心磷脂-细胞色素C体系CD谱的研究

 线粒体内膜中含有特异的心磷脂是细胞色素C氧化酶活性的必需脂。本工作测定了心磷脂脂质体对细胞色素C溶液园二色(CD)谱的影响,发现心磷脂可引起血色素铁的氧化,并使其轴向配位场强的对称性下降。提示心磷脂可能参与酶和底物之间的电子转移过程。     

心磷脂引起细胞色素C的氧化

心磷脂—细胞色素C—细胞色素C氧化酶体系吸收光谱的研究发现:心磷脂与氧化态细胞色素C结合产生230nm吸收峰;心磷脂与还原态细胞色素C作用,230nm吸收值上升,550nm吸收值下降,表明心磷脂可以引起细胞色素C的氧化。     

心磷脂对细胞色素C氧化酶质子泵功能的影响

 用胆酸盐透析法将猪心线粒体细胞色素C氧化酶重组在含心磷脂和二肉豆寇磷脂酰胆碱的脂质体上,以还原态细胞色素C作为酶反应底物,记录脂酶体囊泡外介质液pH的变化,pH下降幅度可以反映细胞色素C氧化酶质子泵的功能。 心磷脂含量不同的细胞色素C氧化酶脂酶体质子泵功能不同。心磷脂含量在10％—40％(w/w)范围内,随心磷脂含量增高,该酶质子泵功能增强;当心磷艏含量超过50％时,该酶质子泵功能却随心磷脂含量的增加表现出下降的趋势。阿霉素可以与心磷脂紧密结合,抑制细胞色素C氧化酶的质子泵功能。然而,少量阿霉素却能增强含70％心磷脂的脂酶体的质子泵功能。     

细胞色素C和含心磷脂的人工脂膜相互作用

 本文报告以芘为荧光探剂,研究细胞色素C和含心磷脂的人工脂膜的相互作用。1.由于芘和细胞色素C的血红素团之间的能量转移,细胞色素C与心磷脂结合引起芘的单体荧光发射峰(395nm)强度下降。这种淬灭效应受脂膜的相行为影响,在液晶相时淬灭效应小于凝胶相;2.氧化态细胞色素C与还原态相比,对心磷脂结合的视和度稍高;3.在以芘的激发二聚体荧光峰(475nm)强度与单体荧光峰强度之比做为脂膜流动性的指标,发现还原态细胞色素C与含心磷脂脂膜结合后引起流动性增加的效应高于氧化态的结合。     

运动性内源自由基对大鼠肝线粒体的影响

采用大鼠耗竭游泳作为动物运动模型,用戊巴比妥酸(TBA)法测定脂质过氧化水平,薄层色谱—定磷法测定心磷脂含量,细胞色素C还原法测定细胞色素C氧化酶活性。结果如下:耗竭运动时,肝线粒体脂质过氧化水平升高24％;心磷脂含量下降21％;细胞色素C氧化酶活性下降25％。上述结果表明:耗竭运动时,机体内源自由基的产生是运动损伤和整体疲劳的原因之一。     

细胞色素C引起心磷脂的还原

本工作采用FT-IR和NMR技术,研究了心磷脂(CL)与还原态细胞色素C作用后其脂肪酸链中双键数目的变化。发现伴随细胞色素C的氧化,CL双键被部分还原为单键,提示CL可能直接参与吸呼链的电子传递。     

心磷脂对细胞凋亡作用的研究进展

心磷脂(cardiolipin, CL)是线粒体特异的一类磷脂,它不仅可以维持线粒体的结构,还对电子传递链复合体有影响。在细胞凋亡时,心磷脂会发生一些重分布,比如线粒体内膜外小叶中心磷脂含量的增加及细胞表面心磷脂的出现。同时,凋亡刺激还可以加速心磷脂的代谢循环,使心磷脂在线粒体内的含量降低。在细胞凋亡发生时,心磷脂作为一个信号整合体,对细胞色素c (cytochrome c, CytC)、胱天蛋白酶-8 (caspase-8)和促凋亡蛋白Bid发挥着重要的协调作用。本文主要就心磷脂在细胞凋亡过程中的重要作用及其在癌症中的研究进展进行综述。     

磷脂对琥珀酸-细胞色素c还原酶的作用

琥珀酸细胞色素c还原酶除去90％以上的磷脂后活力丧失约95％。将去脂琥珀酸细胞色素c还原酶与磷脂和辅酶Q_2保温,可恢复其活性。活力恢复程度依赖于磷脂的组成。当磷脂酰胆碱(PC):心磷脂(CL):磷脂酰乙醇胺(PE)=2:2:1时活力恢复最高,比大豆磷脂的效果更为明显,单组分PC,PE或CL恢复活力较差。与酶蛋白紧密结合的CL和PC在活力可逆恢复中有重要作用。     

分子遗传学术语解释(续前)

Kleinschmidt方法(Kleinschmidt method) 电子显微镜样品制备的一种方法,它有三个主要步骤:(1)将核酸分子与细胞色素C混合;(2)使含有核酸的细胞色素C在水的表面上形成一个膜;(3)将该膜转移到电子显微镜的栅网上。     

二、核磁共振在膜研究中的应用(下)

3.细胞色素氧化酶细胞色素氧化酶位于线粒体内膜上,是呼吸链的最后一个成员。它催化如下反应,从而控制了电子由还原态的细胞色素C向氧化态细胞色素C的传递: 4 细胞色素C(还原态)+O_2+4H~+→ 4 细胞色素C(氧化态)+2H_2O 细胞色素氧化酶的分子量约156,000道尔顿,大小约为50A×50A×80A,形状象一颗牙齿。一般由七个亚基组成,不同来源的酶其亚基数目各不相同。最近对线粒体DNA的研究表明,其中三个最大的亚基在线粒体内部合成,而其余的亚基在细胞质内合成,然后再装配到一起。这是一个关系到膜组装机理的十     

Electrochemical analysis of microsomal cytochromes]

The electrochemical behaviour of the electron transfer proteins -- cytochromes b5, c and P-450 was studied by classical polarography and electrolysis with spectrophotometric monitoring. It was shown electrons are directly transferred from the electrode to oxidized cytochromes c and b5. Cytochrome P-450 is not reduced on the electrodes. However, the reduced inactivated from of cytochrome P-420 was detected at high potential values (-1,5 v).     

Interaction of Horse Heart Cytochrome c with Lipid Bilayer Membranes: Effects on Redox Potentials

Cyclic voltammetry has been used to study the effects of interactions between horse cytochrome c and solid-supported planar lipid membranes, comprised of either egg phosphatidylcholine (PC) or PC plus 20 mol.% cardiolipin (CL), on the redox potential and the electrochemical electron transfer rate between the protein and a semiconductor electrode. Experiments were performed over a wide range of cytochrome c concentrations (0–440 M) at low (20 mM) and medium (160 mM) ionic strengths. Three types of electrochemical behavior were observed, which varied as a function of the experimental conditions. At very low cytochrome c concentration (0.1 M), and under conditions where electrostatic forces dominated the protein–lipid membrane interaction (i.e., low ionic strength with membranes containing CL), a redox potential (265 mV) and an electrochemical electron transfer rate constant (0.09s ^–1)were obtained which compare well with those measured in other laboratories using a variety of different chemical modifications of the working electrode. Two other electrochemical signals (not reported with chemically modified electrodes) were also observed to occur at higher cytochrome c concentrations with this membrane system, as well as with two other systems (membranes containing CL under medium ionic strength conditions, and PC only at low ionic strength). These involved positive shifts of the cytochrome c redox potential (by 40 and 60 mV) and large decreases in the electron transfer rate (to 0.03 and 0.003 s^–1). The observations can be rationalized in terms of a structural model of the cytochrome c–membrane interaction, in which association involves both electrostatic and hydrophobic forces and results in varying degrees of insertion of the protein into the hydrophobic interior of the membrane.     

A reaction cycle for cytochrome c oxidase as an electron-transport-driven proton pump: The effect of electrochemical potential and slips

A detailed reaction cycle for cytochrome oxidase, an electron-transport-driven proton pump, has been presented earlier by our research group. The essential feature of the model is that both cytochrome a and Cu_A must be reduced in order to allow the transition from the electron and proton input state to the output state. The model is thus based on an indirect coupling between electron transfer and proton translocation.In this study, the same model is examined with respect to (1) intrinsic electron and proton leaks and (2) the effect of applying an electrochemical potential gradient on the pump incorporated in a membrane, both with respect to the electrical and chemical components.The model is successfully used to simulate various experimental results. Comparisons of experimental results with simulations based on the model support the existence of electron and proton leaks. The analysis of electron leaks suggests that electron gating is best achieved by varying the reorganization energy rather than by varying the reduction potentials.It is also suggested that both the electrical and chemical components of the electrochemical potential gradient are responsible for the regulation of the enzyme activity. Furthermore, an attempt is made to interpret the seemingly contradictory results obtained when measuring the pH dependence of the reduction potential of cytochrome a. In addition, the simulations support the assumption that protons are pumped by a mechanism that combines a membrane Bohr effect with the transition-state mechanism.Abbreviations R molar gas constant - k _B Boltzmann contant - F Faraday constant - e elementary charge - T absolute temperature - transmembrane electrochemical potential gradient - pH transmembrane pH difference - pH₁ and pH₂ inside (matrix) and outside (cytosol) pH, respectively - transmembrane electrical potential - E _m midpoint potential     

Highly sensitive and selective hydrogen peroxide biosensor based on hemoglobin immobilized at multiwalled carbon nanotubes-zinc oxide composite electrode

A highly sensitive and selective amperometric hydrogen peroxide (H(2)O(2)) biosensor based on immobilization of hemoglobin (Hb) at multiwalled carbon nanotubes-zinc oxide (MWCNT/ZnO) composite modified glassy carbon electrode (GCE) is reported. ZnO microsponges were electrochemically grown on MWCNT surface by the simple, cost-effective, green, electrochemical method at room temperature. The MWCNT/ZnO/Hb composite film showed a pair of well-defined, quasi-reversible redox peaks with a formal potential (E°') of -0.336V, characteristic features of heme redox couple of Hb. The electron transfer rate constant (k(s)) of immobilized Hb was 1.26s(-1). The developed biosensor showed a very fast response (>2s) toward H(2)O(2) with good sensitivity, wide linear range, and low detection limit of 0.02μM. The fabricated biosensor showed interesting features, including high selectivity, acceptable stability, good reproducibility, and repeatability along with excellent conductivity, facile electron mobility of MWCNT, and good biocompatibility of ZnO. The fabrication method of this biosensor is simple and effective for determination of H(2)O(2) in real samples with quick response, good sensitivity, high selectivity, and acceptable recovery.     

The properties of cytochrome f and P700 in chloroplasts suspended in fluid media at sub-zero temperatures.

1. The properties of P700 and cytochrome f have been studied at sub-zero temperatures in chloroplasts suspended in a medium containing 50% (v/v) ethylene glycol. The dark reduction of these components after a period of illumination provided information about the rate-limiting step of photosynthetic electron transport under these conditions. 2. The oxidation of P700 on illumination in the presence of methyl viologen and its subsequent dark reduction can be observed at -35 degrees C. This cycle of reactions could be repeated many times. The rate of reduction was increased by NH4Cl and reduction was inhibited by 3(3,4-dichlorophenyl)-1,1-dimethylurea. 3. The oxidation and reduction of cytochrome f could also be observed under similar conditions. The activation energies for the reduction of cytochrome f and P700 are similar (about 75 kJ mol-1) and the reduction of cytochrome f is also inhibited by dichlorophenyldimethylurea and stimulated by NH4Cl. 4. The reduction of both cytochrome f and P700 seemed to follow first-order kinetics, but the t1/2 for the redcution of the cytochrome was at least three times that for the reduction of P700 at the same temperature. It was concluded that the results were only compatible with a model in which the main pathway of electrons from plastoquinone to P700 involved cytochrome f if the equilibrium constant between the cytochrome and P700 was very much less than that expected from their redox potentials.     

Spectroelectrochemistry of cytochrome P450cam

The spectroelectrochemistry of camphor-bound cytochrome P450cam (P450cam) using gold electrodes is described. The electrodes were modified with either 4,4(')-dithiodipyridin or sodium dithionite. Electrolysis of P450cam was carried out when the enzyme was in solution, while at the same time UV-visible absorption spectra were recorded. Reversible oxidation and reduction could be observed with both 4,4(')-dithiodipyridin and dithionite modified electrodes. A formal potential (E(0')) of -373mV vs Ag/AgCl 1M KCl was determined. The spectra of P450cam complexed with either carbon monoxide or metyrapone, both being inhibitors of P450 catalysis, clearly indicated that the protein retained its native state in the electrochemical cell during electrolysis.     

Cardiolipin-depleted bovine heart cytochrome c oxidase: binding stoichiometry and affinity for cardiolipin derivatives

Detergent-solubilized bovine heart cytochrome c oxidase requires 2 mol of tightly bound cardiolipin (CL) per mole of monomeric complex for functional activity. Four lines of evidence support this conclusion: (1) Phospholipid depletion shows that two tightly bound CL's must remain associated with cytochrome c oxidase in order to maintain full electron transport activity. (2) Removal of the two tightly bound CL's correlates with decreased activity that is restored by reassociation of 2 mol of exogenous CL. (3) CL-depleted cytochrome c oxidase has two high-affinity binding sites for 2-[14C]acetylcardiolipin (AcCL), Kd,app less than 0.1 microM, that are not present in enzyme containing endogenous CL. An additional 2-3 lower affinity AcCL binding sites, Kd,app = 4 microM, are present in the CL-depleted complex, but these sites are also present in enzyme containing endogenous CL. (4) CL, monolysocardiolipin (MLCL), and dilysocardiolipin (DLCL) compete for AcCL binding with approximately the same relative affinities as those measured by the restoration of electron transport activity (MLCL competes much better than DLCL). However, MLCL and DLCL are only 60% and 15% as effective as CL in restoring maximum activity when they are bound to the high-affinity sites. The binding specificity of CL, MLCL, DLCL, and some of their acylated derivatives indicates that the apolar tails are most important for binding, not the polar head group. The presence or absence of hydroxyl groups in CL, MLCL, or DLCL also has little effect upon binding affinities. Binding specificity clearly favors CL since phosphatidylglycerol, phosphatidic acid, and phosphatidylcholine each have very low affinity for the CL binding sites (Kd,app greater than 20 microM). We, therefore, conclude that restoration of activity to CL-depleted cytochrome c oxidase is highly specific and requires the reassociation of CL, or structurally similar compounds, with two high-affinity binding sites.     

Redox properties of mesophilic and hyperthermophilic rubredoxins as a function of pressure and temperature.

The formal equilibrium reduction potentials of recombinant electron transport protein, rubredoxin (MW = 7500 Da), from both the mesophilic Clostridium pasteurianum (Topt = 37 degrees C) and hyperthermophilic Pyrococcus furiosus (Topt = 95 degrees C) were recorded as a function of pressure and temperature. Measurements were made utilizing a specially designed stainless steel electrochemical cell that easily maintains pressures between 1 and 600 atm and a temperature-controlled cell that maintains temperatures between 4 and 100 degrees C. The reduction potential of P. furiosus rubredoxin was determined to be 31 mV at 25 degrees C and 1 atm, -93 mV at 95 degrees C and 1 atm, and 44 mV at 25 degrees C and 400 atm. Thus, the reduction potential of P. furiosus rubredoxin obtained under standard conditions is likely to be dramatically different from the reduction potential obtained under its normal operating conditions. Thermodynamic parameters associated with electron transfer were determined for both rubredoxins (for C. pasteurianum, DeltaV degrees = -27 mL/mol, DeltaS degrees = -36 cal K-1 mol-1, and DeltaH degrees = -10 kcal/mol, and for P. furiosus, DeltaV degrees = -31 mL/mol, DeltaS degrees = -41 cal K-1 mol-1, and DeltaH degrees = -13 kcal/mol) from its pressure- and temperature-reduction potential profiles. The thermodynamic parameters for electron transfer (DeltaV degrees, DeltaS degrees, and DeltaH degrees ) for both proteins were very similar, which is not surprising considering their structural similarities and sequence homology. Despite the fact that these two proteins exhibit dramatic differences in thermostability, it appears that structural changes that confer dramatic differences in thermostability do not significantly alter electron transfer reactivity. The experimental changes in reduction potential as a function of pressure and temperature were simulated using a continuum dielectric electrostatic model (DELPHI). A reasonable estimate of the protein dielectric constant (epsilonprotein) of 6 for both rubredoxins was determined from these simulations. A discussion is presented regarding the analysis of electrostatic interaction energies of biomolecules through pressure- and temperature-controlled electrochemical studies.     

Light-induced proton transport by chloroplasts suspended in fluid media at sub-zero temperatures: kinetics and stoichiometry.

1. Chloroplasts suspended in a medium containing ethanediol and water (1 : 1, v/v) at -16 degrees C show light-induced proton uptake and subsequent dark efflux. Proton uptake in continuous light showed biphasic kinetics. 2. A 1 ms flash caused a single turnover of the photochemical centres at -16 degrees C. Under the same conditions 3H+ were taken up from the external medium in the presence of methyl viologen as electron acceptor. 3. The flash-induced proton uptake was exponential and monophasic with t1/2 = 3 s. The flash-induced proton release into the thylakoid interior was biphasic, with half-times of less than 0.1 s and 3 s. The fast phase represented approximately 30% of the total release and may be correlated with the oxidation of water. 4. The half-time of reduction of cytochrome f in the dark following illumination in the presence of 2 mM NH4Cl (2.5 s) is similar to the half-time of the slow phase of proton release, suggesting a correlation between the kinetics of cytochrome f reduction and plastoquinol oxidation.     

Reagentless biosensor for hydrogen peroxide based on immobilization of protein in zirconia nanoparticles enhanced grafted collagen matrix

A novel matrix, zirconia nanoparticles enhanced grafted collagen (ZrO2-grafted collagen) hybrid composite, for immobilization of protein and biosensing was developed. The scanning electron microscopy, UV-vis and Fourier transform infrared spectra, and electrochemical measurements showed that the matrix was well biocompatible and could retain the bioactivity of immobilized protein to a large extent. The direct electron transfer of the immobilized myoglobin (Mb) exhibited a couple of stable and well-defined redox peaks with the formal potential of -336 mV (versus SCE) in 0.1M pH 7.0 PBS. This matrix could accelerate the electron transfer between Mb and the electrode with a surface-controlled process and an electron transfer rate constant of 3.58+/-0.35s-1 at 10-500 mVs-1. The Mb immobilized in the matrix showed a high thermal stability up to 70 degrees C and an electrocatalytic activity to the reduction of hydrogen peroxide (H2O2) without the help of an electron mediator. The linear response range of the biosensor to H2O2 concentration was from 1.0 to 85.0 microM with the limit of detection of 0.63 microM at a signal-to-noise ratio of 3sigma. The biosensor exhibited high sensitivity, acceptable stability and reproducibility. This work opened a way for the further study on the direct electron transfer and biosensing application of the immobilized protein in collagen-related matrices.