Distance-Dependent Fluorescence Quenching ofp-Bis[2-(5-phenyloxazolyl)]benzene by Various Quenchers

Abstract

We report results of frequency-domain and steady-state measurements of the fluorescence quenching of p-bis-[2-(5-phenyloxazolyl)]benzene (POPOP) when quenched by bromoform (CHBr 3 ), methyl iodide (CH 3 I), potassium iodide (KI), 1,2,4-trimethoxybenzene (TMB), or N,N-diethylaniline (DEA). The quenching efficiency of these compounds decreased in the order DEA, TMB, KI, CH 3 I, CHBr 3 . In the case of DEA and TMB the measurements clearly confirm the applicability of the exponential distance-dependent quenching (DDQ) model, in which the bimolecular quenching rate k(r) depends exponentially on the fluorophore-quencher separation r, k(r) ) k a exp[-(r -a)/r e ], where a is the distance of closest approach. Simultaneous analysis of the frequency-domain and steady-state data significantly improved resolution of the recovered molecular parameters k a and r e . The data for DEA and TMB cannot be satisfactorily fit using either the Smoluchowski or CollinsKimball radiation boundary condition (RBC) model. The quenching behavior of the less efficient quenchers KI, CH 3 I, and CHBr 3 can be adequately described with both the DDQ and RBC models, but this may be a simple consequence of less efficient quenching. The efficiency of quenching is discussed on the basis of the mechanisms of interaction between the fluorophore and quencher molecules, which involves electron transfer and/or heavy atom effects. IntroductionCollisional quenching of fluorescence has been widely used in physical chemistry and biochemistry. Fluorescence quenching of aromatic and heteroaromatic hydrocarbons by aromatic amines, 1-13 aromatic nitriles, 14 haloalkanes, 15-24 and halide ions 25-27 as well as by nitroxides 28,29 and oxygen 30,31 has been studied to determine the bimolecular rate constants, to characterize the solvent effects on quenching, and to determine the mechanisms of interaction between the fluorophore and quencher molecules. Moreover, collisional quenching of tryptophan fluorescence by a variety of ionic and neutral quenchers has been used to study the structure and dynamics of proteins 32-40 and membranes. 41,42 Collisional or dynamic quenching requires contact between the fluorophore and quencher molecules during the lifetime of the excited state. The quenching data can reveal information about accessibility of fluorophores in macromolecules to externally added quenchers and the diffusion of quenchers within proteins and membranes.For many fluorophores in solution, including aromatic and heteroaromatic fluorophores, the intensity decays are often monoexponential. 43 However, in the presence of collisional quenching the monoexponential intensity decays become nonexponential due to transient effects in diffusion. 21,[44][45][46] This effect in collisional quenching of fluorescence is due to a rapid decay of closely spaced fluorophore-quencher pairs, followed by slower diffusion-limited quenching of remaining fluorophores. These effects can be readily detected by using frequency-domain fluorometry. [47][48][49] We have shown for several fl...

p-Cyanophenylalanine and selenomethionine constitute a useful fluorophore–quencher pair for short distance measurements: application to polyproline peptides

“…As there is no overlap between the fluorescence emission spectrum of Phe CN (Figure 1) and the absorption spectrum of SeMet (Figure S1), a prerequisite for FRET to occur, 26 we do not believe that the quenching of the Phe CN fluorescence by SeMet is through the mechanism of FRET. Furthermore, we tentatively rule out the possibility of the heavy atom effect, which is known to increase the intersystem crossing rate of the fluorophore, 41 as no detectable triplet state formation was observed for Phe CN when a high concentration of SeMet (8 mM) was present (Figure S2). Thus, most likely SeMet quenches the Phe CN fluorescence through an electron transfer mechanism.…”

“…To solve for k 0 and β, we numerically fit the Stern-Volmer curve based on the methods described in the literature. 26,37–41 Briefly, the fluorescence decay signal, I ( t ), is described as: $$Ifalse(tfalse)=I_0\text{exp}true(-\frac{1}{{\tau }_0}-C_{\mathrm{Q}}^0{\int }_0^{t}kfalse(t\text{'}false)dt\text{'}true),$$ where τ 0 is the intrinsic fluorescence lifetime of the fluorophore, $C_{\mathrm{Q}}^0$ is the bulk concentration of the quencher, and time-dependent quenching rate, k ( t ), is calculated based on the following equation: $$kfalse(tfalse)=\frac{4\pi }{C_{\mathrm{Q}}^0}{\int }_{a_0}^{\infty }{r}^2{k}_{\mathrm{Q}}false(rfalse)C_{\mathrm{Q}}false(r,tfalse)dr,$$ where C Q ( r , t ) is the quencher concentration at a distance r and time t , which is governed by the rate of diffusion and the rate of quenching, given by: $$\frac{\partial \mathrm{normaly}false(r,tfalse)}{\partial t}=-D{\nabla }^2\mathrm{normaly}false(r,tfalse)-k_{\mathrm{Q}}false(rfalse)\mathrm{normaly}false(r,tfalse),$$ A normalized concentration of quencher, y( r,t ) was defined to simplify the calculation: $$\mathrm{normaly}false(r,tfalse)=C_{\mathrm{Q}}false(r,tfalse)/C_{\mathrm{Q}}^0,$$ Using appropriate initial conditions and boundary conditions, $$\mathrm{normaly}false(r,t=0false)=1,$$ $${true(\frac{\partial \mathrm{normaly}false(r,tfalse)}{\partial t}true)}_{r=a_0}=0,$$ $${\text{lim}}_{r\to \infty }\mathrm{normaly}false(r,tfalse)=...$$…”

“…These parameters are comparable with values obtained for other distance-dependent quenching processes. 36,41–42 For example, for a fixed value of a 0 = 8.0 Å, k 0 and β were determined to be 60 ns −1 and 1.3 Å −1 , respectively, for the fluorescence quenching of p-Bis[2-(5-phenyloxazolyl)]benzene (POPOP) by 1,2,4-Trimethoxybenzene. 41 …”

Analysis of fluorescence quenching of pyronin B and pyronin Y by molecular oxygen in aqueous solution

“…Fluorescence quenching of many aromatic molecules by several molecules in different media has been studied to elucidate the possible mechanisms for the deactivation process [6][7][8]. For this purpose, haloalkanes, halides, molecular oxygen, aromatic and aliphatic amines have been used as quenchers of aromatic hydrocarbons and their derivatives [9][10][11][12][13].…”

Second-harmonic imaging microscopy of living cells

“…͑9͒ A drawback of the ratiometric method described above is that the extent of SHG enhancement and fluorescence quenching due to the gold particle cannot be directly separated. It is well known that fluorescence quenching due to metals is highly distance dependent 47 and that there can also exist a fluorescence enhancement due to surface plasmon resonance. 48 To account for these uncertainties, the fluorescence lifetime was measured for both the normal Di-2-ANEPPA and the dye/nanoparticle conjugate.…”