Fluorescence properties of Ca2+-independent discharged obelin and its application prospects

Discharged obelin, a complex of coelenteramide and polypeptide, is a fluorescent protein produced from the photoprotein obelin, which is responsible for bioluminescence of the marine hydroid Obelia longissima. Discharged obelin is stable and nontoxic and its spectra are variable, and this is why it can be used as a fluorescent biomarker of variable color in vivo and in vitro. Here we examined light-induced fluorescence of Ca²⁺-independent discharged obelin (obtained without addition of Ca²⁺). Its emission and excitation spectra were analyzed under variation of the excitation wavelength (260–390 nm) and the emission wavelength (400–700 nm), as well as the 40 °C exposure time. The emission spectra obtained with excitation at 260–300 nm (tryptophan absorption region) included three peaks with maxima at 355, 498, and 660 nm, corresponding to fluorescence of tryptophan, polypeptide-bound coelenteramide, and a hypothetical indole–coelenteramide exciplex, respectively. The emission spectra obtained with excitation at 310–380 nm (coelenteramide absorption region) did not include the 660-nm maximum. The peak in the red spectral region (λ _max?=?660 nm) has not been previously reported. Exposure to 40 °C under excitation at 310–380 nm shifted the obelin fluorescence spectra to the blue, whereas excitation at 260–300 nm shifted them to the red. Hence, red emission and variation of the excitation wavelength form a basis for development of new medical techniques involving obelin as a colored biomarker. The addition of red color to the battery of known (violet to yellow) colors increases the potential of application of obelin.     

Ochratoxin A in Some French Wines: Application of a Direct Competitive ELISA Based on an OTA–HRP Conjugate

Abstract

In the direct-competitive enzyme-linked immunosorbent assay (dc-ELISA) approach, an ochratoxin A–horseradish peroxidase (OTA–HRP) synthesized conjugate was utilized. Fluorescence studies revealed that when excited at 333 nm, the OTA–HRP conjugate shifted in fluorescence from 438 nm to 444 nm. At the 295-nm excitation wavelength, the tryptophan emission peak blue-shifted from 326 nm to 318 nm. Western blot analysis revealed that anti-OTA antibodies specifically binds and recognize the OTA–HRP conjugate. The dc-ELISA showed an IC₅₀ value of 400 ng/L and a linear working range (LWR) value between 100 and 1500 ng/L. The ELISA results were confirmed with high-performance liquid-chromatography (HPLC) analysis to assess the potential of the OTA–HRP conjugate.     

Determination of tryptophan in bee pollen and royal jelly by high‐performance liquid chromatography with fluorescence detection

A method for tryptophan analysis in bee pollen and royal jelly was developed using HPLC with fluorescence detection. To determine the free tryptophan in bee pollen and royal jelly, ultrasonic extraction was performed using water (pH 6.3)–acetonitrile (10:1, v/v) as extraction solvent. While determining the total tryptophan in these bee products, the method involves alkaline hydrolysis of the proteins with 4 mol/L sodium hydroxide at 110°C for 20 h under anaerobic conditions. The operating conditions for the HPLC analysis were: Symmetry C₁₈ column (4.6 × 250 mm, 5 µm), 0.1% trifluoroacetic acid–methanol (75:25, v/v) as the mobile phase at a flow rate of 1.0 mL/min at 30°C. The fluorescence detector was operated at an excitation wavelength of 280 nm and an emission wavelength of 340 nm. A linear response (r² > 0.9998) was obtained in the range 0.0625–5.0 µg/mL. The method was successfully applied to the determination of the free and total tryptophan contents in bee pollens, which were 0.069 ± 0.003 and 2.693 ± 0.476 mg/g, respectively, while only the total tryptophan was detected in royal jelly, with a content of 1.743 ± 0.066 mg/g. Copyright © 2009 John Wiley & Sons, Ltd.     

含缩醛正离子类脂分子与蛋白质的相互作用

Interactions of a series of dialkyl cationic lipids linking with bovine serum albumin (BSA) through acetal (linker) have been studied by the fluorescence spectroscopy. At low concentrations of cationic lipids, the fluorescence intensity of BSA decreased with binding of cationic lipid, and the maximum of emission wavelength shifted from (344±1)nm to (331±1)nm. It indicates that the BSA goes to uncoiled flexible conformation from its native structure. When the concentrations of lipids increased, the fluorescence intensity increased rapidly and then maintained unchanged. It reveals that two tryptophan residues of BSA are all enwrapped in the bilayer membrane, owing to the hydrophobic interactions between lipids and BSA.     

Synthesis and characterization of a high-efficiency light-emitting alternating copolymer

A new high-efficiency light-emitting alternating copolymer of triphenylamine and pure PPV (TPA–PPV) has been designed and synthesized. The copolymer was highly soluble in common solvents. It could be spin cast onto various substrates to give highly transparent homogeneous thin films without heat treatment. The fluorescence quantum yield in benzene is almost up to 1.00. The maximum fluorescence wavelength for this alternating copolymer appeared around 470 nm. The fluorescence of TPA–PPV solution quenched by C₆₀ was examined, and the result indicated that a strong interaction exists between TPA–PPV and C₆₀ at the exited state. A primary single-layer LED based on ITO/TPA–PPV/Al has been fabricated, and the onset voltage is only 1.5 V and a bright green light was observed. The electroluminescence spectrum gives a peak at 510 nm when the operating voltage of 17 V was applied. The photoluminescence spectrum also appeared at the same wavelength as the electroluminescence, indicating that the same excited states are involved in the two processes. © 1999 John Wiley & Sons, Inc. J Polym Sci A: Polym Chem 37: 2587–2594, 1999     

A NEW FLUORESCENT PHOTOPRODUCT OF TRYPTOPHAN EVIDENCED BY LONG WAVELENGTH EXCITATION OF FLUORESCENCE

Abstract— Besides the normal tryptophan (Trp) fluorescence in aqueous solution (emission maximum at 350 nm), a new emission, peaking around 380 nm, appears by long wavelength excitation. Its fluorescence yield (φ_s 0.24) is higher than that of tryptophan (φ_Trp= 0.13). The growth of this emission is observed under different experimental conditions, mainly under UV anaerobic irradiation. To explain this observation, the formation of a C₃-hydroxylated derivative is tentatively suggested.     

Denaturation of bovine serum albumin under the action of cetyltrimethylammonium bromide, according to data from fluorescence analysis

The tryptophan fluorescence of bovine serum albumin (BSA) in solutions with different concentrations of cationic detergent cetyltrimethylammonium bromide (CTAB) at different pH is investigated, providing information on BSA denaturation under the action of CTAB. It is found that BSA denaturation under the action of CTAB at all of the investigated pH values (3.5–8.0) is a single-stage process, as determined by BSA tryptophan fluorescence quenching, by an increased degree of the BSA tryptophan fluorescence polarization, and by the values of the parameters for the rotational diffusion of BSA molecules in CTAB solutions. It is shown that the cationic detergent CTAB is more efficient for BSA denaturation at pH values higher than the BSA isoelectric point (4.9).     

Polarized fluorescence spectra of poly(ethylene terephthalate) films

The polarized excitation and fluorescence spectra of lowcrystalline, isotropic poly(ethylene terephthalate) (PET) film samples were measured in the glassy state at room temperature. Whereas the emission anisotropy r₀ of the excitation spectrum, recorded at the fluorescence maximum, changes sharply from 0.35 to 0 with decreasing wavelength in the region around 317 nm, the polarization of the fluorescence spectrum of PET is independent of wavelength. The fluorescence polarization of PET remains constant, if the temperature is increased up to 22 °C above T_g until the light scattering due to the crystallization causes complete depolarization. The photophysical behaviour supports the existence of a dilute solution of groundstate - stable sandwich dimers in the non-crystalline regions of PET.     

Ultraviolet laser induced fluorescence of human aorta

Laser induced fluorescence from normal human aorta is studied with u.v. excitations of 305 to 310 nm, observing emission from 320 to 500 nm. In this region LIF lineshapes are strongly dependent on the excitation wavelength, suggesting that at least two fluorophores are being observed. The short wavelength fluorophore, peaking at 34Onm, is identified as tryptophan, while the longer wavelength fluorophore, peaking at 387 nm, is associated with collagen and elastin. In addition, fluorescence time decays of each component are measured with a time correlated photon counting system. A four-exponential fit of each decay is necessary to extract fluorescence lifetimes, which range from 33 ps to 8.6 ns.     

Homogeneous graft copolymerization and characterization of novel artificial glycoprotein: Chitosan‐poly(L‐tryptophan) copolymers with secondary structural side chains

A novel series of artificial glycoprotein, peptide‐chitosan copolymers with secondary structural side chain have been synthesized by ring‐opening polymerization of L ‐tryptophan N‐carboxyanhydride under homogeneous conditions. Their chemical structures and polymerization degree (DP) were characterized by IR, ¹³C NMR, and XRD spectra. Distinctly secondary protein structure has been found in the poly‐L ‐tryptophan side chains of copolymers and with the lengthening of side chain (i.e., the increase of DP at the same time), its conformations could transfer from β‐sheet to α‐helix. The content of α‐helix reaches about 41% when DP of polytryptophan is 22. The solubility of graft copolymers in polar solvent strongly depends on the length of poly‐L ‐tryptophan side chains. Unique fluorescence emission at 360 nm has been observed in the glycopolymers and the intensity shows the positive‐correlation with the increasing of DP of polytryptophan. Importantly, the fluorescence effect can be quenched easily by the coordination with copper ions which provides the possibility on the biosensor design. In comparison with chitosan, glycopolymers also present impressively enhanced compressive strength and elastic modules when it is blended with epoxy E 44 to form epoxy‐copolymer hybrid resin. © 2008 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 47: 925–934, 2009     

Solvent Donicity Influence in the Fluorescence Emission of the Lithium/1,4-Dihydroxyanthraquinone System. Improvement in the Quantification of Lithium.

Abstract

The fluorescence emission from the lithium/1,4-dihydroxyanthraquinone system shows a great enhancement in the presence of certain water-miscible solvents. This is justified from the donicity (nucleophilic properties) of the solvent that facilitates the solvation of the lithium cation in solution and the stabilization of an nondissociated ion-pair between the solvated lithium cation and the 1,4-dihydroxyanthraquinonate anion. A very sensitive analytical method was proposed for the spectrofluorimetric determination of lithium based on its reaction with 1,4-dihydroxyanthraquinone (quinizarin) in a dimethylsulfoxide medium (90%) and in presence of sodium hydroxide. The fluorescence is measured at an excitation wavelength of 602 nm and an emission wavelength of 670 nm and it is stable at 25°C at least 6 h. The calibration curve is linear over the concentration ranges of 2–40 μg/l of lithium in an aqueous matrix and 3–50 μg/l in a serum matrix; the RSD's in the determination of 20 μg/l of Li⁺ were 2.6% and 3.2%, respectively. The proposed procedure was satisfactorily applied to the determination of lithium in drugs, dietetic products and human serum.     

肌红蛋白的同步荧光光谱

首次对肌红蛋白的同步荧光光谱进行了研究,并对肌红蛋白荧光峰予以归属。当△λ为20nm时,308nm处的荧光峰主要为酪氨酸残基的贡献,很小一部分是由色氨酸残基贡献的;△λ为40nm时,分别在322和596nm处观察到两个荧光峰,322nm的荧光峰为酪氨酸和色氨酸残基的共同贡献,596nm的荧光峰则归属为肌红蛋白分子中血红素的贡献。     

Determination of Floctafenine in Presence of its Degradation Product Floctafenic Acid by Direct and Synchronous Spectrofluorimetry

ABSTRACT

A simple, rapid and sensitive spectrofluorimetric method for the determination of floctafenine I (FL) in the presence of its degradation product floctafenic acid II (FLA) is developed. The method involves measuring the fluorescence intensity of FL in acetonitrile and in the presence of triethylamine (TEA) at an emission wavelength of 470 nm (excitation at 360 nm) by direct spectrofluorimetry and at an emission 465 nm (excitation 355 nm) by synchronous spectrofluorimetry. At these conditions FLA does not interfere. FLA is determined alone by measuring the fluorescence intensity of its solution in acetonitrile, without addition of TEA, at emission wavelength 460 nm (excitation 355 nm). The proposed method has been used for the assay of FL in tablets, plasma, urine and in mixtures with FLA. It gave highly accurate results for recovery of FL in the presence of its related acid.     

Two-photon excitation of proteins

Fluorescence emission after two-photon excitation at 532 nm by means of a Nd : YAG laser is observed in apohemoglobin, hemoglobin, albumin and tryptophan at room temperature. The experimental results show that the fluorescence of these proteins originates from tryptophan residues. No fluorescence of a biphotonic nature could be detected from lysozyme and tyrosine.     

Enantiomeric separation of D,L-tryptophan and D,L-kynurenine by HPLC using pre-column fluorescence derivatization with R(-)-DBD-PyNCS

The enantiomeric separation of d ,l ‐tryptophan (Trp) and d ,l ‐kynurenine (KYN) was investigated by high‐performance liquid chromatography using pre‐column fluorescence derivatization with a chiral fluorescent labeling reagent, R(−)‐4‐(3‐isothiocyanatopyrrolidin‐1‐yl)‐7‐ (N,N‐dimethylaminosulfonyl)‐2,1,3‐benzoxadiazole [R(−)‐DBD‐PyNCS]. Using an octadecylsilica column, namely, an Inertsil ODS‐3 column (250 × 2.0 mm; i.d., 3 µm), four fluorescence peaks of D‐ and l ‐Trp as well as d ‐ and l ‐KYN derivatized with R(−)‐DBD‐PyNCS were clearly observed, and their chemical structures were confirmed by HPLC–time‐of‐flight–mass spectrometry. Simultaneous separation was achieved under the mobile phase condition of 1.5% acetic acid in H₂O–CH₃CN (60:40), and the separation factors of d ,l ‐Trp and d ,l ‐KYN derivatized with R(−)‐DBD‐PyNCS were 1.22 and 1.19, respectively. Fluorescence detection was carried out by setting the emission wavelength at 565 nm, and the excitation wavelength at 440 nm, and the detection limits were approximately 0.3–0.5 pmol (signal‐to‐noise ratio of 3). Copyright © 2010 John Wiley & Sons, Ltd.     

FLUORESCENCE INVESTIGATIONS OF ALBUMIN FROM PATIENTS WITH FAMILIAL DYSALBUMINEMIC HYPERTHYROXINEMIA*

Familial dysalbuminemic hyperthyroxinemia (FDH) is an autosomal dominant syndrome in which clinically euthyroid patients have elevated total thyroxine levels. These high serum thyroxine levels are traceable to altered binding of thyroxine to the patient's albumin. Albumin from FDH patients and normal volunteers have been purified. Reverse-phase and ion-exchange high performance liquid chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis on the FDH-human serum albumin (HSA) samples show a single band that comigrates with normal HSA. In both protein solutions the intrinsic fluorescence, upon 280 nm excitation, is predominantly due to the single tryptophan residue. The quantum yield of this intrinsic fluorescence in the FDH-HSA solutions is, however, reduced relative to that of HSA. Furthermore, the “average” lifetime value of the tryptophan emission in the FDH-HSA sample is less than that of normal HSA, consistent with its reduced quantum yield. The binding of thyroxine to both albumins effectively quenches the tryptophan emission probably via a nonradiative energy transfer mechanism. Time-resolved data suggest that the albumin from the dysalbuminemic patients is actually an approximately equimolar mixture of normal HSA and FDH-HSA indicative of heterologous expression. Quenching of the intrinsic HSA and FDH-HSA fluorescence by serial additions of thyroxine showed enhanced quenching of FDH-HSA relative to HSA at any T4 to albumin mole ratio, therefore supporting earlier reports of increased thyroxine affinity to FDH-HSA.     

Fluorimetric Determination of Gatifloxacin in Aqueous,Pure and Pharmaceutical Formulations

Abstract

A spectrofluorimetric method was developed for the determination of gatifloxacin. The emission peak for gatifloxacin was recorded at 495 nm upon excitation at 291 nm. The fluorescence process was pH dependent. The dynamic range for the method was 16–80 ng ml^?1with detection limit of 3.97 ng ml^?1. A linear relationship between the fluorescence intensity and the concentration of gatifloxacin solution was obtained with r ² of 0.9968. The method has successfully applied to the determination of gatifloxacin in pure, authentic and aqueous samples.     

Studies on components 1' (modified monomer) and 2 (dimer) formed during heat-treatment of bovine serum albumin

When the solution of bovine serum albumin at pH 9 is incubated at 65 °C, new components 1' (modified monomer), 2 (dimer) and 3 (probably trimer) are formed. The isoelectric focusing indicated that the isoelectric points of components 1', 2 and 3 were pH 5.9.The hydrogen ion titration curve for the native albumin had well known abnormal steepness at pH near 4, but that for component 1' had no abnormal steepness. The titration curve for component 1' located right side of that for the native albumin.Circular dichroism measurements indicated that some unfolding occurred, when the native albumin was changed to component 1'. The contents of-helix were 74 and 52 % for the native albumin and for component 1', respectively. The contents of -structure were 19 and 23 % for the native albumin and for component 1', respectively. The wavelength of maximum intensity of tryptophan fluorescence shifted to lower wavelength, when the native albumin was changed to component 1'. This suggests that tryptophan residue(s) is transferred to a more hydrophobic environment.The hydrogen ion titration curves, circular dichroism and fluorescence measurements, all supported the possibility that the component 2 is formed by the dimerization of component 1'. Further, it has been found that component 2' (dimer impurity in commercial bovine serum albumin preparations) is formed by the direct dimerization of native bovine serum albumin without conformational change.     

HAS-硅钨杂多酸缔合纳米微粒体系的荧光猝灭

人血清白蛋白;瑞利散射;HAS-硅钨杂多酸缔合纳米微粒体系的荧光猝灭     

Water‐Soluble Red‐Emitting Distyryl‐Borondipyrromethene (BODIPY) Dyes for Biolabeling

A series of water‐soluble red‐emitting distyryl‐borondipyrromethene (BODIPY) dyes were designed and synthesized by using three complementary approaches aimed at introducing water‐solubilizing groups on opposite faces of the fluorescent core to reduce or completely suppress self‐aggregation. An additional carboxylic acid functional group was introduced at the pseudo‐meso position of the BODIPY scaffold for conjugation to amine‐containing biomolecules/biopolymers. The optical properties of these dyes were evaluated under simulated physiological conditions (i.e., phosphate‐buffered saline (PBS), pH 7.5) or in pure water. The emission wavelength (λ_max) of these labels was found in the 640–660 nm range with quantum yields from modest to unprecedentedly high values (4 to 38 %). The bioconjugation of these distyryl‐BODIPY dyes with bovine serum albumin (BSA) and the monoclonal antibody (mAb) 12A5 was successfully performed under mild aqueous conditions.