Time-resolved fluorescence analysis of the mobile flavin cofactor in p -hydroxybenzoate hydroxylase

Conformational heterogeneity of the FAD cofactor in p-hydroxybenzoate hydroxylase (PHBH) was investigated with time-resolved polarized flavin fluorescence. For binary enzyme/substrate (analogue) complexes of wild-type PHBH and Tyr222 mutants, crystallographic studies have revealed two distinct flavin conformations; the ‘in’ conformation with the isoalloxazine ring located in the active site, and the ‘out’ conformation with the isoalloxazine ring disposed towards the protein surface. Fluorescence-lifetime analysis of these complexes revealed similar lifetime distributions for the ‘in’ and ‘out’ conformations. The reason for this is twofold. First, the active site of PHBH contains various potential fluorescence-quenching sites close to the flavin. Fluorescence analysis of uncomplexed PHBH Y222V and Y222A showed that Tyr222 is responsible for picosecond fluorescence quenching free enzyme. In addition, other potential quenching sites, including a tryptophan and two tyrosines involved in substrate binding, are located nearby. Since the shortest distance between these quenching sites and the isoalloxazine ring differs only little on average, these aromatic residues are likely to contribute to fluorescence quenching. Second, the effect of flavin conformation on the fluorescence lifetime distribution is blurred by binding of the aromatic substrates: saturation with aromatic substrates induces highly efficient fluorescence quenching. The flavin conformation is therefore only reflected in the small relative contributions of the longer lifetimes.     

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.     

Energy Transfer between Flavin and Cytochrome c

Energy transfer between photoexcited flavin and cytochrome c has been investigated in order to estimate intermolecular forces between flavin and cytochrome c. The quenching of the fluorescence of flavin by cytochrome c excited at 372 nm was found to be much greater than that excited at 465 nm. This dependence of the quenching on the exciting wavelength is considered to be due to the “prerelaxational” fast energy transfer. From the analysis of the quenching of the fluorescence of FMN and lumiflavin by cytochrome c excited at 465 nm, it was concluded that 1) the quenching is mainly controlled by resonance energy transfer, and 2) the heterogeneous dispersion state of molecules due to electrostatic forces makes the critical transfer distance, R ₀, of the resonance process longer than the real distance. For the quenching of the fluorescence of flavodoxin by cytochrome c, it was found that complex formation is a dominant process and is controlled to a great extent by electrostatic forces. Furthermore, fluorescence decay curves were measured by a single-photon counting method in order to estimate the dynamic processes of flavin fluorescence. The results also showed that the resonance process exists in the energy transfer between flavin and cytochrome c.     

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.     

Kinetics of Pyrene Fluorescence Quenching in the Presence of N,N-Dimethylaniline on the Surface of Titania Silica Colloids

A study was carried out on pyrene (Py) fluorescence quenching in the presence of N,N-dimethylaniline (DMA) on the surface of titania–silica colloids. Py fluorescence quenching is a function of the TiO₂ content in the binary colloids. The coadsorption of DMA molecules is accompanied by an increase in the Py fluorescence intensity due to the competitive adsorption of amine on the surface. Calculation of the formal kinetics of Py fluorescence quenching indicates the existence of two groups of sites differing in adsorption activity within each group.     

Fluorescence Quenching Study on the Interaction of Some Schiff Base Complexes with Bovine Serum Albumin

The interaction of Schiff base ligand A and its three metal complexes [A‐Fe(II), A‐Cu(II), and A‐Zn(II)] with bovine serum albumin (BSA) was investigated using a tryptophan fluorescence quenching method. The Schiff base ligand A and its three metal complexes all showed quenching of BSA fluorescence in a Tris‐HCl buffer. Quenching constants were determined for quenching BSA by the Schiff base ligand A and its metal complexes in a Tris‐HCl buffer (pH=7.4) at different temperatures. The experimental results show that the dynamic quenching constant (K_SV) was increased with increasing temperature, whereas the association constant (K) was decreased with the increase of temperature. The thermodynamic parameters ΔH, ΔG and ΔS at different temperatures were calculated. The ionic strength of the Tris‐HCl buffer had a great influence on the wavelength of maximum emission of BSA. Under low ionic strength, the emission spectra of BSA influenced by A‐Zn(II) had a small blue shift. Compared to A‐Zn(II), the emission spectra of BSA in the presence of the Schiff base ligand A and A‐Cu(II) had no significant λ_em shift. At high ionic strength, the emission spectra of BSA upon addition of the Schiff base A, A‐Fe(II), and A‐Zn(II) all had a red shift, but the emission spectra of BSA had λ_em shift neither at low ionic strength, nor at high ionic strength in the presence of A‐Cu(II). Furthermore, the temperature did not affect the λ_em shift of BSA emission spectra.     

Fluorescence Quenching Study of Zinc Bisporphyrins by Fulleropyrrolidines and Their N-Oxides

Novel phenylene-bridged zinc bisporphyrins （1-4）, fulleropyrrolidines （C60-m, C60-h） and their N-oxides （C60-mo, C60-ho） were synthesized. The fluorescence quenching processes of bisporphyrins in toluene solution by fulleropyrrolidines and their N-oxides were investigated by steady-state fluorescence spectra. The fluorescence quenching constants proved that the fluorescence quenching ability was decreased as reduction of the pyrrolidine functional groups of fullerene surface： C60-h〉C60-m〉C60, and the fluorescence quenching ability was increased about 1.3-7.4 times by utilizing fulleropyrrolidine N-oxides （C60-mo, C60-ho） compared to fulleropyrrolidine compounds （C60-m, C60-h）. The results revealed photoinduced electron transfer （PET） efficiency between bispor-phyrin and fullerene derivatives could be tunable by change of functional groups on fullerene surface.     

Enzyme Assay by Fluorescence Quenching Release: A Novel Fluorometric Method

Abstract

When concentrated fluorescent dye is encapsulated in lecithin liposomes, the fluorescence is Largely self-quenched, The quenching is relieved when the liposomes are disrupted end the escapes. The fluorescence quenching release (FQR) is shown to be proportional to the amount of phospholipase C of C. welchii which hydrolyses lecithin. The FQR method is more sensitive, rapid, and convenient than conventional titrimetric assay and is amenable to automation and Kinetic studies. As a general method, FQR could be adapted to the measurement of other enzymes or agents which disrupt dye-containing microstructures.     

Molecularly Imprinted Silica-Coated CdTe Quantum Dots for Fluorometric Determination of Trace Chloramphenicol

A dual recognition system with a fluorescence quenching of quantum dots (QDs) and specific recognition of molecularly imprinted polymer (MIP) for the detection of chloramphenicol (CAP) was constructed. MIP@SiO₂@QDs was prepared by reverse microemulsion method with 3-aminopropyltriethoxysilane (APTS), tetraethyl orthosilicate (TEOS) and QDs being used as the functional monomer, cross-linker and signal sources, respectively. MIP can specifically recognize CAP, and the fluorescence of QDs can be quenched by CAP due to the photo-induced electron transfer reaction between CAP and QDs. Thus, a method for the trace detection of CAP based on MIP@SiO₂@QDs fluorescence quenching was established. The fluorescence quenching efficiency of MIP@SiO₂@QDs displayed a desirable linear response to the concentration of CAP in the range of 1.00~4.00 × 10² μmol × L⁻¹, and the limit of detection was 0.35 μmol × L⁻¹ (3σ, n = 9). Importantly, MIP@SiO₂@QDs presented good detection selectivity owing to specific recognition for CAP, and was successfully applied to quantify CAP in lake water with the recovery ranging 102.0~104.0%, suggesting this method has the promising potential for the on-site detection of CAP in environmental waters.     

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.     

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。     

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.     

罗丹明6G缔合微粒荧光猝灭法测定痕量碘酸根

研究发现在0.01mol/LHCl-8.0×10-4mol/LKI介质中,罗丹明6G(RhG)在550nm处有1个荧光峰.当有IO-3,I-3与RhG形成缔合微粒,550nm处荧光峰猝灭,在320、400、6103存在时,IO-3与过量的I-反应生成I-nm处有3个共振散射峰,在470nm处有1个同步散射峰.碘酸根浓度在2.0～100×10-7mol/L范围内与荧光猝灭强度成线性关系.据此建立了一个测定食盐中IO-3的荧光猝灭分析法.光谱研究结果表明,(RhG-I3)n缔合微粒和界面的形成是导致体系荧光猝灭的根本原因.     

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.     

Photoinduced Electron Transfer in Host-Guest Complexes of 2-Naphthyl-O(CH2) n -adamantanamines with Mono-6-O-p-nitrobenzoyl-β-cyclodextrin and Mono-6-O-m-nitrobenzoyl-β-cyclodextrin

A number of naphthalene derivatives containing adamantanamine binding moiety and an (CH₂) _n (n=2, 3, 4, 5, 6) spacer were prepared as the electron donor. A supramolecular assembly was fabricated by the inclusion between the donor substrates and the host molecules, i.e., mono-6-O-p-nitrobenzoyl-β-cyclodextrin (pNBCD) and mono-6-O-m-nitrobenzoyl-β-cyclodextrin (mNBCD), in water. The fluorescence quenching in these systems was studied in detail. It revealed efficient photoinduced electron transfers (PET) between the naphthalene donors and the cyclodextrin acceptors. This PET process was partitioned into a dynamic quenching component caused by bimolecule collision reactions and a static quenching component due to hydrophobic binding between the donor and acceptor molecules. Detailed Stern–Volmer constants were measured and they were partitioned into dynamic Stern–Volmer quenching constants (dynamic quenching) and static binding constants (static quenching). In these two pathways, the static quenching was found to be highly efficient and dominant in the presence of NBCD.     

Bioluminophore and Flavin Mononucleotide Fluorescence Quenching of Bacterial Bioluminescence—A Theoretical Study

Bacterial bioluminescence with continuous glow has been applied to the fields of environmental toxin monitoring, drug screening, and in vivo imaging. Nonetheless, the chemical form of the bacterial bioluminophore is still a bone of contention. Flavin mononucleotide (FMN), one of the light‐emitting products, and 4a‐hydroxy‐5‐hydro flavin mononucleotide (HFOH), an intermediate of the chemical reactions, have both been assumed candidates for the light emitter because they have similar molecular structures and fluorescence wavelengths. The latter is preferred in experiments and was assigned in our previous density functional study. HFOH displays weak fluorescence in solutions, but exhibits strong bioluminescence in the bacterial luciferase. FMN shows the opposite behavior; its fluorescence is quenched when it is bound to the luciferase. This is the first example of flavin fluorescence quenching observed in bioluminescent systems and is merely an observation, both the quenching mechanism and quencher are still unclear. Based on theoretical analysis of high‐level quantum mechanics (QM), combined QM and molecular mechanics (QM/MM), and molecular dynamics (MD), this paper confirms that HFOH in its first singlet excited state is the bioluminophore of bacterial bioluminescence. More importantly, the computational results indicate that Tyr110 in the luciferase quenches the FMN fluorescence via an electron‐transfer mechanism.     

Fluorescence resonance energy transfer from tryptophan in human serum albumin to a bioactive indoloquinolizine system

The interaction between a bioactive molecule, 3-acetyl-4-oxo-6,7-dihydro-12H indolo-[2,3-a] quinolizine (AODIQ), with human serum albumin (HSA) has been studied using steady-state absorption and fluorescence techniques. A 1:1 complex formation has been established and the binding constant (K) and free energy change for the process have been reported. The AODIQ-HSA complex results in fluorescence resonance energy transfer (FRET) from the tryptophan moiety of HSA to the probe. The critical energy-transfer distance (R ₀) for FRET and the Stern-Volmer constant (K _sv) for the fluorescence quenching of the donor in the presence of the acceptor have been determined. Importantly, K _SV has been shown to be equal to the binding constant itself, implying that the fluorescence quenching arises only from the FRET process. The study suggests that the donor and the acceptor are bound to the same protein at different locations but within the quenching distance.     

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.     

Synthesis and Characterization of a Water‐Soluble Carboxylated Polyfluorene and Its Fluorescence Quenching by Cationic Quenchers and Proteins

We have developed a new intermediate monomer, 2,7‐[bis(4,4,5,5‐tetramethyl‐1,3,2‐dioxaborolan‐2‐yl)‐9,9‐bis(3‐(tert‐butyl propanoate))]fluorene, that allows the easy synthesis of water‐soluble carboxylated polyfluorenes. As an example, poly[9,9′‐bis(3′′‐propanoate)fluoren‐2,7‐yl] sodium salt was synthesized by the Suzuki coupling reaction, and the properties of the polymer were studied in aqueous solutions of different pH. Fluorescence quenching of the polymer by different cationic quenchers (MV²⁺, MV⁴⁺, and NO₂MV²⁺; MV=methyl viologen) was studied, and the quenching constants were found to be dependent on the charge and electron affinity of the quencher molecule and the pH of the medium. The largest quenching constant was observed to be 1.39×10⁸ M ^?1 for NO₂MV²⁺ at pH 7. The change in polymer fluorescence upon interaction with different proteins was also studied. Strong fluorescence quenching of the polymer was observed in the presence of cytochrome c, whereas weak quenching was observed in the presence of myoglobin and bovine serum albumin. Lysozyme quenched the polymer emission at low protein concentrations, and the quenching became saturated at high protein concentrations. Under similar experimental conditions, the polymer showed improved quenching efficiencies toward cationic quenchers and a more selective response to proteins relative to other carboxylated conjugated polymers.     

Amphiphilic copolymers with pendant ­carbazolylalkyl groups. ­Effect of polymer structure on ­retardation of self‐quenching

A series of amphiphilic polyanions containing (9‐carbazolyl)alkyl methacrylamide or acrylamide were prepared and their fluorescence properties were compared in organic and aqueous solution. In organic solution, all copolymers exhibited a monomer emission which decayed exponentially, indicating no self‐quenching. Decreasing the length of the methylene spacer in the side chain induced the spectral broadening and sharpening, depending on the carbazolyl content in the copolymer. This may be related to the interchromophore interactions and the mobility of chromophores. In H ₂O, by contrast, quenched fluorescence was observed for the copolymers. It should be however noted that acrylamide‐type of amphiphilic copolymers, poly(9‐carbazolylmethyl‐acrylamide‐co‐sodium 2‐acrylamido‐2‐methylpropanesulfonate), are the least quenched polymers among those reported so far. Copyright © 2001 John Wiley & Sons, Ltd.