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1.
蛋白质中二硫键的定位及其质谱分析   总被引:2,自引:0,他引:2  
二硫键是一种常见的蛋白质翻译后修饰,对稳定蛋白质的空间结构,保持及调节其生物活性等都有着非常重要的作用。因此,确定二硫键在蛋白质中的位置是全面了解含二硫键蛋白化学结构的重要方面。在众多实验方法中,现代质谱技术因其操作简单、快速、灵敏等优点而成为分析二硫键的重要手段。本文介绍了目前主要的定位二硫键的方法,以及质谱在二硫键定位分析中的应用与进展。  相似文献   

2.
二硫键是一种与多肽及蛋白质结构和功能密切相关的化学键. 当多肽中存在多个半胱氨酸时, 形成的二硫键可能会存在多种配对方式. 快速且精准地定位多肽中多对二硫键对研究多肽的结构与功能间的关系十分重要. 本文开发了一种基于化学裂解和生物质谱的新方法, 对利那洛肽中3对二硫键进行了精准定位. 通过解析裂解后特异肽段的二级质谱图, 确定利那洛肽中3对二硫键的配对方式分别为Cys1-Cys6, Cys2-Cys10和Cys5-Cys13. 该方法为二硫键的定位研究提供了新思路.  相似文献   

3.
二硫键是维持蛋白质活性构象的最重要因素之一,不正确的二硫键结构常导致蛋白质活性明显降低,甚至完全失活.早期测定二硫键主要采用"酶切-分离纯化-测序"法,过程繁琐,样品需求大.生物质谱为二硫键的测定提供了全新的策略.本文以重组人肝细胞生成素(rhHPO)--这一由我国独立发现和表达成功的、治疗严重肝病的潜在新药--为研究对象,探索了生物质谱进行二硫键定位的方法.  相似文献   

4.
本文用紫外差光谱、紫外二阶导数光谱和圆二色光谱,对二硫键完整和经还原并氨酰羧甲基化(以下简称还原)的核糖核酸酶A(RNase A),牛血清白蛋白(BSA)和溶菌酶在6 mol/L盐酸胍中变性后的结构进行了比较。尽管在圆二色光谱中,二硫键完整和还原的变性蛋白都看不到α-螺旋和β-折叠等有序二级结构的存在,但是,与二硫键还原的变性蛋白相比,含有完整天然二硫键的变性蛋白在芳香族氨基酸残基侧链的暴露程度上明显较低。这些结果说明含有完整天然二硫键的蛋白在6mol/L盐酸胍中变性后可能仍然保留有一定程度的有序空间结构。  相似文献   

5.
以一步法合成了巯丙基功能化的介孔二氧化硅微球,微球的粒径在130~150nm左右,平均孔径为2.79nm.以所合成的微球为载体,在其内、外表面修饰二硫键后,在二硫键的另一端接枝了异硫氰酸荧光素(FITC),并以FITC为标记物研究了二硫键在谷胱甘肽溶液中的断键情况.实验结果表明,微球上的二硫键具有在磷酸盐缓冲溶液(PBS,pH=7.4)中稳定存在,而在一定浓度的谷胱甘肽溶液中却能发生断键反应.以高分辨透射电镜(HR-TEM)、N2吸附-脱附实验表征了介孔二氧化硅微球的表面及孔道情况,用Zeta电位分析、元素分析、热失重分析(TGA)等手段表征了微球上二硫键的接枝情况,用荧光光度计(FLS)及紫外-可见分光光度计(UV-Vis)研究了谷胱甘肽对二硫键的断键情况的影响.  相似文献   

6.
新型含二硫键的环肽的合成   总被引:4,自引:0,他引:4  
黄小毅  王涛  夏传琴  余孝其  谢如刚 《有机化学》2004,24(12):1629-1632
本实验设计从Boc-Glu-OH,Boc-Asp-OH的两个羧基出发,最后通过I2氧化两个半胱氨酸的巯基生成二硫键,得到一类新型含二硫键的环肽,该环肽通过1H NMR,MS等确定了其结构.  相似文献   

7.
利用Fourier红外光谱对二硫键完整和还原的核糖核酸酶A(RNase A),牛血清白蛋白(BSA)和溶菌酶在6mol/L盐酸胍中变性后的结构进行了比较。这三种蛋白质,在二硫键完整和还原状态下变性后的Fourier红外酰胺Ⅰ带光谱都表现了不同程度的差异,但是三种还原蛋白质在完全变性后的光谱却非常相似。说明含有完整天然二硫键的蛋白在6mol/L盐酸胍中变性后仍然有相当程度的残留有序结构,而二硫键断开的蛋白质完全变性后在结构上却是相似的。  相似文献   

8.
氧化型肌酸激酶的研究   总被引:3,自引:0,他引:3  
利用NR/R双向对角线SDS-PAGE作者首次将过去被认为均一的兔肌肌酸激酶分离为含有链内二硫键的和不含任何二硫键的两种肌酸激酶(前者称为“氧化型肌酸激酶”,后者称为“还原型肌酸激酶”)。它们具有相同的化学组成,相同的亚基分子量,相同的等电点及几乎相同的催化活性,因此用一般常用的分离纯化手段很难将它们分离开来。进一步的研究表明,氧化型的肌酸激酶也含有一对表面巯基,并对酶的活性同样是必需的。氧化型肌酸激酶分子中,每一个亚基含有一个链内二硫键,在没有变性的条件下,不能被DTT还原,而还原型肌酸激酶通过某些氧化剂氧化可以形成这个二硫键,因此作者认为该二硫键可能是由内埋较浅的一对巯基氧化而成的。  相似文献   

9.
含巯基/二硫键聚合物生物材料具有多种良好的性能,作为药物、基因等的释放载体在生物医学领域具有广泛的应用前景。随着基因工程和组织工程的发展,含巯基/二硫键聚合物生物材料的可生物降解性得到高度重视,而怎样改善其降解性能成为限制其应用的关键因素。由于二硫键在细胞外环境里保持稳定,在细胞溶质的还原环境中容易发生断裂,因此在制备新型基因、药物等释放载体上,二硫键充当了重要的角色,它的引入为聚合物生物材料的生物降解性能的设计与改善提供了一条重要的途径。本综述重点以聚合物水凝胶、聚合物微胶束、囊泡等为例,从巯基/烯的光聚合反应、Michael加成反应、氧化还原反应的角度,介绍了巯基/烯在聚合物中形成二硫键的不同途径的研究进展,并详细论述了基因载体、蛋白质载体、小分子药物载体三种还原敏感型材料的制备、表面修饰和改性的进展情况,进一步强调含巯基/二硫键聚合物生物材料的研究在生物医学领域应用的重要性。  相似文献   

10.
多肽合成中几种形成二硫键方法的介绍   总被引:6,自引:0,他引:6  
综述了多肽合成中几种形成二硫键的方法。介绍了经典的空气氧化法、铁氰化钾氧化法和碘氧化法及近年来发展起来的二甲亚砜氧化法、三氟乙酸铊氧化法和氯硅烷-亚砜氧化法等方法的特点,同时还介绍了形成两对或多对二硫键的策略。  相似文献   

11.
Disulfide bond‐containing peptides are useful molecular scaffolds with diagnostic and therapeutic applications due to their good biological activity and good target selectivity, but their utility is sometimes limited by the lability of the disulfide moiety under reducing conditions and in the presence of disulfide bond isomerase. The development of disulfide surrogates with improved redox stability has been an area of ongoing research; and one possible strategy is based on a diaminodiacid (DADA) moiety, which can be used to synthesize the disulfide bond replacement peptides with precise structures and enhanced stability through automated solid‐phase peptide synthesis (SPPS). This review summarizes recent developments in the DADA‐based SPPS of peptide disulfide surrogates. Some representative applications and structural studies on the DADA‐based disulfide surrogates are described.  相似文献   

12.
Disulfide bonds have frequently been incorporated into synthetic materials to promote sensitivity of the systems towards different redox environments. Although many strategies have been developed to rationally tune the stability of disulfide linkers, methods to tune their responsiveness towards different redox environments remain elusive. In this work we have developed and explored a disulfide linker bearing two independent disulfide bonds, referred to as a twin‐disulfide linker. We have demonstrated that the twin‐disulfide linker displays an ultrahigh stability at lower concentrations of reducing agent or in weakly reducing environments without a significant compromise in the sensitivity of its response to highly reducing environments such as cytoplasm, a feature that is in remarkable contrast to the traditional single disulfide bonds. Such an extraordinary responsiveness arises from the cooperativity of the twin‐disulfide bonds, which should be of particular interest for applications such as controlled drug delivery and sensing, because relatively large differences in disulfide stability in different redox environments is desired in these applications.  相似文献   

13.
UV‐induced disulfide formation (UV‐DF) and disulfide reduction (UV‐DR) reactions for surface functionalization and dynamic photopatterning are presented. Both photochemical reactions allow for the spatially and temporally controlled, reversible transition between thiol‐ and disulfide‐functionalized surfaces. The dynamic photopatterning strategy was demonstrated by the UV‐induced attachment, exchange, and detachment on thiol‐modified substrates.  相似文献   

14.
Kinetic analyses were carried out for formation of disulfide crosslinkages between thiol groups on linear polymers, poly(acrylamide‐coN‐acrylcysteamine) (P‐SH). Disulfide crosslinkages were formed by auto‐oxidation of pendant thiol groups or through the thiol‐disulfide exchange reaction induced by addition of disulfide compounds gluthathione. In the auto‐oxidation reaction, the rate constant for disulfide formation highly depended on pH values of the reaction mixtures and the P‐SH concentrations. Gelation rate is too slow to enclose living cells into hydrogel under physiological pH 7.4. The hydrogel formation rate can be accelerated by addition of disulfides, such as oxidized glutathione. In the later case, oxygen in the reaction mixture is not consumed. The thiol‐disulfide exchange reaction is much more suitable for the cell encapsulation than the thiol auto‐oxidation reaction. These findings give a basis for enclosure of living cells in a hydrogel. © 2010 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2011  相似文献   

15.
The reaction of sulfur, carbon disulfide, and enamines at room temperature leads mainly or exclusively to 3H-1,2-dithiole-3-thiones; these are occasionally accompanied by 2H-1,3-dithiole-2-thiones, which can also be prepared by a modified procedure. Many enamines react with sulfur at room temperature to form thioamides. At about 50°C, enamines of acetophenone give 2-benzylidene-4-phenyl-2H-1,3-dithiol. The action of isothiocyanates and sulfur on enamines leads to the formation of thiazolidine-2-thiones. 2H-Thiopyran-2-thiones can be prepaAred from enamines or dienamines with carbon disulfide at room temperature. The reaction of ketimines (Schiff bases) with carbon disulfide and sulfur yields 3H-1,2-dithiole-3-thiones or isothiazoline-5-thiones. The reaction of alkynes with sulfur and carbon disulfide leads to 2H-1,3-dithiole-2-thiones. Nitriles containing active methylene groups react with carbon disulfide and sulfur to form 5-amino-3H-1,2-dithiole-3-thiones. When isothiocyanates are used instead of CS2, the reaction leads to δ4-4-amino-thiazoline-2-thiones.  相似文献   

16.
贺红举 《化学研究》2013,(6):622-624
采用毛细管气相色谱法(GC)测定了二乙基二硫的含量.结果表明,二乙基二硫含量在0.010.4g/L范围内具有良好的线性关系,平均回收率为99.91%;该方法简便、准确、重复性好,可方便地用于二乙基二硫含量的测定.  相似文献   

17.
Hydrolysis and oxidation of formamidine disulfide in acidic medium were investigated using high-performance liquid chromatography(HPLC) and mass spectrometry(MS) at 25 °C.By controlling the slow reaction rate and choosing appropriate mobile phase,HPLC provides the unique advantages over other methods(UV-Vis,chemical separation) in species tracking and kinetic study.In addition to thiourea and formamidine sulfinic acid,two unreported products were also detected in the hydrolysis reaction.Mass spectrometry measurement indicates these two products to be formamidine sulfenic acid and thiocyanogen with mass weights of 92.28 and 116.36,respectively.In the oxidation of formamidine disulfide by hydrogen peroxide,besides thiourea,formamidine sulfenic acid,formamidine sulfinic acid,thiocyanogen and urea,formamidine sulfonic acid and sulfate could be detected.The oxidation reaction was found to be first order in both formamidine disulfide and hydrogen peroxide.The rate constants of hydrolysis and oxidation reactions were determined in the pH range of 1.5-3.0.It was found both rate constants are increased with the increasing of pH.Experimental curves of different species can be effectively simulated via a mechanism scheme for formamidine disulfide oxidation,including hydrolysis equilibrium of formamidine disulfide and irreversible hydrolysis of formamidine sulfenic acid.  相似文献   

18.
Defining the plant disulfide proteome   总被引:8,自引:0,他引:8  
Lee K  Lee J  Kim Y  Bae D  Kang KY  Yoon SC  Lim D 《Electrophoresis》2004,25(3):532-541
There is considerable interest in redox regulation and new targets for thioredoxin and glutaredoxin are now being identified. It would be of great benefit to the field to have a list of all possible candidates for redox regulation--that is all disulfide proteins in plant. We developed a simple and very powerful method for identifying proteins with disulfide bonds in vivo. In this method, free thiols in proteins are fully blocked by alkylation, following which disulfide cysteines are converted to sulfhydryl groups by reduction. Finally, proteins with sulfhydryls are isolated by thiol affinity chromatography. Our method is unique in that membrane proteins as well as water-soluble proteins are examined for their disulfide nature. By applying this method to Arabidopsis thaliana we identified 65 putative disulfide proteins, including 20 that had not previously been demonstrated to be regulated by redox state. The newly identified, possibly redox-regulated proteins include: violaxanthin de-epoxidase, two oxygen-evolving enhancer proteins, carbonic anhydrase, photosystem I reaction center subunit N, photosystem I subunit III, S-adenosyl-L-methionine carboxyl methyltransferase, guanylate kinase, and bacterial mutT homolog. Possible functions of disulfide bonding in these proteins are discussed.  相似文献   

19.
Disulfide bridges are important structural elements in proteins. It is well-known that the position of the characteristic disulfide band at ca. 500 cm−1 in the vibrational spectra varies with the conformation around the disulfide unit. In our computational study on the model system diethyl disulfide, both wavenumber and normal mode composition are analyzed simultaneously as a function of conformation. For the disulfide band, a negative correlation between the calculated vibrational wavenumber and the SS stretching contribution is detected. This trend in the normal mode composition provides an explanation for experimentally observed wavenumber shifts of the disulfide band.  相似文献   

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