寡聚核苷酸中鸟苷对四甲基罗丹明的荧光猝灭

具有核苷特异性的荧光猝灭技术在生物领域具有广泛应用.为了更好地理解这一过程的机理及其影响因素,研究了核苷对四甲基罗丹明（TMR）染料的分子间猝灭和在同一条寡聚核苷酸链中的分子内猝灭.与以前的研究结果一致,脱氧单磷酸鸟苷（dGMP）可以有效地猝灭TMR,而其他单磷酸腺苷对其的猝灭可以忽略.由斯特恩-沃尔默图获得TMR和dGMP的双分子猝灭常数为Ks=52.3L/mol.将TMR标记在寡聚核苷酸末端,可以观测到其荧光通过光致电子传递有效地被鸟苷猝灭,我们利用荧光相关光谱的方法测定了这一过程的猝灭速率常数.此外,所得的数据还显示鸟苷附近的碱基会对分子内的猝灭过程产生显著的位阻效应.这些结果将有助于设计寡聚核苷酸荧光探针和理解G猝灭过程.     

Spectroscopic characterization of fluorescein- and tetramethylrhodamine-labeled oligonucleotides and their complexes with a DNA template

We measured absorption and emission spectra, fluorescence quantum yield, anisotropy, fluorescence resonance energy transfer (FRET), and melting temperature to characterize fluorescein- and tetramethylrhodamine (TMR)-labeled oligonucleotides in solution and when hybridized to a common DNA template. Upon hybridization to the template, both the absorption and emission spectra of TMR-labeled duplexes exhibited a shift with respect to those of labeled oligonucleotides, depending on the location of the TMR on the oligonucleotide. Measurements of quantum yield, anisotropy, and melting temperature indicated that TMR interacted with nucleotides within the duplexes in the order (T1>T5>T11, T16) that the oligonucleotide with TMR labeled at the 5' end (T1) is stronger than that labeled at position 5 from the 5' end (T5), which is also stronger than those labeled at the positions, 11 and 16, from the 5' end (T11, T16). In the case of the duplex formed between T1 and the template, fluorescence quenching was observed, which is attributed to the interaction between the dye molecule and guanosines located at the single-stranded portion of the template. A two-state model was suggested to describe the conformational states of TMR in the duplex. The melting temperatures of the four FRET complexes show the same pattern as those of TMR-labeled duplexes. We infer that the interactions between TMR and guanosine persist in the FRET complexes. This interaction may bring the donor and the acceptor molecules closely together, which could cause interaction between the two dye molecules shown in absorbance measurements of the FRET complexes.     

Synthesis and Fluorescence Quenching Study of the Novel Cationic Probe Derived from Luminarosine

A simple and efficient transformation of the zwitterionic luminarosine into a brightly fluorescent cationic analogue, namely 1‐amino‐9‐methoxy‐2,4,10‐triaza‐4b‐azoniaphenanthrene ( 3 ), is reported. The fluorescence quenching of 3 by common nucleotides, calf‐thymus (CT) DNA, and halide ions was investigated by means of spectrophotometric and spectrofluorometric methods. Intermolecular static and dynamic fluorescence‐quenching constants for quenching of 3 by nucleotides and halide ions were determined in aqueous solution. Evidence for formation of nonfluorescent ground‐state complexes of 3 with nucleotides and CT‐DNA is presented. Scatchard analysis of the CT‐DNA quenching data resulted in a binding constant of 2.8×10⁴ M ⁻¹ and a number of binding sites per base pair of 0.049.     

新型尾式卟啉的合成及与人血清白蛋白的相互作用

合成了未见文献报道烟酸分子修饰的自由卟啉o-(niacin)C4O-TPP、p-(niacin)C4O-TPP及锌配合物o-(niacin)C4O-TPPZn、p-(niacin)C4O-TPPZn。通过元素分析、紫外-可见光谱、核磁共振氢谱、红外光谱等多种谱图对结构进行了表征。为模拟金属卟啉的生物功能,采用荧光光谱滴定法测定了金属锌卟啉与人血清白蛋白(HSA)相互作用的光谱性质。按照Stern-Volmer方程、Lineweaver-Burk双倒数方程分析和处理试验数据,得到了反应的猝灭常数、结合常数和热力学参数等。实验结果表明:锌卟啉与人血清白蛋白之间发生了较强的静态荧光猝灭效应,二者之间是以氢键或Van der Waals力结合反应。     

FREE ENERGY DEPENDENCE OF THE QUENCHING OF CHLOROPHYLL a FLUORESCENCE BY SUBSTITUTED QUINONES*

The quenching of chlorophyll a (Chl a) fluorescence hy a series of substituted benzoquinones. naphthoquinones and anthraquinones has been examined employing ethanol and acetonitrile as solvents. All quinones are good quenchers of fluorescence. There is an excellent linear relation between the Stern-Volmer quenching constants, K, and the polarographic half wave potentials (E₁₂) of the quinones, with more oxidizing quinones being better quenchers. The quenching data are consistent with the excited state half wave potential of ?1.31 eV predicted theoretically, demonstrating that the kinetically estimated value of the Chl a excited state reduction potential agrees with that expected on spectroscopic grounds. The results of quenching are not in agreement with the conventional Marcus theory of electron-transfer reactions, as there is no evidence of quenching constant. K_q. decrease vsΔG⁰ even for free energy changes nearly twice that expected for the onset of the Marcus inverted region. However, the kinetically estimated K_q values are in good agreement with the ones calculated by using the Rehm and Weller equation for fluorescence quenching by electron transfer. Our experimental results support the electron transfer mechanism of quenching proposed by Seely.     

Selective quenching of tryptophanyl fluorescence in bovine serum albumin by the iodide ion

Modified Stern-Volmer equation is obeyed by bovine serum albumin (BSA)-iodide system showing selective quenching of tryptophanyl fluorescence of BSA. The fraction of accessible protein fluorescence is 0.56 and the effective Stern-Volmer constant is 290 M^-1 at pH 7.4 in 0.005 M phosphate buffer at 25°C. Collisional quenching is operative both in the BSA -I⁻¹ system and the model system, tryptophan-I⁻¹. It is supported by the observed relationship between the ratio of quenching rate constants (k _q ) and diffusion coefficients and alsok _q with bulk viscosity.     

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.     

Study on the interactions between anti-tumor metal complexes and mononucleotides using trans -[en2Os(η2-H2)(CF3SO3)](CF3SO3) as a 1H NMR probe in a competitive mode

The interaction of several anti-tumor metal complexes with dGMP have been investigated using trans-[en₂Os(η²-H₂)]²⁺ as a ¹H NMR probe in a competitive mode. Me₂SnCl₂, Bu₂SnCl₂, Et₂Sn(phen)Cl₂ and Et₂SnCl₂can bind to dGMP mainly via phosphate; Cp₂TiCl₂ binds to dGMP mainly via phosphate and N₇. The binding constant for (CH₃)₂SnCl₂ binding to phosphate of dGMP exceeds 2.71×10⁴. The binding constant for Cp₂TiCl₂ to phosphate is even greater than that of Sn(IV). Cis-platin has high affinity for both N₇ and phosphate, but mainly for N₇. Binding of the probe to N₇ of dGMP reduces the binding affinity for phosphate of the same dGMP molecule by a factor of 5 to 6. Much the same factor is expected to apply to other metals containing agents interacting with dGMP.     

Fluorescence Quenching of Tris(2,2′-bipyridine)Ruthenium(II) Dichloride by Certain Organic Dyes

Fluorescence quenching of [Ru(bpy)₃]²⁺ by a series of organic dyes has been investigated by using the steady state fluorescence technique in aqueous medium. The dyes used are anthraquinone dyes: uniblue, acid blue 129, alizarin, alizarin red S and the azo dyes: congo red, sunset yellow, methyl orange, tartrazine, acid orange 63, methyl red and erichrome black T. The quenching of [Ru(bpy)₃]²⁺ was found to obey the Stern-Volmer equation and the corresponding Stern-Volmer plots were linear indicating dynamic quenching. The quenching rate constants (k _q) were calculated from the fluorescence data. The mechanism of quenching was discussed on the basis of the quenching rate constants as well as the reduction potential of dyes. The electron transfer mechanism has been proved by the calculation of Gibbs energy changes (ΔG _et) by applying the Rehm-Weller equation.     

Interaction between Xanthoxylin and Bovine Serum Albumin

在模拟生理条件下,采用荧光光谱法、圆二色光谱法和红外光谱法研究了花椒油素(XT)与牛血清白蛋白（BSA）的相互作用。结果表明花椒油素与牛血清白蛋白之间发生动态和静态联合猝灭,二者间的的猝灭常数(K)在286, 298和310 K分别为3.31 × 105, 到2.03 × 105 和 0.94 × 105 L∙mol-1. 热力学参数表明, 花椒油素与牛血清白蛋白间以疏水作用力为主。圆二色光谱和红外光谱法表明加入花椒油素后,牛血清白蛋白的二级结构发生了变化,其中α-螺旋减少了3.9%。另外,我们还研究了共存离子对两者结合的影响。     

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.     

Study on Interactions of Phenolic Acid-Like Drug Candidates with Bovine Serum Albumin by Capillary Electrophoresis and Fluorescence Spectroscopy

The interactions of the phenolic acids cinnamic acid (CNA), ferulic acid (FA), caffeic acid (CA) and chlorogenic acid (CLA) with bovine serum albumin (BSA) were investigated and compared using affinity capillary electrophoresis (ACE) and the fluorescence quenching methods. ACE gives binding constants (K _b) and thermodynamic parameters. The thermodynamic parameters show that each of four phenolic acids bind to BSA mainly by hydrogen bonds, electrostatic and hydrophobic interactions. The fluorescence quenching method provided quenching constant K _sv, binding site number n and K _b. The fluorescence results indicate that BSA fluorescence quenching is mainly a static quenching process. The binding constants (K _b) of CNA, FA, CA and CLA were from 2.52×10⁴ to 7.90×10⁴ L⋅mol⁻¹ from ACE experiments and 1.19×10⁴ to 5.21×10⁴ L⋅mol⁻¹ from fluorescence, their increase corresponded to the increase in the number of hydroxyl groups. These results imply that molecular structure and the number of hydroxyl groups of phenolic acids play act key roles in the affinity of natural phenolic acids towards BSA.     

Fluorescence quenching of para-substitutedstyrenes by tetramethylethylene. Correlationanalysis of the quenching coefficient KSV and thequenching rate constant k_q by the dual-parameter equation with polar and spin-delocalization substituent constants

The fluorescence quenching coefficient (Ksv) and the quenching rate constant kq of ten para-substituted styrenes (1-Ys) have been measured and correlation-analyzed by both the dual-parameter equation (Eq. 1) with (ρxσx+ρ'σ') and the single-parameter equation (Eq. 2) with ρxσx. Ex-cellent results have been obtained for the correlation of KSV against (ρxσmb+ρ'σ'JJ) or (ρxσ+ +ρ'σ'JJ). Our results suggest that, possibly, there might be no need to use excited-state substituent constant for the fluorecence quenching process of excited states of styrenes.     

Studies on the fluorescence quenching of polysilane copolymers by chlorohydrocarbons

The room-temperature solution fluorescence quenching of polysilane copolymers by chlorohydrocarbons such as CCl₄, CHCl₃, C₂Cl₆, and Cl₂CHCHCl₂ was studied. The existence of dynamic quenching was preliminarily demonstrated by the experiment of fluorescence lifetime quenching. The fluorescence quenching data were in conformity with the equation: F₀/F = (1+K_SV[Q])exp(NV[Q]), where F and F₀ are the fluorescence intensity with and without the addition of quencher, K_SV is the Stern-Volmer constant, [Q] is the quencher concentration, N is the Avogadro constant, and V is the volume of the active sphere. The fluorescence quenching by the first three chlorohydrocarbons was attributed to the contemporaneous effect of dynamic quenching and static quenching. There exists, at least mathematically, a critical quencher concentration [Q]_C. When the quencher concentration [Q] < [Q]_C, the fluorescence quenching is dominated by the dynamic quenching part; when [Q] > [Q]_C, it is dominated by the static quenching part. However, the fluorescence quenching by Cl₂CHCHCl₂ was attributed to only static quenching. Furthermore, it was proposed that the dynamic quenching may be related with the electrical positivity of the central carbon nucleus of the quenching molecules while the static quenching may be caused by the “outside heavy atom effect” of the Cl element. © 1996 John Wiley & Sons, Inc.     

Interaction of Co（ll） with Bovine Serum Albumin under UV C Irradiation

The interaction of Co（Ⅱ） with BSA under UV C （253.7 nm） irradiation under physiological conditions has been studied by UV-vis spectrum, ultraviolet second-derivative spectroscopy and fluorescence spectrum. The quenching rate constant kq and the association constant Ka were calculated according to Stern-Volmer equation based on the quenching of the fluorescence of BSA by Co（Ⅱ）.     

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.     

Investigation on the pH-dependent binding of benzocaine and lysozyme by fluorescence and absorbance

The interaction mechanism between benzocaine (BZC) and lysozyme (Lys) has been investigated by fluorescence, synchronous fluorescence, ultraviolet–vis (UV) absorption spectra, and three-dimensional fluorescence (3-D) in various pH medium. The observations of fluorescence spectra were mainly rationalized in terms of a static quenching process at lower concentration of BZC (C_BZC/C_Lys < 9) and a combined quenching process at higher concentration of BZC (C_BZC/C_Lys > 9) at pH 7.4 and 8.4. However, the fluorescence quenching was mainly arisen from static quenching by complex formation in all studied drug concentrations at pH 3.5. The structural characteristics of BZC and Lys were probed, and their binding affinities were determined under different pH conditions (pH 3.5, 7.4, and 8.4). The results indicated that the binding abilities of BZC to Lys decreased at the pH below and above the simulative physiological condition (pH 7.4) due to the alterations of the protein secondary and tertiary structures or the structural change of BZC. The effect of BZC on the conformation of Lys was analyzed using UV, synchronous fluorescence and three-dimensional fluorescence under different pH conditions. These results indicate that the binding of BZC to Lys causes apparent change in the secondary and tertiary structures of Lys. The effect of Zn²⁺ on the binding constant of BZC with Lys under various pH conditions (pH 3.5, 7.4, and 8.4) was also studied.     

Synthesis, characterization and theoretical analysis on a oxygen-sensing phosphorescent copper(I) complex

The fluorescence quenching of 9-aminoacridine by certain substituted uracils in water was studied using absorption, steady state and time resolved measurements. The bimolecular quenching rate constants (k_q), binding constant K and number of binding sites (n) were calculated based on the fluorescence quenching data. The free energy change (ΔG_et) for electron transfer process was calculated by Rehm–Weller equation. From lifetime measurement we observed that the quenching was mainly due to static mechanism involving ground state complex formation.     

Polyfluorophore Labels on DNA: Dramatic Sequence Dependence of Quenching

We describe studies carried out in the DNA context to test how a common fluorescence quencher, dabcyl, interacts with oligodeoxynucleoside fluorophores (ODFs)—a system of stacked, electronically interacting fluorophores built on a DNA scaffold. We tested twenty different tetrameric ODF sequences containing varied combinations and orderings of pyrene (Y), benzopyrene (B), perylene (E), dimethylaminostilbene (D), and spacer (S) monomers conjugated to the 3′ end of a DNA oligomer. Hybridization of this probe sequence to a dabcyl‐labeled complementary strand resulted in strong quenching of fluorescence in 85 % of the twenty ODF sequences. The high efficiency of quenching was also established by their large Stern–Volmer constants (K_SV) of between 2.1×10⁴ and 4.3×10⁵ M ^?1, measured with a free dabcyl quencher. Interestingly, quenching of ODFs displayed strong sequence dependence. This was particularly evident in anagrams of ODF sequences; for example, the sequence BYDS had a K_SV that was approximately two orders of magnitude greater than that of BSDY, which has the same dye composition. Other anagrams, for example EDSY and ESYD, also displayed different responses upon quenching by dabcyl. Analysis of spectra showed that apparent excimer and exciplex emission bands were quenched with much greater efficiency compared to monomer emission bands by at least an order of magnitude. This suggests an important role played by delocalized excited states of the π stack of fluorophores in the amplified quenching of fluorescence.     

Fluorescence Quenching and Binding Interaction of l0-Methylacridinium Iodide to Nucleic Acids

Interaction of 10‐methylacridinium iodide (MAI) as fluorescence probe with nucleobases, nucleosides and nucleic acids has been studied by UV‐visible absorption and fluorescence spectroscopy. It was found that fluorescence of MAI is strongly quenched by the nucleobases, nucleosides and nucleic acids, respectively. The quenching follows the Stern‐Volmer linear equation. The fluorescence quenching rate constant (k_q) was measured to be 10^9‐10¹⁰ (L/mol)/s within the range of diffusion‐controlled rate limit, indicating that the interaction between MAI and nucleic acid and their precursors is characteristic of electron transfer mechanism. In addition, the binding interaction model of MAI to calf thymus DNA (ct‐DNA) was further investigated. Apparent hypochromism in the absorption spectra of MAI was observed when MAI binds to ct‐DNA. Three spectroscopic methods, which include (1) UV spectroscopy, (2) fluorescence quenching of MAI, (3) competitive dual‐probe method of MAI and ethidium bromide (EB), were utilized to determine the affinity binding constants (K) of MAI and ct‐DNA. The binding constants K obtained from the above methods gave consistent data in the same range (1.0–5.5) × 10⁴L/mol, which lend credibility to these measurements. The binding site number was determined to be 1.9. The influence of thermal denaturation and phosphate concentration on the binding was examined. The binding model of MAI to ct‐DNA including intercalation and outside binding was investigated.