Fluorescence quenching in riboflavin-binding protein and its complex with riboflavin

Fluorescence quenching of tryptophan residues in egg-white riboflavin-binding protein by two typical quenchers (charged iodide and uncharged acrylamide) reveals acid-induced changes of protein conformation. At neutralpH, acrylamide flow in macromolecule, (i.e., the quenching effect) is decisive; tryptophan residue accessibility for iodide is small. At lowpH, some tryptophan residues are exposed to the protein surface and become more accessible to iodide. In contrast, acrylamide is less able to permeate this conformational state of RBP. Fluorescence of tryptophan residues in riboflavin-RBP complex and chemically N-bromosucinimide-modified RBP was quenched by iodide and acrylamide.     

Fluorescence quenching method for the determination of bleomycins A5 and A2 with halofluorescein dyes

In weak acidic medium, the anticancer antibiotics bleomycin A₅ (BLMA₅) and bleomycin A₂ (BLMA₂) bind with halofluorescein dyes, such as erythrosin (Ery), eosin Y (EY) and eosin B (EB), to form ion‐association complexes, which causes fluorescence quenching of halofluorescein dyes. The quenching values (ΔF) are directly in proportional to the concentrations of bleomycins over the range 0.09–2.5 µg/mL. Based on this, a fluorescence quenching method for the determination of BLMA₅ and BLMA₂ has been developed. The dynamic range is 0.12–2.5 µg/mL for the determination of BLMA₅ and 0.09–2.0 µg/mL for BLMA₂, with detection limits (3σ) of 0.04 µg/mL for BLMA₅, 0.03 µg/mL for BLMA₂, respectively. It has been applied to determine the two antibiotics in human serum, urine and rabbit serum samples. The recovery is in the range 90–102%. In this work, the optimum reaction conditions and the spectral characteristics of the fluorescence are investigated. The reasons for fluorescence quenching are discussed, based on the fluorescence theory. Copyright © 2007 John Wiley & Sons, Ltd.     

Fluorescence quenching studies of nitrated polycyclic aromatic hydrocarbons

The analysis of nitrated polycyclic aromatic hydrocarbons (NPAHs) is of great importance because of the mutagenicity and possible carcinogenic activity of these compounds, which are distributed widely in the environment. Nitro‐substituents in aromatic compounds are known to quench fluorescence and NPAHs have no intrinsic fluorescence, but they can be determined using their quenching effects on other fluorophores. The quenching effects of several important NPAHs on 1,2,3,4‐ tetrahydro‐1‐naphthol,5,6,7,8‐tetrahydro‐1‐naphthol,4‐(2‐hydroxy‐4‐sulfo‐1‐naphthylazo)‐2‐naphthalene carboxylic acid and 7‐amino‐4‐methyl coumarin have been studied. The singlet emission of these fluorophores is efficiently quenched by all the NPAHs, the quenching following the Stern–Volmer relationship. Quenching constants and the limits of detection and linear ranges of the quenchers have been determined in each case: the limits of detection are ca 1 µm . Copyright © 2010 John Wiley & Son, Ltd.     

Interaction of indole and tryptophan derivatives with sodium dodecyl sulfate micelles measured with ultraviolet absorption and fluorescence quenching

The distribution of indole and tryptophan derivatives between sodium dodecyl sulfate (SDS) micellar and aqueous phases was analyzed using conventional methods of ultraviolet (UV) absorption spectroscopy and measurement of fluorescence quenching by succinimide. On the assumption of a simple pseudo-phase equilibrium between both phases the distribution coefficient was easily obtained by the measurement of the ratioR _pv of the absorbance intensity in the peak to that in the valley of the UV spectra or the fluorescence quenching constant K_sv. The possibilities and limitations of utilizing the ratio of the collisional quenching constant estimating from theK _sv value in the micellar phase to that in the aqueous phase for a measure of the polarity of the microenvironment around the tryptophan derivatives in the SDS micelle is discussed in comparison with theR _pv values for the UV spectra. The indole ring in the derivatives in the SDS micelle is localized near or on the micelle-water interface with its imino group directed toward the aqueous phase. Thus it can serve as a feasible model for interpreting the distribution coefficients andR _pv values obtained for the various indole and tryptophan derivatives.Abbreviations UV ultraviolet - SDS sodium dodecyl sulfate - ATEE N-acetyl-l-tryptophan ethyl ester - ATA N-acetyl-l-tryptophan-amide - CMC critical micelle concentration     

Fluorescence quenching due to mercuric ion interaction with aromatic amino acids and proteins

Mercuric ion interacts with indoles, including tryptophan, to produce complexes whose absorption spectra are broader, less structured, and red-shifted as compared with those of the parent compound. Fluorescence and phosphorescence are totally quenched. In a survey of the effect of transition metal ions on tryptophan fluorescence, the strong quenching by Hg²⁺ was unique among the uncolored ions. Mercuric nitrate quenched the fluorescence of practically every protein tested, but the sensitivity to quenching varied with the protein. Ovalbumin was the most sensitive to quenching by Hg²⁺, over 70% of the intrinsic fluorescence being quenched by 2 moles of mercuric ion. Difference absorption spectra show that sulfhydryl groups are attacked by these reagents and Hg²⁺ is, in addition, perturbing the environment near some tryptophans. In contrast to Hg²⁺, Zn²⁺ had negligible effect on protein fluorescence. The emission spectra of proteins which were partly quenched by mercuric ion showed shifts in their maxima to higher or lower wavelengths. This suggests that mercuric ion quenched certain tryptophans more than others, and supports the idea that protein fluorescence is heterogeneous and arises from tryptophans in different microenvironments.     

Energy transfer of aromatic amino acids in photosystem 2 core antenna complexes CP43 and CP47

Energy transfer of aromatic amino acids in photosystem 2 (PS2) core antenna complexes CP43 and CP47 was studied using absorption spectroscopy, fluorescence spectroscopy, and the 0.35 nm crystal structure of PS2 core complex. The energy of tyrosines (Tyrs) was not effectively transferred to tryptophans (Trps) in CP43 and CP47. The fluorescence emission spectrum of CP43 and CP47 by excitation at 280 nm should be a superposition of the Tyr and Trp fluorescence emission spectra. The aromatic amino acids in CP43 and CP47 could transfer their energy to chlorophyll (Chl) a molecules by the Dexter mechanism and the Föster mechanism, and the energy transfer efficiency in CP47 was much higher than that in CP43. In CP47 the Föster mechanism must be the dominant energy transfer mechanism between aromatic amino acids and Chl a molecules, whereas in CP43 the Dexter mechanism must be the dominant one. Hence solar ultraviolet radiation brings not only damages but also benefits to plants.     

A sensitive fluorescence quenching method for the detection of tartrazine with acriflavine in soft drinks

In this work, a simple, rapid, sensitive, selective spectrofluorimetric method was applied to detect tartrazine. The fluorescence of acriflavine could be efficiently quenched by tartrazine. The method manifested real time response as well as presented satisfied linear relationship to tartrazine. The linear response range of tartrazine (R² = 0.9995) was from 0.056 to 5 μmol L^?1. The detection limit (3σ/k) was 0.017 μmol L^?1, indicating that this method could be applied to detect traces of tartrazine. The accuracy and precision of the method was further assured by recovery studies via a standard addition method, with percentage recoveries in the range of 96.0% to 103.0%. Moreover, a quenching mechanism was investigated systematically by the linear plots at varying temperatures based on the Stern–Volmer equation, fluorescence lifetime and UV–visible absorption spectra, all of which proved to be static quenching. This sensitive, selective assay possessed a great application prospect for the food industry owing to its simplicity and rapidity for the detection of tartrazine.     

Fluorescence quenching studies of Trp repressor-operator interaction

Steady-state quenching and time-resolved fluorescence measurements of L-tryptophan binding to the tryptophan-free mutant W19/99F of the tryptophan repressor of Escherichia coli have been used to observe the coreperessor microenvirnment changes upon ligand binding. Using iodide and acrylamide as quenchers, we have resolved the emission spectra of the corepressor into two components. The bluer component of L-tryptophan buried in the holorepressor exhibits a maximum of the fluorescence emission at 336 nm and can be characterized by a Stern–Volmer quenching constant equal to about 2.0–2.3 M^–1. The second, redder component is exposed to the solvent and possesses the fluorescence emission and Stern–Volmer quenching constant characteristic of L-tryptophan in the solvent. When the Trp holorepressor is bound to the DNA operator, further alterations in the corepressor fluorescence are observed. Acrylamide quenching experiments indicate that the Stern–Volmer quenching constant of the buried component of the corepressor decreases drastically to a value of 0.56 M^–1. The fluorescence lifetimes of L-tryptophan in a complex with Trp repressor decrease substantially upon binding to DNA, which indicates a dynamic mechanism of the quenching process.     

Development responses of symbiotic and aposymbiotic weevils Sitophilus oryzae L. (Coleoptera,curculionidae) to a diet supplemented with aromatic amino acids

Developmental times of symbiotic and aposymbiotic strains of the rice weevil Sitophilus oryzae were compared, in response to changes in concentration of phenylalanine or tyrosine in whole wheat flour pellets. Aposymbiotic insects were shown to require more aromatic amino acids than symbiotic insects, since a very low supply (0.1%) resulted in faster growth (11%). Incorporation results of [³H]-tyrosine during the larval and pupal stages indicated that total tyrosine intake was lower in aposymbiotic insects, but the incorporation into the cuticle of both strains did not significantly differ. It is suggested that the slower growth rate of weevils without symbiotes is due, in part, to a less efficient utilization of exogenous tyrosine (in the food) and to a lack of endogenous tyrosine (supplied by the symbiotes).     

Development of an optical sensor for chlortetracycline detection based on the fluorescence quenching of l‐tryptophan

A simple and selective spectrofluorimetric method for the detection of chlortetracycline (CTC) was studied. In pH 7.4 buffer medium l ‐tryptophan (l ‐Trp), applied as the fluorescence probe, interacted with CTC resulting in fluorescence quenching of the probe. CTC was detected with maximum excitation and emission wavelengths at λ_ex/λ_em = 275/350 nm. Notably, quenching of fluorescence intensities was positively proportional to the CTC concentration over the range of 0.65–30 μmol L^?1 and the limit of detection was 0.2 μmol L^?1. Effect of temperature shown in Stern?Volmer plots, absorption spectra and fluorescence lifetime determination, indicated that fluorescence quenching of l ‐Trp by CTC was mainly by static quenching. The proposed study used practical samples analysis satisfactorily.     

Investigation on the interaction of catalase with sodium lauryl sulfonate and the underlying mechanisms

As a classic type of anionic surfactants, sodium lauryl sulfonate (SLS) might change the structure and function of antioxidant enzyme catalase (CAT) through their direct interactions. However, the underlying molecular mechanism is still unknown. This study investigated the direct interaction of SLS with CAT molecule and the underlying mechanisms using multi‐spectroscopic methods, isothermal titration calorimetry, and molecular docking studies. No obvious effects were observed on CAT structure and activity under low SLS concentration exposure. The particle size of CAT molecule decreased and CAT activity was slightly inhibited under high SLS concentration exposure. SLS prefers to bind to the interface of CAT mainly via van der Waals’ forces and hydrogen bonds. Subsequently, SLS interacts with the amino acid residues around the heme groups of CAT via hydrophobic interactions and might inhibit CAT activity.     

Fluorescence quenching studies of Trp repressor using single-tryptophan mutants

Time-resolved and steady-state fluorescence have been used to resolve the heterogeneous emission of single-tryptophan-containing mutants of Trp repressors W19F and W99F into components. Using iodide as the quencher, the fluorescence-quenching-resolved spectra (FQRS) have been obtained The FQRS method shows that the fluorescence emission of Trp99 can be resolved into two component spectra characterized by maxima of fluorescence emission at 338 and 328 nm. The redder component is exposed to the solvent and participates in about 21% of the total fluorescence emission of TrpR W19F. The second component is inacessible to iodide, but is quenched by acrylamide. The tryptophan residue 19 present in TrpR W99F can be resolved into two component spectra using the FQRS method and iodide as a quencher. Both components of Trp19 exhibit similar maxima of emission at 322–324 nm and both are quenchable by iodide. The component more quenchable by iodide participates in about 38% of the total TrpR W99F emission. The fluorescence lifetime measurements as a function of iodide concentration support the existence of two classes of Trp99 and Trp19 in the Trp repressor. Our results suggest that the Trp aporepressor can exist in the ground state in two distinct conformational states which differ in the microenvironment of the Trp residues.Abbreviations TrpR tryptophan aporepressor fromE. coli - TrpR W19F TrpR mutant with phenylalanine substituted for tryptophan at position 19 - TrpR W99F TrpR mutant with phenylalanine substituted for tryptophan at position 99 - FQRS fluorescence-quenching-resolved spectra - FPLC fast protein liquid chromatography     

Structure-potential dependence of adsorbed enzymes and amino acids revealed by the surface enhanced Raman effect

Surface enhanced Raman scattering (SERS) of some enzymes (alkaline phosphatase, horseradish peroxidase and lactoperoxidase) and some amino acids (tryptophan, tyrosine and phenylalanine) on silver electrodes has been studied. The spectral band intensities of certain amino acids and amino acid residues were determined by their orientation on the surface and depended on the electrode potential (E).Abbreviations SERS surface enhanced Raman scattering - Trp tryptophan - Tyr tyrosine - Phe phenylalanine - E electrode potential - ORC oxidation-reduction cycle     

Separation of amino acids by simulated moving bed using competitive Langmuir isotherm

The separation of two amino acids, phenylalanine and tryptophan, was carried out using laboratory simulated moving bed (SMB) chromatography. The SMB process consisted of four zones, with each zone having 2 columns. The triangle theory was used to obtain the operating conditions for the SMB. The mass transfer coefficients of the two amino acids were obtained from the best-fit values by comparing simulated and experimental pulse data. The competitive adsorption isotherms of the two amino acids were obtained by single and binary frontal analyses, taking into consideration the competition between the two components. A competitive Langmuir isotherm, obtained from single-component frontal chromatography, was used in the first run, and the isotherm from binary frontal chromatography in the second, with the flow rate of zone I modified to improve the purity. Compared to the first and second runs, the competitive Langmuir isotherm from the binary frontal chromatography showed good agreement with the experimental results. Also, adjusting the flow rate in zone I increased the purity of the products. The purities of the phenylalanine in the raffinate and the tryptophan in the extract were 99.84 and 99.99%, respectively.     

Fluorescence quenching studies on the interaction of riboflavin with tryptophan and its analytical application

The ineraction between riboflavin (RBF) and tryptophan (Trp) was investigated using fluorescence spectroscopy and UV–vis absorption spectroscopy under physiological conditions. The fluorescence of Trp was quenched by RBF via dynamic quenching, which was analyzed using the Stern–Volmer relation. The value of the Forster distance R₀ (2.31 nm) was obtained according to the Forster's theory of nonradiative energy transfer. Under physiological conditions, a linear relationship could be established between the quenched fluorescence intensity of Trp and the concentration of RBF in the range of 5.8 × 10^‐7–2.0 × 10^‐5 mol/L. The detection limit was 1.8 × 10^‐7 mol/L. The method was successfully applied to determine riboflavin concentrations in pharmaceutical samples. Copyright © 2012 John Wiley & Sons, Ltd.     

Fluorescence properties of lipoprotein B and apolipoprotein B

The fluorescence properties of apolipoprotein B (ApoB) in various media, including aqueous solutions of three different pH, 6 m urea, 6 m guanidine-HCl and native lipoprotein B (LP-B) particles have been compared by measuring the accessibility of trytophan side chains to iodide ions. The modified Stern-Volmer plots (FΔF vs. 1/[KI]) for LP-B demonstrate heterogeneity of quenching rates at pH 9.0, with a total accessibility of fluorescence to iodide of 43%. At pH 7.3, the total accessibility of LP-B fluorescence to iodide is only 20%. Quenching at pH 2.7 follows a pure Stern-Volmer mechanism. A straight line at this pH intercepting y-axis at 1.0 indicates 100% accessibility of tryptophan residues in LP-B. These results suggest that there are at least three different groups of tryptophan residues present per intact LP-B particle and that each group is situated in a different environment. One group, showing an enhanced quenching rate, is probably near the charged domain; another group, showing a slower quenching rate, is in a relatively hindered environment, and a third group is probably buried in a more hydrophobic environment, inaccessible to iodide at neutral or high pH. But at pH 2.7, all tryptophan residues appear to become situated closer to the surface of the LP-B particle. For isolated ApoB at pH 7.3 and 9.0 in aqueous buffer, about 30% of the fluorescence is relatively easily accessible; another 40% is less easily accessible and the remaining 30% is inaccessible to iodide. These inaccessible tryptophan residues are most likely located in a more hydrophobic matrix and probably in the β-pleated sheet region of ApoB. Similarly to LP-B at pH 2.7, all of the tryptophan residues of ApoB are exposed to the aqueous surface except that one third of them are quenched at a faster rate than the rest. At pH 7.3, in the presence of urea or guanidine-HCl, all of the fluorescence of ApoB is exposed to the aqueous surface, suggesting the presence of random and nonrigid conformation in these media. These results suggest that the conformation of ApoB in aqueous media is pH sensitive. This is true whether the ApoB is present in intact LP-B or as the isolated apolipoprotein. Furthermore, upon removal of lipids from LP-B and passing the ApoB into a denaturing environment, the apolipoprotein loses its ordered structure. When passing ApoB from denaturing agents back to aqueous buffers of neutral or basic pH. ApoB is able to reorient itself to gain an ordered structure, not necessarily identical to that in LP-B, but parallel to it.     

The determination of nitrite by a graphene quantum dot fluorescence quenching method without sample pretreatment

A method for quantitative analysis of nitrite was achieved based on fluorescence quenching of graphene quantum dots. To obtain reliable results, the effects of pH, temperature and reaction time on this fluorescence quenching system were studied. Under optimized conditions, decrease in fluorescence intensity of graphene quantum dots (F₀/F) showed a good linear relationship with nitrite concentration between 0.007692–0.38406 mmol/L and 0.03623–0.13043 μmol/L; the limits of detection were 9.8 μmol/L and 5.4 nmol/L, respectively. Variable temperature experiments, UV absorption spectra and thermodynamic calculations were used to determine the quenching mechanism, and indicated that it was an exothermic, spontaneous dynamic quenching process. This method was used to analyse urine samples, and showed that it could be applied to analyse biological samples.     

The sensitive determination of nucleic acids using a fluorescence-quenching method.

Experiments indicated that nucleic acids can quench the fluorescence of the Eu3+ -2-thenoyltrifluoroacetone (TTA)-1,10-phenanthroline (Phen) system. Based on this, a sensitive method for the determination of nucleic acids was proposed. The experiments indicated that under the optimum conditions, the quenched fluorescence intensity was in proportion to the concentration of nucleic acids in the range 1.0 x 10(-11)-1.0 x 10(-6) g/mL for yeast RNA (yRNA), 5.0 x 10(-11)-5.0 x 10(-7) g/mL for fish sperm (fsDNA) and 1.0 x 10(-10)-1.5 x 10(-6) g/mL for calf thymus DNA (ctDNA). Their detection limits were 3.0 x 10(-12), 4.0 x 10(-12) and 5.0 x 10(-11) g/mL, respectively. Therefore, the proposed method is one of the most sensitive methods available. The interaction between nucleic acids and Eu3+ -TTA-Phen is also discussed.     

The diversity of allosteric controls at the gateway to aromatic amino acid biosynthesis

Present within bacteria, plants, and some lower eukaryotes 3‐deoxy‐D ‐arabino‐heptulosonate 7‐phosphate synthase (DAHPS) catalyzes the first committed step in the synthesis of a number of metabolites, including the three aromatic amino acids phenylalanine, tyrosine, and tryptophan. Catalyzing the first reaction in an important biosynthetic pathway, DAHPS is situated at a critical regulatory checkpoint—at which pathway input can be efficiently modulated to respond to changes in the concentration of pathway outputs. Based on a phylogenetic classification scheme, DAHPSs have been divided into three major subtypes (Iα, Iβ, and II). These subtypes are subjected to an unusually diverse pattern of allosteric regulation, which can be used to further subdivide the enzymes. Crystal structures of most of the regulatory subclasses have been determined. When viewed collectively, these structures illustrate how distinct mechanisms of allostery are applied to a common catalytic scaffold. Here, we review structural revelations regarding DAHPS regulation and make the case that the functional difference between the three major DAHPS subtypes relates to basic distinctions in quaternary structure and mechanism of allostery.     

大豆甙元磺酸钠对应激性胃粘膜损伤的影响及其机制探讨

目的：观察大豆甙元磺酸钠对力竭应激性渍疡的影响,探讨其可能的作用途径。方法：采用小鼠力竭性游泳,计数胃部溃疡点数建立应激溃疡模型,腹腔注射不同剂量的大豆甙元磺酸钠及一氧化氮合酶（NOS）抑制剂（L-NAME）并通过NADPH-黄递酶组织化学法检测胃壁NOS阳性神经元的变化。结果：大豆甙元磺酸钠具有保护胃粘膜的作用,且呈剂量效应;L-NAME可防止应激引起的胃粘膜损伤,L-NAME与有效剂量的大豆甙元磺酸钠联合使用后,大豆甙元磺酸钠对胃粘膜的保护作用明显增强;正常及应激小鼠胃壁NOS神经节数目基本不变,大豆甙元磺酸钠对正常小鼠胃壁NOS神经元影响不明显,而对应激小鼠胃壁单位面积及单个神经节内NOS阳性神经元数目均有显著降低作用。结论：应激时NO升高可导致溃疡,大豆甙元磺酸钠能够保护胃粘膜,其作用是通过抑制应激状态下NOS的升高,限制应激状态下NO过度升高,起到保护胃粘膜的作用。