-12,000. Results with glass of larger pore diameter (25.5 nm) are similar to those reported in the absence of urea in the molecular-weight range 12,000-140,000. Controlled pore glass chromatography also permits the study of the relative importance of conformation free of charge effects for those proteins which deviate from the normal calibration curve for SDS-polyacrylamide gels.     

Continuous matrix assisted refolding of alpha-lactalbumin by ion exchange chromatography with recycling of aggregates combined with ultradiafiltration

Continuous matrix assisted refolding (MAR) can be achieved on a solid support by using a continuous chromatographic system. Recycling the aggregate fraction, simultaneously formed during a refolding reaction, can further increase the refolding yield. Due to the nature of this reaction, aggregates are the main reason for a refolding yield below stoichiometric conversion. A preparative continuous annular chromatographic system (P-CAC) equipped with an ion exchange resin was used to continuously refold the model protein alpha-lactalbumin. For this purpose, this protein was denatured, reduced and adsorbed on the ion exchange resin. Elution was performed with or without redox reagents in the buffer system permitting fast formation of the native disulfide bonds. In the case redox reagents were present, the protein refolds then during its residence time on the matrix. However, aggregate formation is also increased and refolding yields are lower. Tightly bound aggregates were removed from the column by 2M guanidinium hydrochloride. In order to increase the system yield, this aggregate fraction was recycled after lowering the conductivity by ultradiafiltration and adjustment of the protein concentration by dilution. For on-column refolding, recycling of aggregates at a recycling rate of 0.17 increased the system yield from 25% to 30%. An algorithm was developed to show interdependencies of the single influencing parameters. The operability of the system was demonstrated but limitations due to instability of the P-CAC, especially inhomogeneous flow and peak wobbling, have to be considered.     

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.     

温敏型聚合物PNIPAAm辅助的溶菌酶体外复性

合成了 3种具有不同分子量的温敏型聚合物聚 (N 异丙基丙烯酰胺 ) (PNIPAAm) ,测定了其分子量分布以及相应的低临界溶解温度 (LCST) .在溶菌酶复性溶液中加入PNIPAAm可促进溶菌酶复性 ,其中采用中等分子量M—PNIPAAm(Mw 为 2 2× 10 4 g mol)时溶菌酶的复性效果最佳 ,并采用荧光发射光谱技术表征了PMIPAAm分子结构对于溶菌酶结构的影响 .系统考察了采用M—PNIPAAm时 ,复性液中尿素浓度、蛋白质浓度和温度等条件对溶菌酶复性效果影响 .结果显示尿素与M—PNIPAAm对于溶菌酶复性呈现协同效应 ,复性操作温度不仅同溶菌酶自身特性有关 ,而且还受到M—PNIPAAm自身性质变化的影响 .研究结果表明温敏型高聚物在高浓度蛋白质的大规模体外复性中具有很好的应用前景     

Refolding of reduced/denatured bovine pancreatic insulin with ion-exchange chromatography coupled with MALDI-TOF MS

The refolding of the reduced/denatured insulin from bovine pancreas as the model protein was investigated with weak anion exchange chromatography(WAX) coupled with MALDI-TOF MS.The results indicated that the disulfide bonds almost cannot be formed correctly with the common mobile phase by WAX.However,with the urea gradient elution and in the presence of GSSG/ Cyst as the ratio 1:6 in the mobile phase employed,the disulfide exchange of reduced/denatured insulin can be accelerated resulting in forming the ...     

Structure and dynamics of surfactant and hydrocarbon aggregates on graphite: a molecular dynamics simulation study

We have examined the structure and dynamics of sodium dodecyl sulfate (SDS) and dodecane (C12) molecular aggregates at varying surface coverages on the basal plane of graphite via classical molecular dynamics simulations. Our results suggest that graphite-hydrocarbon chain interactions favor specific molecular orientations at the single-molecule level via alignment of the tail along the crystallographic directions. This orientational bias is reduced greatly upon increasing the surface coverage for both molecules due to intermolecular interactions, leading to very weak bias at intermediate surface coverages. Interestingly, for complete monolayers, we find a re-emergent orientational bias. Furthermore, by comparing the SDS behavior with C12, we demonstrate that the charged head group plays a key role in the aggregate structures: SDS molecules display a tendency to form linear file-like aggregates while C12 forms tightly bound planar ones. The observed orientational bias for SDS molecules is in agreement with experimental observations of hemimicelle orientation and provides support for the belief that an initial oriented layer governs the orientation of hemimicellar aggregates.     

Specific glutamic acid residues in targeted proteins induce exaggerated retardations in Phos‐tag SDS‐PAGE migration

We describe two unique proteins, Escherichia coli ClpX and human histone H2A, that show extremely retarded migrations relative to their molecular weights in Phos‐tag SDS‐PAGE, despite being nonphosphorylated. Although ClpX separated into multiple migration bands in Phos‐tag gels, the separation was not due to phosphorylation. The N‐terminal 47–61 region of ClpX was responsible for producing multiple phosphorylation‐independent structural variants, even under denaturing conditions, and some of these variants were detected as highly up‐shifted bands. By systematic Ala‐scanning mutation analysis in the N‐47–61 region, we concluded that the Glu‐51 or Glu‐54 residue was responsible for the appearance of exaggerated mobility‐shifting bands. Histone H2A showed a much slower migration in Phos‐tag gels in comparison with other major histones having similar molecular weights, and we found that the Glu‐62 or Glu‐65 residue caused the retarded migration. In addition, Phos‐tag SDS‐PAGE permitted us to detect a shift in the mobility of the phosphorylated form of histone H2A from that of the nonphosphorylated one. This is the first report showing that exaggerated retardation in the migration of a certain protein in Phos‐tag SDS‐PAGE is induced by interactions between the Phos‐tag molecule and the carboxylate group of a specific Glu residue on the target.     

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。     

The Effects of a Strong Disaggregating Agent on Sec-Page of Aquatic and Soil Humic Matter

Abstract

Size-exclusion chromatographic (SEC) fractionation and electrophoretic separation of aquatic humic matter samples from a Finnish lake using Sephadex G-75 with 7 M urea solution as eluent and 10% polyacrylamide gel (PAGE) with urea and sodium dodecyl sulphate solution (SDS), respectively, were performed and compared to similar analyses performed on a Russian chernozem soil humic acid sample and Nordic reference fulvic and humic acid samples. The integrated whole of aquatic humic solutes and soil humic acids were found to exhibit similar SEC-PAGE behaviours. Humic matter was not excessively disaggregated by the 7 M urea and hence SEC-PAGE can with confidence be applied as a coarse, initial fractionation procedure or for certain predeterminations of the structural composition.