酶的光谱电化学研究（Ⅰ）：辣根过氧化物酶的电化学性质

利用薄层光谱电化学技术研究了辣根过氧化物酶(HRP)及其化合物的氧化还原过程。指出HRP可在固体电极上进行直接电子传递,该电极反应不是酶中二硫键的还原,而是血红素辅基中心金属离子的氧化态转变。测定了HRP(Fe~(3+)/Fe~(2+))电对的标准氧化还原电位和电化学动力学参数,讨论了HRP氧化性中间物的电化学性质。     

麦芽酸性磷酸酶的化学修饰及紫外光谱

本文研究化学修饰剂2,3-丁二酮(Butanedione)、β-巯基乙醇(Mercaptoethanol)、对硝基苯磺酰氟(4-Nitrobenzensulfonyl fluoride,NBSF)和焦碳酸二乙酯(Diethyl pyrocarhonate,DEPC)在不同浓度、不同时间条件下对麦芽酸性磷酸酶(ACPase,E.C.3.1.3.2)相互作用后的活力及紫外光谱的变化。结果表明:色氨酸和精氨酸可能是维系麦芽酸性磷酸酶催化功能的必需基团,酶分子中二硫键和组氨酸残基不参与该酶分子活性中心的构成。     

用X光电子能谱研究人体表皮中二硫键,巯基和黑色素的氧化机理

本文用XPS分析了人体表皮及其黑色素中硫、氮原子的化学状态;研究了表皮中二硫键、巯基的氧化机理;揭示了黑色素与过氧化氢反应后颜色从棕黑色变成黄棕色,是硫的价态从二价氧化成四价和六价的结果.     

毛细管区带电泳监测牛胰岛素的去折叠过程

用毛细管区带电泳监测了二硫苏糖醇作用下二硫键还原引起的牛胰岛素去折叠全过程,同时用离线的基质辅助激光解吸／电离飞行时间的质谱配合确证。从毛细管电泳谱图能直接观察胰岛素去折叠过程中发生的变化,获得蛋白质去折叠信息。结果表明,毛细管区带电泳作为监测蛋白质构象变化的一种有效手段,方法简便、快速、灵敏度高、样品消耗量少。     

马氏钳蝎哺乳动物神经毒素——Ⅱ.N-端氨基酸排列顺序

作者用DABITC/PITC双偶合微量液相手工顺序方法,测定了国产马氏钳蝎哺乳动物神经毒素Ⅱ。N-端部分氨基酸的排列顺序。 蝎毒Ⅱ肽链中S—S二硫桥键的还原,用硫代叔糖醇作为还原试剂。还原所生成的游离-SH基,用α-碘乙酸进行羧甲基化保护,反应混合物用Sephadex G-10凝胶柱层析方法分离。     

含双硫键的自修复交联聚氨酯弹性体的合成与性能

采用预聚法,以多官能度聚醚多元醇和异佛尔酮二异氰酸酯为原料,β-巯基乙醇为封端剂制备多官能度巯基封端聚氨酯低聚物,利用Na I/H2O2将巯基氧化为双硫键,获得含双硫键的交联型聚氨酯。通过FTIR研究了产物的化学结构及组成,采用拉伸测试和对比试验等测试方法对自修复条件、自修复效率以及双硫键和氢键在自修复过程中的作用进行了分析。结果表明,制得的含脂肪族双硫键的交联聚氨酯可以在室温下实现高效自修复,而且不需要额外的催化剂和外界刺激条件。用刀片将试样切成两半后对接紧密在自然光线下放置48h后拉伸强度可恢复到原来的95%,断裂伸长率恢复到原来的100%。其中,聚氨酯的氨基甲酸酯键之间形成的氢键提供30%左右的自修复效率。     

基于聚(L-谷氨酸)树状分子的智能药物释放系统研究

以天然氨基酸L-谷氨酸为原料,通过收敛法合成了聚(L-谷氨酸)树状分子,通过半胱氨酸将抗肿瘤药物甲氨蝶呤( MTX)键合到聚(L-谷氨酸)树状分子上,构建氧化还原敏感的药物传输系统.用核磁(1H～NMR)等对载体以及载药粒子进行了表征.体外释放研究发现,载药粒子具有良好的氧化还原响应性,在不同浓度的还原剂二硫苏糖醇(D...     

牛胰岛素去折叠过程的高效液相色谱法分析

建立了反相高效液相色谱法动态监测牛胰岛素在二硫苏糖醇存在下去折叠的过程。牛胰岛素在二硫苏糖醇作用下,首先发生构象变化,形成稳定的中间体后进一步断裂分子间的二硫键,形成Ａ链和Ｂ链。去折叠过程通过基质辅助激光解吸附质谱得到了鉴定。     

多变鱼腥藻藻胆蛋白共价复合物的合成及其分子内能量传递

利用双功能基偶联剂3-(2-吡啶联巯基)丙酸N-羟基琥珀酰亚胺酯(SPDP)合成了两个藻胆蛋白复合物,藻红蓝蛋白-变藻蓝蛋白复合物PEC-APC和藻红蓝蛋白-藻蓝蛋白复合物PEC-PC.利用吸收光谱和荧光光谱证明了藻胆蛋白构型与构象在反应后得到保持。通过荧光光谱观察到能量传递现象。计算出复合物PEC-APC的分子内能量传递效率约为90%.复合物PEC-PC中藻红蓝蛋白PEC的荧光寿命比PEC本身的寿命大大缩短,证明存在分子内能量传递。二硫苏糖醇(DTT)还原二硫桥键后能量传递被阻断。这进一步证明复合物合成成功及分子内能量传递。     

异丙基溴化镁催化脱氧反应

格氏试剂通常以碳负离子形式对亲电试剂进行加成。但是有催化量的Cp_2TiCl_2存在时,具有β质子的烷基格氏试剂往往能发生还原反应或氢镁化反应。例如,使环氧键、碳氧双键、硅氧键、硅卤键、碳卤键、碳氮双键、碳氮三键、碳碳双键、碳碳三键等还原或氢镁化的反应已有报道。但是,对硫氧双键、氮氧键、磷氧双键和砷氧双键的     

Electrocatalytic cyclization of dithiothreitol on a chemically modified electrode by analogy with protein action

Electrocatalytic oxidative cyclization of dithiothreitol (DTT(SH)2) to a disulfide product was demonstrated on a Nafion/lead-ruthenium oxide pyrochlore chemically modified electrode (NPyCME). The process at the NPyCME with DTT(SH)2 is similar to the behaviour of protein in a disulfide linkage, which can be demonstrated by product analysis using HPLC coupled with UV spectroscopy. A possible electrocatalytic mechanism for DTT(SH)2 oxidation to dihydroxydithiane [i.e. cyclized DTT(S-S)] on the NPyCME was proposed in terms of Py-Ru(IV)/Py-Ru(VI) redox active sites. This physical aspect was further utilized for high precision analytical assays using flow injection analysis (FIA), with a linearity up to 50 microM and a detection limit (S/N = 3) of 28 nM (8.64 pg) in a 20 microL sample loop. This is the most sensitive method ever reported for DTT(SH)2 detection assays. The interference from dissolved oxygen, disulfide and glucose is almost negligible. The present method offers an easy route for extension to redox-related protein studies.     

Reduction-sensitive liposomes from a multifunctional lipid conjugate and natural phospholipids: reduction and release kinetics and cellular uptake

The development of targeted and triggerable delivery systems is of high relevance for anticancer therapies. We report here on reduction-sensitive liposomes composed of a novel multifunctional lipidlike conjugate, containing a disulfide bond and a biotin moiety, and natural phospholipids. The incorporation of the disulfide conjugate into vesicles and the kinetics of their reduction were studied using dansyl-labeled conjugate 1 in using the dansyl fluorescence environmental sensitivity and the Fo?rster resonance energy transfer from dansyl to rhodamine-labeled phospholipids. Cleavage of the disulfide bridge (e.g., by tris(2-carboxyethyl)phosphine (TCEP), dithiothreitol (DTT), l-cysteine, or glutathione (GSH)) removed the hydrophilic headgroup of the conjugate and thus changed the membrane organization leading to the release of entrapped molecules. Upon nonspecific uptake of vesicles by macrophages, calcein release from reduction-sensitive liposomes consisting of the disulfide conjugate and phospholipids was more efficient than from reduction-insensitive liposomes composed only of phospholipids. The binding of streptavidin to the conjugates did not interfere with either the subsequent reduction of the disulfide bond of the conjugate or the release of entrapped molecules. Breast cancer cell line BT-474, overexpressing the HER2 receptor, showed a high uptake of the reduction-sensitive doxorubicin-loaded liposomes functionalized with the biotin-tagged anti-HER2 antibody. The release of the entrapped cargo inside the cells was observed, implying the potential of using our system for active targeting and delivery.     

Determination of glutathione disulfide levels in biological samples using thiol-disulfide exchanging agent, dithiothreitol

A reverse-phase HPLC method incorporating dithiothreitol (DTT) reduction for quantitative determination of oxidized glutathione (GSSG) in biological samples is described here. This method is based on our previous enzymatic reduction technique that uses N-1-(pyrenyl) maleimide (NPM) as a derivatizing agent. In our earlier method, glutathione disulfide (GSSG) was measured by first reducing it to GSH with glutathione reductase (GR) in the presence of NADPH. However, this is a very costly and time-consuming technique. The method described here employs a common and inexpensive thiol-disulfide exchanging agent, DTT, for reduction of GSSG to GSH, followed by derivatization with NPM. The calibration curves are linear over a concentration range of 25-1250 nm (r(2) > 0.995). The coefficients of variations for intra-run precision and inter-run precision range from 0.49 to 5.10% with an accuracy range of 1.78-6.15%. The percentage of relative recovery ranges from 97.3 to 103.2%. This new method provides a simple, efficient, and cost-effective way of determining glutathione disulfide levels with a 2.5 nm limit of detection per 5 microL injection volume.     

Molecular tensile machines: intrinsic acceleration of disulfide reduction by dithiothreitol

Significant tension on the order of 1 nN is self-generated along the backbone of bottlebrush macromolecules due to steric repulsion between densely grafted side chains. The intrinsic tension is amplified upon adsorption of bottlebrush molecules onto a substrate and increases with grafting density, side chain length, and strength of adhesion to the substrate. These molecules were employed as miniature tensile machines to study the effect of mechanical force on the kinetics of disulfide reduction by dithiothreitol (DTT). For this purpose, bottlebrush macromolecules containing a disulfide linker in the middle of the backbone were synthesized by atom transfer radical polymerization (ATRP). The scission reaction was monitored through molecular imaging by atomic force microscopy (AFM). The scission rate constant increases linearly with the concentration of DTT and exponentially with mechanical tension along the disulfide bond. Moreover, the rate constant at zero force is found to be significantly lower than the reduction rate constant in bulk solution, which suggests an acidic composition of the water surface with pH = 3.7. This work demonstrates the ability of branched macromolecules to accelerate chemical reactions at specific covalent bonds without applying an external force.     

Alkaline cleavage of covalent bonds in chicken insulin and bovine alpha-lactalbumin analyzed by matrix-assisted laser desorption/ionization-mass spectrometry

In the course of searching methods to extract proteins from Coomassie blue-stained polyacrylamide gels, we found proteins are extracted in relatively high recovery when the gel pieces are soaked in alkaline solutions. However, alkaline conditions are known to cause decomposition of proteins, especially peptide bond cleavage and disulfide degradation. We studied the effects of alkaline on two purified proteins, chicken insulin and bovine alpha-lactalbumin, both containing four disulfide bonds in their structure. The process of covalent bond cleavage was traced by analyzing the mass spectra of the proteins using matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS). When the proteins are kept at pH 13 in the presence of 0.1% dithithreitol (DTT), peptide bonds at the C-terminal side of asparaginyl residues are preferably cleaved producing succinimides, whereas cysteinyl residues are not decomposed. In the absence of DTT, the disulfide bonds of the proteins are decomposed by alkaline and the cleavage of the peptide bonds are less obvious, possibly because the conformation of the proteins are partially retained until the full decomposition of disulfide bonds. These results identified for the first time the cleavage sites of proteins under alkaline treatment and further suggested the general tendency of the reactions, both in the presence and absence of DTT.     

Role of thiol-disulfide exchange in insulin binding to its receptor.

The effects of reduction by DTT, oxidation by DTNB and treatment with NEM on the thiol contents and insulin binding to its receptor in mice liver membranes were studied. Reduction with DTT leads to a parallel increase in the thiol content and the specific binding of insulin to the membrane. Scatchard analysis of the results shows little change in the number of binding sites but a twofold increase of the binding constant. Washing the membrane with bound insulin by a DTT containing buffer results in a more marked increase in the release of bound insulin than washing with buffer alone, suggesting that part of the insulin is bound to its receptor by covalent disulfide linkages through a thiol-disulfide exchange reaction and reduction with DTT leads to a marked increase in this "disulfide-linked" insulin. Treatment with DTNB or NEM of the DTT-reduced membrane seems to reverse the effect of DTT reduction, although the reaction of the untreated membrane with DTNB or NEM had little or no effect on the specific binding of insulin. It is suggested that initially, part of the thiols responsible for the exchange reaction may not be available for reaction with DTNB and reduction with DTT generates further thiols leading to increased specific binding in general and increased insulin binding to the receptor through covalent disulfide linkages in particular.     

ROLE OF THIOL-DISULFIDE EXCHANGE IN INSULIN BINDING TO ITS RECEPTOR

The effects of reduction by DTT, oxidation by DTNB and treatment with NEM on the thiol contents and insulin binding to its receptor in mice liver membranes were studied. Reduction with DTT leads to a parallel increase in the thiol content and the speciflc binding of insulin to the membrane. Scatchard analysis of the results shows little change in the number of binding sites but a twofold increase of the binding constant. Washing the membrane with bound insulin by a DTT containing buffer results in a more marked increase in the release of bound insulin than washing with buffer alone, suggesting that part of the insulin is bound to its receptor by covalent disulfide linkages through a thIol-disulfide exchange reaction and reduction with DTT leads to a marked increase in this "disulfide-linked" insulin. Treatment with DTNB or NEM of the DTT-reduced membrane seems to reverse the effect of DTT reduction, although the reaction of the untreated membrane with DTNB or NEM had little or no effect on the specific     

Disulfide proteinoid micelles responsive to reduction

Abstract

Proteinoid composed of aspartic acid (Asp) and Leucine (Leu) (Prot(Asp-Leu)) and of Asp, Leu, and dithiopropionic acid (DTPA) (Prot(Asp-Leu-DTPA)) were prepared by melt-condensation method. ¹H NMR spectroscopy confirmed the successful synthesis of the proteinoids. The air/water interfacial tension of Prot(Asp-Leu-DTPA) solution increased when dithiothreitol (DTT, a reducing agent) was in the solution, possibly due to the breakdown of the disulfide bond of the proteinoid. On transmission electron micrograph, the proteinoid micelles were round and the diameter was less than 200?nm. Sulfur signal was found in the energy-dispersive X-ray spectrum of Prot(Asp-Leu-DTPA) micelle, indicating that the disulfide compound (i.e., DTPA) was successfully included in the proteinoid. As the pH value increased, the mean hydrodynamic diameter of the proteinoid micelle increased. The release degree of doxorubicin (DOX) loaded in Prot(Asp-Leu-DTPA) micelle was relatively low (3.5%–5.8%) and it was not affected by a reducing agent (i.e., DTT), possibly because of electrostatic attraction between DOX and the proteinoid. DTT had a significant effect on the release degree of amaranth (a negatively charged dye) loaded in Prot(Asp-Leu-DTPA) micelle. Prot(Asp-Leu-DTPA) has disulfide bonds so it can be broken into thiol proteinoids via DTT-caused reduction, giving rise to the micellar shell loosening and the promoted release.     

A potent, versatile disulfide-reducing agent from aspartic acid

Dithiothreitol (DTT) is the standard reagent for reducing disulfide bonds between and within biological molecules. At neutral pH, however, >99% of DTT thiol groups are protonated and thus unreactive. Herein, we report on (2S)-2-amino-1,4-dimercaptobutane (dithiobutylamine or DTBA), a dithiol that can be synthesized from l-aspartic acid in a few high-yielding steps that are amenable to a large-scale process. DTBA has thiol pK(a) values that are ~1 unit lower than those of DTT and forms a disulfide with a similar E°' value. DTBA reduces disulfide bonds in both small molecules and proteins faster than does DTT. The amino group of DTBA enables its isolation by cation-exchange and facilitates its conjugation. These attributes indicate that DTBA is a superior reagent for reducing disulfide bonds in aqueous solution.     

Controllable exploding microcapsules as drug carriers

Controllable exploding polyelectrolyte microcapsules were developed by layer-by-layer assembly of poly(allylamine hydrochloride) (PAH) and poly(sodium 4-styrenesulfonate) (PSS) on a dextran microgel core containing a cleavable disulfide bond fabricated via click chemistry. The microcapsules can explode upon the injection of DTT with an explosive release of the drug.