Fluorescence quenching method for the determination of carbazochrome sodium sulfonate with aromatic amino acids

In Britton‐Robinson (BR) buffer medium (pH 3.3), carbazochrome sodium sulfonate (CSS) can react with some aromatic amino acids such as tryptophan (Trp), tyrosine (Tyr) and phenylalanine (Phe) to form a 1:1 complex by electrostatic attraction, aromatic stacking interaction and Van der Waals' force, resulting in fluorescence quenching of these amino acids. Maximum quenching wavelengths were located at 352 nm (CSS‐Trp system), 303 nm (CSS‐Tyr system) and 284 nm (CSS‐Phe system), respectively. The fluorescence quenching value (ΔF) was proportional to the concentration of CSS in a certain range. The fluorescence quenching method for the determination of CSS showed high sensitivity, with detection limits of 31.3 ng/mL (CSS‐Trp system), 44.6 ng/mL (CSS‐Tyr system) and 315.0 ng/mL (CSS‐Phe system), respectively. The optimum conditions of the reaction conditions and the effect of coexisting substances were investigated and results showed that the method had good selectivity. The method was successfully applied for the rapid determination of CSS in blood and urine samples. Based on the bimolecular quenching constant K_q, the effect of temperature and Stern‐Volmer plots, this study showed that quenching of fluorescence of amino acids by CSS was a static quenching process. Copyright © 2012 John Wiley & Sons, Ltd.     

Effects of the interaction between Na2Wo4‐6‐benzylaminopurine anionic chelate and rhodamine 6G on the resonance Rayleigh scattering and fluorescence spectra and their analytical applications

In pH 4.99‐6.06 Britton‐Robinson (BR) buffer medium, 6‐benzylaminopurine (6‐BA) reacted with Na₂WO₄ to form 1:1 anionic chelate (6‐BA·WO₄)^2‐, which further reacted with rhodamine 6G to form ternary ion complexes at room temperature. This resulted in a significant enhancement of resonance Rayleigh scattering (RRS) with a maximum RRS wavelength of 316 nm. Meanwhile, the fluorescence of the solution was quenched and excitation (λ_ex) and emission (λ_em) wavelengths of the fluorescence were 290 and 559 nm, respectively. Intensities of RRS enhancing (ΔI_RRS) and fluorescence quenching (ΔI_F) were directly proportional to concentrations of 6‐BA. As a result, RRS and fluorescence quenching for determination of trace amounts of 6‐BA were developed. Under optimal conditions, linear ranges and detection limits of the two methods were 0.05‐15.00 µg/mL and 8.2 ng/mL (RRS), 0.50‐15.00 µg/mL and 17.0 ng/mL, respectively. It was found that the RRS method was superior to fluorescence quenching. The influence of these methods were investigated and results showed that RRS had good selectivity. RRS was applied to determine 6‐BA in vegetable samples with satisfactory results. Furthermore, the reaction mechanisms of the ternary ion‐association system are discussed. In addition, the polarization experiment revealed that the resonance light scattering (RLS) peak of Na₂WO₄‐6‐BA‐R6G consisted mainly of depolarized resonance fluorescence and resonance scattering. It was speculated that light emission fluorescence energy (E_L) transformed into resonance light scattering energy (E_RLS), which was a key reason for enhancement of RRS. Copyright © 2012 John Wiley & Sons, Ltd.     

Sodium 4‐mercaptophenolate capped CdSe/ZnS quantum dots as a fluorescent probe for pH detection in acidic aqueous media

Development of the fluorescent pH detection method is promising due to the sensitivity, easy operation, and low‐cost, etc. However, traditional organic fluorophores have still some disadvantages such as the tedious preparation and purification as well as low photostability and water solubility, which limits the rapid detection application. Semiconductor quantum dots (QDs) have recently risen to prominence as an alternative for organic fluorophores in fluorescence analysis by virtue of their convenient synthesis and superior optical properties. In this study, we report on sodium 4‐mercaptophenolate functionalized CdSe/ZnS QDs (denoted as ^?OPhS‐QDs), which can serve as a selective “on–off” fluorescence probe for aqueous media pH. ^?OPhS‐QDs exhibit strong fluorescence in near neutral medium. As a Lewis organic base, ^?OPhS‐ moieties on QDs surface easily binds to proton under acidic conditions to yield 4‐mercaptophenol capped QDs (i.e. HOPhS‐QDs), which acts as an efficient hole trapper. As a result, the QDs photoluminescence (PL) is switched off. Under optimal conditions, the present probe exhibits a good linear relationship between fluorescence response and pH values in the pH range 3.0–5.2. Furthermore, the present probe exhibits a high selectivity for proton over other common cations and has been successfully used for pH detection in real water samples.     

Binding of caffeine with caffeic acid and chlorogenic acid using fluorescence quenching,UV/vis and FTIR spectroscopic techniques

The interactions of caffeine (CF) with chlorogenic acid (CGA) and caffeic acid (CFA) were investigated by fluorescence quenching, UV/vis and Fourier transform infrared (FTIR) spectroscopic techniques. The results of the study indicated that the fluorescence quenching between caffeine and hydroxycinnamic acids could be rationalized in terms of static quenching or the formation of non‐fluorescent CF–CFA and CF–CGA complexes. From fluorescence quenching spectral analysis, the quenching constant (K_SV), quenching rate constant (k_q), number of binding sites (n), thermodynamic properties and conformational changes of the interaction were determined. The quenching constants (K_SV) between CF and CGA, CFA are 1.84 × 10⁴ and 1.04 × 10⁴ L/mol at 298 K and their binding site n is ~ 1. Thermodynamic parameters determined using the Van't Hoff equation indicated that hydrogen bonds and van der Waal's forces have a major role in the reaction of caffeine with caffeic acid and chlorogenic acid. The 3D fluorescence, UV/vis and FTIR spectra also showed that the binding of CF with CFA and CGA induces conformational changes in CFA and CGA. Copyright © 2015 John Wiley & Sons, Ltd.     

Comparison of interactions between human serum albumin and silver nanoparticles of different sizes using spectroscopic methods

Three different sizes (15.9 ± 2.1 nm, 26.4 ± 3.2 nm and 39.8 ± 4.0 nm, respectively) of citrate‐coated silver nanoparticles (SNPs) have been synthesized and characterized. The interactions of the synthesized SNPs with human serum albumin (HSA) at physiological pH have been systematically studied by UV‐vis absorption spectroscopy, fluorescence spectroscopy, synchronous fluorescence spectroscopy, three‐dimensional fluorescence spectroscopy and circular dichroism (CD) spectroscopy. The results indicate that the SNPs can bind to HSA with high affinity and quench the intrinsic fluorescence of HSA. The binding constants and quenching rate constants were calculated. The apparent association constants (K_app) values are 2.14 × 10⁴ M^–1 for 15.9 nm SNP, 1.65 × 10⁴ M^–1 for 26.4 nm SNP and 1.37 × 10⁴ M^–1 for 39.8 nm SNP, respectively. The values of binding constant obtained from the fluorescence quenching data match well with that determined from the absorption spectral changes. These results suggest that the smaller SNPs have stronger interactions to HSA than the larger ones at the same concentrations. Synchronous fluorescence, three‐dimensional fluorescence and CD spectroscopy studies show that the synthesized SNPs can induce slight conformational changes in HSA. Copyright © 2014 John Wiley & Sons, Ltd.     

Cooligopeptides containing aromatic residues spaced by glycyl residues. IX. Fluorescence properties of tryptophan-containing peptides

The fluorescence properties of several cooligopeptides of glycine, phenylalanine, and tryptophan, containing one or two aromatic residues, are investigated. In particular, a detailed analysis is made of the influence of pH upon the quantum yield and the position of the emission maximum (λ_max) in H-Trp-Trp-OH, H-Trp-Gly-OH, H-Gly-Trp-OH, H-Gly-Trp-Gly-OH, H-Trp-Trp-OH, H-Trp-Trp-Gly-OH, H-Gly-Trp-Trp-OH, H-Phe-Trp-OH, H-Phe-Trp-Gly-OH, H-Gly-Phe-Trp-OH, and H-Gly-X-(Gly)_n-Trp-Gly-OH, with X = Phe or Trp, and n = 0,1,2. It is shown that raising the pH from ca. 2 to 11 results in a red shift of λ_max, and an increase in the quantum yield. These changes, mostly structure dependent, are in most cases attributable to electronic perturbations acting directly upon the λ_max of the fluorophore(s) and upon the quenching efficiency of the free amino and carbonyl groups. For the compounds having two adjacent tryptophyl residues, it is shown that the two fluorophores do not appear to have the same emission properties and the quantum yield is lower than expected. The causes of this behavior are discussed in terms of conformational effects, stacking interactions, and radiationless energy transfer. Finally, an attempt is made to correlate fluorescence data with previous circular dichroism data which had indicated the occurrence of a conformationally rigid structure for some of the compounds having two adjacent aromatic residues.     

Studies on the synthesis,characterization, human serum albumin binding and biological activity of single chain surfactant–cobalt(III) complexes

The interaction of surfactant–cobalt(III) complexes [Co(bpy)(dien)TA](ClO₄)₃ · 3H₂O (1) and [Co(dien)(phen)TA](ClO₄)₃ · 4H₂O (2), where bpy = 2,2′‐bipyridine, dien = diethylenetriamine, phen = 1,10‐phenanthroline and TA = tetradecylamine with human serum albumin (HSA) under physiological conditions was analyzed using steady state, synchronous, 3D fluorescence, UV/visabsorption and circular dichroism spectroscopic techniques. The results show that these complexes cause the fluorescence quenching of HSA through a static mechanism. The binding constant (K_b) and number of binding‐sites (n) were obtained at different temperatures. The corresponding thermodynamic parameters (?G°, ?H° and ?S°) and E_a were also obtained. According to Förster's non‐radiation energy transfer theory, the binding distance (r) between the complexes and HSA were calculated. The results of synchronous and 3D fluorescence spectroscopy indicate that the binding process has changed considerably the polarity around the fluorophores, along with changes in the conformation of the protein. The antimicrobial and anticancer activities of the complexes were tested and the results show that the complexes have good activities against pathogenic microorganisms and cancer cells. Copyright © 2015 John Wiley & Sons, Ltd.     

Study of the interaction between sodium salts of (2E)‐3‐(4'‐halophenyl)prop‐2‐enoyl sulfachloropyrazine and bovine serum albumin by fluorescence spectroscopy

Three sodium salts of (2E)‐3‐(4'‐halophenyl)prop‐2‐enoyl sulfachloropyrazine (CCSCP) were synthesized and their structures were determined by ¹H and ¹³C NMR, LC‐MS and IR. The binding properties between CCSCPs and bovine serum albumin (BSA) were studied using fluorescence spectroscopy in combination with UV–vis absorbance spectroscopy. The results indicate that the fluorescence quenching mechanisms between BSA and CCSCPs were static quenching at low concentrations of CCSCPs or combined quenching (static and dynamic) at higher CCSCP concentrations of 298, 303 and 308 K. The binding constants, binding sites and corresponding thermodynamic parameters (ΔH, ΔS, ΔG) were calculated at different temperatures. All ΔG values were negative, which revealed that the binding processes were spontaneous. Although all CCSCPs had negative ΔH and positive ΔS, the contributions of ΔH and ΔS to ΔG values were different. When the 4'‐substituent was fluorine or chlorine, van der Waals interactions and hydrogen bonds were the main interaction forces. However, when the halogen was bromine, ionic interaction and proton transfer controlled the overall energetics. The binding distances between CCSCPs and BSA were determined using the Förster non‐radiation energy transfer theory and the effects of CCSCPs on the conformation of BSA were analyzed by synchronous fluorescence spectroscopy. Copyright © 2012 John Wiley & Sons, Ltd.     

Solvent effect on the relative quantum yield and fluorescence quenching of a newly synthesized coumarin derivative

We estimated the relative florescence quantum yield (Φ) of 8‐methoxy‐3‐[1‐(4,5‐dicarbomethoxy‐1,2,3‐triazoloacetyl)]coumarin [8MDTC] using a single‐point method with quinine sulfate in 0.1 M of sulfuric acid used as a standard reference. The fluorescence lifetimes, radiative and non‐radiative decay rate constants are calculated. Relative quantum yields were found to be less in the non‐polar solvents, indicating that the solute exhibits less fluorescence in a non‐polar environment. The fluorescence quenching of [8MDTC] by aniline was studied at room temperature by examining the steady state in five different solvents in order to explore various possible quenching mechanisms. The experimental results show a positive deviation in Stern–Volmer plots in all solvents. Ground state complex and sphere of action static quenching models were used to interpret the results. Many quenching rate parameters were calculated using these models. The values of these parameters suggest that the sphere of action static quenching model agrees well with the experimental results. Further, a finite sink approximation model was used to check whether these bimolecular reactions were diffusion limited or not. The values of the distance parameter R′ and the diffusion coefficient D were determined and are compared with the values of the encounter distance R and diffusion coefficient D calculated using the Stokes–Einstein equation. Copyright © 2014 John Wiley & Sons, Ltd.     

Radiative decay engineering 6: Fluorescence on one-dimensional photonic crystals

During the past decade the interactions of fluorophores with metallic particles and surfaces has become an active area of research. These near-field interactions of fluorophores with surface plasmons have resulted in increased brightness and directional emission. However, using metals has some disadvantages such as quenching at short fluorophore–metal distances and increased rates of energy dissipation due to lossy metals. These unfavorable effects are not expected in dielectrics. In this article, we describe the interactions of fluorophores with one-dimensional (1D) photonic crystals (PCs), which have alternating layers of dielectrics with dimensions that create a photonic band gap (PBG). Freely propagating light at the PBG wavelength will be reflected. However, similar to metals, we show that fluorophores within near-field distances of the 1DPC interacts with the structure. Our results demonstrate that these fluorophores can interact with both internal modes and Bloch surface waves (BSWs) of the 1DPC. For fluorophores on the surface of the 1DPC, the emission dominantly occurs through the 1DPC and into the substrate. We refer to these two phenomena together as Bragg grating-coupled emission (BGCE). Here we describe our preliminary results on BGCE. 1DPCs are simple to fabricate and can be handled and reused without damage. We believe that BGCE provides opportunities for new formats for fluorescence detection and sensing.     

A temperature‐dependent conformational change of NADH oxidase from Thermus thermophilus HB8

Using molecular dynamics simulations and steady‐state fluorescence spectroscopy, we have identified a conformational change in the active site of a thermophilic flavoenzyme, NADH oxidase from Thermus thermophilus HB8 (NOX). The enzyme's far‐UV circular dichroism spectrum, intrinsic tryptophan fluorescence, and apparent molecular weight measured by dynamic light scattering varied little between 25 and 75°C. However, the fluorescence of the tightly bound FAD cofactor increased approximately fourfold over this temperature range. This effect appears not to be due to aggregation, unfolding, cofactor dissociation, or changes in quaternary structure. We therefore attribute the change in flavin fluorescence to a temperature‐dependent conformational change involving the NOX active site. Molecular dynamics simulations and the effects of mutating aromatic residues near the flavin suggest that the change in fluorescence results from a decrease in quenching by electron transfer from tyrosine 137 to the flavin. Proteins 2012. © 2011 Wiley Periodicals, Inc.     

Simultaneous multicolor fluorescence cross-correlation spectroscopy to detect higher order molecular interactions using single wavelength laser excitation

Fluorescence cross-correlation spectroscopy is a powerful method for the study of molecular interactions and dynamics in solution and even in living cells. Usually, in the optical setup, either two laser beams have to be superimposed in their respective confocal volumes or two-photon excitation is used for a dual-color detection system. It has been shown recently that fluorescence cross correlation can be achieved with spectrally similar fluorophores using single wavelength excitation fluorescence cross-correlation spectroscopy (SW-FCCS). In this study, we show that SW-FCCS allows the simultaneous excitation of up to three fluorophores in which the cross correlation of their fluctuation signals is detected separately in three detection channels. The experimental and theoretical model to describe triple pairwise cross correlations incorporating cross talk and possible changes in emission characteristics such as quenching upon binding are outlined. The effectiveness of SW-FCCS to detect binding of three interacting partners is experimentally verified with a standard ligand-receptor model, biotin-streptavidin, where differently labeled biotin ligands and their binding to a third-color labeled streptavidin are studied. The cross-correlation amplitudes and their changes with stoichiometric binding are analyzed and the upper limits of dissociation constants are determined. Performed with appropriate negative controls, SW-FCCS can determine interaction patterns between ligands and receptors.     

Synthesis, X-ray crystal structure and optical properties of novel 2-aryl-3-ethoxycarbonyl-4-phenylpyrido[1,2-a]benzimidazoles

A series of novel 2‐aryl‐3‐ethoxycarbonyl‐4‐phenylpyrido[1,2‐a]benzimidazole derivatives were synthesized by the tandem reaction of 2‐benzoyl benzimidazole and (Z)‐ethyl 4‐bromo‐3‐arylbut‐2‐enoate in the presence of potassium carbonate. The compounds were characterized using IR, ¹H‐NMR, ¹³C‐NMR, HRMS and the structure of 6f was further determined by X‐ray crystallography. Both absorption and fluorescence spectra characteristics of the compounds were investigated in acetonitrile and dichloromethane. The results showed that the absorption maxima of the compounds varied from 220 to 284 nm, depending on the structure of 2‐aryl group. The fluorescence results revealed that these compounds exhibited blue‐green fluorescence (463–475 nm) in dilute solutions and showed acceptable fluorescence quantum yields (Ф_PL = 0.13–0.73) in dichloromethane. Copyright © 2011 John Wiley & Sons, Ltd.     

New phthalimide‐appended Schiff bases: Studies of DNA binding,molecular docking and antioxidant activities

Herein, we investigated new phthalimide‐based Schiff base molecules as promising DNA‐binding and free radical scavenging agents. Physicochemical properties of these molecules were demonstrated on the basis of elemental analysis, ultraviolet–visible (UV–Vis), infra‐red (IR), ¹H and ¹³C nuclear magnetic resonance (NMR) spectroscopy. All spectral data are agreed well with the proposed Schiff base framework. The DNA‐binding potential of synthesized compounds were investigated by means of UV–visible, fluorescence, iodide quenching, circular dichroism, viscosity and thermal denaturation studies. The intrinsic binding constants (K _b) were calculated from absorption studies were found to be 1.1 × 10⁴ and 1.0 × 10⁴ M^?1 for compounds 2a and 2b suggesting that compound 2a binding abilities with DNA were stronger than the compound 2b. Our studies showed that the presented compounds interact with DNA through groove binding. Molecular docking studies were carried out to predict the binding between Ct‐DNA and test compounds. Interestingly, in silico predictions were corroborated with in vitro DNA‐binding conclusions. Furthermore, the title compounds displayed remarkable antioxidant activity compared with reference standard.     

Insights into the binding of paclitaxel to human serum albumin: multispectroscopic studies

The interaction of paclitaxel with human serum albumin (HSA) was studied using fluorescence, resonance light scattering, ultraviolet‐visible, circular dichroism and Fourier transform infrared spectroscopy at pH 7.4. Fluorescence data revealed that the fluorescence quenching of HSA by paclitaxel was a static quenching procedure. Time‐resolved fluorescence data also confirmed the quenching mode, which present a constant decay time of about 5 ns. The binding sites were approximately 1 and the binding constant suggested a weak association (324/M at 298 K), which is helpful for the release of the drug to targeted organs. The thermodynamic parameters, ΔG^○, ΔH° and ΔS° were calculated as – 1.06 × 10⁴ J/mol, 361 J/mol per K and 9.7 × 10⁴ J/mol respectively at 298 K, suggesting that binding was spontaneous and was driven mainly by hydrophobic interactions. The binding distance between HSA and paclitaxel was determined to be 2.23 nm based on the Förster theory. Analysis of circular dichroism, ultraviolet‐visible, three‐dimensional fluorescence, Fourier transform infrared and resonance light scattering spectra demonstrated that HSA conformation was slightly altered in the presence of paclitaxel and dimension of the individual HSA molecules were larger after interacting with paclitaxel. These results were confirmed by a molecular docking study. Copyright © 2013 John Wiley & Sons, Ltd.     

Structural and photophysical properties of novel dual fluorescent compounds, 1‐aryl‐substituted 6‐methoxy‐4‐quinolones

Dual fluorescent compounds are useful platforms for the development of ratiometric probes and sensors. We have developed a new series of dual fluorescent compounds, 1‐aryl‐substituted 6‐methoxy‐4‐quinolones, and investigated their structural and photophysical properties. The X‐ray crystallographic analysis and ab initio quantum chemical calculations revealed that the developed compounds exhibited 60–75° twisted structures. The dual fluorescence of the compounds were observed in polar solvents, and the ratiometric fluorescence responses to alterations in the acidity and temperature were obtained. Copyright © 2008 John Wiley & Sons, Ltd.     

Fluorescence quenching method for the determination of bleomycins A5 and A2 with halofluorescein dyes

In weak acidic medium, the anticancer antibiotics bleomycin A₅ (BLMA₅) and bleomycin A₂ (BLMA₂) bind with halofluorescein dyes, such as erythrosin (Ery), eosin Y (EY) and eosin B (EB), to form ion‐association complexes, which causes fluorescence quenching of halofluorescein dyes. The quenching values (ΔF) are directly in proportional to the concentrations of bleomycins over the range 0.09–2.5 µg/mL. Based on this, a fluorescence quenching method for the determination of BLMA₅ and BLMA₂ has been developed. The dynamic range is 0.12–2.5 µg/mL for the determination of BLMA₅ and 0.09–2.0 µg/mL for BLMA₂, with detection limits (3σ) of 0.04 µg/mL for BLMA₅, 0.03 µg/mL for BLMA₂, respectively. It has been applied to determine the two antibiotics in human serum, urine and rabbit serum samples. The recovery is in the range 90–102%. In this work, the optimum reaction conditions and the spectral characteristics of the fluorescence are investigated. The reasons for fluorescence quenching are discussed, based on the fluorescence theory. Copyright © 2007 John Wiley & Sons, Ltd.     

Chlorinated hydrocarbon-cell membrane interactions studied by the fluorescence quenching of carbazole-labeled phospholipids: Probe synthesis and characterization of the quenching methodology

In recognition of the need to understand better the interactions of the chlorinated hydrocarbon insecticides with cell membranes we investigated the use of fluorescence quenching of membrane-bound fluorophores by these chlorinated hydrocarbons. An extensive survey of potential fluorophores identified the N-alkyl derivatives of carbazole as being especially suitable fluorophores. The fluorescence emission of these derivatives is quenched by a wide variety of commonly-used chlorinated hydrocarbons. This quenching is collisional and does not result in significant photodecomposition.Four structurally distinct carbazole-labeled phospholipids were synthesized, and their structures were confirmed by 270 MHz proton NMR and by chromatographic and chemical means. The carbazole moiety of each labeled phospholipid should be localized at a different depth in lipid bilayer. However, water soluble quenchers indicate that the fluorophores are inaccessible to the aqueous phase, irrespective of their point of attachment to the phospholipids.When incorporated into lipid bilayers, the fluorescence lifetime of these carbazole-labeled phospholipids reveals the collisional frequency between the fluorophore and the chlorinated hydrocarbon. As a result quenching of membrane-bound fluorophores may be used to measure: (1) the diffusional rate of the chlorinated hydrocarbon in the bilayer; (2) the lipid-water partition coefficient; (3) the maximum binding capacity of the membrane for the chlorinated hydrocarbon. Examples of all these measurements are given, and the fluorometric results are confirmed by direct chemical analysis.     

Fluorescence resonance energy transfer in a novel cyclodextrin-peptide conjugate for detecting steroid molecules

A novel cyclodextrin conjugated peptide, 1, having two different fluorophores, coumarin and pyrene, in the side chains has been designed and synthesized. The circular dichroism study reveals that 1 shows typical -helix pattern, and forms intramolecular inclusion complex with coumarin. The fluorescence emission study shows that the peptide exhibits intramolecular fluorescence resonance energy transfer (FRET) without quenching of two fluorophores. We have determined the binding constants of 1 for various biologically important steroid molecules as guests using the guest-responsive variation in the fluorescence emission intensity of coumarin.     

Quenching of the intrinsic tryptophan fluorescence of mitochondrial ubiquinol--cytochrome-c reductase by the binding of ubiquinone

1. The quenching by ubiquinone (Q) of the intrinsic fluorescence of tryptophan residues within ubiquinol--cytochrome-c reductase (complex III) has been exploited to provide direct information on the interaction between these two components of the mitochondrial respiratory chain. 2. The fluorescence quenching data have been corrected for inner filter effects and interpreted using the classical Stern-Volmer and modified Stern-Volmer plots. The latter of these plots allows computation of both the dissociation constant (Kd) of complex formation between ubiquinone and complex III, and the percentage of fluorophores accessible to quenching. 3. It is found that different Q homologues bind to complex III with different affinities depending upon the length of the isoprenoid chain: 2,3-dimethoxy-5-methyl-6-decyl-1,4-benzoquinone, an analogue of Q2, exhibits the same Kd as Q2. Furthermore, the accessibility of fluorophores to quenching was lower for Q1 than for the other quinones tested. 4. The binding affinity of Q2 to complex III depends upon the redox state of the enzyme. 5. Addition of the complex III inhibitor, antimycin, has very little effect on the binding affinity or on the accessibility of fluorophores to the quencher. 6. Addition of the inhibitor myxothiazol has a similar effect to reducing complex III with ascorbate. 7. Reconstitution of complex III into asolectin lipid vesicles gives similar qualitative results to the enzyme in solution regarding both the redox state and the addition of inhibitors.