Direct and Cytochrome c Mediated Electrochemistry of Bilirubin Oxidase on Gold

Direct electron transfer (DET) of bilirubin oxidase from Myrothecium verrucaria (BOD) was established on promoter‐modified gold electrodes. The electrochemical behavior of the enzyme in solution was studied by means of cyclic voltammetry evaluating the biocatalytic reduction of dioxygen. The reaction of BOD at Au electrodes was shown to be efficient only at low pH. In addition, a novel interaction between BOD and cytochrome c (cyt.c) was found. It was shown that BOD efficiently accepts cyt.c as an electron donor in both cases when cyt.c is in solution and electrostatically adsorbed. The results suggest that cyt.c can play the role of a mediator facilitating electron transfer in a pH range where no DET could be observed between the enzyme and the electrode. For the interaction between cyt.c and BOD in solution the reaction kinetics has been studied electrochemically and spectrophotometrically.     

Electrochemical Investigation of Cytochrome c Immobilized onto Self‐Assembled Monolayer of Captopril

The electrochemical behavior of cytochrome c (cyt‐c) that was electrostatically immobilized onto a self‐assembled monolayer (SAM) of captopril (capt) on a gold electrode has been investigated. Cyclic voltammetry, scanning electrochemical microscopy (SECM) and electrochemical impedance spectroscopy were employed to evaluate the blocking property of the capt SAM. SECM was used to measure the bimolecular electron transfer (ET) kinetics (k_BI) between a solution‐based redox probe and the immobilized protein. In addition, the tunneling ET between the immobilized protein and the underlying gold electrode was calculated. A k_BI value of (5.0±0.6)×10⁸ mol^?1 cm³ s^?1 for the bimolecular ET and a standard tunneling rate constant (k⁰) of 46.4±0.2 s^?1 for the tunneling ET have been obtained.     

细胞色素c在单壁碳纳米管表面的固定、直接电子转移及电催化

应用吸附法将细胞色素c(Cytoc)固定在单壁碳纳米管(SWNT)表面.红外光谱(IR)显示被固定的Cytoc能保持原有的空间结构,没有发生变性.循环伏安测试表明,Cytoc在SWNT表面能发生稳定的直接电子转移,其i~E曲线上出现一对良好的、几乎对称的氧化还原峰.式量电位E0’基本不随扫速的增加而变化(在20 mV~120 mV/s的扫速范围内,E0’平均值为0.165±0.001V).实验同时给出,吸附在SWNT表面的Cytoc仍能保持其对H2O2电化学还原的生物电催化活性.     

细胞色素c在微-液/液界面上电化学行为的研究

本文采用电化学技术,研究了细胞色素c（Cyt c）在玻璃微米管尖端处形成的微-水/1,2-二氯乙烷（W/DCE）界面上的电化学行为.选用四丁基铵四苯硼（TBAT-PB）、四丁基铵四氯代苯硼（TBATPBCl）以及四丁基铵四氟代苯硼（TBATPBF）三种不同的有机相支持电解质来研究Cyt c在W/DCE界面上的反应.在电势窗较窄的含TBATPB体系中只能够观察到吸附过程;在电势窗较宽的含TBATPBCl和TBATPBF的体系中,可以同时观察到吸附与离子转移过程.当Cyt c浓度较低时,两种过程都可以观察到;当Cyt c浓度较高时,主要是吸附.文中对这些过程的机理进行了探讨.     

表面带负电荷氨基酸残基的突变对细胞色素b5与细胞色素c结合和识别的影响

利用核磁共振方法研究表面带不同负电荷氨基酸残基突变后的细胞色素b₅与细胞色素c的结合与识别.结果表明,静电作用在细胞色素b₅与细胞色素c的结合过程中有着重要的贡献,而且这些静电贡献在一定程度上具有累加性,E48的贡献略大于E44.同时还证明Browniandynamicssimulations优化出的Glu48-Lys13,Glu56-Lys₈₇,Asp60-Lys86和heme6-propionate-Tml₇₂(细胞色素b₅的残基排在前面)的结合方式在溶液中的确存在.细胞色素b₅突变体(E48,E56/A,D60/A)及[Cr(oxalate)₃]^3-对细胞色素c的表面结合竞争实验表明,细胞色素c表面结合区Site仍然同细胞色素b₅突变体(E48,E56/A,D60/A)有结合作用,只是结合强度上相对于野生细胞色素b₅同细胞色素c的结合有所降低.这表明除上述的Brownian dynamics simulations模型外,尚有其它如Salemme模型等的结合方式,这也揭示出细胞色素b₅和细胞色素c之间的结合是比较动态的.     

新型簇合物离子K+(KNO3)n和NO-3(KNO3)m

轴向配体在决定血红素蛋白结构和性能方面的作用引起人们的兴趣[1] .细胞色素 c是一个重要的电子传递蛋白 ,在天然状态下轴向配体 His 1 8和 Met80与血红素的 Fe原子配位 . UV光谱和 NMR谱显示氧化态细胞色素 c配位的 Met80在 p H大于 9或强外源配体存在时较易被取代[2 ] .人们对外源配体配位引发细胞色素 c的构象的研究得到一些重要的结构特征 [3,4 ] .但对整个蛋白溶液结构完整精确的描述和血红素电子结构的研究还很少 .此外 ,细胞色素 c在重折叠过程中形成组氨酸配位的中间体 ,而咪唑能捕获和阻断中间体的形成 .为此 ,本文对咪唑 -…     

Structure and Evolution of the “Atmungsferment” Cytochrome c Oxidase

Approximately 50 years ago, Otto Warburg received the Nobel Prize for his fundamental work on the “Atmungsferment”. But not until the end of the fifties was it possible to isolate this complicated membrane enzyme, which is necessary for respiration and energy production in most living organisms on earth. Since then, intensive research has been performed to elucidate the mechanism of reduction of oxygen to water and the coupled translocation of protons across the membrane which is involved in ATP synthesis. Until now the results have been unsatisfactory because the four catalytic heavy-metal redox centers are bound to proteins, the structures of which have begun to be studied only recently. In the course of this research, it was discovered that cytochrome c oxidase from bacteria contains only two or three, whereas the enzyme complex from animals contains thirteen different protein components. The present article analyzes the possible functions of the various protein subunits and, using the example of cytochrome c oxidase, shows that biochemical evolution proceeds in such a way as to increase regulatory capacity.     

基于磁球表面的抗原决定基印迹法用于选择性识别细胞色素c的研究

本工作合成了一种核壳型的抗原决定基磁性分子印迹聚合物,并用于选择性识别目标蛋白细胞色素c(Cytochrome c,Cyt c)。制备过程中先用溶剂热法合成Fe_3O_4磁性纳米粒子,然后加入Cyt c其C端的九肽作为模板,进行一段时间的预组装,最后加入多巴胺盐酸盐(DA)溶液,调节反应体系的p H使多巴胺聚合在磁球表面。洗脱掉模板后,即得到分子印迹聚合物。优化DA的用量使聚合物达到最佳的吸附效果。在最优条件下,制得的印迹聚合物对目标蛋白有较好的吸附选择性,并且有良好的重复利用性。此外,用抗原决定基做模板制得的聚合物的吸附容量和印迹因子明显优于用相应蛋白质做模板的情况。     

蚕丝蛋白固相萃取分离富集细胞色素c的研究

在特定实验条件下, 蚕丝蛋白对细胞色素c表现出选择性吸附. 以蚕丝蛋白微填充柱为载体, 在流动系统中建立了细胞色素c的分离富集方法, 以分光光度法在410 nm处检测分离富集过程. 在进样流速低于10 μL/s时, 2 mL样品溶液(pH=5.6的水溶液)中5 μg/mL的细胞色素c可被蚕丝蛋白微柱完全吸附, 而在洗脱流速低于15 μL/s时, 200 μL NaCl溶液(1.0 mol/L)可将吸附的细胞色素c完全洗脱, 分离富集系数为10. 用本方法测定细胞色素c的线性范围为1.0~10.0 μg/mL, 检出限为0.33 μg/mL, 精密度RSD为2.5%(5 μg/mL, n=9). 此外, 还采用本文方法对人全血中的蛋白质进行了分离富集, 并用SDS-聚丙烯酰胺凝胶电泳验证了分离后蛋白的纯度.     

细胞色素C电化学反应的静电作用模型

首次发现,简单的阴离子Ⅰ~-是细胞色素c电化学反应的良好促进剂。在碘离子修饰的金电极上能观察到细胞色素c的准可逆电化学反应。根据细胞色素c的结构特点和碘离子的吸附特性,提出了一个以静电作用为基础的细胞色素c的电化学反应的模型。     

Cytochrome c biosensor for determination of trace levels of cyanide and arsenic compounds

An electrochemical method based on a cytochrome c biosensor was developed, for the detection of selected arsenic and cyanide compounds. Boron doped diamond (BDD) electrode was used as a transducer, onto which cytochrome c was immobilised and used for direct determination of Prussian blue, potassium cyanide and arsenic trioxide. The sensitivity as calculated from cyclic voltammetry (CV) and square wave voltammetry (SWV), for each analyte in phosphate buffer (pH = 7) was found to be in the range of (1.1–4.5) × 10⁻⁸ A μM⁻¹ and the detection limits ranged from 4.3 to 9.1 μM. The biosensor is therefore able to measure significantly lower than current Environmental Protection Agency (EPA) and World Health Organisation (WHO) guidelines, for these types of analytes. The protein binding was monitored as a decrease in biosensor peak currents by SWV and as an increase in biosensor charge transfer resistance by electrochemical impedance spectroscopy (EIS). EIS provided evidence that the electrocatalytic advantage of BDD electrode was not lost upon immobilisation of cytochrome c. The interfacial kinetics of the biosensor was modelled as equivalent electrical circuit based on electrochemical impedance spectroscopy data. UV–vis spectroscopy was used to confirm the binding of the protein in solution by monitoring the intensity of the soret bands and the Q bands. FTIR was used to characterise the protein in the immobilised state and to confirm that the protein was not denatured upon binding to the pre-treated bare BDD electrode. SNFTIR of cyt c immobilised at platinum electrode, was used to study the effect of oxidation state on the surface bond vibrations. The spherical morphology of the immobilised protein, which is typical of native cytochrome c, was observed using scanning electron microscopy (SEM) and confirmed the immobilisation of the cytochrome c without denaturisation.     

适配体功能化多壁碳纳米管整体柱分离富集细胞色素c

本文结合核酸适配体的特异性吸附及碳纳米管比表面积大的特点,研制了核酸适配体功能化碳纳米管/聚合物复合整体柱(AMMC),并将其应用于细胞色素c(Cyt c)选择性分离。结果表明,AMMC具有良好的通透性和稳定性,可选择性富集Cyt c,富集量可达1.47 mg。结合顺序注射系统,AMMC成功用于猪心样品中Cyt c的选择性分离富集,在生物样品分析中具有良好的应用前景。     

An Electrochemical Study of Immobilized Ruthenocene in Aqueous Media

The voltammetric exploration of ruthenocene RuCp₂ in aqueous solution via abrasive modification on a basal plane pyrolytic graphite electrode is reported. It is found that ruthenocene undergoes a one‐electron electrochemically irreversible oxidation to ruthenocenium, which then rapidly dimerizes to form [RuCp₂] . Then, depending on the anion in solution, either disproportionates to [RuCp₂]²⁺ and subsequently decomposes to ruthenium oxide, or is anion stabilized. SEM images and cyclic voltammetry of the electrode surface indicates that in the presence of smaller anions the dimer is not stabilized, whereas for larger anions, such as sodium toluene sulfonate, the dimer is stabilized and decomposition is inhibited.     

自组装膜上细胞色素c的电化学石英晶体微天平实时表征和定量检测

采用电化学石英晶体微天平(EQCM)实时表征和定量检测细胞色素c(Cytc).在压电石英晶振表面上自组装巯基十一酸(MUA)单层膜,以盐酸1-乙基-3-(3-二甲基氨基丙基)碳二亚胺(EDC)和N-羟基琥珀酰亚胺(NHS)活化羧基,将Cytc共价固化到电极表面.EQCM实时监测了MUA的自组装和Cytc的固化过程,测定了二者在电极表面的覆盖度和Cytc的固化量.结果表明,Cytc在0.03～3.00μmol/L浓度范围内呈线性变化,检测限可达到1.19×10^-9mol/L.     

基于ITO电极的细胞色素c吸附层的直接电化学

采用未经修饰的铟锡氧化物(ITO)工作电极直接探测到了细胞色素c(Cytc)吸附层的氧化还原峰,并得出了Cytc的表面浓度,随着溶液浓度从2μmo·lL-1增大到10μmo·lL-1,Cytc的表面浓度相应地从0.35×10-12mo·lcm-2增大到1.53×10-12mo·lcm-2.实验获得的表面浓度倒数与溶液浓度倒数的准线性关系说明Cytc在ITO表面的吸附基本满足Langmuir等温吸附理论.对Cytc溶液的循环伏安测试结果表明参与电极反应的Cytc包括游离分子和吸附分子,前者的贡献大于后者,电极反应主要受扩散控制并呈准可逆过程.根据Nicholson方法估算得到反应物的标准异相速率常数的平均值为1.65×10-3cm·s-1.实验结果显示在室温下放置1h后Cytc吸附层电化学活性部分丧失,在80℃下放置1h后吸附层完全失活.失活的Cytc吸附层对铁氰化钾溶液在Au电极上的电极反应具有明显的阻碍作用.     

Assessment of Aqueous Extraction Methods on Extractable Organic Matter and Hydrophobic/Hydrophilic Fractions of Virgin Forest Soils

The assessment of water-extractable organic matter using an autoclave can provide useful information on physical, chemical, and biological changes within the soil. The present study used virgin forest soils from Chini Forest Reserve, Langkawi Island, and Kenyir Forest Reserve (Malaysia), extracted using different extraction methods. The dissolved organic carbon (DOC), total dissolved nitrogen (TDN), total dissolved phosphorus (TDP), and ammonium-nitrate content were higher in the autoclave treatments, up to 3.0, 1.3, 1.2, and 1.4 times more than by natural extraction (extracted for 24 h at room temperature). Overall, the highest extractable DOC, TDN, TDP, ammonium and nitrate could be seen under autoclaved conditions 121 °C 2×, up to 146.74 mg C/L, 8.97 mg N/L, 0.23 mg P/L, 5.43 mg N mg/L and 3.47 N mg/L, respectively. The soil extracts became slightly acidic with a higher temperature and longer duration. Similar trends were observed in the humic and nonhumic substances, where different types of soil extract treatments influenced the concentrations of the fractions. Different soil extraction methods can provide further details, thus widening the application of soil extracts, especially in microbes.     

大分子拥挤条件下光诱导细胞色素C还原的光谱研究

稀溶液中高铁细胞色素C（ferric cytochrome c,Fe（Ⅲ）-Cyt c）能被光照还原成亚铁细胞色素C（ferrous cytochrome c,Fe（Ⅱ）-Cyt c）,但这一研究忽略了生物体细胞的真实环境是高度拥挤的,因此采用紫外可见、同步荧光及圆二色谱光谱法研究了大分子拥挤环境中光诱导Cyt c的还原过程及其对外界环境的依赖情况. UV-Vis光谱和同步荧光光谱结果表明,拥挤试剂葡聚糖70（Dextran70）和聚蔗糖70（Ficoll70）的加入利于Cyt c光还原的进行,且Ficoll70存在时Cyt c光还原程度远高于Dextran70存在时;Dextran70和Ficoll70环境中光诱导Cyt c还原的最适温度分别为37 ℃和25 ℃,定波长280 nm光照射利于Cyt c还原;Cyt c的光还原程度随着Dextran70浓度增加而增大,Ficoll70环境中Cyt c的最适光还原浓度为100 g·L^-1;拥挤环境下Met,Trp,Tyr和Phe的存在对光诱导Cyt c的还原过程依Phe> Tyr> Trp> Met顺序有催化促进作用;CD光谱结果表明光照射稀溶液中Cyt c蛋白还原后,其α-螺旋含量降低,β-折叠含量升高,而光照射拥挤环境中Cyt c还原后蛋白的二级结构基本没变化. 结果表明拥挤试剂具有稳定蛋白质二级结构的作用,且影响光还原Cyt c的电子传递过程及效率.     

Interaction Between Cytochrome c and the Hapten 2,4-Dinitro-fluorobenzene by Electrospray Ionization Mass Spectrometry

Allergic contact dermatitis is a delayed hypersensitivity reaction, which results from skin exposure to low molecular weight chemicals such as haptens. To clarify the pathogenic mechanism, electrospray ionization mass spectrometry (ESI-MS) and hydrogen/deuterium (H/D) exchange, as well as UV spectroscopy, were applied to determine the interaction between the model protein cytochrome c (cyt c) and the hapten 2,4-dinitro-fluorobenzene (DNFB). The ESI-MS results demonstrate that the conformation of cyt c can change from native folded state into partially unfolded state with the increase of DNFB. The equilibrium state H/D exchange followed by ESI-MS further confirms the above results. UV spectroscopy indicates that the strongfield coordination between iron of heme (prosthetic group) and His18 or Met80 of cyt c is not obviously affected by the hapten.     

Structure of Cytochromec and Its Platinum-modified Derivatives by Fourier Transform Infrared Spectroscopy

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