Luminescence properties of pyron red—A new molecular probe for protein research

The dependence of the luminescence of the new anionic dye Pyron Red (PR) on the polarity of the medium is investigated. Upon passage from an aqueous phase to a nonpolar phase, PR shows a shortwave shift of the fluorescence emission maximum from 675 to 650 nm and an increase in the fluorescence quantum yield from 0.03 to 0.54–0.70. When complexed with human serum albumin, PR shows fluorescence excitation and emission maxima at 525 and 625 nm and a fluorescence quantum yield of 0.8. In a comparison of the luminescence properties of PR with those of the well-known probes ANS and K35 in water and a complex with albumin, PR is shown to have the maximum absolute sensitivity but a lower fluorescence enhancement upon binding with a protein compared to ANS. A convenient criterion of the probe sensitivity toward binding with a protein that is defined as the ratio of the fluorescence intensities of the protein-bound and the free probe A_F=F_b/F_f is proposed. The value of A_F(35) for the PR probe ranks between those for the K35 probe with a low A_F(18) and ANS with a high A_F(105). Translated from Zhurnal Prikladnoi Spektroskopii, Vol. 66, No. 3, pp. 369–374, May–June, 1999.     

Exploration of twisted intramolecular charge transfer fluorescence properties of trans-2-[4-(dimethylamino)styryl]benzothiazole to characterize the protein–surfactant aggregates

The characterization of aggregates of an anionic surfactant, sodium dodecyl sulphate (SDS) with bovine serum albumin (BSA) in various regions of binding isotherm of SDS to BSA with increasing concentration of the former have been done by exploring the twisted intramolecular charge transfer (TICT) fluorescence properties of a probe, trans-2-[4-(dimethylamino)styryl] benzothiazole (DMASBT). The TICT fluorescence, steady-state fluorescence anisotropy and time-resolved fluorescence of DMASBT, and the fluorescence resonance energy transfer (FRET) study reveal the characteristics of the native protein as well as the protein–surfactant aggregates viz., micropolarity, microviscosity, locations of probe, denaturation of protein in various regions of binding isotherm, and also the validation of necklace-bead model. The changes in the polarity and the viscosity of the microenvironment around the probe from one binding region of SDS to other have been reflected in the highly sensitive fluorescence properties of DMASBT. The study of FRET between the DMASBT and the tryptophan residue (Trp) of BSA has identified the locations of the probe molecule in the native protein as well as that in various BSA–SDS aggregates. The energy transfer efficiency decreases, whereas the distance between the DMASBT and the Trp residue increases with increasing concentration of SDS. The significant change in the conformations of protein molecules during the non-cooperative binding region of SDS is evidenced by the fluorescence anisotropic behavior of DMASBT in the same region.     

Role of the head group on the mixed micellization process in binary systems containing a sugar-based surfactant: decanoyl- N -methylglucamide

The mixed micelles of nonionic decanoyl-N-methylglucamide (MEGA-10) with the anionic sodium dodecyl sulphate (SDS), the cationic dodecyltrimetylammonium bromide (DTAB), and the nonionic octaoxyethylene monododecyl ether (C₁₂E₈) have been studied using the fluorescence probe technique. The critical micelle concentration of the three mixed systems in the whole composition range were determined by the pyrene 1:3 ratio method, and the experimental results were analysed in the context of the pseudophase separation model, by using the regular solution theory. It was found that the mixed micelles containing the anionic surfactant are more stable than the pure micelles. This fact was attributed to the occurrence of ion–dipole interactions between the head groups of the component surfactants in the mixed micelle. The static quenching method was used to determine the mean aggregation number of pure and mixed micelles. It was found that whereas mixed micelles containing SDS show a positive deviation from the ideal behaviour, those constituted by DTAB deviate negatively. This different tendency was interpreted on the basis of both steric and electrostatic interactions. The evolution of the microstructure of the mixed micelles upon the participation of the co-surfactant was followed through the micropolarity and microviscosity of the mixed systems. Although the micropolarity studies do not allow definite conclusions, the microviscosity assays indicate that the participation of the co-surfactant induces the formation of less ordered micelles, this effect being more pronounced in the case of mixtures with the anionic surfactant.     

Luminescence and anisotropy decays ofN-3-pyrene maleimide labeling IgG proteins and cells

The Py.M (N-3-Pyrene Maleimide) is a dye that covalently binds to reactive amino or sulfhycryl groups to give highly fluorescent protein conjugates. Measurements of luminescence lifetimes and anisotropy decays have been performed with a Phase and Modulation Fluorometer. Complexes of Py.M-antibody (IgG antimouse) and tumoral cells C6 labeled with Py.M have been investigated. The Py.M fluorescence in buffer solution and the protein and cells natural fluorescence have been checked. For Py.M-IgG and labeled cells, the fluorescence decays present interesting behaviours. The least-squares analysis of the experimental results on Py.M-IgG complex points out two lorentzian distributions centered at 74 ns and 11 ns, on the contrary, for the labeled cells, a discrete component at 100 ns and a lorentzian distribution centered at 5 ns are shown. In both systems a weak component lower than 1 ns is observed. The fluorescence decays, mainly the long lifetime one, are very sensitive to oxygen quenching, showing the high efficiency of O₂ quenching. For samples N₂ bubbled, the lifetime experimental resuits show a decrease of the oxygen accessibility from free probe in solution to Py.M-IgG complex and to labeled cells, compatible with a more compact packing of the probe binding site. The experimental results of anisotropy decays of degassed samples show for Py.M-IgG complexes a long rotation correlation time of about 200 ns at T=5°C, assigned to overall rotation of the protein, besides shorter correlation times attributable to inner protein motions. For labeled cells, the long rotation correlation time becomes of the order of 580 ns confirming a progressive increase of the stabilization of the binding site.     

Ethenyl indoles as neutral hydrophobic fluorescence probes

3‐(4‐Nitrophenylethenyl‐E)‐NH‐indole ( 1 ), 3‐(4‐nitrophenyl ethenyl‐E)‐N‐acetylindole ( 2 ), and 3‐(4‐nitrophenylethenyl‐E)‐N‐benzenesulfonylindole ( 3 ) are relatively less fluorescent in organic solvents, with fluorescence quantum yield (Φ_f) in the range of 0.002 to 0.066 depending on the solvent polarity. However, in bovine serum albumin (BSA)‐phosphate buffer, the fluorescence of these compounds gets drastically enhanced with Φ_f in the range of 0.21 to 0.67, depending on the substituent on the indolic nitrogen atom. Additionally, linear increase in the fluorescence intensity of 2 and 3 occurs on increasing the BSA concentration. These fluorescence properties together with the neutral, hydrophobic nature of these compounds make these fluorophores good fluorescence probe for studying the micropolarity of proteins like BSA and in general the ligand–protein interactions. Copyright © 2007 John Wiley & Sons, Ltd.     

Spectroscopic investigations on the interaction of an anionic probe with nonionic micelles of Igepal surfactants in aqueous media

The behaviour of the anionic dye 8-anilino-1-napthalenesulfonic acid ammonium salt, or ANS, in aqueous solutions containing the Igepal series of polyoxyethylene nonionic surfactants was investigated using fluorescence spectroscopic technique. The interactions of the dye with the nonionic surfactants were examined in micellar media, to prevent dye aggregate formation and to ensure maximum dye and surfactant interaction. From the relative fluorescence enhancements, binding constants of the dye to the surfactant micelles and aggregation numbers of the micelles were determined. The aggregation numbers were also separately determined by static fluorescence quenching of pyrene by cetylpyridinium chloride in aqueous surfactant mixtures at a fixed concentration of surfactant, and compared with the value obtained from the present investigation of the interaction of the micelles with the ANS probe. The values of binding constants, micropolarity values sensed by pyrene and the Stern–Volmer constants for quenching of pyrene fluorescence by cetylpyridinium chloride were correlated with the number of ethylene oxide groups in the Igepal series.     

Fluorescence Enhancement of Carbendazim Fungicide in Cucurbit[6]uril

The potential increase in fluorescence of a benzimidazole-type fungicide (carbendazim) due to complexation with cucurbit[6]uril is reported. The fluorescence of the probe carbendazim in aqueous Na₂SO₄ solution (pH=7.61) at room temperature is found to increase by a maximum factor of ∼10.0 and blue-shifted up to ∼11±1 nm with the increase in cucurbit[6]uril concentration up to ∼5 mM. This fluorescence enhancement is the result of formation of a 1:1 guest-host inclusion complex, in which the guest carbendazim is incorporated inside the hydrophobic cavity of the host curbit[6]uril through the amido-ester part. Such mode of inclusion is supported by NMR spectral measurements, in which upon encapsulation, the resonance of the methyl-protons of the amido-ester moiety is shifted significantly to upfield in the ¹H NMR spectrum. Also, to assess the formation of inclusion complex, solid samples prepared by co-evaporation have been studied, using differential scanning calorimetry (DSC). Measurement of the enhancement as a function of cucurbit[6]uril concentrations yielded a value of the equilibrium constant (K _a ) of 271±10 M⁻¹ at 25°C. From the temperature dependence of the equilibrium constants, ΔH and ΔS values have been negative in sign, indicating the dipole-dipole interactions and the steric factors associated with the formation of this inclusion complex. It might be proposed that the spectral changes due to the inclusion of carbendazim are the result of decrease in the polarity of the surrounded media rather than the loss of carbendazim rotational mobility.     

The effect of molecular structure on the photophysical behavior of substituted styryl pyrazine derivatives

Six styryl pyrazine compounds, some of which have steric hindrance to rotate and some have twist freedom, were synthesized in this work. The effects of solvent polarity and viscosity on the photophysical and luminescent behavior of these compounds were preliminarily studied. Results indicated that the blocking of the double-bond twist by a sufficiently rigid bridge increases fluorescence quantum yields dramatically and the rotation of a single bond connecting the double bond and the phenyl group is favorable to form a single-bond twisted state (T*), which is the state responsible for the main channel of fluorescence emission. The reason for the higher fluorescence quantum yield of distyryl pyrazine was thought to be involved with the lower probability of transition from the Frank-Condon state (S ₁) to the phantom state (P*). Studies were also extended to develop a novel probe to detect the special microviscosity.     

Light scattering and fluorescence probe studies on micellar properties of Triton X-100 in KCl solutions

The effect of KCl on micelle formation and structure of Triton X-100 (TX-100) was investigated by using combined static and dynamic light scattering measurements, together with the fluorescence probe technique. An analysis of the light scattering data, including hydrodynamic radius and micellar aggregation number, accounted for both micelle growth and hydration. Fluorescence studies using pyrene as a probe were carried out to determine the critical micelle concentration (CMC) as a function of solution composition. In addition, with the aim of gaining information on the possible changes in the micro-environmental properties of TX-100 micelles, fluorescence probe studies, including intermolecular pyrene excimer formation and fluorescence polarization of coumarin 6 associated with micelles, were carried out. It was found that the addition of electrolyte induces a decrease in the CMC and an increase in both aggregation number and hydration. However, complementary data of partial specific volume and cloud point of the surfactant suggested that the main contribution to micellar hydration is due to water mechanically trapped in the micelle. Fluorescence measurements do not indicate changes in the micellar micropolarity, probably due to modifications of the solubilization site of the probe caused by the micellar growth. Both pyrene excimer formation and fluorescence polarization of coumarin 6 revealed an increase in microviscosity with electrolyte addition, which is consistent with increased micellar hydration.     

Effect of Calcium Ion Concentration on Small Molecule Desorption from Alginate Beads

Spherical alginate beads were prepared by ionotropic gelation of sodium alginate through the use of calcium ions. Pyranine (Py) was added to the alginate solution as a small molecule probe for fluorescence studies. Desorption of Py in water from the alginate beads cross-linked with calcium ions was studied by using the steady state fluorescence technique. The fluorescence emission intensity (I) from Py was monitored during the desorption process at 512 nm using the time drive mode of the spectrofluorometer. The increase in I was attributed to Py release from the beads. The Fickian diffusion model was used to calculate the desorption coefficients, D_, which were found to be increased up to 3% (w/v) CaCl₂ concentration in the beads, and then decreased with a further increase of CaCl₂ content. On the other hand, the encapsulation efficiency of Py in the calcium alginate beads presented the reverse behavior compared to D. It was observed that, when the content of CaCl₂ was increased, the incubation time, t_0, for the start of desorption increased.     

Probing the Sol-Gel Transition in SiO2 Hydrogels—A New Application of Near-Infrared Fluorescence

Picosecond time-resolved fluorescence spectroscopy has enabled us to use a near-infrared fluorescent dye to probe the sol-gel transition in SiO₂ hydrogels, polymerized from sulfuric acid and sodium silicate solution, for the first time. We compare the microviscosity surrounding the probe during the sol-to-gel transition as predicted by two alternative models which both describe the decay of fluorescence anisotropy well. The results for one rotational time and a residual anisotropy imply that macrogelation of the sol leads to relatively small changes in the mobility of the fluorophore caused by small changes in microviscosity, but after much longer times, e.g., 1500 min, the mobility of the fluorophore decreases, reflecting a rapid increase in microviscosity of over several orders in magnitude. In sharp contrast, analysis of the anisotropy in terms of two rotational times predicts little change in microviscosity over the whole polymerization process.     

Investigation of the Structure of Polyelectrolyte–Micelle Complexes by the Luminescing Probe Technique

Using the luminescing probe technique, we have investigated the process of the formation of polyelectrolyte–micelle complexes and their structure. We have studied the interaction between macroions on the micelle–polyelectrolyte boundary. It has been established that, when the complex is formed in this way, the polarity in the surface layer of the micelle is increased. On increase in the concentration of polyelectrolyte and decrease in the length of its chain the micelle–polyelectrolyte complex becomes stabilized.     

A Fluorescence Study of New Angular Polycyclic Blue Light-Emitting pyrazolo[3,4-<Emphasis Type="Italic">h</Emphasis>][1,6]naphthyridine and their Interaction with Bovine Serum Albumin (BSA)

The blue light-emitting pyrazolo[3,4-h][1,6]naphthyridines has been synthesized by Friedländer condensation of 4-amino-3-(4-phenyl)-1-phenyl-1H-pyrazolo[3,4-b]pyridine-5-carbaldehyde (o-aminoaldehyde) 1 with different cyclic ketones and 1,3-diketones. The synthesized angular polycyclic naphthyridine derivatives were studied for Semi-empirical, thermal, UV–vis and fluorescence spectroscopic properties on binding with bovin serum albumin (BSA). These fluorescence properties together with the neutral, hydrophobic nature of these compounds make these fluorophores good fluorescence probe for studying the micropolarity of proteins like BSA and in general the ligand-protein interactions. All of them displays bright absorption at 394 nm &; emission in visible region (491 nm). Quantum yields of all synthesized compounds were calculated.     

A Novel Water-soluble Ratiometric Fluorescent Probe Based on FRET for Sensing Lysosomal pH

A new ratiometric fluorescent probe based on Förster resonance energy transfer (FRET) for sensing lysosomal pH has been developed. The probe (RMPM) was composed of imidazo[1,5-α]pyridine quaternary ammonium salt fluorophore as the FRET donor and the rhodamine moiety as the FRET acceptor. It’s the first time to report that imidazo[1,5-α]pyridine quaternary ammonium salt acts as the FRET donor. The ratio of fluorescence intensity of the probe at two wavelengths (I₄₂₄/I₅₈₁) changed significantly and responded linearly toward minor pH changes in the range of 5.4–6.6. It should be noted that it’s rare to report that a ratiometric pH probe could detect so weak acidic pH with pKa = 6.31. In addition, probe RMPM exhibited excellent water-solubility, fast-response, all-right selectivity and brilliant reversibility. Moreover, RMPM has been successfully applied to sensing lysosomal pH in HeLa cells and has low cytotoxicity.     

若丹明6G溶液体系的荧光量子效率

本文对八种若丹明6G溶液的荧光量子效率进行了测量,并着重讨论了染料若丹明6G溶液体系的量子效率的溶剂效应.在研究中发现:若丹明6G溶渡的荧光量子效率与溶液极性(ε-1)/(2ε+1)之间存在线性关系,体系的荧光量子效率随溶剂极性的增加而下降.由于染料分子与成氢键溶剂作用愈强,能量散逸愈快将是导致这一结果的主要原因.这一规律也适用于ANS染料溶液体系中.     

Determining the binding affinity and binding site of bensulfuron-methyl to human serum albumin by quenching of the intrinsic tryptophan fluorescence

Bensulfuron-methyl (BM) is a highly active sulfonylurea herbicide for use on paddy rice. Steady state fluorescence, UV/vis absorption, circular dichroism (CD), time-resolved fluorescence and molecular modeling methods have been exploited to determine the binding affinity and binding site of BM to human serum albumin (HSA). From the synchronous fluorescence, UV/vis, CD and three-dimensional fluorescence spectra, it was evident that the interaction between BM and HSA induced a conformational change in the protein. Steady state and time-resolved fluorescence data illustrates that the fluorescence quenching of HSA by BM was the formation of HSA-BM complex at 1:1 molar ratio. Site marker competitive experiments demonstrated that the binding of BM to HSA primarily took place in subdomain IIIA (Sudlow’s site II), this corroborates the hydrophobic probe ANS displacement and molecular modeling results. Thermodynamic analysis displays hydrophobic, electrostatic and hydrogen bonds interactions are the major acting forces in stabilizing the HSA-BM complex.     

Effect of a protein on the light energy conversion in dual fluorophore-nitroxide probes studied by ESR and fluorescence spectroscopy

Probing biological environment with dual fluorophore-nitroxide (FN) molecules in which fluorophore is tethered with nitroxide, a fluorescence quencher, opens unique opportunities to study molecular dynamics and micropolarity of the medium which affect intramolecular fluorescence quenching (IFQ), electron transfer, photoreduction and light energy conversion. In such molecules, the excited fragment of the chromophore can serve as an electron donor, and the nitroxide fragment as an electron acceptor. The same groups allow monitoring of molecular dynamics and also make it possible to measure micropolarity of the medium in the vicinity of the donor (by fluorescence technique) and acceptor (by electron spin resonance [ESR]) moieties. In the present work, two dual dansyl-nitroxide probes were incorporated in a binding site of bovine serum albumin. Their interactions with the protein, mobility, and photoreduction, as well as micropolarity of the media, have been studied by ESR and fluorescence methods. IFQ and spectral relaxation shift of the dansyl fragment have been monitored by time-resolved fluorescence technique. In parallel, computational studies on intramolecular dynamics of the FN probe were performed. On the basis of the Marcus model of the electron transfer between the excited dansyl fluorophore (donor) and nitroxide group (acceptor) and our experimental data, the mechanism of the electron transfer in the dual molecules incorporated into bovine serum albumin was established. It was shown that dual FN molecules in the protein meet main requirements for an efficient light energy conversion system.     

Confinement of 4,4-Diaminodiphenyl Sulfone by γ –CD in Micellar Environment: A Spectroscopic Investigation

This paper reports the double confinement of 4,4-diaminodiphenyl sulfone (Dapsone) inside γ–cyclodextrin (CD) in presence of surfactants (cationic, anionic and nonionic) using steady-state and time-resolved fluorescence spectroscopy. Interpretation of fluorescence spectra, fluorescence anisotropy and time resolved fluorescence decay of the γ-CD?Dapsone?micellar system hints at lesser microviscosity and the partial release of the probe molecule from the supramolecular host–guest complex in ionic micelles, of which greater in cationic micelles, but due to greater restriction and rigidity in presence of non-ionic micelle makes the probe more rigidly inside CD. Changes in computed rotational decay also corroborate the above findings.

Aggregation and microenvironmental properties of gemini and conventional mixed surfactants systems: A fluorometric study

Aggregation behavior of cationic gemini (hexanediyl-1,5-bis(dimethylcettylammonium bromide) (16-5-16)) surfactant with conventional single chain surfactants cetyltrimethylammonium bromide (CTAB) and tetradecyltrimethylammonium bromide (TTAB) were studied with the help of fluorescence measurements. Fluorescence probe is a proficient technique for examining the surfactant-surfactant interaction and aggregation. The micelle aggregation number (N _agg) was measured using steady-state fluorescence quenching method. The micelle aggregation numbers of binary combinations fall between those of constituent surfactants. The micropolarity (I ₁/I ₃), binding constant (K _sv) and dielectric constant (D _exp) of mixed systems were determined from the ratio of peaks intensity in the pyrene fluorescence spectrum. The I ₁/I ₃ values were found to be more than >1, showing more polar environment around pyrene in the mixed micelle as compared to the pure micelles.