Quenching of tryptophan fluorescence in various proteins by a series of small nickel complexes

A series of twelve anionic, cationic, and neutral nickel(II) complexes have been synthesized and characterized. The interaction of these complexes with bovine serum albumin (BSA), human serum albumin (HSA), lysozyme (Lyso), and tryptophan (Trp) has been studied using steady-state fluorescence spectroscopy. Dynamic and static quenching constants have been calculated, and the role played in quenching by the ligand and complex charge investigated. The nickel complexes showed selectivity towards the different proteins based on the environment surrounding the Trp residue(s). Only small neutral complexes with hydrophobic ligands effectively quenched protein fluorescence via static quenching, with association constants ranging from 10(2) M(-1) (free Trp) to 10(10) M(-1) (lysozyme), indicating a spontaneous and thermodynamically favorable interaction. The number of binding sites, on average, was determined to be one in BSA, HSA and free Trp, and two in lysozyme.     

Fluorescence quenching of dyes by tryptophan: interactions at atomic detail from combination of experiment and computer simulation

Fluorescence spectroscopy and molecular dynamics (MD) simulation are combined to characterize the interaction of two organic fluorescent dyes, rhodamine 6G (R6G) and an oxazine derivative (MR121), with the amino acid tryptophan in aqueous solution. Steady-state and time-resolved fluorescence quenching experiments reveal the formation of essentially nonfluorescent ground-state dye/Trp complexes. The MD simulations are used to elucidate the molecular interaction geometries involved. The MD-derived probability distribution of the distance r between the centers of geometry of the dye and quencher ring systems, P(r), extends to higher distances for R6G than for MR121 due to population in the R6G/Trp system of fluorescent interaction geometries between Trp and the phenyl ring and ester group of the dye. The consequence of this is the experimental finding that under the conditions used in the simulations about 25% of the R6G dye is fluorescent in comparison with 10% of the MR121. Combining the above findings allows determination of the "quenching distance", r, above which no quenching occurs. r is found to be very similar (approximately 5.5 A) for both dye/Trp systems, corresponding to close to van der Waals contact. Both experimental dynamic Stern-Volmer analysis and the MD trajectories demonstrate that the main determinant of the fluorescence intensity is static quenching. The approach presented is likely to be useful in the structural interpretation of data obtained from fluorescent conjugates commonly used for monitoring the binding and dynamics of biomolecular systems.     

Dye induced quenching of firefly luciferase-luciferin bioluminescence

The quenching of firefly bioluminescence (BL) in presence of xanthene dyes and tetratolylporphyrin was investigated. The BL intensity was quenched with an altered decay pattern in presence of xanthene dyes and tetratolylporphyrin. The electronic absorption spectra indicate that there is no significant interaction occurring between the dyes and the BL components in the ground state. The BL quenching decay rate and fluorescence quenching studies of luciferin by the dyes suggest an energy transfer through an exciplex, involving oxyluciferin, in the excited state and the dyes, in the ground state. The bimolecular quenching rate constant (K(q)) values obtained from fluorescence studies varied between 7.7 x 10(12) and 19.8 x 10(12)M(-1)s(-1). The magnitude of the bimolecular quenching rate constants confirmed the complex formation between dye and excited oxyluciferin. The exciplex subsequently undergoes a non-radiative decay to the ground state via a combination of heavy atom induced and F?rster-type energy transfer. The decay rate constants in presence and in absence of dyes vary between 7.47 x 10(-4) and 7.6 x 10(-2)s(-1). In the presence of dyes the effective decay rate constants (k(eff)) increased while the lifetime of light emitting species decreased. The kinetic studies in presence of singlet oxygen scavengers, like beta-carotene and NaN(3), prove that there is no significant quenching of the firefly BL due to the formation of singlet oxygen.     

Pd(Ⅱ)与色氨酸、酪氨酸及苯丙氨酸相互作用的荧光光谱

当Pd(Ⅱ)与色氨酸(Trp)、酪氨酸(Tyr)及苯丙氨酸(Phe)等芳香族氨基酸相互作用时,能观察到3种氨基酸的荧光均发生猝灭. 从吸收光谱的变化,温度对猝灭作用的影响以及猝灭常数Ksv,可以判定荧光猝灭作用是由于Pd(Ⅱ)与上述氨基酸形成基态配合物而导致的静态猝灭过程. 并认为在一定浓度的Cl-存在下,Pd(Ⅱ)与氨基酸分别以N, N配位和N, O配位形成以下混配型三元配合物Pd(HR)Cl2 (Trp和Phe体系)和Pd(H2R)Cl2(Tyr体系),并推测了配合物相应的结构. 该荧光猝灭体系不仅可用于研究钯(Ⅱ)与上述芳香族氨基酸的相互作用,也可成为以氨基酸(特别是Trp)作探针高灵敏荧光猝灭法测定钯的基础.     

Fluorescence quenching of meso-tetrakis (4-sulfonatophenyl) porphyrin by colloidal TiO(2)

A stable colloidal TiO(2) has been prepared. The interaction of meso-tetrakis (4-sulfonatophenyl) porphyrin (TSPP) with colloidal TiO(2) was studied by absorption and fluorescence spectroscopy. Upon excitation of its absorption band, the fluorescence emission of TSPP was quenched by colloidal TiO(2). The bimolecular quenching rate constant (k(q)) is 1.78 x 10(11)M(-1)s(-1). The porphyrin can participate in the quenching process by injecting electrons from its excited states into the conduction band of TiO(2). The quenching mechanism is discussed on the basis of the quenching rate constant as well as the reduction potential of the colloidal TiO(2). Rehm-Weller equation was applied for the calculation of free energy change (DeltaG(et)).     

速灭威与牛血清白蛋白作用的研究

在pH=7.4的生理条件下,应用荧光光谱法研究了速灭威与牛血清白蛋白间相互作用。结果表明:速灭威对牛血清白蛋白的荧光有较强的猝灭作用,测定不同温度下的猝灭常数,证实了速灭威对牛血清白蛋白的荧光猝灭过程机理为静态猝灭。根据猝灭结果计算了不同温度下的结合位点数、结合常数。应用同步荧光光谱法探讨了速灭威对牛血清白蛋白构象的影响。依据f ster非辐射能量转移理论确定受体间的结合距离和能量转移效率。     

钇配合物荧光猝灭法测定姜黄素含量

本文利用荧光光度法,吸收光谱法和共振光散射光谱法研究了钇(Y3+)-姜黄素(CU)-色氨酸(Trp)-十二烷基苯磺酸钠(SDBS)体系的荧光猝灭作用,研究发现,姜黄素能猝灭Y3+-Trp-SDBS体系的荧光强度。在最佳实验条件下,体系的荧光猝灭程度与CU的浓度在73.68～442.1,0.074～5.3μg.mL-1范围内呈良好的线性关系,其检出限为0.0185μg.mL-1,据此建立了姜黄素的测定方法,并且将其成功应用于实际样品的测定中。机理研究表明,Y3+-Trp-SDBS体系中的Y3+与姜黄素结合形成缔合物,拉近了Trp与姜黄素之间的距离,Trp通过分子间能量转移将所吸收能量转移到CU上,使体系的荧光猝灭。     

Exploring Reversible Quenching of Fluorescence from a Pyrazolo[3,4‐b]quinoline Derivative by Protonation

Pyrazolo[3,4‐b]quinoline derivatives are reported to be highly efficient organic fluorescent materials suitable for applications in light‐emitting devices. Although their fluorescence remains stable in organic solvents or in aqueous solution even in the presence of H₂O, halide salts (LiCl), alkali (NaOH) and weak acid (acetic acid), it suffers an efficient quenching process in the presence of protic acid (HCl) in aqueous or ethanolic solution. This quenching process is accompanied by a change in the UV spectrum, but it is reversible and can be fully recovered. Both steady‐state and transient fluorescence spectra of 1‐phenyl‐3,4‐dimethyl‐1H‐pyrazolo‐[3,4‐b]quinoline (PAQ5) during quenching are measured and analyzed. It is found that a combined dynamic and static quenching mechanism is responsible for the quenching processes. The ground‐state proton‐transfer complex [PAQ5 ??? H⁺] is responsible for static quenching. It changes linearly with proton concentration [H⁺] with a bimolecular association constant K_S=1.95 M ^?1 controlled by the equilibrium dissociation of HCl in ethanol. A dynamic quenching constant K_D=22.4 M ^?1 is obtained by fitting to the Stern–Volmer equation, with a bimolecular dynamic quenching rate constant k_d=1.03×10⁹ s^?1 M ^?1 under ambient conditions. A change in electron distribution is simulated and explains the experiment results.     

Binding Interaction of Captopril with Metal Ions: A Fluorescence Quenching Study

The binding interaction of captopril (CPL) with biologically active metal ions Mg²⁺, Ca²⁺, Mn²⁺, Co²⁺, Ni²⁺, Cu²⁺ and Zn²⁺ was investigated in an aqueous acidic medium by fluorescence spectroscopy. The experimental results showed that the metal ions quenched the intrinsic fluorescence of CPL by forming CPL‐metal complexes. It was found that static quenching was the main reason for the fluorescence quenching. The quenching constant in the case of Cu²⁺ was highest among all quenchers, perhaps due to its high nuclear charge and small size. Quenching of CPL by metal ions follows the order Cu²⁺>Ni²⁺>Co²⁺>Ca²⁺>Zn²⁺>Mn²⁺>Mg²⁺. The quenching constant K_sv, bimolecular quenching constant K_q, binding constant K and the binding sites "n" were determined together with their thermodynamic parameters at 27 and 37°C. The positive entropy change indicated the gain in configurational entropy as a result of chelation. The process of interaction was spontaneous and mainly ΔS‐driven.     

家蝇幼虫抗菌肽MDL-1的荧光特性分析

本文研究了家蝇幼虫抗菌肽MDL-1的荧光光谱和淬灭剂对内源性荧光的影响。家蝇幼虫抗菌肽MDL-1在激发波长280 nm时,其荧光光谱为酪氨酸（Tyr）残基和色氨酸（Trp）残基共同提供。结果表明,KI不能淬灭抗菌肽MDL-1的Trp残基的荧光,而丙烯酰胺（Acr）能淬灭几乎所有的Trp残基的荧光（f-0．92）;这说明,Trp残基不是位于抗菌肽分子的表面,而是位于分子的内部。     

Amplified quenching of a conjugated polyelectrolyte by cyanine dyes

The conjugated polyelectrolyte PPESO3 features a poly(phenylene ethynylene) backbone substituted with anionic 3-sulfonatopropyloxy groups. PPESO3 is quenched very efficiently (KSV > 10(6) M(-1)) by cationic energy transfer quenchers in an amplified quenching process. In the present investigation, steady-state and picosecond time-resolved fluorescence spectroscopy are used to examine amplified quenching of PPESO3 by a series of cyanine dyes via singlet-singlet energy transfer. The goal of this work is to understand the mechanism of amplified quenching and to characterize important parameters that govern the amplification process. Steady-state fluorescence quenching of PPESO3 by three cationic oxacarbocyanine dyes in methanol solution shows that the quenching efficiency does not correlate with the Forster radius computed from spectral overlap of the PPESO3 fluorescence with the cyanines' absorption. The quenching efficiency is controlled by the stability of the polymer-dye association complex. When quenching studies are carried out in water where PPESO3 is aggregated, changes observed in the absorption and fluorescence spectra of 1,1',3,3,3',3'-hexamethylindotricarbocyanine iodide (HMIDC) indicate that the polymer templates the formation of a J-aggregate of the dye. The fluorescence dynamics in the PPESO3/HMIDC system were probed by time-resolved upconversion and the results show that PPESO3 to HMIDC energy transfer occurs on two distinctive time scales. At low HMIDC concentration, the dynamics are dominated by an energy transfer pathway with a time scale faster than 4 ps. With increasing HMIDC concentration, an energy pathway with a time scale of 0.1-1 ns is active. The prompt pathway (tau < 4 ps) is attributed to quenching of delocalized PPESO3 excitons created near the HMIDC association site, whereas the slow phase is attributed to intra- and interchain exciton diffusion to the HMIDC.     

岩白菜素与牛血清蛋白相互作用的荧光光谱研究

应用荧光光谱研究了岩白菜素与牛血清白蛋白(BSA)分子间的相互作用.结果表明,岩白菜素对BSA内源荧光的猝灭机制属于形成化合物所引起的静态猝灭,猝灭常数Ksv为1.905×104L.mol-1;岩白菜素与BSA反应的结合常数为2.083×104,结合位点数为1.由热力学参数确定了岩白菜素与牛血清白蛋白的结合作用主要为静电作用.实验还发现随着岩白菜素的加入,BSA的猝灭值与岩白菜素浓度在1.5×10-5~1.5×10-4mol.L-1的范围内呈良好的线性关系,检出限2.0×10-6mol.L-1,可用于岩白菜素的测定.     

Spectroscopic analyses on interaction of o-Vanillin-D-Phenylalanine, o-Vanillin-L-Tyrosine and o-Vanillin-L-Levodopa Schiff Bases with bovine serum albumin (BSA)

In this work, three o-Vanillin Schiff Bases (o-VSB: o-Vanillin-D-Phenylalanine (o-VDP), o-Vanillin-L-Tyrosine (o-VLT) and o-Vanillin-L-Levodopa (o-VLL)) with alanine constituent were synthesized by direct reflux method in ethanol solution, and then were used to study the interaction to bovine serum albumin (BSA) molecules by fluorescence spectroscopy. Based on the fluorescence quenching calculation, the bimolecular quenching constant (K(q)), apparent quenching constant (K(sv)), effective binding constant (K(A)) and corresponding dissociation constant (K(D)) as well as binding site number (n) were obtained. In addition, the binding distance (r) was also calculated according to Foster's non-radioactive energy transfer theory. The results show that these three o-VSB can efficiently bind to BSA molecules, but the binding array order is o-VDP-BSA>o-VLT-BSA>o-VLL-BSA. Synchronous fluorescence spectroscopy indicates that the o-VDP is more accessibility to tryptophan (Trp) residues of BSA molecules than to tyrosine (Tyr) residues. Nevertheless, the o-VLT and o-VLL are more accessibility to Tyr residues than to Trp residues.     

色氨酸与功能单体自组装体系的分子间非共价相互作用

利用荧光猝灭分析、 三维荧光光谱和计算机模拟等方法分别研究色氨酸(L-Trp)与己二酸二酰肼(ADH)、 双丙酮丙烯酰胺(DAAM)和丙烯酸(AA)3种功能单体间的非共价相互作用. 考察了荧光猝灭机理、 色氨酸-功能单体的相互作用强弱以及作用力类型. 研究结果表明, 功能单体对L-Trp的荧光猝灭过程是由于形成不发射荧光的复合物而引起的静态猝灭; L-Trp与功能单体的结合常数较大, 所形成的复合物较稳定, 非共价作用中以氢键和静电作用贡献最大; L-Trp主要通过羧基与功能单体产生相互作用, 芳杂环与功能单体间的相互作用相对较弱; L-Trp与功能单体相互作用的强弱顺序为L-Trp-ADH > L-Trp-DAAM > L-Trp-AA.     

具抗凝血作用的三种钕配合物与人血清白蛋白的相互作用

采用荧光光谱法,紫外光谱法以及圆二色谱法研究了具抗凝血作用的水杨酸钕((NdL′3.2H2O,L′=水杨酸离子))、华法灵钕(NdL3.2H2O,L=华法灵离子)和华法灵水杨酸钕(NdL2L′.2H2O)3种配合物与人血清白蛋白的相互作用。结果表明:配合物对人血清白蛋白(HSA)的荧光产生猝灭现象;配合物的存在使得HSA紫外吸收光谱的强度增加;配合物的存在也对HSA的构象产生影响。水杨酸钕的猝灭方式为动态与静态猝灭,而华法灵钕和华法灵水杨酸钕的猝灭方式属于两者之间生成了不发荧光的复合物而导致的静态猝灭。并分别确定了它们的结合力类型:华法灵钕与HSA之间主要作用力是静电作用力;水杨酸钕与HSA之间主要作用力为典型的疏水作用力;华法灵水杨酸钕与HSA之间为氢键和范德华力。计算了配合物与人血清白蛋白的结合常数K和结合位点数n。     

铱(IV)离子与人血丙种球蛋白的作用研究

在0.1 mol•L^－1醋酸-醋酸钠(pH 5.0)体系中, 采用紫外吸收光谱、荧光光谱及同步荧光光谱法研究了人血丙种球蛋白(gamma seroglobulinum humanum, 简称GSH)与铱(IV)离子的相互作用. 结果表明, Ir(IV)离子使人血丙种球蛋白的构象发生了改变, α-螺旋含量减少, 并且用同步荧光光谱发现Ir(IV)离子与人血丙种球蛋白的作用位点更接近于色氨酸, 从而使色氨酸残基的疏水性略有减小. 荧光光谱结果表明Ir(IV)对人血丙种球蛋白内源荧光(342 nm)产生了较强的荧光猝灭作用, 根据不同温度下Ir(IV)对人血丙种球蛋白的荧光猝灭作用, 证明了这种荧光猝灭为静态猝灭机制, 计算了其结合常数和结合位点数, 从而得出了静电作用力为其主要的作用力.     

Study on the interaction between methyl violet and bovine serum albumin by spectral analyses

In this article the interaction between methyl violet (MV) and bovine serum albumin (BSA) was studied with spectroscopy. The results indicated that the fluorescence intensity of BSA was quenched strongly by MV through a static quenching procedure. The association constants, the number of binding sites and basic thermodynamic parameters were obtained based on fluorescence quenching data. The effect of MV on the conformation of BSA had been investigated with synchronous fluorescence spectroscopy and circular dichroism (CD) spectrum.     

绞股蓝皂苷与牛血清白蛋白相互作用的荧光光谱研究

用荧光光谱技术研究了绞股蓝皂苷与牛血清白蛋白(BSA)在pH=7.40的Tris-HCl缓冲溶液中的相互作用;通过计算确定了绞股蓝皂苷与BSA的结合位点数和结合常数,利用热力学分析探讨了绞股蓝皂苷与BSA之间的结合方式;同时采用同步荧光技术考察了绞股蓝皂苷对BSA构象的影响.结果表明,绞股蓝皂苷对牛血清白蛋白的荧光猝灭过程为静态猝灭;二者主要靠疏水作用和静电引力结合.     

Fluorescence of the single tryptophan of cutinase: temperature and pH effect on protein conformation and dynamics

The cutinase from Fusarium solani pisi is an enzyme with a single L-tryptophan (Trp) involved in a hydrogen bond with an alanine (Ala) residue and located close to a cystine formed by a disulfide bridge between two cysteine (Cys) residues. The Cys strongly quenches the fluorescence of Trp by both static and dynamic quenching mechanisms. The Trp fluorescence intensity increases by about fourfold on protein melting because of the disruption of the Ala-Trp hydrogen bond that releases the Trp from the vicinity of the cystine residue. The Trp forms charge-transfer complexes with the disulfide bridge, which is disrupted by UV light irradiation of the protein. This results in a 10-fold increase of the Trp fluorescence quantum yield because of the suppression of the static quenching by the cystine residue. The Trp fluorescence anisotropy decays are similar to those in other proteins and were interpreted in terms of the wobbling-in-cone model. The long relaxation time is attributed to the Brownian rotational correlation time of the protein as a whole below the protein-melting temperature and to protein-backbone dynamics above it. The short relaxation time is related to the local motion of the Trp, whose mobility increases on protein denaturation.     

溴百里酚蓝与牛血清白蛋白的相互作用研究

在模拟动物体生理条件和不同温度下,用荧光光谱和紫外-可见吸收光谱法研究了溴百里酚蓝(BTB)与牛血清白蛋白(BSA)结合反应的光谱行为。用Stern-Volmer和Lineweaver-Burk方程分别处理试验数据,发现BSA与BTB发生反应生成了新的复合物,属于静态荧光猝灭。求出了反应时复合物的形成常数KLB(2.792×105L.mol-1)、热力学参数(ΔHθ=(20.24 kJ.mol-1,ΔSθ=37.22J.K-1,ΔGθ=(31.25kJ.mol-1)与结合位点数(1.1578)。根据F rster偶极-偶极非辐射能量转移理论计算出结合位置距离212位色氨酸残基2.60nm,证明二者主要靠静电作用力结合。同时用同步荧光光谱和三维荧光光谱法探讨了BTB对BSA构象的影响,表明BTB使色氨酸残基所处微环境的极性减弱、疏水作用增强,为阐明BTB的染色机理、毒理效应和生物学效应提供重要信息。