Fluorometric Study Research Articles

Mixed Micellization of Gemini Surfactant with Nonionic Surfactant in Aqueous Media: A Fluorometric Study

Mixed Micellization of Gemini Surfactant with Nonionic Surfactant in Aqueous Media: A Fluorometric Study

Affinity of two highly preorganized ligands for the base metal ions Co(II), Ni(II) and Cu(II): A thermodynamic, crystallographic and fluorometric study

The metal ion complexing properties of 1,10-phenanthroline-2,9-dicarboxylic acid (PDA), with cobalt(II) and nickel(II) are reported. These were determined by UV–Vis spectroscopy in 0.1M NaClO4 at 25°C and the logK values are 12.6±0.05 and 13.6±0.05 respectively. These two values are compared to that of the previously reported value of copper(II), as well as the previously reported values of the ligand 2,9-bis(hydroxymethyl)-1,10-Phenanthroline (PDALC). Crystal structures of {[Ni(PDALC)2]Cℓ2}2·5H2O, {[Co(PDALC)2]Cℓ2}2·5H2O and {[Ni(PDAMOD)2](H2O)2}2 are reported, all showing distorted octahedral geometry around the metal centre. Furthermore, all three structures have four nitrogens and two oxygen atoms from alternate ligands coordinated to the metal ion. The crystal structure of the free ligand PDALC is also reported showing π–π stacking, hydrogen bonding with the solvent and also the extent of its preorganization. Lastly, a fluorescence study of divalent metal ions with these two ligands was carried out which included the three metal ions of interest. These results illustrate that Ca(II) produces the largest CHEF (chelation enhanced fluorescence) effects, followed by Mg(II) and Ba(II) in the PDA complexes. Metal ions with non-filled d-subshells produce very large quenching of the fluorescence. For the PDALC complexes, the largest CHEF was with Zn(II), followed by Ca(II), then Mg(II) which is the same as that of Ba(II), then closely followed by Cd(II). Both ligands show quenching effects with Co(II), Ni(II) and Cu(II) which are in the order: Co(II)>Ni(II) >Cu(II) for both ligands.

Fluorometric study of fluoxetine DNA binding

Fluoxetine (FLX), a selective serotonin reuptake inhibitor (SSRI), is commonly prescribed to treat depression. The interaction between FLX antidepressant and calf thymus DNA (ctDNA) was investigated under simulated physiological conditions (Tris–HCl buffer at pH 7.4) using methylene blue [3,7-bis(dimethylamino) pheno-thrazin-5-ium chloride] (MB) dye as a probe using fluorescence spectroscopy. A strong fluorescence quenching reaction of DNA to FLX was observed. The corresponding numbers of binding sites (n) and binding constants (Kf) of DNA with FLX at 281, 310 and 318K were calculated 2.1×105, 6.7×105 and 9.7×105 respectively. It can be concluded that FLX molecules could interact with ctDNA via outside, non-intercalative, binding as evidenced by quenching study with I−, ionic strength with NaCl and competitive investigation with MB. Thermodynamic parameters, enthalpy (ΔH) and entropy (ΔS) changes were calculated according to Van’t Hoff equation, which indicated that reaction is entropically driven. Furthermore, the interaction of FLX with poly A–T and poly C–G were carried out in order to comprehend the binding location of drug to DNA.

Solvatochromic Studies of Fluorescent Azo Dyes: Kamlet-Taft (π*, α and β) and Catalan (Spp, SA and SB) Solvent Scales Approach

A new metal ion-responsive azo-based fluorescent probes have been synthesized and characterized by NMR spectral techniques. Steady-state fluorometric study has been used to analyze the spectroscopic and photophysical characteristics of dye derivatives in various solvents. The fluorescence properties of these dyes are strongly solvent dependent, the wavelength of maximum fluorescence emission shifts to the red. The Kamlet-Taft and Catalan's solvent scales were found to be the most suitable for describing the solvatochromic shifts of the absorption and fluorescence emission. The hydroxy substituted azo dye formed complexes with several metal ions (Co(2+), Hg(2+), Ni(2+) and Cu(2+)) and fluorescence quenching with metal ions reveal that it can be used as a new fluorescence sensor to detect the Cu(2+) ion.

Analysis of surface cavity in serpin family reveals potential binding sites for chemical chaperone to reduce polymerization

Serpin constitute about 10% of blood protein and are associated with mutations that results in aberrant intermolecular linkages which leads to polymer formation. Studies with short peptides have shown promise in depolymerization of serpins however a reactive center loop based peptide also makes the serpin inactive. A chemical chaperone based approach is a better option in terms of maintaining activity and retarding polymerization but not much is known about its binding and mechanism. Specific target for chemical chaperones and its effectiveness across many serpin is not known. We did an analysis of serpin cavity using CASTp and show that cavities are distributed throughout the molecule where the largest cavities are generally present in areas of major conformational change like shutter region, helix D and helix F. An analysis of different conformational states of serpins showed that this large cavity undergoes increase in size in latent and cleaved states as compared to native state. We targeted serpins with a variety of carbohydrate, methylamine and amino acid based chemical chaperones and selected those that have highest binding energy across different serpins to assess their ability to bind large cavities. The results show that carbohydrate based chemical chaperone like sorbitol, sucrose, arabitol and trehalose and amino acid based chaperones like dopamine, phenylalanine, arginine and glutamic acid are the most effective in binding serpins. Most of these chemical chaperone interacted with residues in the shutter region and the helix D arm at the C-terminal which are part of the largest cavities. We selected the carbohydrate based chemical chaperone with best binding energies and did experimental study under the condition that induce polymerization and show that indeed they were able to retard polymer formation with moderate effect on inhibition rates. However a fluorometric study with native antithrombin showed that chemical chaperone may effect the conformation of the proteins. Our study shows that chemical chaperones have the best binding affinities for the cavities around shutter region and helix D and that a cavity targeting based approach seems to be a better option for retarding polymerization in serpins, but a thorough analysis of its effect on folding, inhibition and cofactor binding is required.

Compromise between compactness of micelle and overlap integral toward Förster resonance energy transfer from an indoloquinoline derivative to fluorescein: A fluorometric study

Förster resonance energy transfer (FRET) from an indoloquinoline derivative, 2,8-Dichloro-5-methyl-5H-indolo[3,2-c]quinoline (DCMIQ) to fluorescein (FL) is studied in anionic micelles formed by surfactants of varying chain lengths by steady state fluorescence spectroscopy. The Förster distance is found to change in different media. However, the calculated FRET efficiency for the donor–acceptor pair does not follow the trend observed for the overlap integrals. The aggregation number and hence the micellization kinetics on the dynamic surface properties of the surfactants seem to be responsible for such heterogeneity. Compromise between compactness of the micelle and overlap integral toward FRET has been proposed with experimental evidence.

Fluorometric Study for the Reaction Between Sertraline and 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole: Kinetics, Mechanism and Application for the Determination of Sertraline in Tablets

A fluorometric study has been carried out, for the first time, to investigate the reaction of the new generation antidepressant sertraline (SRT) with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl). In an alkaline buffered medium (pH 8.0), a green fluorescent product exhibiting maximum fluorescence intensity at 532 nm after excitation at 470 nm was produced. The factors affecting the reaction were carefully studied and the conditions were optimized. The kinetics of the reaction was investigated, the stoichiometry of the reaction was determined, and the mechanism was postulated. The activation energy of the reaction was determined and found to be 27.34 KJ mole(-1). Under the optimum reaction conditions, a linear relationship with good correlation coefficient (r = 0.9998, n = 6) was found between the fluorescence intensity of the reaction product and SRT concentrations in the range of 0.3-20.0 microg ml(-1). The limit of detection and limit of quantitation were 0.07 and 0.21 microg ml(-1), respectively. The intra- and inter-assay precisions were satisfactory; the relative standard deviations did not exceed 2.61%. The proposed method was successfully applied to the determination of SRT in its pharmaceutical tablets with good accuracy; the recovery percentages were 96.97-102.23 +/- 1.01-1.62%. The results were compared favorably with those of the reported method.

Involvement of Na+/Ca2+ exchanger in migration and contraction of rat cultured tendon fibroblasts

In response to injury and inflammation of tendons, tendon fibroblasts are activated, migrate to the wound, and eventually induce contraction of the extracellular matrices to repair the tissue. Under such conditions, Ca(2+) signalling is involved in motility and contractility of tendon fibroblasts. Using cultured tendon fibroblasts isolated from rat Achilles tendons, we investigated functional expression of Na(+)/Ca(2+) exchangers (NCX). The fluorometric study showed that the intracellular Ca(2+) concentration ([Ca(2+)](i)) was increased by reducing extracellular Na(+) concentration ([Na(+)](o)) in tendon fibroblasts. Selective NCX inhibitors, KB-R7943 and SEA0400, both attenuated [Na(+)](o)-dependent [Ca(2+)](i) elevation and the resting [Ca(2+)](i) in tendon fibroblasts. RT-PCR, Western blots and sequence analyses revealed that NCX1.3 and NCX1.7 were expressed in cultured tendon fibroblasts. NCX2 mRNA was undetected. NCX3 expression was negligibly low. Immunofluorescence microscopy indicated that NCX1 protein localized in the plasma membrane especially at the microspikes of tendon fibroblasts. In the wound-healing scratch assay, the cells migrated toward the space created by a scratch and almost completely filled the space within 48 h. This phenomenon was significantly suppressed by KB-R7943 and SEA0400. Furthermore, the NCX inhibitors abrogated the tendon fibroblast-mediated collagen-matrix contractions. Two types of siRNAs for NCX1 also suppressed the migration and contraction of tendon fibroblasts. We conclude that NCX is expressed and mediates Ca(2+) influx in cultured tendon fibroblasts. Since the pharmacological inhibitors and siRNA for NCX1 suppressed motility and contractility of tendon fibroblasts, NCX may play an important role in the function of tendon fibroblasts in the wound healing.

Possible Steric Control of the Relative Strength of Chelation Enhanced Fluorescence for Zinc(II) Compared to Cadmium(II): Metal Ion Complexing Properties of Tris(2-quinolylmethyl)amine, a Crystallographic, UV−Visible, and Fluorometric Study

The idea is examined that steric crowding in ligands can lead to diminution of the chelation enhanced fluorescence (CHEF) effect in complexes of the small Zn(II) ion as compared to the larger Cd(II) ion. Steric crowding is less severe for the larger ion and for the smaller Zn(II) ion leads to Zn-N bond length distortion, which allows some quenching of fluorescence by the photoinduced electron transfer (PET) mechanism. Some metal ion complexing properties of the ligand tris(2-quinolylmethyl)amine (TQA) are presented in support of the idea that more sterically efficient ligands, which lead to less M-N bond length distortion with the small Zn(II) ion, will lead to a greater CHEF effect with Zn(II) than Cd(II). The structures of [Zn(TQA)H(2)O](ClO(4))(2).1.5 H(2)O (1), ([Pb(TQA)(NO(3))(2)].C(2)H(5)OH) (2), ([Ag(TQA)(ClO(4))]) (3), and (TQA).C(2)H(5)OH (4) are reported. In 1, the Zn(II) is 5-coordinate, with four N-donors from the ligand and a water molecule making up the coordination sphere. The Zn-N bonds are all of normal length, showing that the level of steric crowding in 1 is not sufficient to cause significant Zn-N bond length distortion. This leads to the observation that, as expected, the CHEF effect in the Zn(II)/TQA complex is much stronger than that in the Cd(II)/TQA complex, in contrast to similar but more sterically crowded ligands, where the CHEF effect is stronger in the Cd(II) complex. The CHEF effect for TQA with the metal ions examined varies as Zn(II) >> Cd(II) >> Ni(II) > Pb(II) > Hg(II) > Cu(II). The structure of 2 shows an 8-coordinate Pb(II), with evidence of a stereochemically active lone pair, and normal Pb-N bond lengths. In 3, the Ag(I) is 5-coordinate, with four N-donors from the TQA and an oxygen from the perchlorate. The Ag(I) shows no distortion toward linear 2-coordinate geometry, and the Ag-N bonds fall slightly into the upper range for Ag-N bonds in 5-coordinate complexes. The structure of 4 shows the TQA ligand to be involved in pi-stacking between quinolyl groups from adjacent TQA molecules. Formation constants determined by UV-visible spectroscopy are reported in 0.1 M NaClO(4) at 25 degrees C for TQA with Zn(II), Cd(II), and Pb(II). When compared with other similar ligands, one sees that, as the level of steric crowding increases, the stability decreases most with the small Zn(II) ion and least with the large Pb(II) ion. This is in accordance with the idea that TQA has a moderate level of steric crowding and that steric crowding increases for TQA analogs tris(2-pyridylmethyl)amine (TPyA) < TQA < tris(6-methyl-2-pyridyl)amine (TMPyA).

Preferential Solvation of 1,4-Dimethoxy-2,3-Dimethyl-9,10-Anthraquinone-A Spectrophotometric and Fluorometric Study

Electronic absorption and fluorescence emission spectra of DMDMAQ (1,4-dimethoxy-2,3-dimethyl-9,10-anthraquinone) have been studied as a function of solvent composition in some binary mixtures and in different neat solvents. The binary mixtures consist CCl(4) (Carbon tetrachloride)-DMSO (Dimethylsulfoxide), EtOH (Ethanol)-DMSO, and CCl(4)-EtOH combination of single solvents. The wavelength maxima of the absorption band for DMDMAQ are quite solvent sensitive in aprotic solvents. But, in protic solvent, there is no marked shift in absorption and emission maximum which shows the absence of specific interaction. Excited state shows increasing shift with increasing solvent polarity compared to ground state. The ratio of dipole moment in the excited state to that in the ground state was calculated. Different criteria were considered to analyse preferential solvation characteristics in different binary mixtures, viz., local mole fraction (X(2)(L)), solvation index (delta(S2)) and exchange constant (K(12)).

Interaction of Fusarium solani Lectin with Monosaccharides and Oligosaccharides: A Fluorometric Study

The intrinsic fluorescence intensity of Fusarium solani lectin was quenched upon binding to mono- and oligosaccharides without any change in the emission maximum and it was used to determine association constants for several sugars and glycans. The lectin interacted very poorly with monosaccharides but well with disaccharides (T-antigen and LacNAc) with a distinction between beta1-->4 and beta1-->3 linkages. Among the monosaccharides, the interaction was observed only with Gal/GalNAc derivatives and not with Glc/Man derivatives. Thermodynamic studies revealed that the binding of the lectin with all the saccharides is enthalpically driven and exothermic in nature. Asialo-triantennary N-glycan and asialo-biantennary N-glycan showed higher affinity than monovalent LacNAc with significant increase in binding enthalpy, pointing towards the importance of multivalency in the lectin-ligand interactions. Time-resolved fluorescence measurement indicated the lectin has two lifetimes for tryptophan and the shorter lifetime is affected on ligand binding.

Fluorometric Study on the Interaction between Lomefloxacin and Bovine Lactoferrin

The binding of lomefloxacin to bovine lactoferrin (BLf) in a dilute aqueous solution was studied using fluorescence spectra. The binding constant (K) and the number of binding sites (n) were obtained by a fluorescence quenching method. The binding distance (r) and energy-transfer efficiency (E) between lomefloxacin and bovine lactoferrin were also obtained according to the mechanism of Foörster-type dipole-dipole nonradiative energy-transfer. The effect of lomefloxacin on the conformation of bovine lactoferrin was also analyzed by synchronous fluorescence spectroscopy. The interaction between lomefloxacin and bovine lactoferrin is strong. Lomefloxacin can affect the conformation of bovine lactoferrin to some degree.

Chromatographic and fluorometric study of interactions between thiophilic and hydrophobic ligands and tryptophan peptide homologues

The interactions of tryptophan and its peptide homologues with thiophilic ligands were studied in terms of their chromatographic retention and steady-state fluorescence under various conditions, and compared with non-polar structures typically regarded as pure hydrophobic ligands. The experimental results show that both non-polar and polar interactions are involved in what has been termed “thiophilic adsorption chromatography”.

Interaction of pyrene-end-capped poly(ethylene oxide) with bovine serum albumin and human serum albumin in aqueous buffer medium: A fluorometric study

The photophysical behavior of a hydrophobically tailored water-soluble polymer, pyrene-end-capped poly(ethylene oxide) (PYPY), has been studied in aqueous buffered bovine serum albumin (BSA) and human serum albumin (HSA) media. In buffered aqueous solution the polymer shows dual emission corresponding to the monomer and the excimer of pyrene moiety. The relative intensity of the monomer to the excimer emission shows interesting variation with the addition of BSA and HSA and is indicative of significant interaction of these albumin proteins with the polymer. The binding interaction has been shown to have a prominent role on the steady state fluorescence anisotropy of the two emission bands. Attempt has been made to determine the micropolarities of the protein microenvironments from a comparison of the variation of the monomer to excimer relative fluorescence intensities of the probe in water–dioxane mixtures with varying composition.

The GUS reporter-aided analysis of the promoter activities of Arabidopsis ACC synthase genes AtACS4, AtACS5, and AtACS7 induced by hormones and stresses

Ethylene biosynthesis in higher plants is regulated developmentally and environmentally. To investigate the regulation of ACC synthase gene expression, the promoters of Arabidopsis ACS genes, AtACS4, AtACS5, and AtACS7, were fused to a GUS reporter gene, and the recombinant transgenes were introduced into Arabidopsis to produce three groups of AtACS::GUS transgenic plants. Histochemic and fluorometric study of these transgenic plants revealed that promoters of AtACS4, AtACS, and AtACS7 are all active in dark-germinated seedlings. AtACS5 has the highest promoter activity in leaves of 2-week-old light-grown seedlings among the three AtACS genes studied. In the mature leaves, AtACS4 and AtACS7 genes are expressed in both veins and areoles, whereas AtACS5 is expressed at a higher level in the areoles and epidermal cells surrounding trichomes. The promoter activities of all these AtACS genes are found in the reproductive organs. AtACS5 and AtACS7 are highly expressed in petals, sepals, carpels, stamens, cauline leaves, inflorescence stems, and siliques, while AtACS4 expression is undetectable in the petals of open flowers. All three AtACS genes are expressed in root tissue. In the 2-week-old light-grown Arabidopsis, the AtACS4 promoter is responsive to the plant hormones IAA, ethylene, and ABA, and to darkness and wounding; the AtACS5 promoter to IAA, ABA, salt, high temperature, and wounding; and the AtACS7 promoter to GA3, ethylene, and ABA, and to darkness and salt. Low-temperature treatment abolishes the darkness-induced AtACS7 gene expression, but not that of AtACS4. Each AtACS gene has a unique expression profile during growth and development. It appears that at any developmental stage or any growth period of Arabidopsis, there is always a member of AtACS multigene family that is actively expressed.

Structural Stability of Azurin Encapsulated in Sol-Gel Glasses: A Fluorometric Study

In this study we investigated the structural features of azurin, a blue copper-containing enzyme, upon encapsulation in tetramethoxysilane derived sol-gel glasses. Fluorescence spectroscopy revealed that gelation of inorganic networks does not affect the protein tertiary structure and only after two months solvent phase loss altered protein stability. In case of organically modified sol-gel matrices, the protein stability was reduced after encapsulation into hosts modified by adding 3-Mercaptopropyl-trimethoxysilane, 3-Glycidyloxypropyl-trimethoxysilane and Trimethoxy octylsilane, while it was found to be enhanced in networks doped with 3-Trimethoxysilyl-propyl methacrylate and 3-Aminopropyl-trimethoxysilane. In order to better investigate the effects of silica glasses on azurin stability, unfolding experiments of the protein, in solution or entrapped, were also performed in the presence of both methanol and guanidinium hydrochloride (GdHCl). Our results suggest that the matrix protects azurin against the aggregation induced by alcohol, and that the free energy change value upon unfolding by GdHCl was lower than the value calculated for azurin in solution and was dependent on the surface chemistry of silica matrix.

Photophysics of norharmane in micellar environments: a fluorometric study

Steady state photophysics of norharmane (NHM) has been studied in different aqueous micellar environments. In aqueous solution at pH 7, excitation of the neutral species promotes a rapid transfer of proton giving rise to the corresponding cationic emission. Aqueous micelles differing in their surface charge characteristics interact with the fluorophore differently. The dependence of the fluorescence of the probe molecule on different micelles has been exploited to determine the critical micellar concentrations (CMCs) of the surfactants. The binding constant ( K) and free energy change (Δ G) for the interaction of norharmane with the micelles have been evaluated from the fluorescence data. The probable location of the probe in the micelles has also been suggested. Polarity of the microenvironment around the probe has been determined for CTAB and TX-100 micellar systems from a comparison of the variation of fluorescence properties of the two prototropic species in water–dioxane mixture with varying composition.

Fluorometric study of rabbit sperm head membrane phospholipid asymmetry during capacitation and acrosome reaction using Annexin-V FITC.

This study was conducted to evaluate phosphatidylserine translocation in head plasma membrane of Percoll-gradient purified of rabbit cauda epididymal sperm during capacitation and acrosome reaction (AR) using Annexin-V. Propidium iodide was used as control to reject dead or dying cells. The presence and distribution of Annexin-V binding sites were analyzed using flow fluorocytometry and confocal microscopy. After 6 h of incubation of sperm in capacitation medium, the number of cells positively stained with Annexin-V showed a small but significant increment. The Annexin-V binding sites produced during capacitation were found mainly in the post-acrosomal region of the sperm head plasma membrane. After AR induction with progesterone, the localization of phosphatidylserine was changed and the Annexin-V binding sites were found almost only in the acrosomal region, but with higher number of binding sites in the equatorial area. On the contrary, after AR induction with A23187, phosphatidylserine translocation, although predominant over the acrosomal region, was also observed in the post-acrosomal region. Plasma membrane destabilization during capacitation and AR may be important for sperm-oocyte fusion.

Fluorometric study of the acid-induced denaturation of Staphylococcal nuclease and its mutant forms.

The acid-induced denaturation of wild-type Staphylococcal nuclease (WT) and its eight mutant forms, L25A, V66L, G79S, A90S, G88V, H124L, V66L/G88V, and V66L/G79S/G88V, was investigated using Trp140 fluorescence as a probe at 30 degrees C. The values of pH(1/2), at which the denaturation is half completed, and n, the apparent number of protons which trigger the denaturation and are taken up by the proteins upon denaturation at pH(1/2), were evaluated from the pH dependence of the fluorescence intensity. The values of pH(1/2) and n for WT were 3.8 and 1.8 respectively. The amino acid replacements changed the pH(1/2) values to a range between 3.0 (H124L) and 4.4 (G79S) and also changed the n values to a range between 1.0 (A90S) and 3.0 (G88V). There was a negative correlation between the values of pH(1/2) and n. It was suggested that the amino acid replacements may change the energy levels of the native state and/or the denatured state mainly in the neutral (stable) pH region, not in the acidic (unstable) region, resulting in the correlative changes in pH(1/2) and n.

Fluorometric study of the inclusion interaction of β-cyclodextrin derivatives with tetraphenylporphyrin and its analytical application

The effects of native β-cyclodextrin (β-CD) and four kinds of alkylated β-cyclodextrin (β-CDs), i.e. heptakis (2,6-di-O-isobutyl-β-cyclodextrin) (I), heptakis (2,6-di-O-octyl-β-cyclodextrin) (II), heptakis (2,6-di-O-dodecyl-β-cyclodextrin) (III), and heptakis (2,6-di-O-hexadecyl-β-cyclodextrin) (IV), on the fluorescence behaviors of tetraphenylporphyrin (TPP) are investigated. An obvious fluorescence enhancement is observed from TPP by using alkylated derivatives compared to that obtained in β-CD aqueous or in water. A 114-N fluorescence emission intensity enhancement is found for the complex with 2,6-di-O-octyl-β-cyclodextrin relative to the free analyte. The exact stoichiometric ratios and the formation constants of the inclusion complexes have been examined by application of curve fitting method. The linear calibration plots between fluorescence intensity and TPP concentration are determined in the 1.14×10 −8–5.06×10 −6 mol l −1 range.