Steady-state fluorescence and phosphorescence spectroscopic studies of bacterial luciferase tryptophan mutants

Bacterial luciferase, which catalyzes the bioluminescence reaction in luminous bacteria, consists of two nonidentical polypeptides, and . Eight mutants of luciferase with each of the tryptophans replaced by tyrosine were generated by site-directed mutagenesis and purified to homogeneity. The steady-state tryptophan fluorescence and low-temperature phosphorescence spectroscopic properties of these mutants were characterized. In some instances, mutation of only a single tryptophan residue resulted in large spectral changes. The tryptophan residues conserved in both the and the subunits exhibited distinct fluorescence emission properties, suggesting that these tryptophans have different local enviroments. The low-temperature phosphorescence data suggest that the tryptophans conserved in bot the and the subunits are not located at the subunit interface and/or involved in subunit interactions. The differences in the spectral properties of the mutants have provided useful information on the local environment of the individual tryptophan residues as well as on the quaternary structure of the protein.     

Tryptophan phosphorescence of ribonuclease T1 as a probe of protein flexibility

The phosphorescence properties of Trp-59 of ribonuclease T₁ fromAspergillus oryzae were monitored as a function of temperature, pH, salt concentration, and complex formation with substrate analogues and, also, in the presence of glycerol as viscogenic cosolvent. The results establish a rough correlation between the internal flexibility of the macromolecule, as derived from the triplet lifetime, and its stability (G orT _m ) toward unfolding. Below 10°C or in 70% glycerol the triplet probe distinguishes at least two gross conformations for the protein, which are characterized by a large difference in phosphorescence lifetime. It is pointed out that such structural heterogeneity does not correspond with the heterogeneity inferred from fluorescence decays and acrylamide quenching rates. Further, implications of the phosphorescence data with regard to the interpretation of acrylamide quenching of fluorescence are discussed.     

X-ray excited fluorescence and phosphorescence of CaSO4: Sm phosphors

Calcium sulphate phosphors activated with samarium as an impurity with various percentage composition have been prepared and their fluorescence rise and phosphorescence decay behaviours have been studied. The growth curve is investigated to study the density of traps. The process of filling of traps during x-ray excitation is revealed. The decay curves are analysed to study the distribution of traps, the nature of decay and kinetics involved in the luminescence process. The concentration dependence of fluorescence emission of the phosphors is also discussed.     

Room-temperature phosphorescence of anilinonaphthalenesulfonate “fluorescence probe” compounds

2,6-anilinonaphthalenesulfonate (2,6-ANS) and 2,6-toluidinonaphthalenesulfonate (2,6-TNS) exhibit room-temperature phosphorescence when absorbed on filter paper, but the corresponding 1,8-isomers (1,8-ANS and 1,8-TNS) do not. The phosphorescence of the 2,6-isomers and nonphosphorescence of the 1,8-isomers is also found to hold in rigid glass at 77 K. No phosphorescence was detectable from N-phenyl-1-napthylamine at room temperature or 77 K, indicating that intramolecular hydrogen bonding in the 1,8-isomers isomers is not an essential feature inhibiting phosphorescence. Very effective heavy-atom enhancement of the phosphorescences of 2,6-ANS and 2,6-TNS by sodium iodide is observed.     

Davydov splitting and two-photon excitation of cadmium tungstate (CdWO4) single crystal phosphorescence

Phosphorescence characteristics of CdWO₄ excited by one-photon (λ = 308 nm) and two-photon (λ = 570–590 nm) processes were measured. A Davydov splitting of 120 ± 20 cm⁻¹ was obtained in the phosphorescence spectra, suggesting a diffusion coefficient of about 1.2 × 10⁻² cm² s⁻¹, and a diffusion length of about 3.1 × 10⁻⁴ cm for the room temperature measured lifetime of 8μs. The phosphorescence quantum efficiency was less than 2% at low temperatures (only 0.25% at room temperature), indicating that the dominant decay mechanism was radiationless. The radiative lifetime was thus estimated as 1–2 ms. The two-photon phosphorescence excitation is characterized by an absorption cross-section of the order of 10⁻⁴⁹cm⁴s.     

One-step aqueous synthesis of CdS nanoparticles as a novel fluorescence probe for the ultrasensitive detection of DNA

One-step aqueous synthesis of CdS nanoparticles as a novel fluorescence probe for sensitive and selective determination of DNA with synchronous fluorescence spectrometric method has been developed. Different from the traditional organometallic route, in which toxic precursors or solvents might be used, the wet chemical approach demonstrated in this paper is superior in terms of simplicity, using of nontoxic materials, mild synthetic condition and good reproducibility. When Δλ=255 nm, maximum synchronous fluorescence is produced at 264 nm, the synchronous fluorescence intensity of the composite nanoparticles is significantly decreased in the presence of trace DNA at PH 0.91. Under optimal conditions, the linear ranges of the calibration curves are 0.08-30.0 μg mL⁻¹ for ctDNA and 0.05-35.0 μg mL⁻¹ for hsDNA, respectively. The detection limits are 1.5 ng mL⁻¹ for ctDNA and 2.2 ng mL⁻¹ for hsDNA, respectively. Furthermore, the method is successfully applied to the quantification of DNA in synthetic samples. The results show that this proposed method is stable, sensitive and practical for the determination of trace DNA.     

Nanoscaled ZnO films used as enhanced substrates for fluorescence detection of dyes

The ability of nanoscaled ZnO films to enhance fluorescence was studied. We found that the fluorescence intensities of Cy5, rhodamine 6G, and fluorescein can be enhanced about 10-fold on nanoscaled ZnO films as compared to that on glass substrates. The lifetimes of all samples were measured, and no obvious change in lifetime was observed for dyes on different substrates. The mechanism for the nanoscaled ZnO film enhanced fluorescence appears to be different from that for the metal-fluorophore systems.     

Nanoscaled ZnO films used as enhanced substrates for fluorescence detection of dyes

The ability of nanoscaled ZnO films to enhance fluorescence was studied.We found that the fluorescence intensities of Cy5,rhodamine 6G,and fluorescein can be enhanced about 10-fold on nanoscaled ZnO films as compared to that on glass substrates.The lifetimes of all samples were measured,and no obvious change in lifetime was observed for dyes on different substrates.The mechanism for the nanoscaled ZnO film enhanced fluorescence appears to be different from that for the metal-fluorophore systems.     

Poly(thymine)-templated copper nanoparticles as a fluorescence probe for highly selective and rapid detection of cysteine

A highly selective and rapid analytical method was proposed to detect cysteine in aqueous solution by using poly(thymine)-templated copper nanoparticles. In a neutral aqueous condition, the fluorescence of poly(thymine)-templated copper nanoparticles could be quenched by cysteine in 10?min effectively coexisting with common biological small molecules. Under the optimal experimental condition, the present assay allowed for the selective determination of cysteine in the range of 12.5–100?µM, with a detection limit of 7.3?µM. The results indicated that the poly(thymine)-templated copper nanoparticles probe would find potential application in bioanalysis.     

Induced phosphorescence of some aza- and thio-stilbenes embedded in thallium-exchanged zeolites

The emission properties of some aza-stilbenes (2-, 3- and 4-styrylpyridine) and thio-stilbenes [2- and 3-styrylthiophene and 1,2-di-(3-thienyl)ethene] have been investigated after inclusion in commercial (NaY) and cation-exchanged (TlY) faujasite zeolites to get information on the triplet properties through population of the T₁ state induced by the heavy atom effect. The fluorescence properties in NaY and TlY were compared with those reported in solution. The phosphorescence spectra, observed in TlY at liquid nitrogen temperature, allowed the energy levels of the T₁ states to be obtained. Phosphorescence lifetimes were also measured. Their comparison with the lifetime known for stilbene showed that the radiative decay is little affected by the heteroatoms.     

Mechanism of singlet-oxygen induced delayed fluorescence of bacteriopheophytin in laser excitation

The relative intensity of photosensitized phosphorescence of singlet oxygen (¹O₂) at 1270 nm (L₁₂₇₀) and¹O₂-induced delayed fluorescence (L_df) of bacteriopheophytin a (BPh) (770 nm) in air-saturated solutions of BPh in hexafluorobenzene in excitation by 337-nm pulses of a nitrogen laso is investigated. It is established that L_df≪L₁₂₇₀. The ratio of the initial intensity of delayed fluorescence and phosphorescence of¹O₂(L_df)₀/(L₁₂₇₀)₀ changed from 0.02 to 0.30 as a function of the energies of laser pulses (2.5–5.0 mJ/cm¹) and the BPh concentration (6–18 μM). As the index of quantum efficiency of the delayed fluorescence, the authors used the coefficient

Two-line atomic fluorescence as a temperature probe for highly sooting flames

We investigate the applicability of two-line atomic fluorescence (TLAF) from seeded indium atoms for temperature measurements in highly sooting flames. The results show that TLAF holds promise for two-dimensional temperature measurements in sooting and fuel-rich flames under conditions in which other thermometry techniques fail, a result that is attributed to the superior characteristics of the indium atomization process. Furthermore, no native species was found to interfere spectrally with the detected TLAF wavelengths. Advantages of and problems with the technique are discussed.     

Luminescence of biologically active 24-epicastasterone and a model compound

Biologically active brassinosteroid 24-epicastasterone, ring B of which contains a C=O group and has the nπ*-configuration for a low-lying electronic excited state, exhibits rapid fluorescence. The wavelengths of the fluorescence maxima of the steroid dissolved in hexane and acetonitrile are equal to 332 and 394 nm, respectively. The fluorescence lifetime of the steroid dissolved in acetonitrile is τ = 9.9 nsec. Solutions of 24-epibrassinolide do not luminesce. The long-wavelength electronic absorption band λ_max^abs = 340 nm in the absorption spectrum of an ethanol solution of model compound 2, ring D of which contains a C=O group π*-conjugated with the C=C double bond of ring C, like in the spectrum of the steroid, has a low extinction coefficient. An ethanol solution of 2 does not fluoresce. 24-Epicastasterone at 77 K in ethanol solution exhibits phosphorescence with λ_max^phos = 447 nm. The phosphorescence decay is exponential with τ = 0.79 msec. Compound 2 also phosphoresces. The phosphorescence spectrum of its ethanol solution has a maximum at 490 nm. The phosphorescence decay is nonexponential in the early stage. The phosphorescence lifetime is 25 msec in the exponential decay region. __________ Translated from Zhurnal Prikladnoi Spektroskopii, Vol. 75, No. 2, pp. 182–186, March–April, 2008.     

Delayed fluorescence ODMR as a probe of triplet-triplet fusion probability

A kinetic scheme is presented to rationalize recent observations of delayed fluorescence ODMR in isotopically mixed naphthalene crystals, in which the sign of the signal changes from negative to positive as the critical concentration for triplet-triplet transport among the perprotonated traps is approached. The effects of chopped optical excitation, prolonged microwave pumping, temperature and light intensity are considered. The observation of positive DF-ODMR in the high concentration case is used to obtain a lower limit for the ratio of the annihilation constantk ₂ to the pair dissociation constantk _?1.     

Conformational differences of oxytocin and vasopressin as observed by fluorescence anisotropy decays and transient effects in collisional quenching of tyrosine fluorescence

We used gigahertz frequency-domain fluorometry to examine the tyrosyl fluorescence intensity and anisotropy decays of the single-tyrosine cyclic peptide hormones oxytocin and vasopressin. Acrylamide quenching and a distance-dependent quenching model for collisional quenching were used to evaluate the extent of tyrosyl exposure to the quencher and to provide increased resolution of the picosecond anisotropy decays. Analysis of the intensity decays using a lifetime distribution model shows different distributions for oxytocin and vasopressin. We found that the tyrosyl fluorescence of lysine-vasopressin, as revealed both by the lifetime Stern-Volmer plots and from the quenching analysis, is quenched more effectively than oxytocin. ForN-acetyltyrosinamide (NATyrA), oxytocin, and lysine-vasopressin, we recovered apparent diffusion coefficients for quenching of 4.7×10^–6, 0.44×10^–6, and 4.3×10^–6 cm²/s, respectively, the lower value for oxytocin suggesting a shielded environment for its tyrosyl residue. Tyrosyl anisotropy decays were recovered by global analysis of progressively quenched samples. Compared with oxytocin, vasopressin displayed a longer correlation time for overall rotational diffusion and a higher amplitude for picosecond segmented motions of its tyrosyl residue. All the data are consistent with a more extended and flexible solution structure for vasopressin than for oxytocin.Dedicated to Professor Alfons Kawski on the occasion of his 65th birthday.     

Simultaneous Determination of Plasma Enrichments of 1-13C- and 15N-Labelled Phenylalanine and Tyrosine

Abstract

A methylchloroformate derivative was used for the simultaneous determination of plasma enrichments of 1-¹³C-phenylalanine, 1-¹³C-tyrosine, ¹⁵N-phenylalanine and ¹⁵N-tyrosine by gas chromatography/mass spectrometry. All four tracer enrichments could be measured in a single GC run. A specific ion fragment was obtained for each tracer. This approach allowed an easy determination of the “tracer to tracee ratios”. Each ion fragment could be measured with an appropriate single-to-noise ratio and precision in samples obtained from 100 μl plasma. The derivatization consists of a fast one-step reaction. Therefore it is well suited for studies involving a large number of samples, such as non-steady state bolus studies.     

农药荧光寿命测试系统的原理与设计

介绍荧光的产生、荧光寿命的产生机理以及荧光寿命测量的基本原理。设计了一种利用直接记录法(光子计数法)测量农药荧光寿命的测试系统。该系统针对待测样品的特性，选用了相应的脉冲光源、光学元件和半导体探测器等器件，优化了各器件的工作参数，进行了简易而又科学的模块化设计，并对西维因农药的荧光寿命在无激励光干扰情况下进行了实际测试，测得了西维因溶液在500μg／L浓度时的荧光衰减曲线和荧光寿命(0.30～0.40ns)。结果表明，该系统具有结构简易、操作方便的优点，能测量100ps级的荧光寿命，适合于对能发荧光的农药进行荧光寿命的定量测量。     

129Xe-NMR of physisorbed xenon used as a probe for the study of microporous solids

¹²⁹Xe-NMR of adsorbed xenon is of increasing interest for studying the physical properties of solids: cavity or channel dimensions; short-range crystallinity; nature of structural defects, polymer heterogeneity, etc. This review, which does not claim to be exhaustive, gives a critical account of the various ways of using this technique for studying the porosity of solids.     

Extrinsic fluorescence probe study of human serum albumin using Nile red

Nile red bound to human serum albumin (HSA) shows an order of magnitude increase in the probe's fluorescence intensity. Here, we report on the fluorescence characteristics of the probe-protein complex in Trizma buffer (pH 7.1), urea, guanidine hydrochloride, and AOT/isooctane/buffer reverse micelles using both steady—state and time-resolved fluorescence techniques. With a view to illustrating the use of extrinsic probe fluorescence spectroscopy in protein research, we demonstrate that protein unfolding can be observed through measurements of the probe's time-resolved anisotropy and steady-state fluorescence spectrum. Moreover, this shows that thermal unfolding is fundamentally different from using denaturant, with respect to changes in both the nanosecond diffusional rotation of the probe at intermediate stages and in the denatured protein's structure. Also, the large Stokes shift of Nile red allows the changes in the environment of the probe-protein complex in reverse micelles of varying waterpool size to be easily identified in the steady-state fluorescence. This was not seen in earlier work exploiting the intrinsic tryptophan fluorescence of HSA and further demonstrates the complementary information that extrinsic fluorescence probe studies can offer protein science. We discuss the complex acrylamide quenching characteristics of Nile red bound to HSA in terms of the possibility of at least two binding sites for the probe and the effect of acrylamide on the probe-protein structure at very high quencher concentrations.