参数Z对疏水色谱中胍变蛋白质分子构象亦化的表征

用计量置换参数Ｚ对疏水色谱流动相中存在盐酸胍时蛋白质分子构象变化进行了表征。除溶菌酶外，其余４种蛋白质的Ｚ值随盐酸胍浓度的增大先增大，而后减小。蛋白质的Ｚ值随盐酸及脲浓度的增大，埋藏在蛋白质分子内     

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的构象变化的表征。     

液相色谱Z值对溶菌酶分子构象变化的定量表征

目前, 研究蛋白分子的构象变化用得最多的是光谱法, 如荧光光谱、圆二色谱和核磁共振谱等[1]. 由于蛋白质分子构象变化与其在色谱中的保留行为直接相关[2], 所以色谱法也成为研究蛋白质分子构象变化的一种新方法[3]. 此外, 计量置换理论中的Z值也已被成功地用于表征生物大分子的构象变化[4]. 用Z值研究蛋白质分子构象变化的优点是可以使用不纯的样品, 这是因为在测定Z值的过程中会与其它组分相分离. 使用Z值还会对蛋白分子构象变化进行定量表征[5,6]. 本文以溶菌酶(Lys)为目标蛋白, 用色谱法[反相液相色谱(RPLC)和弱阳离子交换色谱(WCX)]系统地研究了Lys在不同变性(胍变非还原、脲变非还原、胍变还原和脲变还原)环境中因疏水性及电荷分布的不同对Lys分子构象变化的影响, 并用Z值进行了定量表征.     

液相色谱中一个新的表征参数Z：Ⅳ.反相液相色谱中甲酸浓度与生物大…

反相高效液相色谱中蛋白质分子的Ｚ值可以为平衡状态下蛋白分子构象变化的表征，它与蛋白分子起始状态构象无关，在异丙醇－甲醇－水体系中蛋白的Ｘ值会因流动相中甲酸浓度的增大而增小，除蛋白分子构象变化因素外，蛋白Ｚ值的变化也与甲酸参与蛋白与置换剂分子间的讲师置换过程有关，在蛋白分子未完全失去分子立体结构时，其Ｚ值与分子的立方根成正比，当甲酸浓度足够大量，例如大于４０％（Ｖ／Ｖ）时，蛋白分子完全失去三维结构。     

脲、盐酸胍对碳氢链疏水作用的影响

本文测定了不同脲、盐酸胍浓度下,Triton X-100溶液的临界胶团浓度(cmc)和肌酸激酶分子在溶液中的暴露巯基数.通过计算脲、盐酸胍引起的Trito X-100胶团化过程中碳氢链的疏水能改变值,得到了评价脲、盐酸胍对碳氢链疏水作用影响的参数,盐酸胍降低碳氢链疏水能的能力是脲的3.5倍.实验结果还表明,盐酸胍引起肌酸激酶内坦巯基暴露的能力约为脲的3.2倍,这意味首在一定浓主工范围内脲、盐酸胍引起肌酸激酶变性的主要因素是它们降低了碳氢链的疏水作用.     

反相液相色谱中温度对蛋白分子构象变化的新表征

液相色谱中溶质计量置换保留模型的Ｚ值可用来对反相高效液相色谱中热变蛋白的分子构象进行表片。发现在异丙醇－甲酸４４％（Ｖ／Ｖ）－水体系为流动相对完全变性蛋白的Ｚ值随温度升高而降低。且Ｚ值与绝对温度例数（１／Ｔ）成正比，因此蛋白分子构象变化可用Ｚ对１／Ｔ作图所得两条直线的斜率差值来表征，差值愈大，蛋白分子构象变化愈甚。该两直线的交点即为蛋白分子构象的突变温度。与通常采用的１ｇＫ＇对１／Ｔ的作图相比，用     

盐酸胍对疏水色谱中蛋白质保留行为的影响

研究了变性剂酸胍对几种蛋白质在高效疏水色谱中保留行为的影响，用液相色谱中溶质的计量置换保留模型和测流动相表面张力及蛋白质紫外吸收光谱方法研究的结果表明：在低浓度范围内，盐酸胍主要以疏水作用影响蛋白质的保留；而在高浓度区域内，盐酸胍的存在则显著影响蛋白质分子的构象，使其保留行为发生变化。     

液相色谱中一个新的表征参数Z Ⅳ.反相液相色谱中甲酸浓度与生物大分子的构象变化

反相高效液相色谱中蛋白质分子的Z值可以作为平衡状态下蛋白分子构象变化的表征,它与蛋白分子起始状态构象无关。在异丙醇-甲酸-水体系中蛋白的Z值会因流动相中甲酸浓度的增大而减小。除蛋白分子构象变化因素外,蛋白Z值的变化也与甲酸参与蛋白与置换剂分子间的计量置换过程有关。在蛋白分子未完全失去分子立体结构时,其Z值与分子量的立方根成正比。当甲酸浓度足够大时,例如大于40％(V/V)时,蛋白分子完全失去三维结构。     

溶菌酶热变性的DSC研究

用差示扫描量热法研究了固体溶菌酶的热变性以及水溶液中不同变性剂与浓度对溶菌酶变性的影响. 结果表明, 溶剂水的存在及变性剂尿素和盐酸胍的加入使溶菌酶的变性温度降低, 变性焓减小; 同时, 在一定的浓度范围内, 溶菌酶的变性温度和变性焓随变性剂浓度的增大而降低. 盐酸胍的变性效果较尿素强, 这是由于盐酸胍与蛋白质分子间除了氢键作用外还存在着静电作用.     

液相色谱中的一个新的表征参数Z

研究了在反相高效液相色谱中的烷基醇－甲酸－水、烷基醇－三氟醋酸－水和烷基醇－磷酸盐－水三种流动相，９种生物大分子的分离体系中Ｚ的表征，发现当蛋白质完全变性时，Ｚ值与其分子量呈正比，而与置换剂分子大小呈反比。Ｚ值会因流动相中离子对试剂的存在而减小，且浓度愈大，减小程序愈甚。对于在异丙醇－水溶液中的不同种类离子对试剂而言，对蛋白构象变性影响的减小顺序为４４％甲酸＞０．０５ｍｏｌ／Ｌ磷酸盐缓冲液（ｐＨ＞     

脲和盐酸胍诱导过氧化氢酶去折叠的研究

用荧光相图法分别研究了脲和盐酸胍诱导牛肝过氧化氢酶去折叠的过程。当脲 浓度从0依次增大至0.50,4.5和8.0 mol/L时,过氧化氢酶从天然四聚体依次转变 为蓬松的四聚体、部分折叠的无活性二聚体和去折叠态,而当盐酸胍浓度从0依次 变化至0.65,2.5和6.0 mol/L时,过氧化氢酶则从天然四聚体集资转变为部分折叠 的激活二聚体、部分折叠的单体和去折叠态,这表明无论是用脲还是用盐酸胍作为 变性剂,该蛋白的变性过程都符合“四态模型”,但这两种变性剂诱导该蛋白去折 叠的途径和机制有较大差异。实验结果表明荧光相图法可以检测蛋白质去折叠的中 间态。用等温滴定量去热法研究了盐酸胍诱导过氧化氢酶去折叠过程的热力学, 25.0 ℃时低浓度盐酸胍诱导该蛋白从天然四聚体转变为部分折叠的激活二聚体的 本征摩尔构象变化焓、Gibbs自由能和熵分别为-69.2 kJ·mol~(-1),6.43 kJ· mol~(-1)和-254 J·K~(-1)·mol~(-1),据此推断盐酸胍通过熵效应和静电效应来 稳定和激活该二聚体。     

Spectral Studies on the Conformational Transitions of Bovine Insulin During Denaturant-induced Unfolding

Transitions among various molecule states and conformational changes of bovine insulin were investigated under different denaturing conditions by means of fluorescence phase diagrams,fluorescence quenching,1-anilinonaphthalene-8-sulfonate（ANS） binding assay and circular dichroism（CD） spectra.In both guanidine hydrochloride（GuHCl）-and urea-denatured procedures,the spatial structure of insulin molecules changed from ordered states to relative unordered ones with the increasing of denaturant concentration.The GuHCl-denatured process followed a four-state model,for there were two intermediates existed in 2.0 and 6.0 mol/L GuHC1,respectively.Intermediate I1 is more compact than the normal protein.And intermediate I2 has lost most of the secondary structures.When GuHCl concentration was above 6.0 mol/L,the fluorophores originally existed in the internal of insulin molecules would expose to the surface.However,the urea-denatured process followed a three-state model,only one intermediate existed in 2.5 mol/L urea.During the urea-denatured procedure,the fluorophores originally existed in theinternal of insulin molecules didn＇t expose to the surface.     

Viscosities and apparent molar volumes of some amino acids in water and in 6M guanidine hydrochloride at 25°C

Apparent molar volumes and viscosity B- and D- coefficients of amino acids glycine, valine, proline, serine and arginine have been determined in water and in aqueous 6M guanidine hydrochloride (GuHCl) solution at 25°C. Transfer volumes and transfer viscosity B-coefficients were evaluated for the amino acids studied in going from water to 6M GuHCl. These transfer properties which were all positive were interpreted in terms of strong interactions of GuHCl molecules with the charged centers of amino acid molecules. A comparison of results obtained in this work for GuHCl and those obtained from literature for urea has shown that GuHCl has stronger interactions than urea with amino acids. This finding explained the previous experimental observations on GuHCl being a stronger denaturing agent than urea for proteins.     

Identification of Folding Intermediates of Streblin,The Most Stable Serine Protease: Biophysical Analysis

Streblin, a serine proteinase from plant Streblus asper, has been used to investigate the conformational changes induced by pH, temperature, and chaotropes. The near/far UV circular dichroism activities under fluorescence emission spectroscopy and 8-aniline-1-naphthalene sulfonate (ANS) binding have been carried out to understand the unfolding of the protein in the presence of denaturants. Spectroscopic studies reveal that streblin belongs to the α+β class of proteins and exhibits stability towards chemical denaturants, guanidine hydrochloride (GuHCl). The pH-induced transition of this protein is noncooperative for transition phases between pH 0.5 and 2.5 (midpoint, 1.5) and pH 2.5 and 10.0 (midpoint, 6.5). At pH 1.0 or lower, the protein unfolds to form acid-unfolded state, and for pH 7.5 and above, protein turns into an alkaline denatured state characterized by the absence of ANS binding. At pH 2.0 (1 M GuHCl), streblin exists in a partially unfolded state with characteristics of a molten globule state. The protein is found to exhibit strong and predominant ANS binding. In total, six different intermediate states has been identified to show protein folding pathways.     

胍变及脲变α-淀粉酶的研究 Ⅰ.用高效疏水色谱法研究变性机理和复性效率

用疏水性强弱不同的两种色谱柱对７．０ｍｏｌ／Ｌ盐酸胍及８．０ｍｏｌ／Ｌ脲变性的α－淀粉酶变体和在疏水色谱介质表面上折叠的中间体进行了分离和复性。通过研究和比较发现,两者的变性机理和形成折叠中间体的个数以及复性效率均不相同。在用疏水性较弱的疏水色谱柱对脲变α－淀粉酶的折叠中间体进行分离时,得到了疏水性接近连续的、数目很多的中间体。用疏水性较强的疏水色谱柱对胍变α－淀粉酶进行复性的效果较好。还研究了柱温变化对其折叠、分离效果和复性效率的影响。     

Thermodynamic characterisation of RNAase a in the presence of urea and GuHCl

It is presented a study concerning the influence of guanidinium chloride (GuHCl) and urea on thermal stability of Bovine Pancreatic Ribonuclease A (RNAase A) at differentpH values. As expected, at increasing the denaturant concentration, the protein thermostability decreases. This is shown by a decrease of both the thermodynamic parameters, temperature and heat effect, characterising the denaturation process. In order to analyse the calorimetric curves we adopt a statistical thermodynamic approach. The individual one-dimensional DSC profiles have been expanded into another dimension by varying the GuHCl concentration, so that a heat capacity surface is defined for eachpH. By means of the ICARUS program, developed in our laboratory, we accomplish a two dimensional deconvolution of the experimental data linking the binding equilibrium to the denaturation process. This analysis provides a well founded and complete statistical thermodynamic characterisation of denaturation process of RNAase A in the presence of GuHCl and allows to calculate the thermodynamic parameters associated to the binding of denaturant molecule.