脲和盐酸胍诱导溶菌酶去折叠的荧光相图法研究

用荧光相图法分别研究了脲和盐酸胍诱导卵清溶菌酶去抓叠的过程。当变性体 系中无还原剂2－巯基乙醇存在、脲浓度从0变化至4.0 mol/L（或盐酸胍浓度从0变 化至3.0 mol/L）时,溶菌酶从天然态转变为部分折叠中间态,当脲浓度从4.0 mol/L变化至8.0 mol/L（或盐酸胍浓度从3.0 mol/L变化至6.0 mol/L）时,溶菌 酶从中间态转变为去折叠态,此时该蛋白的变性过程符合“三态模型”。而当变性 体系中有该还原剂存在时,溶菌酶则由天然态直接转变为去折叠态,此时脲诱导该 蛋白去折叠的过程符合曲型的“二态模型”。实难结果表明荧光相图法可以检测蛋 白南去抓叠的中间态。     

荧光相图法研究脲诱导牛血清白蛋白的去折叠过程

作为从分子水平上阐明生命奥秘的中心课题之一,蛋白质的折叠问题一直受到生物化学、生物物理学和结构生物学等领域研究工作者的高度关注。在蛋白质的变性过程中,它们往往达不到完全去折叠,而是会形成不同的部分折叠中间态[1-3],这些部分折叠中间态在蛋白质折叠过程中起着重要作     

氧氟沙星与脲诱导牛血清白蛋白结合的机制研究

摘要 利用荧光光谱和紫外光谱研究了脲（Urea）对牛血清白蛋白（BSA）结构的影响以及氧氟沙星（Oflxacin）与脲诱导的BSA结合的情况。结果显示：Urea诱导BSA变性历经两步、三态过程,且伴随中间态的形成。随着Urea浓度的增大,BSA荧光强度降低并先蓝移（344 nm～336 nm）,后又红移至350 nm。Urea浓度在4.6～5.2 mol/L范围时,Oflx对BSA中间态有强的猝灭作用（KQ=10.46×104 L/mol, Urea 4.8 mol/L）和较大的结合常数（KA=3.8807×105 L/mol, Urea 4.8 mol/L）,但是结合位点数小（n=0.76, Urea 5.0 mol/L）,能量传递效率低（E=0.3002, Urea 4.8 mol/L）。同步荧光光谱显示：Urea诱导BSA去折叠时,Trp-212残基微环境并未发生改变,而Tyr的最大荧光发射峰蓝移,Oflx的加入诱导Trp-212的微环境更具疏水性。Oflx加速了Urea对BSA的失活作用。     

水稻谷胱甘肽磷脂氢过氧化物酶的表达、纯化及晶体生长条件初筛

将水稻PHGPx(OsPHGPx)的编码序列克隆到表达载体pGEX-6P-1上, 并转化为大肠杆菌进行表达. 通过GST亲和层析、离子交换层析和凝胶过滤层析, 制备了可用于晶体学研究的OsPHGPx, 其纯度超过95%, 具备明显的PHGPx活性. 质谱显示OsPHGPx的精确分子量为19642.5553, 与理论分子量基本一致. OsPHGPx在多个晶体生长条件下出现微晶. 三维结构同源建模显示 OsPHGPx的结构为硫氧还蛋白折叠形式.     

Monitoring folding/unfolding transitions of proteins by capillary zone electrophoresis: measurement of deltaG and its variation along the pH scale.

Free-solution capillary zone electrophoresis (CZE) can be used to monitor folding/unfolding transitions of proteins and to construct the classical sigmoidal transition curve describing this isomerization process. By performing a series of CZE experiments along the pH scale (here between pH 2.5 and 6.0) it is possible to measure the parameter [urea]1/2, which represents the concentration of urea at the midpoint of each transition curve, and its dependence from the local pH value. The [urea]1/2 parameter provides an idea of the stability of the protein at a given pH; in the case of cytochrome c, for example, it shows that at and below pH 2 the protein will spontaneously unfold even in the absence of a denaturant. The equation describing the sigmoidal folding/unfolding transition can be used for deriving the term deltaG degrees, which refers to the intrinsic difference in the Gibb's free energy between the (total or partial) denatured state and the reference state, taken usually as the native configuration of a protein. The variation of deltaG degrees between the two extremes of our measurements (pH 2.5 and 6.0) along the stated pH interval has been measured (and theoretically calculated) to be of the order of 7-10 kcal/mol and is here interpreted by assuming that at pH 2.5 and below there is an additionally stretching of the polypeptide coil due to coulombic repulsion, as the unfolded chain looses its zwitterionic character and assumes a pure (or very nearly so) cationic surface. Given the minute amounts of sample required, the fully automated state of the analysis, the rapidity and ease of operation, it is hoped that the CZE technique will become more and more popular in the years to come for monitoring folding/unfolding transitions of proteins.     

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。     

脲和盐酸胍诱导的卵清溶菌酶分子去折叠过程中各稳定构象态的分布和过渡

以内源荧光光谱和荧光相图法研究了脲和盐酸胍诱导的卵清溶菌酶分子的去折叠过程,结果表明,当变性液中脲和盐酸胍的浓度分别约为4.0和3.0 mol/L时,卵清溶菌酶分子的去折叠过程均存在一个折叠中间态,这两个去折叠过程均符合"三态模型".在卵清溶菌酶分子"三态"去折叠过程的基础上,通过变性剂分子和卵清溶菌酶分子之间的缔合一...     

Denaturing Effects of Urea and Guanidine Hydrochloride on Hyperthermophilic Esterase from Aeropyrum pernix K1

Introduction Esterases(EC3.1.1.x)representadiversegroup ofhydrolasescatalyzingthecleavageandformationof esterbonds.Theyarewidelydistributedinanimals,plantsandmicroorganisms.Becauseoftheiractivities inbothaqueousandnonaqueoussolventsystems,ester aseshavebe…     

Unfolding of Bovine Heart Cytochrome c Induced by Urea and Guanidine Hydrochloride

The unfolding of bovine heart cytochrome c induced by urea and guanidine hydrochloride was studied through their intrinsic fluorescence emission spectra, fluorescence phase diagrams, fluorescence quenching, size‐exclusion chromatographies, native polyacrylamide gel electrophoreses and deactivation profiles. The results showed that during their unfolding in urea and guanidine hydrochloride solutions, bovine heart cytochrome c molecules existed only in a unimolecular form and their bi‐molecular and/or poly‐molecular aggregates and aggregate precipitates were not formed all along. When the urea and guanidine hydrochloride concentrations in denaturation solution were separately about 6.0 and 3.0 mol/L, they could be completely deactivated and almost all of the tryptophan residues originally embedded in the interior of their molecules were exposed to the surface of their molecules. Different from the unfolding of the most often used horse heart cytochrome c, that of bovine heart cytochrome c induced by urea and guanidine hydrochloride was separately a completely co‐operative procedure and followed a two‐state model.     

Urea Unfolding of Opsin in Phospholipid Bicelles†

Opsin is the unstable apo‐protein of the light‐activated G protein‐coupled receptor rhodopsin. We investigated the stability of bovine opsin, solubilized in 1,2‐dimyristoyl‐sn‐glycero‐3‐phosphocholine (DMPC)/detergent bicelles, against urea‐induced unfolding. A single irreversible protein unfolding transition was observed from changes in intrinsic tryptophan fluorescence and far‐UV circular dichroism. This unfolding transition correlated with loss of protein activity. Changes in tertiary structure, as indicated by fluorescence measurements, were concomitant with an approximate 50% reduction in α‐helical content of opsin, indicating that global unfolding had been induced by urea. The urea concentration at the midpoint of unfolding was dependent on the lipid/detergent environment, occurring at approximately 1.2 m urea in DMPC/1,2‐dihexanoyl‐sn‐glycero‐3‐phosphocholine bicelles, while being significantly stabilized to approximately 3.5 m urea in DMPC/3‐[(cholamidopropyl)dimethylammonio]‐1‐propanesulfonate bicelles. These findings demonstrate that interactions with the surrounding lipids and detergent are highly influential in the unfolding of membrane protein structure. The urea/bicelle system offers the possibility for a more detailed understanding of the structural changes that take place upon irreversible unfolding of opsin.     

Pyrene excimer fluorescence of yeast alcohol dehydrogenase: a sensitive probe to investigate ligand binding and unfolding pathway of the enzyme

The cysteine residues of yeast alcohol dehydrogenase (YADH) were covalently modified by N-(1-pyrenyl) maleimide (PM). A maximum of 3.4 cysteines per YADH monomer could be modified by PM. The secondary structure of PM-YADH was found to be similar to that of the native YADH using far-UV circular dichroism. The covalent modification of YADH by PM inhibited the enzymatic activity indicating that the active site of the enzyme was altered. PM-YADH displayed maximum excimer fluorescence at an incorporation ratio of 2.6 mol of PM per monomeric subunit of YADH. Nucleotide adenine dinucleotide (NAD) divalent zinc and ethanol reduced the excimer fluorescence of PM-YADH indicating that these agents induce conformational changes in the enzyme. Guanidinium hydrochloride (GdnHCl)-induced unfolding of YADH was analyzed using tryptophan fluorescence, pyrene excimer fluorescence and enzymatic activity. The unfolding of YADH was found to occur in a stepwise manner. The loss of enzymatic activity preceded the global unfolding of the protein. Further, changes in tryptophan fluorescence with increasing GdnHCl suggested that YADH was completely unfolded by 2.5 M GdnHCl. Interestingly, residual structures of YADH were detected even in the presence of 5 M GdnHCl using the excimer fluorescence of PM-YADH.     

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.     

Distribution and Transition of Native and Completely Unfolded Conformations in the Unfolding of Bovine Heart Cytochrome c Induced by Urea and Guanidine Hydrochloride

The unfolding of bovine heart cytochrome c induced by urea and guanidine hydrochloride was first studied through intrinsic fluorescence emission spectra and fluorescence phase diagram and the results showed that both of them separately followed a two‐state model. As the simplest sample of the unfolding of protein molecules induced by denaturants, an equation was presented to show the effect of the denaturant concentrations in denaturation solution on the residual activity ratios of bovine heart cytochrome c in their two‐state unfolding. There are two characteristic unfolding parameters K and m in this equation. The former is the thermodynamic equilibrium constant of the unfolding of bovine heart cytochrome c induced by denaturants, the latter is the number of denaturant molecules associated with a bovine heart cytochrome c molecule during the unfolding procedure, and through them the distribution and transition of native and completely unfolded bovine heart cytochrome c conformations under different concentrations of urea or guanidine hydrochloride in denaturation solution can be accurately described.     

硫酸铜诱导烟草多酚氧化酶抗变性的研究

In order to investigate the interaction between exogenous Cu2+ and Polyphenoloxidase (PPO) from Nictiana Tobacum, the Cu2+-induced resistant-denaturation against guanidine hydrochloride(Gdn-HCl) was studied by the following enzymatic activity assay, fluorescence spectroscopy and circular dichroism (CD). The results show that the GdnHCl-induced unfolding is a two-state process with no detectable inter mediate state in the absence of exogenous Cu2+, while the GdnHCl-induced unfolding in the presence of 10.0 mmol/L exogenous Cu2+ follows a three-state transition with an intermediate state, which results from the fact that Cu2+ increases the structural stability of native PPO and its intermediate. In terms of enzymatic activities, 6 mol/L GdnHCl makes PPO lose 81.4% of its original activity after 5 min, inactivate completely after 30 min, while in the presence of 10.0 mmol/L exogenous Cu2+, only 39.4% and 75.1%, after 5 and 30 min respectively. According to the CD measurements, the relative average fractions of α-helix, anti-parallel2-sheet, 2-turn/parallel 2-sheet, aromatic residues and disulfide bond, and random coil/γ-turn are 1.1%, 3.8%, 3.3%, 7.5% and 84.3%, respectively, in the 6.0mol/L GdnHCl containing no CuSO4, but34.2%, 13.7%, 21.0%, 9.5% and 21.6%, respectively, in the same concentration of Gdn-HCl containing 10mmol/L CuSO4.     

盐酸胍诱导的淀粉液化芽孢杆菌α-淀粉酶去折叠过程的研究

分别用内源荧光光谱法、荧光相图法、荧光探针法、荧光猝灭法、蛋白质电泳法以及体积排阻色谱法研究了盐酸胍诱导的淀粉液化芽孢杆菌a-淀粉酶的去折叠过程. 内源荧光光谱和荧光相图结果表明, 当变性液中盐酸胍浓度约为1.0 mol/L时, 芽孢杆菌a-淀粉酶的去折叠过程中出现一个部分折叠中间体, 其去折叠过程符合“三态模型”; 荧光探针结果表明, 在溶液中盐酸胍浓度约为1.0 mol/L时, 中间态芽孢杆菌a-淀粉酶分子中存在着能够与探针分子1-苯胺 基-8-萘磺酸(ANS)结合的稳定的疏水区域; 荧光猝灭研究给出了不同程度变性的淀粉液化芽孢杆菌a-淀粉酶中的Trp的分布情况, 结果表明中间态芽孢杆菌a-淀粉酶分子中能够被碘化钾猝灭的位于分子表面的色氨酸残基数目达到最大的8个; 蛋白电泳和体积排阻色谱结果表明, 在盐酸胍诱导的芽孢杆菌a-淀粉酶分子的整个去折叠过程中, 不会以共价键或非共价键形式形成芽孢杆菌a-淀粉酶分子之间的集聚体或集聚体沉淀. 在此基础上, 对盐酸胍诱导的淀粉液化芽孢杆菌a-淀粉酶的去折叠过程进行了描述.     

具有GPX活力的抗体片段Fv的制备和性质

具有谷胱甘肽(GSH)结合部位的鼠抗体3H₄(IgM)经胃蛋白酶水解,产生分子量为25000的抗体Fv片段,用荧光滴定法测定了它与GSH的亲和常数K_a=1.17×10₇L/mol.该片段经苯甲基磺酰氟活化,再经NaHSe作用,其结合部位的丝氨酸被突变为谷胱甘肽过氧化物酶(GPX)的催化基团硒代半胱氨酸.突变后的Fv片段表现出很高的GPX活性,其活力高达2500U/μmol,称为Fv抗体酶.动力学分析表明,Fv酶的最适温度为55℃,最适pH为7.0,催化机制为乒乓机制,米氏常数分别为:K_m(GSH)=4.16×10^-3mol/L,K_m(H₂O₂)=2.8×10^-4mol/L.     

Effect of Native Disulfide Bonds on the Denaturation of Proteins in 6 mol/L Guanidinium Chloride——A Fourier Transformed Infrared Study

The unfolding of bovine serum albumin, lysozyme and ribonuclease A denatured in 6 mol/L GuHCl with their disulfide bridges intact and reduced have been compared by FTIR studies. The peak positions and heights in the deconvolved spectra of amide I bands of the above denatured proteins with native disulfide bonds show marked differences whereas those for the denatured proteins without disulfide linkages are closely similar. The above and other evidence suggest that denatured proteins with intact disulfides still have considerable ordered conformation even in 6 mol/L GuHCl.     

基于石墨烯基复合物和聚天青Ⅰ的HRP传感器检测有机过氧化物的研究

利用电化学还原氧化石墨烯(GO)的方法将石墨烯(rGO)固定在电极表面上,然后电沉积氢氧化铜和氢氧化镍复合物,构成石墨烯/金属氢氧化物复合纳米材料修饰的玻碳电极(GCE),并通过电聚合天青Ⅰ将辣根过氧化酶(HRP)固定在GCE/rGO/Cu(OH)_2-Ni(OH)_2表面,制得GCE/rGO/Cu(OH)_2-Ni(OH)_2/HRP-PA。对石墨烯/金属氢氧化物复合纳米材料进行了SEM和能谱表征。通过电化学阻抗法和循环伏安法对传感器的制备过程和电化学性能进行了研究,并进一步分别对过氧化氢叔丁基(BHP)及过氧化氢异丙苯(CHP)进行了分析测定。该传感器对BHP和CHP具有良好的检测效果,在2.0×10~(-5)~9.2×10~(-4)mol/L范围内响应电流与BHP浓度呈良好的线性关系,检出限为9.9×10~(-6)mol/L;在3.0×10~(-6)~1.0×10~(-4)mol/L范围内响应电流与CHP浓度呈良好的线性关系,检出限为6.9×10~(-7)mol/L。     

Study on the Fluorescence System of Oxolinic Acid‐Tb(III) and the Determination of Oxolinic Acid

Abstract

In Tris‐HCl buffer (pH=7.43), Tb³⁺ can react with oxolinic acid (OA) and form a 1:2 complex, which emits the intrinsic fluorescence of Tb³⁺. Based on this, a new fluorimetric method of determination of OA is developed. Under the optimum conditions, the enhanced fluorescence intensity of the system is proportional to the concentration of OA in the range of 1.5×10^?7～2.5×10^?5mol/L, and the detection limit is 5.5×10^?9 mol/L. Recovery test was also satisfactory. The experiments indicated that the luminescence mechanism was attributed to the M*–M luminescence.     

Analysis of serotonin in brain microdialysates using capillary electrophoresis and native laser-induced fluorescence detection

Serotonin or 5-hydroxytryptamine (5-HT) is a major neurotransmitter in the central nervous system. In this work, a method for analyzing 5-HT in brain microdialysis samples using a commercially available capillary electrophoresis (CE) system has been developed. A pH-mediated in-capillary preconcentration of samples was performed, and after separation by capillary zone electrophoresis, native fluorescence of 5-HT was detected by a 266 nm solid-state laser. The separation conditions for the analysis of 5-HT in standard solutions and microdialysates have been optimized, and this method has been validated on both pharmacological and analytical bases. Separation of 5-HT was performed using a 80 mmol/L citrate buffer, pH 2.5, containing 20 mmol/L hydroxypropyl-beta-cyclodextrin (HP-beta-CD) and +30 kV voltage. The detection limit was 2.5 x 10(-10) mol/L. This method allows the in vivo brain monitoring of 5-HT using a simple, accurate CE measurement in underivatized microdialysis samples.