Time-resolved spectral observations of cadmium-enriched cadmium sulfide nanoparticles and the effects of DNA oligomer binding

We measured the steady-state and time-resolved fluorescence spectral properties of cadmium-enriched nanoparticles (CdS-Cd2+). These particles displayed two emission maxima, at 460 and 580 nm. The emission spectra were independent of excitation wavelength. Surprisingly, the intensity decays were strongly dependent on the observation wavelength, with longer decay times being observed at longer wavelengths. The mean lifetime increased from 150 to 370 ns as the emission wavelength was increased from 460 to 650 nm. The wavelength-dependent lifetimes were used to construct the time-resolved emission spectra, which showed a growth of the long-wavelength emission at longer times, and decay-associated spectra, which showed the longer wavelength emission associated with the longer decay time. These nanoparticles displayed anisotropy values as high as 0.35, depending on the excitation and emission wavelengths. Such high anisotropies are unexpected for presumably spherical nanoparticles. The anisotropy decayed with two correlation times near 5 and 370 ns, with the larger value probably due to overall rotational diffusion of the nanoparticles. Addition of a 32-base pair oligomer selectively quenched the 460-nm emission, with less quenching being observed at longer wavelengths. The time-resolved intensity decays were minimally affected by the DNA, suggesting a static quenching mechanism. The wavelength-selected quenching shown by the nanoparticles may make them useful for DNA analysis.     

Interaction of aromatic compounds with Photobacterium leiognathi luciferase: fluorescence anisotropy study

The time‐resolved and steady‐state fluorescence techniques were employed to elucidate possible interactions of four aromatic compounds (anthracene, POPOP, MSB and 1,4‐naphthalendiol) with bacterial luciferase. Fluorescence spectra and fluorescence anisotropy decays of these compounds were studied in ethanol, water–ethanol solutions and in the presence of bacterial luciferase. Shifts of fluorescent spectra and differences in rotational correlation times are interpreted in terms of weak (hydrophobic) interactions of the molecules with the enzyme. These interactions suggest the feasibility of intermolecular energy transfer by an exchange resonance mechanism with a collision‐interaction radius as a way of excitation of these compounds in the reaction catalysed by bacterial luciferase. Copyright © 2003 John Wiley & Sons, Ltd.     

Mechanism of the down regulation of photosynthesis by blue light in the Cyanobacterium synechocystis sp. PCC 6803

Exposure to blue light has previously been shown to induce the reversible quenching of fluorescence in cyanobacteria, indicative of a photoprotective mechanism responsible for the down regulation of photosynthesis. We have investigated the molecular mechanism behind fluorescence quenching by characterizing changes in excitation energy transfer through the phycobilin pigments of the phycobilisome to chlorophyll with steady-state and time-resolved fluorescence excitation and emission spectroscopy. Quenching was investigated in both a photosystem II-less mutant, and DCMU-poisoned wild-type Synechocystis sp. PCC 6803. The action spectra for blue-light-induced quenching was identical in both cell types and was dominated by a band in the blue region, peaking at 480 nm. Fluorescence quenching and its dark recovery was inhibited by the protein cross-linking agent glutaraldehyde, which could maintain cells in either the quenched or the unquenched state. We found that high phosphate concentrations that inhibit phycobilisome mobility and the regulation of energy transfer by the light-state transition did not affect blue-light-induced fluorescence quenching. Both room temperature and 77 K fluorescence emission spectra revealed that fluorescence quenching was associated with phycobilin emission. Quenching was characterized by a decrease in the emission of allophycocyanin and long wavelength phycobilisome terminal emitters relative to that of phycocyanin. A global analysis of the room-temperature fluorescence decay kinetics revealed that phycocyanin and photosystem I decay components were unaffected by quenching, whereas the decay components originating from allophycocyanin and phycobilisome terminal emitters were altered. Our data support a regulatory mechanism involving a protein conformational change and/or change in protein-protein interaction which quenches excitation energy at the core of the phycobilisome.     

Resolution of low-energy chlorophylls in Photosystem I of Synechocystis sp. PCC 6803 at 77 and 295 K through fluorescence excitation anisotropy

Fluorescence excitation spectra of highly anisotropic emission from Photosystem I (PS I) were measured at 295 and 77 K on a PS II-less mutant of the cyanobacterium Synechocystis sp. PCC 6803 (S. 6803). When PS I was excited with light at wavelengths greater than 715 nm, fluorescence observed at 745 nm was highly polarized with anisotropies of 0.32 and 0.20 at 77 and 295 K, respectively. Upon excitation at shorter wavelengths, the 745-nm fluorescence had low anisotropy. The highly anisotropic emission observed at both 77 and 295 K is interpreted as evidence for low-energy chlorophylls (Chls) in cyanobacteria at room temperature. This indicates that low-energy Chls, defined as Chls with first excited singlet-state energy levels below or near that of the reaction center, P700, are not artifacts of low-temperature measurements.If the low-energy Chls are a distinct subset of Chls and a simple two-pool model describes the excitation transfer network adequately, one can take advantage of the low-energy Chls' high anisotropy to approximate their fluorescence excitation spectra. Maxima at 703 and 708 nm were calculated from 295 and 77 K data, respectively. Upper limits for the number of low-energy Chls per P700 in PS I from S. 6803 were calculated to be 8 (295 K) and 11 (77 K).Abbreviations Chl - chlorophyll - BChl - bacteriochlorophyll - LHC - light-harvesting chlorophyll - PS - Photosystem - RC - reaction center - S. 6803 - Synechocystis sp. PCC 6803     

Analysis of internal motion of single tryptophan in Streptomyces subtilisin inhibitor from its picosecond time-resolved fluorescence.

A mode of internal motion of single tryptophan, Trp 86, of Streptomyces subtilisin inhibitor, was analyzed from its time-resolved fluorescence. The intensity and anisotropy decays of Trp 86 were measured in the picosecond range. These decays were analyzed with theoretical expressions derived assuming that the indole ring of tryptophan as an asymmetric rotor rotates around covalent bonds connecting indole with the peptide chain and an effective quencher of fluorescence of Trp 86 is the nearby SS bond of Cys 35-Cys 50. First, the intensity decays at 6 degrees, 20 degrees, and 40 degrees C were analyzed, and then the both decays of the intensity and anisotropy at 20 degrees C were simultaneously simulated with common parameters. Constants concerning geometrical structures of the protein used for the analysis were obtained from x-ray crystallographic data. Best fit between the observed and calculated decay curves was obtained by a nonlinear least squares method by adjusting a quenching constant averaged over the rotational angles, koq height of the potential energy, p, and three of six diffusion coefficients, Dxx, Dyy, Dzz, Dxy, Dyz, and Dzx, as variable parameters. The obtained results revealed that the internal motion of the indole ring became faster, the quenching rate of the fluorescence of Trp 86 was enhanced and the height of potential energy became lower at higher temperatures, and suggested that Trp 86 was wobbling around the long axis of the indole ring in the protein.     

Fluorimetric determination of febuxostat in dosage forms and in real human plasma via Förster resonance energy transfer

A rapid, simple, selective and precise fluorimetric method was developed and validated for determination of a selective xanthine oxidase inhibitor; febuxostat (FBX) in pharmaceutical formulations and in human plasma. The proposed method is based on quenching effect of FBX on the fluorescence intensity of terbium (Tb³⁺) through fluorescence resonance energy transfer (FRET) from Tb³⁺ to FBX. The formed complex was measured at λ_ex. 320 nm/λ_em. 490 nm against a reagent blank. Fluorescence intensity of Tb³⁺ was diminished when FBX was added. A linear relationship between the fluorescence quenching value of the formed complex and the concentration of FBX was investigated. The reaction conditions and the fluorescence spectral properties of the complex have been studied. The linearity range of the developed method was 1.0–16.0 μg/ml. The suggested method was applied successfully for the estimation of FBX in bulk powder, dosage forms and spiked plasma samples with excellent recoveries (96.79–98.89%). In addition, the developed method has been successfully applied for determination of FBX in real plasma samples collected from healthy volunteers with good recoveries (82.06–85.65%). All obtained results of the developed method were statistically analyzed and validated according to ICH (International Conference on Harmonization) guidelines.     

三种藻胆蛋白在复合累积LB膜中的能量传递

制备了R-藻红蛋白(R-PE)、C-藻蓝蛋白(C-PC)及变藻蓝蛋白(APC)的单组分累积LB膜及三组分复合累积LB膜. 吸收及荧光光谱测定表明三种蛋白的LB膜的光谱重叠性质与他们在水溶液中的性质相同. 结构测定表明, 三种蛋白在其LB膜中排列有序, 且由它们组装的复合累积LB膜结构类似于藻类植物中的藻胆体结构. 通过稳态荧光光谱及其光谱解叠, 观察到了三种蛋白在复合累积LB膜中的激发能量传递现象. 根据给体荧光峰的猝灭, 计算出在复合累积LB膜中从给体R-PE经C-PC到受体APC的能量传递效率为51%.     

槐定碱与牛血清白蛋白的相互作用研究

在模拟动物体生理条件下,用荧光猝灭、荧光偏振和紫外-可见吸收光谱法研究了槐定碱与牛血清白蛋白(BSA)结合作用。荧光猝灭数据显示,槐定碱与BSA发生反应生成了新的复合物,属于静态荧光猝灭。求出了不同温度(19、25、31、37℃)下槐定碱与BSA作用的结合常数分别为1.219×106,1.164×106,1.110×106和1.057×106L/mol,由van’tHoff方程式计算槐定碱与BSA反应的热力学参数:焓变ΔH和熵变ΔS值分别为-5.97kJ/mol和96.11J/(mol.K),表明槐定碱与BSA间的作用力以静电引力为主。以华法林和布洛芬(分别为siteI和siteII探针)为标记药物研究槐定碱在BSA上的结合位点,结果表明,槐定碱结合在BSA疏水空腔的siteI位点。     

Evaluation of binding and thermodynamic characteristics of interactions between a citrus flavonoid hesperitin with protein and effects of metal ions on binding

The mechanism of interaction of a non-glycosidic citrus flavonoid, hesperitin (HES) with bovine serum albumin (BSA) was studied by UV-vis absorption, fluorescence, FT-IR, circular dichroism, fluorescence anisotropy and synchronous fluorescence spectroscopy in phosphate buffer of pH 7.4. Fluorescence data revealed that the fluorescence quenching of BSA by HES was the result of the formed complex of HES-BSA. The binding constants and thermodynamic parameters at four different temperatures, the location of binding, and the nature of binding force were determined. The hydrogen bonds interactions were found to be the predominant intermolecular forces to stabilize the complex. The conformation of BSA was discussed by synchronous fluorescence and CD methods. The alterations of protein secondary structure upon complexation with HES were evident from the gradual decrease in α-helicity. The distance between the donor (BSA) and acceptor (flavonoid) was calculated from the fluorescence resonance energy transfer and found to be 1.978 nm. Common ions viz., Zn(2+), K(+), Cu(2+), Ni(2+), Mn(2+) and Co(2+) were found to influence the binding of flavonoid to protein.     

Eco-friendly approach for the determination of moxifloxacin in pharmaceutical formulations and biological fluids based on fluorescence quenching of l-tryptophan

A rapid, novel and cost-effective spectrofluorimetric method developed to determine moxifloxacin (MFX) in pharmaceutical preparations because MFX in a pH 10 medium could reduce the fluorescence intensity of l -tryptophan. The maximum fluorescence excitation and emission wavelengths were found to be 280 and 363 nm respectively. A range of factors affecting fluorescence quenching and the effect of co-existing substances were investigated. Fluorescence quenching values (ΔF = F_L-tryptophan − F_{Moxi-L-tryptophan}) displayed a strong linear relationship with the MFX concentration ranging from 0.2 to 8.0 μg/ml under optimum conditions. The limit of detection was found to be 6.1 × 10⁻⁴ μg/ml. The proposed method was shown to be suitable for MFX determination in pharmaceutical tablets and biological fluids by the linearity, recovery and limit of detection. The spectrofluorimetric approach that has been developed is extremely eco-friendly, as evidenced by the fact that all the experimental components and solvents were safe for the environment.     

A non-linear least-squares approach to the resolution of heterogeneous fluorescence from multitryptophan proteins

Protein heterogeneous fluorescence results from the different microenvironment of each emitting chromophore. The structural and dynamic information contained in this emission can be extracted to some extent by selective quenching experiments. In this work, graphical and numerical methods are described for the analysis of protein emission in terms of three separated contributions: a fluorescence fraction which is not accessible to the quencher and two additional fractions with different solvent exposure. ‘Static quenching’ deviations from Stern-Volmer behaviour are also discussed. The application of these methods is exemplified on simulated quenching experiments and real data on acrylamide quenching of lysozyme fluorescence.     

Fluorescence properties of free and protein bound fluorescein dyes. I. Macrospectrofluorometric measurements.

Excitation and emission properties of fluorescein derivatives were studied macrofluorometrically. Measurements were performed with solutions of various concentrations (0.07-100 microgram/ml) of free sodium fluorescein prepared from fluorescein diacetate (FDA), fluorescein isothiocyanate (FITC) and FITC bound to rabbit gamma-globulin. Both excitation and emission spectra as well as fluorescence intensities at constant excitation/emission wavelengths (496/515 nm) were recorded. The findings indicate that (1) FDA gives about twice the fluorescence intensity compared to equal concentrations of FITC. (2) The fluorescence properties of FITC upon excitation with blue light (lambda = 496 nm) are only slightly altered by the conjugation to rabbit gamma-globulin. (3) Considerable quenching due to conjugation could, however, be shown to occur upon UV excitation (lambda = 340 nm). (4) Fluorescence emission excited by visible blue light (496 nm) increases linearly to dye concentration in a range of 0.07-2.5 microgram/ml. Beginning at 5 microgram/ml (10-(5) M/1) all three compounds show a sharp decrease of fluorescence intensity with further increasing concentration. Practical aspects of these data for the immunofluorescence method are discussed.     

Time resolved fluorescence and phosphorescence properties of the individual tryptophan residues of barnase: evidence for protein-protein interactions.

Steady-state and time-resolved fluorescence, as well as phosphorescence measurements, were used to resolve the luminescence properties of the three individual tryptophan residues of barnase. Assignment of the fluorescence properties was performed using single-tryptophan-containing mutants and the results were compared with the information available from the study of wild-type and two-tryptophan-containing mutants (Willaert, Lowenthal, Sancho, Froeyen, Fersht, Engelborghs, Biochemistry 1992;31:711-716). The fluorescence and the phosphorescence emission of wild-type barnase is dominated by Trp35, although Trp71 has the strongest intrinsic fluorescence when present alone. Fluorescence emission of these two tryptophan residues is blue-shifted and pH-independent. The fluorescence decay parameters of Trp94 are pH-dependent, and an intramolecular collision frequency of 2 to 5 x 10(9) s(-1) between Trp94 and His18 is calculated. Fluorescence emission of Trp94 is red-shifted. Fluorescence anisotropy decay reveals the local mobility of the individual tryptophan residues and this result correlates well with their phosphorescence properties. Trp35 and Trp71 display a single phosphorescence lifetime, which reflects the rigidity of their environment. Surface Trp94 does not exhibit detectable phosphorescence emission. The existence of energy transfer between Trp71 and Trp94, as previously detected by fluorescence measurements, is also observed in the phosphorescence emission of barnase. Dynamic quenching causes the phosphorescence intensity to be protein-concentration dependent. In addition, fluorescence anisotropy shows concentration dependency, and this can be described by the formation of trimers in solution.     

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.     

Fluorescence analysis of the Hansenula polymorpha peroxisomal targeting signal-1 receptor, Pex5p.

Correct sorting of newly synthesized peroxisomal matrix proteins is dependent on a peroxisomal targeting signal (PTS). So far two PTSs are known. PTS1 consists of a tripeptide that is located at the extreme C terminus of matrix proteins and is specifically recognized by the PTS1-receptor Pex5p. We studied Hansenula polymorpha Pex5p (HpPex5p) using fluorescence spectroscopy. The intensity of Trp fluorescence of purified HpPex5p increased by 25% upon shifting the pH from pH 6.0 to pH 7.2. Together with the results of fluorescence quenching by acrylamide, these data suggest that the conformation of HpPex5p differs at these two pH values. Fluorescence anisotropy decay measurements revealed that the pH affected the oligomeric state of HpPex5p, possibly from monomers/dimers at pH 6.0 to larger oligomeric forms at pH 7.2. Addition of dansylated peptides containing a PTS1, caused some shortening of the average fluorescence lifetime of the Trp residues, which was most pronounced at pH 7.2. Our data are discussed in relation to a molecular model of HpPex5p based on the three-dimensional structure of human Pex5p.     

Localization and environment of tryptophans in soluble and membrane-bound states of a pore-forming toxin from Staphylococcus aureus

The location and environment of tryptophans in the soluble and membrane-bound forms of Staphylococcus aureus alpha-toxin were monitored using intrinsic tryptophan fluorescence. Fluorescence quenching of the toxin monomer in solution indicated varying degrees of tryptophan burial within the protein interior. N-Bromosuccinimide readily abolished 80% of the fluorescence in solution. The residual fluorescence of the modified toxin showed a blue-shifted emission maximum, a longer fluorescence lifetime as compared to the unmodified and membrane-bound alpha-toxin, and a 5- to 6-nm red edge excitation shift, all indicating a restricted tryptophan environment and deeply buried tryptophans. In the membrane-bound form, the fluorescence of alpha-toxin was quenched by iodide, indicating a conformational change leading to exposure of some tryptophans. A shorter average lifetime of tryptophans in the membrane-bound alpha-toxin as compared to the native toxin supported the conclusions based on iodide quenching of the membrane-bound toxin. Fluorescence quenching of membrane-bound alpha-toxin using brominated and spin-labeled fatty acids showed no quenching of fluorescence using brominated lipids. However, significant quenching was observed using 5- and 12-doxyl stearic acids. An average depth calculation using the parallax method indicated that the doxyl-quenchable tryptophans are located at an average depth of 10 A from the center of the bilayer close to the membrane interface. This was found to be in striking agreement with the recently described structure of the membrane-bound form of alpha-toxin.     

Evidence for a novel mechanism of time-resolved flavin fluorescence depolarization in glutathione reductase

Time-resolved flavin fluorescence anisotropy studies on glutathione reductase (GR) have revealed a remarkable new phenomenon: wild-type GR displays a rapid process of fluorescence depolarization, that is absent in mutant enzymes lacking a nearby tyrosine residue that blocks the NADPH-binding cleft. Fluorescence lifetime data, however, have shown a more rigid active-site structure for wild-type GR than for the tyrosine mutants. These results suggest that the rapid depolarization in wild-type GR originates from an interaction with the flavin-shielding tyrosine, and not from restricted reorientational motion of the flavin. A novel mechanism of fluorescence depolarization is proposed that involves a transient charge-transfer complex between the tyrosine and the light-excited flavin, with a concomitant change in the direction of the emission dipole moment of the flavin. This interaction is likely to result from side-chain relaxation of the tyrosine in the minor fraction of enzyme molecules in which this residue is in an unsuitable position for immediate fluorescence quenching at the moment of excitation. Support for this mechanism is provided by binding studies with NADP+ and 2'P-5'ADP-ribose that can intercalate between the flavin and tyrosine and/or block the latter. Fluorescence depolarization analyses as a function of temperature and viscosity confirm the dynamic nature of the process. A comparison with fluorescence depolarization effects in a related flavoenzyme indicates that this mechanism of flavin fluorescence depolarization is more generally applicable.     

Allosteric transition of aspartokinase I-homoserine dehydrogenase I studied by time-resolved fluorescence

The allosteric transition of threonine-sensitive aspartokinase I-homoserine dehydrogenase I from Escherichia coli has been studied by time-resolved fluorescence spectroscopy. Fluorescence decay can be resolved into 2 distinct classes of tryptophan emitters: a fast component, with a lifetime of about 1.5 ns; and a slow component, with a lifetime of about 4.5 ns. The fluorescence properties of the slow component are modified by the allosteric transition. In the T-form of the enzyme stabilized by threonine, the lifetime of the slow component is longer, with a red-shifted spectrum; its accessibility to quenching by acrylamide becomes slightly higher without any decrease of fluorescence anisotropy. These results indicate a change in polarity of the slow component environment. The quaternary structure change associated with the allosteric transition probably involves global movements of structural domains without leading to any local mobility on the nanosecond time-scale. We suggest that the slow component corresponds to the unique tryptophan of the buried kinase domain.     

A fluorescence study of dehydroergosterol in phosphatidylcholine bilayer vesicles

The fluorescent sterol delta 5,7,9(11),22-ergostatetraen-3 beta-ol (dehydroergosterol) was incorporated into 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) small unilamellar vesicles (SUV) with and without cholesterol in order to monitor sterol-sterol interactions in model membranes. In the range 0-5 mol % fluorescent sterol, dehydroergosterol underwent a concentration-dependent relaxation characterized by red-shifted wavelengths of maximum absorption as well as altered ratios of absorbance maxima and fluorescence excitation maxima at 338 nm/324 nm. Fluorescence intensity per mole of dehydroergosterol increased up to 5 mol % in POPC vesicles. In contrast, quantum yield, steady-state anisotropy, limiting anisotropy, lifetime, and rotational rate remained relatively constant in this concentration range. Similarly, addition of increasing cholesterol in the range 0-5 mol % in the presence of 3 mol % dehydroergosterol also increased the fluorescence intensity per mole of dehydroergosterol, red-shifted wavelengths of maximum absorption, and altered ratios of absorbance maxima. In POPC vesicles containing between 5 and 33 mol % dehydroergosterol, the fluorescent dehydroergosterol interacted to self-quench, thereby decreasing the fluorescence intensity, quantum yield, steady-state anisotropy, and limiting anisotropy and increasing the rotational rate (decreased rotational relaxation time) of the fluorescent sterol. The fluorescence lifetime of dehydroergosterol remained unchanged. The results were in accord with the interpretation that below 5 mol% sterol, the sterols behaved as monomers exposed to some degree to the aqueous solvent in POPC bilayers.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fluidity of intact erythrocyte membranes. Correction for fluorescence energy transfer from diphenylhexatriene to hemoglobin

Membranes of intact erythrocytes were labeled by the fluorescent probe 1,6-diphenyl-1,3,5-hexatriene (DPH) using an improved labeling procedure described previously (Plásek, J. and Jarolím, P. (1987) Gen. Physiol. Biophys. 6, 425-437). The relationship between the steady-state DPH fluorescence anisotropy r and the mean corpuscular hemoglobin concentration (MCHC) was studied. Fluorescence anisotropy increased with increasing MCHC. A linear dependence of r = 0.0026 (MCHC) + 0.113 was obtained which enabled us to measure the fluidity of intact red cell membranes. Without this correction for fluorescence quenching by hemoglobin, incorrect conclusions about membrane fluidity could be made. This fact is demonstrated in a group of pyruvate kinase deficient patients compared with a group of healthy blood donors.