Quencher Molecules Research Articles

Theory of diffusion-influenced fluorescence quenching. Effects of static quenching on the Stern-Volmer curve

A theory for describing the effects of static quenching in the diffusion-influenced fluorescence quenching is presented, which is based on a hierarchical system of many-body Smoluchowski equations for the reactant molecule distribution functions. The present theory describes the dependence of fluorescence quenching dynamics on the complex formation between the ground-state fluorophor and quencher molecules and on the intensity of external illumination within a unified framework. In the conventional Smoluchowski approach, it has been simply assumed that the intensity of illumination should be quite weak, and the influence of the complex formation has been considered only from a static viewpoint. A model calculation shows that the light intensity effect on Stern-Volmer curves should be more pronounced in the presence of static quenching.

Refined Perrin equation for the analysis of fluorescence quenching by electron transfer

Fluorescence quenching by electron transfer was studied theoretically in the case where diffusion of fluorescence and quencher molecules can be neglected. Starting from an exact expression, a simple equation was derived on the basis of physically reasonable assumptions which is similar to the Perrin equation but refines it. This equation closely follows the exact one. By using this equation, the parameters of fluorescence quenching can be obtained from experiment with reasonable accuracy.

Chiral recognition between dissymmetric Ln(dpa)33- and cobalt(III)-nucleotide complexes in aqueous solution. Enantioselective luminescence quenching as a probe of intermolecular chiral discrimination

Time-resolved chiroptical luminescence spectroscopy is used to investigate enantioselective excited-state quenching processes in solution samples that contain a racemic mixture of chiral luminophores and a small concentration of dissymmetric quencher molecules. The luminophores are Ln(dpa) 3 3- complexes (Ln≡Eu 3+ or Tb 3+ , dpa≡a dipicolinate dianion) that have tris(terdentate) chelate structures of D 3 symmetry with either left-handed (Λ) or right-handed (4) configurational chirality about the trigonal axis. The quenchers are 6-coordinate Co(III) complexes with the general stoichiometric formula Co(NH 3 ) 4 (ndp) or Co(NH 3 ) 4 (ntp), were ndp denotes a diphosphate nuceotide ligand and ntp denotes a triphosphate nucleotide ligand.

On the Mechanism of Excitation Energy Transfer Involving Long and Short Range Interaction in Dilute Organic Liquid Scintillator Systems

Tle rate parameter K3 of solvent-solute energy transfer and the rate parameter Κ7b for solvent-quencher energy transfer are determined experimentally under ultraviolet excitation for a system Ethyl 1 butyl-2-methyl-5 ethoxycarboxy methoxy indole-3-carboxylate (EBMEC) in a deoxygenated system comprising 1:9 mixture of toluene—cyclohexane as a function of temperature in the range of 20-70°C, using bromobenzene as a solvent quencher. The data is analysed in terms of compact equation formed b y combining Voltz et al. and Birks and Conte models. The interaction distance for the energy transfer from the excited solvent to solute molecules and solvent to quencher molecules are determined using this equation. The magnitude of the interaction distance indicates that the excitation energy transfer takes place due to long-range interaction in the case of solvent-solute energy transfer and short-range interaction in the case of solvent quencher energy transfer in dilute systems.

Viscosity dependence of acrylamide quenching of ribonuclease T 1 fluorescence. The gating mechanism

The structural regulation of the access of acrylamide molecules, as quenchers, to the buried tryptophans of a protein can be modelled by a simple gate concept. Such a gate, when open, allows transient exposure of the fluorophore to the quencher molecule in solution. We have previously shown that the observed viscosity dependence of acrylamide quenching process in ribonuclease T1 (RNAse T1) is not reconcilable with the gating mechanism. However, on that occasion, we neglected the effect of changes in the activity of the quencher molecule and the possible presence of static quenching. The experimental observation of a considerable contribution by static quenching and the realization that static quenching might produce dramatic effects in steady state measurements led us to reexamine the question. It is shown that in a gating model the static component can also influence the apparent dynamic quenching. In this paper, we present derived equations for the gated quenching mechanism including possible contributions from the static component. We also carefully remeasured the acrylamide quenching of RNAase T1 as a function of increasing glycerol concentration. Computer simulations were carried out to compare the experimental data set to the generalized model. We reach the conclusion that even the new, quite complex equations fail to predict the qualitative and quantitative features of the observed quenching experiments. We arrived at the conclusion that the fluorophore is never the target of the quencher molecules in solution.

Non-destructive laser technique for the measurement of viscosities in fluid flows: application to a Newtonian fluid

A new laser technique to measure the viscosities in fluid flows without disturbing the flow has been developed. Picosecond laser pulses are used for the measurement of the lifetimes of excited probe molecules as a function of added quencher molecules and the rotational relaxation times of probe molecules. As a test case, the technique has been applied to the Newtonian fluid water for a flow over a flat plate. As expected for Newtonian fluids, the viscosity has been found to be constant in the various flow regions indicating that this technique should be a reliable method for the measurement of more complex fluid flow involving non-Newtonian fluids.

Theory of diffusion-influenced fluorescence quenching: Dependence of the Stern-Volmer curve on light intensity

A theory for describing the effects of quencher molecules on the intensity of fluorescence stimulated by steady-state illumination is presented, which is based on a hierarchical system of many-body Smoluchowski equations for the reactant molecule distribution functions. An important aspect of the present theory is that it describes the dependence of fluorescence quenching dynamics on the intensity of external illumination. In the conventional Smoluchowski approach it has been simply assumed that the intensity of illumination should be quite weak. Hence the validity of the present theory may be tested by a fluorescence quenching experiment under intense illumination.

Organization and dynamics of pyrene and pyrene lipids in intact lipid bilayers. Photo-induced charge transfer processes

The dynamics of fluorescence quenching and the organization of a series of pyrene derivatives anchored in various depths in bilayers of phosphatidylcholine small unilamellar vesicles was studied and compared with their behavior in homogeneous solvent systems. The studies include characterization of the environmental polarity of the pyrene fluorophore based on its vibronic peaks, as well as the interaction with three collisional quenchers: the two membrane-soluble quenchers, diethylaniline and bromobenzene, and the water soluble quencher potassium iodide. The system of diethylaniline-pyrene derivatives in the membrane of phosphatidylcholine vesicles was characterized in detail. The diethylaniline partition coefficient between the lipid bilayers and the buffer is approximately 5,800. Up to a diethylaniline/phospholipid mole ratio of 1:3 the perturbation to membrane structure is minimal so that all photophysical studies were performed below this mole ratio. The quenching reaction, in all cases, was shown to take place in the lipid bilayer interior and the relative quenching efficiencies of the various probe molecules was used to provide information on the distribution of both fluorescent probes and quencher molecules in the lipid bilayer. The quenching efficiency by diethylaniline in the lipid bilayer was found to be essentially independent on the length of the methylene chain of the pyrene moiety. These findings suggest that the quenching process, being a diffusion controlled reaction, is determined by the mobility of the diethylaniline quencher (with an effective diffusion coefficient D approximately 10(-7) cm2 s-1) which appears to be homogeneously distributed throughout the lipid bilayer. The pulsed laser photolysis products of the charge-transfer quenching reaction were examined. No exciplex (excited-complex) formation was observed and the yield of the separated radical ions was shown to be tenfold smaller than in homogenous polar solutions. The decay of the radical ions is considerably faster than the corresponding process in homogenous solutions. Relatively high intersystem crossing yields are observed. The results are explained on the basis of the intrinsic properties of a lipid bilayer, primarily, its rigid spatial organization. It is suggested that such properties favor ion-pair formation over exciplex generation. They also enhance primary geminate recombination of initially formed (solvent-shared) ion pairs. Triplet states are generated via secondary geminate recombination of ion pairs in the membrane interior. The results bear on the general mechanism of electron transfer processes in biomembranes.

Energy dependence of the relaxation of highly excited NO2 donors under single collision conditions: Vibrational and rotational state dependence and translational recoil of CO2 quencher molecules

The rotational state distribution and velocity recoil of CO2 bath molecules excited to the asymmetric stretch (ν3=2349 cm−1) vibrational level by collisions with high energy NO2 molecules have been determined. The vibrational excitation process occurs with very little rotational or translational excitation of the CO2 and its probability is independent of NO2 internal energy over the range 22 200–16 600 cm−1.

Chiral discrimination in electronic energy-transfer processes between dissymmetric metal complexes in solution. Time-resolved chiroptical luminescence measurements of enantioselective excited-state quenching kinetics

Steady-state and time-resolved chiroptical luminescence measurements are used to investigate enantioselective excited-state quenching processes in solution. Theory and measurement methodology are presented for the case in which an initially racemic excited-state population of chiral luminophores evolves to a nonracemic population in the presence of chiral quencher molecules. Generation of enantiomeric excess in the luminophore excited-state population produces differential (spontaneous) emission of left and right circularly polarized light, and the time dependence of this chiroptical luminescence provides a direct measure of the differential excited-state quenching kinetics associated with homochiral versus heterochiral luminophore-quencher interactions. Experimental results are presented for a series of systems in which the luminophores are dissymmetric, tris(terdentate) lanthanide complexes and the quenchers are dissymmetric, tris(bidentate) transition-metal complexes.

Ligand-dependent quenching of tryptophan fluorescence in human erythrocyte hexose transport protein

D-Glucose transport by the 492-residue human erythrocyte hexose transport protein may involve ligand-mediated conformational/positional changes. To examine this possibility, hydrophilic quencher molecules [potassium iodide and acrylamide (ACR)] were used to monitor the quenching of the total protein intrinsic fluorescence exhibited by the six protein tryptophan (Trp) residues in the presence and absence of substrate D-glucose, and in the presence of the inhibitors maltose and cytochalasin B. Protein fluorescence was found to be quenched under various conditions, ca. 14-24% by KI and ca. 25-33% by ACR, indicating that the bulk of the Trp residue population occurs in normally inaccessible hydrophobic regions of the erythrocyte membrane. However, in the presence of D-glucose, quenching by KI and ACR decreased an average of -3.4% and -4.4%, respectively; Stern-Volmer plots displayed decreased slopes in the presence of D-glucose, confirming the relatively reduced quenching. In contrast, quenching efficiency increased in the presence of maltose (+5.9%, +3.3%), while addition of cytochalasin B had no effect on fluorescence quenching. The overall results are interpreted in terms of ligand-activated movement of an initially aqueous-located protein segment containing a Trp residue into, or toward, the cellular membrane. Relocation of this segment, in effect, opens the D-glucose channel; maltose and cytochalasin B would thus inhibit transport by mechanisms which block this positional change. Conformational and hydropathy analyses suggested that the region surrounding Trp-388 is an optimal "dynamic segment" which, in response to ligand activation, could undergo the experimentally deduced aqueous/membrane domain transfer.

Quenching of fluorescence of pyrene-substituted lecithin by tetracyanoquinodimethane in liposomes

In this work we have applied a kinetic scheme derived from fluorescence kinetics of pyrene-labeled phosphatidylcholine in phosphatidylcholine membrane to explain the fluorescence quenching of 1-palmitoyl-2-(10-[pyrenl-yl]-sn-glycerol-3-phosphatidylchol ine (PPDPC) liposomes by tetracyanoquinodimethane (TCNQ). The scheme was also found to be applicable to neat PPDPC and the effect of the quencher could be attributed to certain steps of the proposed mechanism. The TCNQ molecules influence the fluorescence of pyrene moieties in PPDPC liposome in two ways. Firstly, an interaction between the quencher molecule and the pyrene monomer in the excited state quenches monomer fluorescence and effectively prevents the diffusional formation of the excimer. Secondly, an interaction between the quencher molecule and the excited dimer quenches the excimer fluorescence. The TCNQ molecule does not prevent the formation of the excimer in pyrene moieties aggregated in such a way that they require only a small rotational motion to attain excimer configuration. The diffusional quenching rate constant is calculated to be 1.0 x 10(8) M-1 s-1 for the pyrene monomer quenching and 1.3 x 10(7) M-1 s-1 for the pyrene excimer quenching. The diffusion constant of TCNQ is 1.5 x 10(-7) cm2 s-1 for the interaction radii of 0.8-0.9 nm. The TCNQ molecules are practically totally partitioned in the membrane phase.

Simultaneous enhancement of fluorescence and thermal lensing by reversed micelles

ABSTRACT Aerosol-OT (AOT) reversed micelles have been used to simultaneously enhance the fluores­ cence as well as the thermal lens effect in the analysis of solubilized lanthanide ions. The fluorescence is enhanced because the reversed micelles isolate and protect the analyte from quencher molecules while the thermal lens enhancement is due to the modification of the thermal physical properties of the solvent, i.e., increased dn/dT and decreased thermal conductivity values. Compared to that in water, AOT/CCla reversed micelles increased fluorescence and thermal lens enhancement factors of Tb3* by factors of 2 and 38, respec­ tively. The mechanism for the fluorescence and thermal lens enhancement is discussed. 1. INTRODUCTION Fluorescence and thermal lens are often used in trace chemical analysis1 4. The fluo­ rescence technique is based on the measurement of emitted photons of an excited analyte. It is, therefore, particularly well suited for the detection of substances which have high fluorescence quantum yields. Conversely, thermal lens technique is based on the measurement of the heat generated by the nonradiative relaxation of an excited analyte. It has been successfully employed for trace detection of nonf1uorescent substances. Thus, the two techniques are complementary and have been used to determine fluorescent as well as non- fluorescent substances at concentrations as low as 10~11 M2~5.Fluorescence can be enhanced by increasing the radiative processes of a molecule. In particular, isolation of the fluorescence molecule from quenching impurities and/or solvent molecules can be used to achieve this enhancement.The thermal lens technique is based on the temperature rise that is produced in an illu­ minated sample by nonradiative relaxation of the energy absorbed from a laser. Thus, its intensity can be enhanced by improving the nonradiative relaxation processes of the analyte and more significantly the thermal physical properties of the solvent. When a modulated laser beam, which has a Gaussian intensity distribution is used to excite the sample, the heat generated will be strongest at the center of the beam since this is where the beam intensity is greatest. Consequently, a lens-like optical element is formed in the sample due to the temperature gradient between the center of the beam and the bulk sample. The effect of the thermal lens is generally measured as a relative change in the beam center intensity, Albc/Ibc , in the far field. When a sample is located 3l'2 Zc beyond the beam waist, this change is given by:A*bc V

Photoluminescence and photocatalytic activity of MgO powders

Photoluminescence properties and photocatalysed isomerization reaction of 2-butenes have been investigated using a standard JRC-MgO-I sample. The effects of the degassing temperature of the MgO sample and of the added quencher molecules such as O 2, CO, and H 2 indicated that the photoluminescence with a lifetime of 10 −4 sec is the radiative decay process from the charge transfer excited complex, (Mg LC + − O LC − ) ★ associated with the lower co-ordinated surface ions. It has been found that U.V. irradiation of degassed MgO samples in the presence of 2-butenes leads catalytically to trans — cis geometrical isomerization reaction with high efficiency, together with less efficient double bond shift isomerization reaction. The addition of CO or O 2 led to the inhibition of this photocatalysed isomerization as well as the photoluminescence of MgO samples. A complete parallel between the intensity of photoluminescence and the rate of photocatalysed isomerization of butenes indicated that the lower co-ordinated surface ions play a significant role in the photocatalytic activity of degassed MgO powders.

Benzylidene fluorene ozonolysis under chemiluminescence mimetic conditions in homogeneous and micellar media II. Rate constants of fluorenone quenching by the leaving moiety

The rate constants for the quenching of the fluorenone fluorescence by substituted benzaldehydes and benzoic acids (substitutents: p-nitro, p-cyano, p-chloro, p-bromo, p-H, p-methyl, p-methoxy, p-methoxy- m-methyl, and p-dimethylamino) are reported in ethanolic and micellar media. These are compared with the diffusion rate constant and evidence is presented that, under chemiluminescence mimetic conditions, the quenching leaving moiety in close proximity to the emitter is responsible for most of the quenching observed. The binding constants and partition coefficients of the above compounds as well as those of the corresponding substituted 9-benzylidene fluorenes in cetyltrimethylammonium chloride are also reported together with quencher molecules per micelle. The influence of the micellar medium on the quenching rate constants is discussed and the position of the above compounds inside the micelle is reported.

Computer programs for the analysis of protein fluorescence quenching data

Educational computer programs for analysis of fluorescence quenching studies of proteins have been developed. The program is written according to the classical Stern-Volmer equation and some modified Stern-Volmer equations. The static and dynamic quenching constants as well as the accessibility of the quencher molecules to the fluorescence groups are calculated. The experimental data are plotted on a high resolution graph. The calculated data or the graphs can be printed out to obtain a hard copy for filing.

Fluorescence quenching studies of apolipoprotein A-I in solution and in lipid-protein complexes: protein dynamics.

Fluorescence lifetime and intensity quenching studies of human plasma apolipoprotein A-I (apo A-I) in aqueous solution and in recombinant lipoprotein complexes with dimyristoylphosphatidylcholine (DMPC) indicate differences in conformational dynamics. In aqueous solution, the bimolecular quenching constants (k*) for lipid-free apo A-I fluorescence quenching by oxygen and acrylamide are 2.4 X 10(9) and 0.38 X 10(9) M-1 s-1, respectively. These values are independent of the oligomeric form of the protein. There is no correlation between the relatively small k* for apo A-I, which reflects rapid, low-amplitude protein fluctuations, and the labile conformational changes of apo A-I folding reactions, like denaturation, which occur on a slower time scale. In recombinant DMPC/apo A-I complexes (100:1 molar ratio) the protein increases in amphiphilic alpha-helical structure as it blankets the lipid matrix. The apparent k* for oxygen quenching of apo A-I fluorescence in the complex is large and increases in a temperature-dependent manner. We have introduced a two-compartment model, which discriminates the source of quencher molecules as aqueous or lipid, to describe oxygen quenching of DMPC/apo A-I fluorescence. The magnitude and temperature dependence of the apparent k* predominantly reflect the partitioning of oxygen between the two phases rather than being a probe of the lipid physical state. Calculations of the helical hydrophobic moment in apo A-I indicate that tryptophan residues 8 and 72 occur at the lipid-protein interface of amphiphilic alpha-helices, whereas the other two tryptophan residues (50, 108) lie on the nonpolar faces of amphiphilic helices.(ABSTRACT TRUNCATED AT 250 WORDS)

NH A 3Πi quenching at 1400 K

We have measured electronic quenching rate constants, kQ, in the NH A 3Πi, v′=0 state for several quencher molecules at 1400 K. The NH radicals were produced in a laser pyrolysis apparatus when a mixture of SF6, CF4, NH3, and added quencher was irradiated by a CO2 laser. The ground state NH radicals were then excited to the A state by a pulsed tunable dye laser. The fluorescence decay rates as a function of added quencher pressure yielded the rate constants. The thermally averaged cross sections, σQ=kQ/〈v〉, for NH at 1400 K are all smaller than those observed for OH at 1200 K in this laboratory. In most cases, our high temperature σQ’s are the same as those observed elsewhere at room temperature. These experimental σQ’s are all less than those calculated with an attractive forces ‘‘collision complex’’ model.

Untersuchungen zu den Sondeneigenschaften von 7‐Hydroxy‐cumarinen in micellarer Cetyl‐triethyl‐ammoniumbromid‐Lösung – Hilfsmittel zur Beschreibung von micellaren Systemen

Investigation on Probe Properties of 7‐Hydroxy‐coumarines in Micellar Cetyl‐triethyl‐ammoniumbromide Solution – Means for Characterization of Micellar SystemsAnions of 3‐substituted 7‐hydroxy‐coumarines (1a–c) as fluorescence probes have been studied in this work. Acid‐base equilibria in water and micellar solution of cetyl‐triethylammoniumbromide (cmc = 0,72 mmol l−1), fluorescence properties and fluorescence quenching by octacyano‐molybdate‐IV have been investigated. It has been confirmed that probe and quencher are completely micellized. Fluorescence life times and quenching constants per one quencher molecule in the micell of about 108 s−1 have been determined. The kinetics of saponification of the probe 3‐acetyl‐7‐hydroxy‐coumarine at pH = 12 in micellar solution has been followed by probe fluorescence.

Interactions of excited-state porphyrin–quinone in reversed micelles studied by time-resolved fluorescence spectroscopy

Interactions in reversed micelles of benzyldimethyl-n-hexadecylammonium chloride (BHDC) between excited-state porphyrin (ZnTPP) and quinones [duroquinone (DQ) and anthraquinone-2-sulphonate (AQS)] have been investigated by time-resolved spectroscopy. Essentially dynamic quenching is observed when the quencher DQ is solubilised in the organic phase, although this is affected by the distribution of quencher molecules at the micellar interface.When the quencher (AQS) is bound at the interface, the decay of the singlet excited state of porphyrin (ZnTPP) also bound at the interface in the absence of water (ω0=[H2O]/[surf]= 0) shows two components: one with a mean lifetime 〈τ〉≈ 1 ns, which corresponds to unquenched porphyrin, and a much shorter 〈τ〉= 100 ps, which accounts for the decay of quenched porphyrin. For quencher concentrations in the range of [AQS]= 1–4 mmol dm–3 the observed decay data may be interpreted in terms of a static active-sphere quenching model, where only bound quencher molecules within a certain volume around the bound porphyrin molecule lead to quenching. The lifetimes obtained in these studies indicate a pseudounimolecular electron-transfer rate constant for P*i⋯Qi of the order of 1010 s–1.