Cu2＋和铜锌超氧化物歧化酶作用的光谱学研究

利用邻苯三酚自氧化法监测在磷酸盐缓冲体系中Cu^2＋对猪肝铜锌超氧化物歧化酶(CuZnSOD)活力的影响, 认为Cu^2＋与猪肝CuZnSOD存在直接相互作用. 通过荧光光谱方法研究了这种相互作用, 内源荧光的猝灭实验表明Cu^2＋与CuZnSOD形成1∶1型稳定配合物; 荧光猝灭的动力学分析表明配合物形成过程由两个独立步骤完成: 第一步是双分子快速缔合过程, 形成了结合疏松的配合物, 第二步是单分子慢速过程, 即松散的配合物“异构化”成为结合紧密的配合物. FTIR和CD证实相互作用过程伴随了蛋白分子构象的变化.     

Cu2+与烟草多酚氧化酶相互作用研究

本文通过酶活性测定，荧光光谱和紫外光谱研究了外加Cu²⁺与烟草多酚氧化酶(简称PPO)的相互作用。结果表明，微量铜的加入能增加酶的活性，[Cu²⁺]/[PPO]为0.20左右时酶活性最大，[Cu²⁺]/[PPO]为0.91时，Cu²⁺开始表现出对PPO活性的抑制;Cu²⁺对PPO内源荧光的猝灭机制属于形成络合物所引起的静态猝灭，猝灭常数K_sv为8.0375×10³L·mol^-1;Cu²⁺的加入使PPO蛋白质分子构象发生变化，α-螺旋含量增加，多肽链及Trp和Tyr残基的芳杂环进一步向分子内收缩，疏水基团之间的疏水作用增强。     

邻菲咯啉乳酸镍配合物的合成与表征及其与牛血清白蛋白相互作用的荧光分析

利用溶液法合成了配合物[Ni(Hlact)₂(phen)]·2H₂O(1),并对该配合物进行了元素分析、红外光谱和X-射线单晶衍射表征。通过荧光光谱法研究了不同温度下配合物1与牛血清白蛋白相互作用的荧光强度的变化,计算在不同温度下,配合物1与牛血清白蛋白(BSA)的结合常数、结合位点数以及热力学函数,进一步讨论了配合物1与BSA相互作用的作用力类型和两者之间的距离。结果表明,配合物1对牛血清白蛋白的荧光猝灭为静态猝灭过程,它与牛血清白蛋白的相互作用有一个位点,结合常数的平均值5.06×10⁵ L·mol^-1,作用距离为2.35 nm,相互作用力表现为氢键和范德华相互作用。     

新型双核配合物的形成、与DNA的作用机制及荧光性质研究

利用紫外、荧光和粘度等方法研究了含不同配体的钌(II)配合物[Ru(phen)₂CImP]^2＋(CImP＝3,4-二羟基-咪唑并[4,5-i][1,10]邻菲咯啉)和[Ru(phen)₂TPPZ]^2＋(TPPZ＝四吡啶[3,2-a:2',3'-c:3',2'-h:2'',3''-j]吩嗪)与DNA的作用机制, 并研究了配合物与Zn^2＋配合后荧光性质变化. 结果表明[Ru(phen)₂TPPZ]^2＋与DNA以插入模式作用, 而[Ru(phen)₂CImP]^2＋与DNA则以沟面结合模式作用. 向配合物溶液中滴加Zn^2＋后, 配合物[Ru(phen)₂TPPZ]^2＋和[Ru(phen)₂CImP]^2＋均可以与Zn^2＋形成双核配合物[Ru(phen)₂(TPPZ)Zn]^4＋和[Ru(phen)₂(CImP)Zn]^4＋, 配合物的荧光减弱. 与DNA作用后, 配合物仍可以与Zn^2＋配位形成双核配合物, 但[Ru(phen)₂(TPPZ)Zn]^4＋保持插入模式与DNA作用, 配合物的荧光减弱. 而[Ru(phen)₂(CImP)Zn]^4＋与DNA则由沟面结合改为插入结合, 配合物的荧光增强.     

外源Cu2+与烟草CuZnSODⅢ的相互作用

In this paper， we have studied the interaction of CuZnSODⅢ and Outer-Copper， enzyme activity experiments expressed that 0.1mmol·L^-1 Cu²⁺ addition reduced the enzyme activity sharply， but this reduced action had not been found for the additions of 0.1mmol·L^-1 1∶1 Cu²⁺ and Zn²⁺， and 0.1mmol·L^-1 Zn²⁺， respectively. This was due to the Cu²⁺ exchanged the Zn²⁺ in CuZnSODⅢ，and it was proved by the experiment of determination of metal content. Meanwhile， the static fluorescence quenching mechanism revealed the exist of molecular complex of CuZnSOD with Cu²⁺. The binding constant was obtained from lineweaver-burk and double-lg plot. The distance of active site to Trp is about 2.83nm， was calculated according to F?rster theory.     

邻二氮菲-Cd2+与DNA的相互作用研究

本文以邻二氮菲作为分子探针,在磷酸盐-氯化钠中性介质中(pH=7.2),用荧光光谱和吸收光谱法研究了邻二氮菲-Cd²⁺与DNA之间的相互作用。结果表明,Cd²⁺离子与邻二氮菲形成1∶2型的配合物,该配合物与DNA有较强的相互作用,这是位于配合物中心的Cd²⁺离子与DNA发生键合。实验发现,在邻二氮菲-Cd²⁺-DNA体系中加入EDTA或柠檬酸,邻二氮菲-Cd²⁺与DNA的相互作用减弱,这表明EDTA或柠檬酸对Cd²⁺的毒性有一定的缓解作用。     

手性方酰化哒嗪衍生物的合成及荧光特性研究

以3,6-二甲酰基哒嗪和丙氨酸、α-氨基苯酚、方酸为原料合成了一种新的手性方酰化哒嗪胺衍生物, 利用IR, ¹H NMR及元素分析对其结构进行了表征, 并研究了金属离子对化合物的荧光性的影响. 结果表明, Cu^2＋, Ni^2＋等金属离子对化合物产生明显的荧光猝灭作用.     

罗布麻活性成分与人血清白蛋白结合的光谱学研究

应用荧光和紫外光谱研究了人血清白蛋白与罗布麻活性成分槲皮素(QUE)、芸香苷(RUT)和儿茶素(CAT)的结合机理. 在QUE与蛋白质浓度比小于3.5时, 其荧光猝灭机理主要是静态猝灭, 在药物浓度较高时动态猝灭所占的比例增加; RUT在整个实验浓度范围内对蛋白质的荧光猝灭机理为静态猝灭; CAT与蛋白质之间不能形成复合物, 其荧光猝灭主要由动态猝灭产生. QUE和RUT分别与蛋白质形成1∶1的复合物, 结合常数分别为(1.51±0.13)×10⁵和(0.81±0.08)×10⁵ L•mol^－1. 由于激发态质子转移, 与蛋白质的相互作用引起QUE和RUT内源荧光发射峰强度的明显增加, 进一步证实了它们与蛋白质的结合. 与蛋白质的结合也引起了QUE紫外吸收带的明显红移, 说明药物分子中的酚羟基发生了解离, 以离子形式与蛋白质发生作用. RUT的紫外吸收谱带没有明显移动, 说明它主要以中性状态与蛋白质结合. 应用与蛋白质作用后药物分子紫外吸收光谱的二阶导数谱, 对药物与蛋白质的结合模式进行了深入探讨.     

苯乙烯三嗪衍生物的合成及在重金属离子识别中的应用

合成了2,4-二(2-噻吩乙烯基)-6-(4'-N,N-二甲氨基苯乙烯基)-1,3,5-均三嗪(2)并鉴定了其结构。在乙腈-水混合介质中,化合物2在355和416nm处呈现双吸收峰,加入Cu²⁺,Hg²⁺ 和Fe³⁺ 后,均在520nm附近形成新的吸收峰。化合物2与Cu²⁺、Hg²⁺ 和Fe³⁺ 均形成1:1型配合物,其结合常数分别为1.9×10⁵L·mol^-1,6.6×10³L·mol^-1,2.7×10³L·mol^-1。对照化合物4与金属离子的光谱响应与化合物2相似,仅吸收峰的位置不同。因此,可认为化合物2和4中三嗪环中的N和噻吩环中的S与Cu²⁺、Hg²⁺ 和Fe³⁺ 共同配位形成了稳定的金属配合物。     

微波辅助合成双1,2,4-三嗪化合物及其荧光特性研究

以联苯甲酰、二甲酰肼为原料, 采用微波技术合成了5个双1,2,4-三嗪化合物3, 产率72%～85%, 利用元素分析、MS, IR, ¹H NMR和荧光光谱对其结构进行了表征, 并研究了金属离子对化合物3a的荧光性质的影响. 结果表明, 微波合成具有缩短反应时间、提高产率的优点; Cu^2＋, Ni^2＋等金属离子对化合物3a产生明显的荧光猝灭作用.     

Determination of the concentration of active sites and the catalytic rate constant of recombinant formate dehydrogenase from <Emphasis Type="Italic">Glycine max</Emphasis>

The analysis of a formate dehydrogenase (FDH) structure in the apo form and in a complex with nicotinamide (NAD⁺) and azide ion has shown a high probability of efficient fluorescence quenching during the formation of such a triple complex. The excitation and fluorescence spectra indicated that the enzyme fluorescence is determined by tryptophan residues. The dependence of FDH fluorescence quenching on the NAD⁺ and azide concentrations was studied. The obtained data were used to determine the concentration of active sites and the catalytic rate constant of recombinant FDH from Glycine max.     

A fluorescence “on–off” sensor for the highly selective and sensitive detection of Cu2+ ion

In an attempt to obtain a model of copper(II) ion-selective sensors, a new 1,8-naphthalimide-based fluorescence chemosensor, N-allylamine-4-[(E)-4-(([2-aminoethyl]imino)methyl) benzene-1,3-diol]-1,8-naphthalimide (NABN), was designed and synthesized. The sensor NABN is fully characterized by melting point analysis, fourier transform infrared spectra, Ultraviolet–visible (UV–vis) spectra, fluorescence spectra, ¹H NMR and ¹³C NMR spectroscopy, and mass spectrometry. NABN showed an unrivaled sensing behavior and an ardent selectivity toward copper(II) ion over other competitive metal ions tested in solution (N,N-Dimethylformamide (DMF)/Tris–HCl buffer, 1:1, v/v, pH = 7.2). The sensor showed a linear fluorescence quenching toward copper(II) ion in the range 0–50 μM, with a detection limit of 1.92 × 10⁻⁷ M estimated. Job's method indicated the formation of a 2:1 coordinative mode of the sensor with copper(II) ion with a high threshold of binding constant of 4 × 10¹² M⁻¹. Combining the above results, the quenching response of NABN toward Cu(II) ions could be ascribed to the strong, intrinsic paramagnetic behavior of Cu(II).     

Determination of Cysteine using the Fluorescence from a L-Tyrosine-Copper(II) Complex

A simple and sensitive method for the determination of cysteine is reported based on fluorescence quenching and recovery of L-tyrosine. At pH 10, copper(II) reacted with L-tyrosine to form a 1:1 complex that resulted in the quenching of L-tyrosine. However, the quenched fluorescence of L-tyrosine was recovered upon adding cysteine due to the strong affinity between these components. Under the optimized conditions, the recovered fluorescence was linearly proportional to the concentration of cysteine from 6.5?×?10^?7 to 4?×?10^?5?mol?L^?1 with a detection limit of 7.32?×?10^?8?mol?L^?1, demonstrating high sensitivity for the determination of cysteine. The mechanisms of fluorescence quenching and recovery were characterized and the method was used to determine cysteine in a pharmaceutical product with satisfactory results.     

Cu, Zn Superoxide dismutase: distorted active site binds substrate without significant energetic cost

Copper, Zinc superoxide dismutase (CuZnSOD) catalyzes the dismutation of the toxic superoxide radical into molecular oxygen and hydrogen peroxide. Dismutation is achieved by reduction and re-oxidation of the active site copper ion, where the superoxide substrate binds. This enzyme is considered to be a perfect enzyme, as the catalytic rate is very high and diffusion controlled. The redox active copper ion is coordinated by four histidine residues in a distorted square planar geometry. Much has been written about the biological significance of the geometry distortion. It is sometimes considered that it should help to tune the redox potential of the copper ion in order to efficiently reduce the first superoxide molecule and oxidize the second one. In this work we present a series of high level theoretical calculations using realistic models, which demonstrate that the distorted geometry is fundamental for the catalytic efficiency of the enzyme by allowing substrate binding without extensive geometric reorganization of the copper complex, upon changing from four to five ligands. A lower limit for the reorganization energy is calculated here in 22 kcal/mol, which would slow down the reaction kinetics by more than 13 orders of magnitude, transforming a perfect enzyme into an inefficient one.     

天然水中痕量铜的荧光测定

8-苯胺基-1-萘磺酸(ANS)可作为蛋白质研究的荧光探针^[1~3],ANS与可溶性的聚乙烯亚胺在微酸性溶液中以缔合物的形式结合,产生强烈的荧光并使ANS在水中的荧光激发和发射光谱分别产生红移和紫移,斯托克斯位移减小.当有痕量铜存在时,聚乙烯亚胺将与铜离子键合成聚合阳离子,它同ANS阴离子生成离子对后,由于ANS微环境极性的增强,导致荧光熄灭^[4],利用这一性质将水中痕量和超痕量的铜用巯基棉分离富集后进行测定,获得了满意结果,铜的检测下限为4.7ppb,对5至100ppb含量范围内铜的测定,相对标准偏不大于2.0%。     

流动注射-抑制化学发光法测定克百威

基于在碱性介质中,克百威抑制鲁米诺-过氧化氢-叶绿素铜钠体系的化学发光,提出了流动注射-抑制化学发光法测定克百威含量的方法。试验结果表明:叶绿素铜钠对克百威荧光猝灭过程是静态猝灭过程,叶绿素铜钠与克百威结合形成物质的量比为1比1的稳定配合物,平衡常数(K0)为3.41×105L.mol-1(25℃),结合距离(r)为0.39 nm。克百威质量浓度在0.08～2.00 mg.L-1范围内与其发光强度呈线性关系,方法检出限(3σ/k)为0.03 mg.L-1。此法用于克百威杀虫剂样品的分析,测得方法的平均加标回收率为101.5%。     

Photophysical Properties of Safranine T in Aqueous Glycol Oligomer Mixtures

The photophyscial behavior of the dye Safranine T in the ground and excited states in aqueous mixtures of glycol oligomers was investigated by absorption and fluorescence spectroscopic techniques. The formation of a 1:1 complex in the excited state was inferred from fluorescence studies. The calculated equilibrium constants of the complex are directly proportional to the molar mass of the glycol. Microviscosity values of the aqueous glycol mixtures were determined by employing Auromine O as a fluorescence probe. Stokes shift values were correlated with the bulk dielectric constant and the microviscosity of the media. Also employing Stokes shifts, solvent parameters such as the Kowoser Z values and the intramolecular charge transfer energy, E _T (30), were evaluated. The fluorescence quenching of Safranine T by the inorganic ions [Fe(CN)₆]³⁻, [Fe(CN)₆]⁴⁻, and [I]⁻ in aqueous oligomer mixtures at a fixed concentration was investigated. The ions influenced the quenching process to different extents, and the efficiency of quenching of the dye in the glycol media for [Fe(CN)₆]⁴⁻ and [I]⁻ ions follows the order EG > DEG > TEG > TTEG. In the case of ferricyanide ion the reverse order was observed. The microviscosity and bulk dielectric constant of the media played a major role in the collisional quenching process.     

含氟菲咯啉的设计合成及其杂配铜配合物的光解水性能

设计合成新型含氟菲咯啉二齿配体,与六氟磷酸四乙腈合铜及磷配体Xantphos进行配位,得到相应的铜基配合物（CP1~CP4）,再以此配合物作为光敏剂用于光催化水解制氢,发现其在均相光解水体系中表现出较好的光敏活性,制氢总转换数（TON）可达896。结合对所选的配合物进行吸收光谱和荧光发射光谱的测试结果可知,此配合物在溶液中也具有很好的稳定性,不会发生配体的重组现象。荧光淬灭实验表明,氧化淬灭为光解水过程中主要的淬灭途径。此外,对这类配合物作为光敏剂的构效关系及其内在机理进行了初步的解释与探讨。     

Synthesis and Photophysical Properties of a Deazaflavin‐Bridged Porphyrinatoiron(III) That Mimics the Interaction of a Deazaflavin Inhibitor with the Heme‐Thiolate Cofactor of Cytochrome P450 3A4

Cytochrome P450 3A4 metabolizes a majority of administered therapeutic agents in the human liver. We recently reported the synthesis of a new inhibitor, 1 , whose binding to and displacement from the active site of CYP 3A4 can be conveniently followed by the associated changes in fluorescence intensity. Here we report the synthesis of a bichromophoric compound, 6 , in which deazaflavin was strapped over the distal side of a porphyrinatoiron(III) complex to mimic the envisaged enzyme–inhibitor interaction within the active site. Femtosecond pump–probe and fluorescence spectroscopies were used to study the photophysical processes of 6 . Rapid intramolecular energy transfer and enhanced intersystem‐crossing processes induced by the high‐spin Fe^III central ion are responsible for the complete suppression of deazaflavin fluorescence in 6 . Fluorescence quenching is less efficient in the iron‐free analogue of 6 , i.e., in 21 .     

Determination of Sodium Hexametaphosphate in Beverages Based on the Fluorescence Quenching of Acridine Orange

Sodium hexametaphosphate was shown to form a complex with acridine orange by electrostatic interactions that induce fluorescence quenching. Analysis of fluorescence intensity showed that the process was dominated by static quenching, which was confirmed by absorption spectra and lifetime of the excited state. The decreased fluorescence intensity was directly proportional to the concentration of sodium hexametaphosphate between 8.0 × 10^?7 and 1.1 × 10^?5 mol L^?1 with a limit of detection of 5.3 × 10^?7 mol L^?1. Beverages were analyzed for sodium hexametaphosphate with recoveries between 91.7% and 108.3%.