曲酸与胰蛋白酶相互作用的光谱学研究

研究了曲酸对胰蛋白酶活性的影响,以及曲酸处理后胰蛋白酶紫外光谱、荧光淬灭光谱、同步荧光光谱和三维荧光光谱的变化．结果显示曲酸对胰蛋白酶具有激活作用;二者相互作用后,曲酸可以使胰蛋白酶的紫外吸收强度增强,但却使得蛋白内源荧光强度下降;Stern-Volmer作图显示曲酸对胰蛋白酶的荧光淬灭为静态淬灭作用;用同步荧光光谱和三维荧光光谱探讨了曲酸对胰蛋白酶构象的影响．     

光谱法研究nC60纳米颗粒与牛血红蛋白的相互作用

用几种光谱方法研究了n C60纳米颗粒与牛血红蛋白之间的相互作用。用溶剂置换法制备n C60的水分散液,并用紫外-可见分光光度法、动态光散射和透射电子显微镜技术对n C60纳米颗粒进行表征;用紫外-可见分光光谱、荧光光谱、同步荧光光谱研究n C60与牛血红蛋白之间的作用。结果表明,n C60纳米颗粒对牛血红蛋白的紫外-可见吸收光谱有一定程度的改变作用;n C60可猝灭牛血红蛋白的内源荧光,且最大发射波长发生明显的蓝移现象;随着n C60浓度的增加,酪氨酸残基和色氨酸残基的同步荧光强度降低,其中酪氨酸残基峰位发生蓝移,而色氨酸残基峰位基本保持不变。研究表明n C60与牛血红蛋白之间存在一定的相互作用;n C60可引起牛血红蛋白构象的改变,酪氨酸残基所处微环境的极性减小,n C60与牛血红蛋白的作用位置更接近酪氨酸残基。     

荧光光谱法研究乙基麦芽酚与胰α-淀粉酶相互作用特征

研究了乙基麦芽酚对胰α-淀粉酶活性的影响,以及二者相互作用的荧光猝灭光谱、同步荧光光谱和三维荧光光谱特征．证实了乙基麦芽酚对胰α-淀粉酶活性具有激活作用．证实了乙基麦芽酚与胰α-淀粉酶间的相互作用为静态猝灭,求出了猝灭常数以及乙基麦芽酚与胰α-淀粉酶的结合常数K和结合位点数n．由求得的热力学参数,根据Ross 判断生物大分子和小分子结合力性质的规律推测,乙基麦芽酚与胰α-淀粉酶之间的作用力主要为疏水作用力．用同步荧光光谱和三维荧光光谱技术探讨了乙基麦芽酚对胰α-淀粉酶蛋白构象的影响．     

异烟肼与牛血清蛋白相互作用的光谱研究

用紫外吸收光谱和荧光光谱研究了异烟肼(INH)与牛血清蛋白(BSA)的相互作用.荧光光谱表明,INH对BSA的荧光有较强的猝灭作用.通过结合常数和结合位点数的计算,证明这种荧光猝灭机理符合静态机制,INH和BSA形成了1∶1稳定复合物;考察不同温度和酸度下的猝灭作用,进一步证实其静态猝灭行为和疏水作用机制.紫外吸收光谱和同步荧光光谱表明,INH与BSA的相互作用使蛋白质的分子构象发生变化,而同步荧光光谱显示两者的结合位点更接近于色氨酸.     

竹红菌甲素和马心肌红蛋白相互作用的光谱研究

利用紫外可见吸收光谱和荧光光谱研究了竹红菌甲素(HA)与肌红蛋白(Mb)的相互作用．结果表明两者的相互作用主要存在于竹红菌甲素与马心肌红蛋白分子中的氨基酸残基之间．     

光谱法研究灿烂甲酚蓝与血浆蛋白质结合性质及蛋白质构象变化

在模拟的生理条件下,采用荧光发射光谱、紫外-可见分光光度法、同步荧光光谱、三维荧光光谱和圆二色(Circular dichroism,CD)光谱等技术研究了灿烂甲酚蓝(Brilliant cresyl blue,BCB)与人血清白蛋白(Human serum albumin,HSA)的结合性质以及药物的结合对HSA构象的影响.结果表明,BCB和能猝灭HSA的内源荧光,BCB对HSA的荧光猝灭是形成HSA-BCB复合物的静态猝灭过程,紫外-可见吸收光谱的结果也进一步证实了BCB对HSA荧光的静态猝灭.BCB与HSA分子Trp残基的距离r小于7 nm,说明HSA与BCB之间能够发生非辐射能量转移,但由于rR0值,这说明形成HSA-BCB复合物导致的静态猝灭是HSA荧光猝灭的主要原因.热力学参数计算结果表明BCB与HSA相互作用形成HSA-BCB复合物的过程是自发进行的,疏水作用力和氢键在形成HSA-BCB复合物的过程中发挥主要作用.同步荧光光谱、三维荧光光谱和CD光谱的结果说明BCB与HSA的相互作用形成的HSA-BCB复合物导致了HSA的构象发生了变化.     

LSPcZn与马心肌红蛋白相互作用的研究

本文研究了在人体生理条件下β-四(5-磺酸萘氧基)锌酞菁(LSPcZn)与马心肌红蛋白分子(FePP-Mb)的相互作用,LSPcZn能够显著地猝灭FePP-Mb分子的荧光,说明两者之间有着很强的相互作用.变温荧光光谱实验的结果验证该作用可以导致LSPcZn分子和FePP-Mb分子之间形成复合物,使得LSPcZn可以通过静态猝灭有效地猝灭FePP-Mb的荧光.通过对荧光光谱的数据进行处理,得到LSPcZn分子和马心肌红蛋白(FePP-Mb)在不同温度下形成复合物的结合常数和结合位点.另外,LSPcZn与FePP-Mb分子之间相互作用可以改变FePP-Mb的构象.     

细胞色素C在汞表面生长性质的AFM研究——吸附自组装机理

利用原子力显微镜对细胞色素C在汞表面生长过程进行研究,发现细胞色素C在汞表面首先形成的是高度稍大于1nm的吸附单分子层,随着生长时间的增加,吸附在汞表面的细胞色素C分子和溶液中的分子问相互作用,自组装形成生长活性点,生长速度明显加快,生长时间达到8min时,细胞色素C能形成底部直径达527nm,高度达138nm,分布密度约为1．2～2．3个/μm^2的细胞色素C分子集聚体．生长机理是吸附自组装机理．细胞色素C的浓度较大地影响其在Hg表面的自组装平衡,随着浓度的减少,细胞色素C所能形成的最大分子集聚体的高度和数目都随之减少。浓度与平均表现高度是非线性的关系．     

乙醇对细胞色素c和脱辅基细胞色素c的结构转换影响

用紫外－可见吸收光谱,荧光光谱和圆二色谱对细胞色素c和脱辅基细胞色素c在不同的φ（乙醇）溶液中的结构进行了表征。结果表明,乙醇对细胞色素c的α－螺旋二级结构影响很小,而对血红素配位的影响十分明显,可导致血红素Fe-Met80配位键断裂,另一方面,乙醇对脱辅基细胞色素c有明显的结构转换诱导作用,在φ（C2H5OH）＝0．55（乙醇／水）溶液中,脱辅基细胞色素c的α－螺旋结构含量可以达到全蛋白的89％,研究结果表明：负电荷微环境的诱导对脱辅基细胞色素c的结构转换并不是必要的,改变溶剂与脱辅基细胞色素c之间的疏水和氢键相互作用力也可以诱导脱辅基细胞色素cα-螺旋的形成。     

光谱法研究痂囊腔菌素A与血红蛋白的相互作用

利用紫外-可见吸收光谱和荧光光谱系统地研究了痂囊腔菌素A(EA)与血红蛋白(Hb)之间的相互作用.实验结果表明,EA与Hb之间的相互作用影响Hb分子的构象,且该影响在避光与光照等两种情况下存在明显差异,避光时EA仅与Hb的氨基酸残基发生作用,而光照时EA与Hb的氨基酸残基和血红素辅基均发生作用.避光实验条件下的荧光猝灭常数、热力学参数等计算结果表明Hb荧光被猝灭方式为动态猝灭,EA对Hb之间的作用力为疏水作用力.同步荧光进一步研究了EA对Hb微环境的影响.     

纳米火棉胶膜自组装细胞色素c的直接电化学研究

用纳米火棉胶膜将细胞色素c固定在玻碳电极表面,制备了细胞色素c-火棉胶膜修饰电极.吸附在火棉胶膜上的细胞色素c可以与电极发生直接电子传递.在pH=7.0的0.1mol/LPBS缓冲溶液中可得到一对准可逆的细胞色素c的血红素辅基Fe(Ⅲ)/Fe(Ⅱ)电对氧化还原峰,实验求得细胞色素c异相电子传递速率常数k0为65.4μm/s.进一步考察了扫速、溶液pH值等因素对细胞色素c电子传递的影响,并用电化学阻抗法研究了修饰电极的电化学行为.     

细胞色素C在SAM修饰电极上的电化学行为

应用循环伏安法研究了细胞色素c在2-氨基乙硫醇自组装膜修饰金电极上的电化学行为。结果表明，细胞色素c在2-氨基乙硫醇修饰的金电极上的电子传递过程为—扩散控制的可逆反应，2-氨基乙硫醇自组装膜可用作细胞色素c电子传递的有效促进剂。依据电化学石英晶体微天平和电化学交流阻抗谱的测量结果，讨论了单分子膜的组装过程及其对促进细胞色素c电子传递的作用机制。     

Structural insights into electron transfer in caa3-type cytochrome oxidase

Cytochrome c oxidase is a member of the haem copper oxidase superfamily (HCO). HCOs function as the terminal enzymes in the respiratory chain of mitochondria and aerobic prokaryotes, coupling molecular oxygen reduction to transmembrane proton pumping. Integral to the enzyme's function is the transfer of electrons from cytochrome c to the oxidase via a transient association of the two proteins. Electron entry and exit are proposed to occur from the same site on cytochrome c. Here we report the crystal structure of the caa3-type cytochrome oxidase from Thermus thermophilus, which has a covalently tethered cytochrome c domain. Crystals were grown in a bicontinuous mesophase using a synthetic short-chain monoacylglycerol as the hosting lipid. From the electron density map, at 2.36?? resolution, a novel integral membrane subunit and a native glycoglycerophospholipid embedded in the complex were identified. Contrary to previous electron transfer mechanisms observed for soluble cytochrome c, the structure reveals the architecture of the electron transfer complex for the fused cupredoxin/cytochrome c domain, which implicates different sites on cytochrome c for electron entry and exit. Support for an alternative to the classical proton gate characteristic of this HCO class is presented.     

Synthesis and Characterization of Porphyrin-Trisbenzimidazole Dinucleating Ligand and Its Heterodinuclear Complex as CcO Active Site Model

A new dinucleating ligand having two metal-binding sites has been designed and synthesized as model ligand for Cytochrome c Oxidase. The corresponding heterodi-nuclear complex, as an active site model of Cytochrome c Oxidase, consisting of a porphyrinatocobalt compound covalently linked with a copper derivative of tris(2-benzimidazylmethyl) amine bearing three benzimidazole ligands for copper was synthesized and spectroscopically characterized. The spectra data suggest that there are interactions between the cobalt and copper coordination units. The cobalt is coordinated to four central nitrogens of the porphyrin and the copper has pentaco-ordinate geometry with the four tertiary amine nitrogens and a chloride.     

细胞色素C在醇修饰金电极上的直接电化学

细胞色素Ｃ是一种金属蛋白酶，在生物体内起着电子传递体的作用，细胞色素Ｃ在金属电极上的氧化还原反应极不可逆，但是它在促进剂修饰的电极上却显示出较高的可逆性。本对细胞色素Ｃ在丙三醇，乙二醇，甲醇等醇修饰金电极上的直接电化学反应进行了研究，发现不同的醇分子对细胞色素Ｃ的促进作用各不相同，结合糖分子的促进作用，探讨了醇上羟在数的多少对其促进作用的影响。     

Docking and molecular dynamics studies on CYP2D6

Drug-metabolizing enzymes, also known as cytochrome P450s, are a superfamily of hemoglobin responsible for metabolizing more than 90% clinical drugs. Cytochrome P450 2D6 (CYP2D6) is a significant member of cytochrome P450s for the reason of metabolizing about 20% clinical drugs. In this paper, molecular docking and molecular dynamic simulations are used to investi- gate the active site of CYP2D6, roles of essential amino acids within the active site and time-dependent protein energy changes. The results suggest that amino acids Glu216, Asp301, Ser304 and Ala305 in the active site are likely to form hydrogen bonding interac-tions with substrates; the benzene ring of Phe120 and aromatic ring in the substrates form ∏-∏ interactions. In addition, molecular dynamics simulations prove that the catalytic conformation of CYP2D6 without ligands can be obtained by their own atomic fluctuations. The impact of ligands on protein system energy and large conformational shift is not very large. Cytochrome P450s is known for their genetic polymorphisms, which will result in severe adverse drug reactions. Ideally, we hope to use mo- lecular modeling to investigate the differences between the substrates of wild-type and mutants while they are bonded with drugs, and predict the drug metabolizing ability of mutants. Reduce the possibility for people taking drugs that they can not metabolize, therefore reduce the rate of adverse drug reactions, and eventually establish a platform of personalized drugs to largely benefit human health.     

Directional electron transfer in ruthenium-modified horse heart cytochrome c

Cytochrome c can be modified by [(NH3)5RuII/III-] specifically at the imidazole moiety of histidine 33, and we have recently discussed the thermodynamics and kinetics of electron transfer within this modified protein. X-ray crystal structures of the oxidized and reduced forms of tuna cytochrome c indicate that the separation between the haem group of cytochrome c and the ruthenium label is 12-16 A. Internal electron transfer from the [(NH3)5RuII-] centre to the Fe(III) haem centre occurs with a rate constant k congruent to 53 s-1 (25 degrees C) (delta H = 3.5 kcal mol-1, delta S = -39 EU), as measured by pulse radiolysis. The measured unimolecular rate constant, k congruent to 53 s-1, is on the same timescale as a number of conformational changes that occur within the cytochrome c molecule. These results raise the question of whether electron transfer or protein conformational change is the rate limiting step in this process. We describe here an experiment that probes this intramolecular electron transfer step further. It involves reversing the direction of electron transfer by changing the redox potential of the ruthenium label. Electron transfer in the new ruthenium-cytochrome c derivative described here is from haem(II) to the Ru(III) label, whereas in (NH3)5Ru-cytochrome c the electron transfer is from Ru(II) to haem(III). Intramolecular electron transfer from haem(II) to Ru(III) in the new ruthenium-cytochrome c described here proceeds much slower (greater than 10(5) times) than the electron transfer from Ru(II) to haem(III) in the (NH3)5Ru-cytochrome c. We therefore conclude that electron transfer in cytochrome c is directional, with the protein envelope presumably involved in this directionality.     

人细胞色素P450氧化还原酶的克隆表达和抗体的制备

细胞色素P450还原酶(CPR)是人细胞色素P450酶系的电子供给者,对后者活性的发挥起到重要作用.本研究将从人胚胎cDNA文库中得到的人CPR的编码基因,连接入pT7450表达载体中,在大肠杆菌BL21(DE3)中高效表达并纯化.每升发酵液可得到纯化蛋白49 mg,占总蛋白含量的10%,以细胞色素C为底物检测酶活性,比活为65 u/mg.利用纯化的CPR作为抗原,常规免疫纯系大耳家兔,获得高效价、高特异性的抗血清,抗体滴度为1∶200 000(ELISA法),纯化后抗体应用于不同组织中CPR含量的评价,证明重组CPR及其抗体可用于细胞色素P450的体外药物代谢及细胞色素P450与CPR作用机理的研究.     

细胞色素P450酶生物转化及新催化反应的最新进展

细胞色素P450酶(P450s或CYPs)是一类广泛存在于生命体中,依赖于亚铁血红素催化多种底物的单加氧酶。一方面,它涉及许多高活性天然产物生物合成的关键步骤,对其生物转化研究有助于提高活性分子的产率和活性;另一方面,其催化涉及金属元素以及辅助因子,其蛋白质改造和新催化反应的研发也不断取得新突破。本文就细胞色素P450酶这两方面的最近研究进展进行了总结与论述。