MECHANISMS OF QUENCHING OF THE FLUORESCENCE OF A BENZO[a]PYRENE TETRAOL METABOLITE MODEL COMPOUND BY 2'-DEOXYNUCLEOSIDES

Abstract— The hydrophobic interactions of bulky polycyclic aromatic hydrocarbons with nucleic acid bases and the formation of noncovalent complexes with DNA are important in the expressions of the mutagenic and carcinogenic potentials of this class of compounds. The fluorescence of the polycyclic aromatic residues can be employed as a probe of these interactions. In this work, the interactions of the (+)-trans stereoisomer of the tetraol 7,8,9,10-tetrahydroxytetrahydrobenzo[a]pyrene (BPT), a hydrolysis product of a highly mutagenic and carcinogenic diol epoxide derivative of benzo[a]pyrene, were studied with 2′-deoxynucleosides in aqueous solution by fluorescence and UV spectroscopic techniques. Ground-state complexes between BPT and the purine derivatives 2′-deoxyguanosine (dG), 2′-deoxyadenosine (dA), and 2′-deoxyinosine (dI) are formed with association constants in the range of ～40–130 M^?1 Complex formation with the pyrimidine derivatives 2′-deoxythymidine (dT), 2′-deoxycytidine (dC), and 2′-deoxyuridine (dU) is significantly weaker. Whereas dG is a strong quencher of the fluorescence of BPT by both static and dynamic mechanisms (dynamic quenching rate constant k_dyn= [2.5 ± 0.41 × 10⁹M¹ s ¹, which is close to the estimated diffusion-controlled value of ～ 5 × 10⁹M^{? 1} s^?1), both dA and dI are weak quenchers and form fluorescenceemitting complexes with BPT. The pyrimidine derivatives dC, dU, and dT are efficient dynamic fluorescence quenchers (K_dyn～ [1.5–3.0] × 10⁹M^?1 s^?1), with a small static quenching component due to complex formation evident only in the case of dT. None of the four nucleosidcs dG, dA, dC and dT are dynamic quenchers of BPT in the triplet excited state; the observed lower yields of triplets are attributed to the quenching of single excited states of BPT by 2′-deoxynucleosides without passing through the triplet manifold of BPT. Possible fluorescence quenching mechanisms involving photoinduced electron transfer are discussed. The strong quenching of the fluorescence of BPT by dG, dC and dT accounts for the low fluorescence yields of BPT-native DNA and of pyrene-DNA complexes.     

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）.     

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.     

CHLOROPHYLL a FLUORESCENCE OF GONYAULAX POLYEDRA GROWN ON A LIGHT-DARK CYCLE AND AFTER TRANSFER TO CONSTANT LIGHT

Abstract— The chlorophyll a fluorescence properties of Gonyaulax polyedra cells before and after transfer from a lightdark cycle (LD) to constant dim light (LL) were investigated. The latter display a faster fluorescence transient from the level ‘I’ (intermediary peak) to ‘D’ (dip) to ‘P’ (peak) than the former (3 s as compared to 10 s), and a different pattern of decline in fluorescence from ‘I’ to ‘D’ and from ‘P’ to the steady state level with no clearly separable second wave of slow fluorescence change, referred to as ‘s' (quasi steady state)→‘M’ (maximum) →‘T’ (terminal steady state). The above differences are constant features of cells in LD and LL, and are not dependent on the time of day. They are interpreted as evidence for a greater ratio of photosystem II/photosystem I activity in cells in LL. After an initial photoadaptive response following transfer from LD to LL, the cell absorbance at room temperature and fluorescence emission spectra at 77 K for cells in LL and LD are comparable. The major emission peak is at 685–688 nm (from an antenna Chl a 680, perhaps Chl a-c complex), but, unlike higher plants and other algae, the emission bands at 696–698 nm (from Chl a_II complex, Chl a 685, close to reaction center II) and 710–720 nm (from Chl a₁, complexes, Chl a 695, close to reaction center I) are very minor and could be observed only in the fluorescence emission difference spectra of LL minus LD cells and in the ratio spectra of DCMU-treated to non-treated cells. Comparison of emission spectra of cells in LL and LD suggested that, in LL, there is a slightly greater net excitation energy transfer from the light-harvesting peridinin-Chl a (Chl a 670) complex, fluorescing at 675 nm, to the other antenna chlorophyll a complex fluorescing at 685–688 nm, and from the Chl a., complex to the reaction center II. Comparison of excitation spectra of fluorescence of LL and LD cells, in the presence of DCMU, confirmed that cells in LL transfer energy more extensively from the peridinin-Chl a complex to other Chl a complexes than do cells in LD.     

Dipole-dipole transfer between acetone solvates of chlorophyll a and chlorophyll a dihydrate dimers in water/acetone mixtures. A model for P680 sensitized excitation

We describe a simple model for P680 sensitized excitation in photosynthesis. Chl a fluorescence quenching effects observed when water is added to Chl a solutions in acetone are shown to be the result of resonant transfer between acetone solvates of monomeric Chl a, Chl a·Ac, and dimers of Chl a dihydrate. The presence of (Chl a·2H₂O)₂ is evidenced by a 678 nm difference absorbance (ΔA band obtained on conversion of a 680 nm absorption shoulder to polycrystalline Chl a precipitate, (Chl a·H₂O)_n. The equilibration between (Chl a·2H₂O)₂ and Chl a·Ac as a principal mechanism for Chl a·Ac fluorescence quenching is supported by theoretical fits of the data.     

Über das «pK» elektronisch angeregter Azulenium-Kationen

It is shown that azulenium cations in the first electronically excited state S₁ are stronger acids than in the ground state S₀. Their apparent pK″*, obtained from the H₀-dependence of the quenching of the azulenium cation S₁ → S₀ fluorescence does not correspond to a true acid-base equilibrium in the electronically excited state S₁. The pK″* values are kinetically controlled, the rate of reprotonation of azulene in the S₁ state being too low to compete with the internal conversion to S₀.     

Theoretical Study of Fluorescence Spectra Utilizing the pKa Values of Acids in Their Excited States

Assignment of the fluorescence spectrum of firefly luciferin in aqueous solutions was achieved by utilizing not only emission energies but also theoretical absorption spectra and relative concentrations as estimated by pK_a values. Calculated Gibbs free energies were utilized to estimate pK_a values. These pK_a values were then corrected by employing the experimental results. It was previously thought that the main peak near 550 nm observed in the experimental fluorescence spectra at all pH values corresponds to emission from the first excited state of the luciferin dianion [Ando et al. (2010) Jpn. J. Appl. Phys. 49, 117002–117008]. However, we found that the peak near 550 nm at low pH corresponds to emission from the first excited state of the phenolate monoanion of luciferin. Furthermore, we found that the causes of the red fluorescence at pH 1–2 are not only the emission from phenol monoanion but also the emission from the protonated species at nitrogen atom in the thiazoline ring of dianion.     

The cation binding of benzo crown ethers in acetonitrile using fluorescence spectroscopy

The effects of Na⁺, K⁺ and Li⁺ cations on the fluorescence spectra of benzo[15]crown-5, benzo[18]crown-6 and dibenzo[18]crown-6 were investigated in acetonitrile. The alkali cation role observed was usually the complexation-enhanced quenching fluorescence effect (CEQF) in acetonitrile due to the increased fluorescence quenching rate of the complexed fluoroionophore. The association constants for 1 :1 stoichiometry InK _a have been obtained using the relationship 1/K _a[L ₀] = (1 –P)²/P. It was shown that the preferential interaction rule of compatibility of cationic radii and macrocyclic ring size is in excellent agreement with the association constants obtained by fluorescence spectroscopy. The order of InK _a found for benzo[15]crown-5 complexation was Li⁺ > Na⁺ > K⁺ and K⁺ > Na⁺ > Li⁺ for benzo[18]crown-6 in acetonitrile.Presented at the Sixth International Seminar on Inclusion Compounds, Istanbul, Turkey, 27–31 August, 1995.     

一种新型pH荧光探针：中位-吡啶取代的硼二吡咯亚甲基(BDP)染料的合成、结构与光谱性质研究

本文报道了一种新型中位-吡啶取代的硼-二吡咯亚甲基(BDP)染料的合成与晶体结构表征,发现中位-吡啶环与因达省平面几乎正交,其二面角为88.2°。同时对它的吸收和稳态荧光性质研究表明：当溶液的酸性增强时,化合物的荧光减弱,pK_a为2.24。该化合物在可见光激发下,可以作为比较灵敏的pH荧光探针。     

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.     

EFFECT OF THE BROMINE ATOM UPON THE DISSOCIATION CONSTANTS OF BROMOPYRIDINES,BROMOQUINOLINES AND 4-BROMOISOQUINOLINE IN THEIR LOWEST EXCITED SINGLET AND TRIPLET STATES*

Abstract— The lowest excited singlet-state dissociation constants (pK^S_a) of bromosubstituted pyridines, quinolines, and isoquinolines were determined from the pH-dependent shifts in their electronic absorption spectra. The lowest excited triplet-state dissociation constants (pK^T_a) of bromosubstituted quinolines and 4-bromoisoquinoline were obtained from the shifts of the 0–0 phosphorescence bands measured in rigid aqueous solution at 77 K. The pK^S_a values indicate that the basicity of these brominated nitrogen heterocycles is increased in the lowest excited singlet state by 2 to 10 orders of magnitude as compared with the ground state. The pK^T_a values are found to be significantly different from the corresponding ground-state pK_a values, indicating that the basicity of bromoquinolines is increased in the lowest excited triplet state by 1.7 to 3.0 pK units. The enhancement of the excited singlet-and triplet-state basicity of brominated nitrogen heterocycle derivatives as compared with the unsuhstituted parent compounds is attributed to the increased electron-donor conjugative interactions of the bromine atom pπ orbitals with π orbitals in the lowest excited singlet and triplet state.     

CHLOROPHYLL PHOTOCHEMISTRY IN CONDENSED MEDIA—II. TRIPLET STATE QUENCHING AND ELECTRON TRANSFER TO QUINONE IN LIPOSOMES*

The fate of excitation energy and electron transfer to quinones within Chl-a-containing phosphatidyl choline liposomes has been investigated. The bilayer membrane of the liposome stabilizes the Chl triplet state, as evidenced by a three-fold increase in the lifetime over that observed in ethanol solution. The relative triplet yield follows the relative fluorescence yield, indicative of quenching at the singlet level. Triplet state lifetimes are markedly shortened as the Chl concentration is increased, demonstrating that quenching occurs at the triplet level as well. This process is shown to be due to a collisional de-excitation. In the presence of quinones, the Chl triplet reduces the quinone resulting in production of long-lived electron transfer products. The percent conversion of Chl triplet to cation radical when benzoquinone is employed as acceptor is approximately 60 ± 10%, which is slightly less than in ethanol solution (70 ± 10%). The lifetime of the radical, however, can be as much as 1900 times longer. With respect to potentially useful photochemical energy conversion, the magnitude of this increased lifetime is far more significant than is the decreased radical yield.     

Electron Transfer Fluorescence Quenching of Blepharisma japonicum Photoreceptor Pigments

Abstract— The hypericin analogs blepharismin (BP), oxyblepharismin (OxyBP) and stentorin (ST), the photosensing chromophores responsible for photomotile reactions in the ciliates Blepharisma japonicum (red and blue cells) and Stentor coeruleus, represent a new class of photoreceptor pigments whose chemical structures have recently been determined. In the case of ST it has been shown that the first excited singlet state can be deactivated by donation of an electron to an appropriate acceptor molecule (e.g. a quinone molecule). This charge transfer can be considered a possible mechanism for the primary photoprocess for the photomotile responses in S. coeruleus. To determine whether an electron transfer process also occurs in the deactivation of excited blepharismin, we studied the fluorescence quenching of OxyBP in dimethyl-sulfoxide (DMSO) and in ethanol using electron acceptors with different reduction potentials. Under our experimental conditions ground state and excited state complexes (like fluorescent exciplexes) are not formed between the fluorophore and the quenchers. In DMSO the bimolecular quenching constant values (k_q) calculated on the basis of the best fitting procedures clearly show that the quenching efficiency decreases with the quencher negative reduction potential, E⁰. The k_q (M^-1 s^-1) and E⁰ (V) values are, respectively, 7.8 times 10⁹ and -0.134 for 1,4-benzoquinone, 8.9 times 10⁹ and -0.309 for 1,4-naphthoquinone, 2.4 times 10⁹ and -0.8 for nitrobenzene, 0.009 times 10⁹ and -1.022 for azobenzene and 0 and -1.448 for benzophenone. These findings point to the conclusion that upon formation of the encounter complex between OxyBP and the quencher, an electron is released from excited OxyBP to the quencher, similar to what happens in ST. It is suggested that in the pigment granules such a light-induced charge transfer from excited blepharismin to a suitable electron acceptor triggers sensory transduction processes in B. japonicum.     

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.     

Quantifying Protein-Fatty Acid Interactions Using Electrospray Ionization Mass Spectrometry

The application of the direct electrospray ionization mass spectrometry (ESI-MS) assay to quantify interactions between bovine β-lactoglobulin (Lg) and a series of fatty acids (FA), CH₃(CH₂)_xCOOH, where x = 6 (caprylic acid, CpA), 8 (capric acid, CA), 10 (lauric acid, LA), 12 (myristic acid, MA), 14 (palmitic acid, PA) and 16 (stearic acid, SA), is described. Control ESI-MS binding measurements performed on the Lg-PA interaction revealed that both the protonated and deprotonated gas phase ions of the (Lg + PA) complex are prone to dissociate in the ion source, which leads to artificially small association constants (K _a ). The addition of imidazole, a stabilizing solution additive, at high concentration (10 mM) increased the relative abundance of (Lg + PA) complex measured by ESI-MS in both positive and negative ion modes. The K _a value measured in negative ion mode and using sampling conditions that minimize in-source dissociation is in good agreement with a value determined using a competitive fluorescence assay. The K _a values measured by ESI-MS for the Lg interactions with MA and SA are also consistent with values expected based on the fluorescence measurements. However, the K _a values measured using optimal sampling conditions in positive ion mode are significantly lower than those measured in negative ion mode for all of the FAs investigated. It is concluded that the protonated gaseous ions of the (Lg + FA) complexes are kinetically less stable than the deprotonated ions. In-source dissociation was significant for the complexes of Lg with the shorter FAs (CpA, CA, and LA) in both modes and, in the case of CpA, no binding could be detected by ESI-MS. The affinities of Lg for CpA, CA, and LA determined using the reference ligand ESI-MS assay, a method for quantifying labile protein–ligand complexes that are prone to in-source dissociation, were found to be in good agreement with reported values.     

DETERMINATION OF THE ACRYLAMIDE QUENCHING CONSTANT FOR PROTEIN AND MODEL INDOLE TRIPLETS

Abstract— The decay of the indole triplet of single tryptophan-containing proteins and model compounds can be readily measured at room temperature in aqueous solution by monitoring the triplet-triplet absorption or phosphorescence emission following a 265 nm exciting laser pulse. The quenching action of acrylamide on the triplet excited state of indole side chains was studied in an analogous fashion to that previously done at the singlet level (Eftink and Ghiron, 1977). The acrylamide triplet quenching constant (^tk_q) ranged from a high of 7.8 times 10⁸M^-1 s^-1 for the exterior indole of corticotropin (ACTH) to a low of 2 times 10⁵ Af^-1 s^-1 for the interior indole of ribonuclease T, (RNase T,). The ratio (7) of these values with their respective acrylamide singlet quenching constants (^tk_q),(γ=^tk_q⁸K_q) ranged from a high of 0.22 for ACTH to a low of 0.001 for RNase T₁,. Acrylamide is also an inefficient quencher of model indoles in various solvents (i.e. it has a γ less than 1). The magnitude of γ varied from a high of 0.3 in H₂0 to a low of 0.02 in acetonitrile, but did not correlate with viscosity, dielectric constant or polarity. The lower efficiency observed for internal indole groups can not be explained by that class of models which predict the presence of static quenching at the triplet level, since none was observed. The present results confirm the observation of Calhoun et al. of a large discrepancy between acrylamide's singlet and triplet quenching constants for buried indole side chains, but suggest that it may be largely explained by the fact that acrylamide is an inefficient quencher of the indole triplet state (1983). The magnitude of this inefficiency is probably determined by specific microenvironmental factors. Thus, unlike ⁸K_q, the environmentally sensitive ^lk_H cannot be easily used to characterize the dynamics of proteins.     

THE REACTIVITY OF CITRONELLOL and α-THUJENE WITH SINGLET OXYGEN. RATE CONSTANTS OF CHEMICAL REACTION and PHYSICAL QUENCHING*

The quantum yields of Rose Bengal sensitized photooxidation of citronellol and α-thujene have been determined as a function of added acceptor and compared with those of furfuryl alcohol as a standard. The results permitted the calculation of the corresponding rate constants of chemical reaction (k_T) and physical quenching (K_q) of singlet oxygen. The sum (k_T+ k_q) has been verified independently by a Stern-Volmer analysis of the singlet oxygen luminescence quenching. α-Thujene reacts faster with singlet oxygen than citronellol, physical quenching being negligible in both cases.     

AMPHIPHILIC COPOLYMERS AS MEDIA FOR LIGHT-INDUCED ELECTRON TRANSFER–I. ELECTROSTATIC EFFECT ON THE FORWARD REACTION AS STUDIED BY FLUORESCENCE QUENCHING

Abstract— Fluorescence quenching of amphiphilic copolymers, poly(9-vinylphenanthrene-co-sodium 2-acrylamido-2-methylpropanesulfonate) (APh) and poly(9-vinylphenanthrene-co-3-methacrylamidopropyltrimethylammonium methyl sulfate) (QPh), in aqueous solution, was studied using methyl viologen (MV²⁺) or 4,4'-bipyridinium-1, 1'-bis(trimethylenesulfonate) (SPV) as oxidative quenchers. The fluorescence of the excited phenanthrene groups in APh was found to be efficiently quenched by MV²⁺. The apparent second-order rate constant for the quenching, k_q, ranged in the magnitude of 10¹¹ -10¹²M^-1 s^-1, which are well beyond the diffusion-controlled limit. This is presumably due to an increase of the effective concentration of MV²⁺ around the fluorophore in the copolymer resulting from electrostatic attraction between MV²⁺ and anionic segments of APh. This strong electrostatic interaction also favors the formation of ground-state EDA (electron donor acceptor) complex between the phenanthrene residue and MV²⁺. Such striking behaviors were not observed with the related model compound. Unexpectedly, the quenching with SPV, a zwitterionic quencher, was also enhanced in the polymer system (k_q= 2–6 × 10¹⁰M^-1 s^-1), suggesting the presence of some attractive interaction between APh and SPV. Contrary to the APh system, the fluorescence quenching of the corresponding cationic polymer (QPh) with MV²⁺ was strongly diminished (k_q= 5 × 10⁸M^-1 s^-1). This indicates that the polycation of QPh effectively prevents the access of MV²⁺ to the polymer.     

INTERACTION OF THE PEPTIDE HORMONE ADRENOCORTICOTROPIC ACTH(l-24), WITH A MEMBRANE MODEL SYSTEM: A FLUORESCENCE STUDY*

The peptide hormone adrenocorticotropin and a related peptide were studied in solution and in interaction with a model system of membranes (small unilamellar vesicles of dipalmitoylphosphatidylcholine and 17% dimyristoylphosphatidylglycerol) via fluorescence spectroscopy. In aqueous solution, intramolecular distances between the fluorescent residues R(Tyr²-Trp⁹) = 9.2 Å and R(Trp⁹-Tyr²³) 18 Å were obtained, in agreement with molecular models. Interaction of the peptide with the negatively charged membrane is evident from the alteration of the Trp photophysical parameters (quantum yield, fluorescence spectra and anisotropy), with a partition constant between the lipidic and aqueous phase of Kp =1–2 times 10³. The existence of two populations of Trp in the membrane, which are distinctly accessed by acrylamide, was concluded from the tryptophan fluorescence quenching study; the two fractions are located near the membrane interface as inferred from its fluorescence quenching by the 5-doxylstearate and 16-doxylstearate lipophilic quenchers. This result is further supported by energy transfer experiments to the 3-(9-anthroyloxyl)stearic acid and 12-(9-anthroyloxyl)stearic acid probes.     

SPECTROSCOPIC STUDIES OF CHLOROPHYLL a AGGREGATES FORMED BY AQUEOUS DIMETHYL SULFOXIDE

Characteristic chlorophyll (Chl) a aggregates formed in aqueous dimethyl sulfoxide (DMSO) were investigated spectroscopically. Four chlorophyll forms were found with increasing DMSO concentration; they are called A-672, A-683, A-695 and A-665 according to the wavelengths of their absorption maxima. Transformation occurred only in this order. Reverse transformation could not be realized. A-683 and A-695 were apparently formed by the interaction of Chl a with DMSO in the linear dimer and linear polymer arrangements, respectively. Coordination of the Mg atom with a DMSO O atom and interaction between the S atom of one DMSO molecule and the O atom of an other DMSO molecule should lead to formation of a sandwich-type complex of partially overlapping chlorophyll macrocycles (Chl a-DMSO)_n. A-672 and A-665 were assigned to Chl a micelles and to dissolved monomeric Chl a in DMSO, respectively. Fluorescence spectra showed that the A-683 was highly fluorescent, while the A-695 was less fluorescent. Energy migration within the A-695 form to a trap with a low fluorescence yield might be responsible for this difference in the emission intensity.