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1.
Abstract— Steady-state and multifrequency phase fluorometry were used to characterize the conformational state and conformational dynamics of recombinant tick anticoagulant peptide ( Ornithodorus moubata ) (TAP). The TAP contains two tryptophan residues at positions 11 and 37. The fluorescence emission varies sigmoidally as a function of pH with a pKa of 6.01 ± 0.07. This pH dependency suggests that tryptophan fluorescence is quenched by His43 at low pH. This is confirmed by modification of the his-tidine with diethylpyrocarbonate. At pH 9 the fluorescence decay is well described by a sum of three exponentials (0.52,1.9 and 5.4 ns), which decrease all three at pH 4 (0.25, 1.61 and 4.4 ns). From the reactivity of the fluorescence lifetimes toward N -bromosuccinimide and from the calculation of the accessibility we can attribute the long lifetime to Trpll, the short one to Trp37 and the middle one to both. The anisotropy decay was resolved into two components of 3.85 ns and 0.27 ns at pH 4 and 4.5 ns and 0.6 ns at pH 9. The long anisotropy decay time corresponds to the rotational correlation time of the protein, the short one to local mobility of the tryptophan residues.  相似文献   

2.
Abstract— Fluorescence lifetimes are reported for intact human lenses in vitro. Two spectral regions were investigated: The first was excited at 296nm and detected at 332 or 370nm and corresponds to emission from tryptophan residues in the lens proteins. The second spectral region was excited at 359 nm and detected at 435 nm and corresponds to non-tryptophan 'fluorogen' fluorescence. The latter displayed a constant lifetime, 3.8 ns, independent of the anatomical part of the lens excited. This value was compared with measured lifetimes for some model fluorogens. The tryptophan fluorescence lifetime (332 nm detection) was found to vary from 1.8 to 2.8 ns depending on the anatomical part of the lens excited.  相似文献   

3.
The excitation and emission spectra have been determined for the fluorescence from trypto-phan residues in dry keratin. The fluorescence decay was also measured and shown to be a single exponential with a rather long lifetime of 6.9 ns. It is suggested that the emission takes place from a state formed by interaction between the 1La state of the tryptophan residues and neighbouring polar or polarizable groups in the protein. The fluorescence excitation spectrum displays a peak at 290 nm, and its appearance at this position rather than at 280 nm, which is the absorption maximum of tryptophan, is believed to arise from inner filtering by the tyrosine residues in keratin.  相似文献   

4.
Studies of acrylamide quenching of tryptophan (Trp) fluorescence, photochemistry, and photoionization have been conducted. Quenching of Trp fluorescence in aqueous solution by addition of acrylamide in the concentration range 0.0-0.5 M was measured and resulted in a Stern-Volmer quenching constant of KSV = 21 +/- 3 M-1. Photolysis experiments were performed in which Trp was photolyzed at 295 nm in the presence of varying concentrations of acrylamide. The loss of Trp was monitored using reverse-phase high performance liquid chromatography (RP-HPLC) and was observed to follow first order kinetics. Production of N-formylkynurenine (NFK) was observed by RP-HPLC in irradiated Trp samples both in the presence and absence of added acrylamide. In addition, no new photochemical product was detected. This was taken as evidence that acrylamide did not alter the photochemical pathway but just reduced the reaction rate as expected for a physical quenching mechanism. Plotting the reciprocal of photolysis rate constant versus acrylamide concentration produced a Stern-Volmer constant for quenching of Trp photochemistry of KSV = 6 +/- 2 M-1. The KSV values for both fluorescence quenching and photolysis quenching were thus large, implying efficient quenching of both processes by acrylamide. Assuming an excited singlet state lifetime of 2.8 ns, the calculated second-order quenching rate constants for fluorescence and photolysis were kq = 7.5 x 10(9) and 2.1 x 10(9) M-1 s-1 respectively. The possible involvement of photoionization in the photolysis mechanism was investigated by studies of acrylamide quenching of voltage transients produced by xenon flash lamp excitation of Trp at aqueous/teflon or aqueous/mica interfaces.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

5.
采用荧光猝灭和同步荧光法,研究了磷酸缓冲溶液(PBS, pH=7.4)中有无碳纳米管(CNTs)共存时,荧光活性物质槲皮素(Qct)与牛血清白蛋白(BSA)和酪蛋白(Cas)的相互作用. 推导了方法1(固定蛋白质浓度, 改变Qct浓度, 测量蛋白质荧光改变)和方法2(固定Qct浓度, 改变蛋白质浓度, 测量Qct荧光改变)研究分子间作用的一般方程, 由非线性最小二乘拟合法测算了结合常数K和摩尔结合比n, 并藉此定量评估了“光内滤所致猝灭”效应的影响. 研究了共存CNTs或Qct对BSA或Cas的荧光猝灭效应, 及CNTs对Qct-BSA和Qct-Cas相互作用的影响. 以同步荧光法考察了CNTs或Qct对BSA或Cas构象的影响, 并测算了CNTs或Qct与蛋白质中酪氨酸(Tyr)或色氨酸(Trp)残基相关的K和n. 结果表明, CNTs主要与处于蛋白质分子表面附近的Trp残基作用, 而小分子Qct则还可与处于蛋白质分子内部的Tyr残基作用.  相似文献   

6.
A tryptophan analog, dehydro-N-acetyl-L-tryptophanamide (delta-NATA), which is produced enzymatically via L-tryptophan 2',3'-oxidase from Chromobacterium violaceum, is newly used for time-resolved fluorescence. The absorption and emission maxima of delta-NATA at 332 and 417 nm, respectively, in 20% dimethylformamide-water are significantly shifted to the red with respect to those of tryptophan in water, permitting us to measure its fluorescence in the presence of tryptophan residues. We demonstrate that the steady-state spectra and the fluorescence decay of delta-NATA are very sensitive to environment, changing dramatically with solvent as the chromophore is localized within a protein and when this tagged protein binds to a peptide. The tryptophan oxidase was also used to modify the single Trp of a neurotoxin from snake (Naja nigricollis) venom. Modification of the toxin alpha (dehydrotryptophan-toxin alpha) permitted its investigation in complex with a synthetic 15-amino acid peptide corresponding to a loop of the agonist-binding site of acetylcholine receptor (AchR) from Torpedo marmorata species. The peptide alpha-185 possesses a single Trp at the third position (Trp187 of AchR) and a disulfide bridge between Cys192 and Cys193. A single-exponential rotational diffusion time with a constant of 1.65 ns is measured for the isolated 15-amino acid peptide. This suggests that Trp motion in the peptide in solution is strongly correlated with the residues downstream the peptide sequence, which may in part be attributed to long-range order imposed by the disulfide bond. The dynamics of the bound peptide are very different: the presence of two correlation times indicates that the Trp187 of the peptide has a fast motion (taur1 = 140 ps and r(0)1 = 0.14) relative to the overall rotation of the complex (taur2 = 3.4 ns and r(0)2 = 0.04). The correlation of the Trp residue with its neighboring amino acid residues and with the overall motion of the peptide is lost, giving rise to its rapid restricted motion. Thus, the internal dynamics of interacting peptides change on binding.  相似文献   

7.
The access to oxygen concentration is of importance in various organelles of living cells, especially in mitochondria. A new probe, (1"-pyrene butyl)-2-rhodamine ester, was designed to target this organelle. We present here the properties of the probe in such an environment. Microspectrofluorometry confirms the localization of the probe in the mitochondrial environment at low doses (1 microM). At these doses, the cell toxicity experiments show no effect on the cell growth. The emission spectrum measured at an excitation wavelength of 340 nm (pyrene centered) indicates energy transfer from the pyrene to the rhodamine chromophore, as also observed in an ethanol solution. With excitation at 337 nm, the excited state decays biexponentially with lifetime decays of 6-9 ns and 90 ns. The first corresponds to the intrinsic fluorescence of the cell and the latter corresponds to the pyrene chromophore. In degassed conditions the pyrene lifetime decay increases up to 130 ns. Under an oxygen atmosphere the lifetime decays decrease to 62 ns. The lifetime changes with the oxygen concentration were compared with the range obtained during our previous study in ethanol solution (5-220 ns). The observed differences were interpreted by studying the lifetime of the probe in simplified environments, liposome suspensions and protein solutions. In this paper we show that the new probe can be used to measure the fluctuation of oxygen concentration in the surroundings of mitochondria.  相似文献   

8.
Qiu B  Guo L  Chen M  Lin Z  Chen G 《The Analyst》2011,136(5):973-978
A new fluorescence reagent, 2-methylbenzo[b][1,10]phenanthrolin-7(12H)-one (mBPO), synthesized in our laboratory was used as the probe for protein and its interaction with Bovine Serum Albumin (BSA) was investigated in detail in this paper. It was found that BSA had the ability to quench the fluorescence of mBPO at 411 nm (λ(ex) = 286 nm), and the quenched intensity of fluorescence was proportional to the concentration of BSA. Based on this fact, mBPO has been used as a fluorescence probe for the detection of BSA. Under the optimal conditions, the calibration graph is linear up to 0.5 mg L(-1) for BSA and the limit of detection (LOD) was 0.06 mg L(-1). The regression equation is y = 1048.8x + 7.2093 with R(2) = 0.9913. The mechanism for the interaction of mBPO with BSA was also studied, while the binding constant and the number of binding sites were calculated. According to the thermodynamics parameter, the binding mode between mBPO and BSA was deduced. The results suggested the interaction between mBPO and BSA to be hydrophobic force in nature. It also proved that the fluorescence quenching reaction was affected by the tryptophan residue of BSA. For there are two tryptophan (Trp) residues, in site 134 and site 212 of BSA, and mBPO maybe has interaction with them respectively.  相似文献   

9.
The interaction between lysozyme (LYSO) and puerarin has been studied at three temperatures (294, 302 and 310K) through/using fluorescence spectroscopy and circular dichroism (CD). The LYSO fluorescence was quenched by the binding of puerarin to LYSO. The binding constants and the number of binding sites can be calculated from the data obtained from fluorescence quenching experiments. According to the van't Hoff equation, the standard enthalpy change (DeltaH degrees ) and standard entropy change (DeltaS degrees ) for the reaction were calculated to be 17.47kJ/mol and 163.5J/molK. It indicated that the hydrophobic interactions play a main role in the binding of puerarin to LYSO. In addition, the distance between puerarin (acceptor) and tryptophan residues of LYSO (donor) was estimated to be 1.47nm on the basis of fluorescence energy transfer. The changes of LYSO secondary structure in the presence of puerarin were observed from CD spectroscopy.  相似文献   

10.
Fluorescence quenching and synchronous fluorescence methods were used to study the interactions of fluore-scence-active quercetin (Qct) with casein (Cas) and bovine serum albumin (BSA) in phosphate buffer solution (PBS, pH=7.4) with or without coexisting carbon nanotubes (CNTs). Formulae for binding constant (K) and molar binding ratio (n) were established for methods 1 (fixing protein concentration, changing Qct concentration, and monitoring the fluorescence of protein) and 2 (fixing Qct concentration, changing protein concentration, and monitoring the fluorescence of Qct), to which values of K and n were calculated via nonlinear least-square fitting of the experimental data, and the “optical inner filtering induced fluorescence quenching” effect was thus quantitatively evaluated. The quenching effects of coexisting CNTs on the fluorescence of Qct, BSA, and Cas, as well as the effects of coexisting CNTs on Qct-BSA and Qct-Cas interactions, were examined. Synchronous fluorescence was also used to examine the effects of coexisting CNTs and Qct on the conformations of BSA and Cas, with relevant K and n values for tyrosine (Tyr) and tryptophan (Trp) residues estimated. It was concluded that the CNTs mainly interacted with the Trp residues locating near the protein surfaces, but small-sized Qct molecules could further interact with the Tyr residues locating inside the protein molecules.  相似文献   

11.
ANS is widely used as a probe for locating binding sites of proteins and studying structural changes under various external conditions. However, the nature of ANS-binding sites in proteins and the accompanying changes in fluorescence properties are controversial. We examined the steady-state and time-resolved fluorescence of the ANS-protein complexes for tear lipocalin (TL) and its mutants in order to discern the origin of lifetime components via analysis that included the multiexponential decay and the model-free maximum entropy methods. Fluorescence lifetimes of ANS-TL complexes can be grouped into two species, 14.01-17.42 ns and 2.72-4.37 ns. The log-normal analyses of fluorescence spectral shapes reveal the heterogeneous nature of both long- and short-lifetime species. The constructed time-resolved emission, amplitude (TRES) and area normalized (TRANES), and decay-associated spectra are consistent with a model that includes heterogeneous modes of ANS binding with two separate lifetime components. The two lifetime components are not derived from solvent relaxation, but rather may represent different binding modes.  相似文献   

12.
Bovine beta-lactoglobulin A (BLGA) is a well characterized globular protein whose tertiary structure has been investigated in detail. BLGA undergoes a pH-dependent conformational change which X-ray data described as involving mostly the loop connecting strands E and F and the deprotonation of a glutamic acid residue (Glu89). These structural changes have been investigated using, among other techniques, fluorescence spectroscopy. The intrinsic fluorescence of BLGA is dominated by two Trp residues. These residues are located far from the EF loop and would not be expected to probe the pH-induced conformational change of the protein. Trp19 is located at the bottom of the interior beta-barrel, whereas Trp61 is located at the aperture of the barrel near the CD loop and is "silent" in the emission of native BLGA because of the proximity of a disulfide moiety. Our study suggests that, surprisingly, the fluorescence of Trp19 has the characteristic of a more polar environment than structural models from X-ray data would suggest and that at least two distinct conformations (or rotamers) of Trp19 contribute to the fluorescence of the protein. The less populated rotamer (relative amplitude (alpha) approximately 20%, tau approximately 3 ns) probes a more polar environment and a pH-dependent conformational change of BLGA in the region of Trp19 which X-ray data do not detect. Finally, our study provides the estimate of the fluorescence lifetime of Trp61 in the "unquenched" form.  相似文献   

13.
The amino acid composition of the superoxide dismutase(SOD) from camellia pollen was measured and the tryptophan(Trp) residues were modified by using N-bromosuccinimide(NBS). The results show that there are 21 Trp residues in an SOD molecule and seven of which are located on the surface of the enzyme. By researching the fluorescence spectra of the native SOD and the modified SOD, we have found that the emission wavelength of Trp is at 335 nm and the fluorescence intensity will decrease when the enzyme is modified. The results also show that potassium iodide(KI) can significantly quench the fluorescence of the native SOD, but it has a less pronounced effect on the modified enzyme. Glycerin as a surface activation reagent can stabilize the fluorescence of the modified enzyme.  相似文献   

14.
Ni(2+)-induced intramolecular excimer formation of a naphthalene-based novel fluorescent probe, 1-[(naphthalen-3-yl)methylthio]-2-[(naphthalen-6-yl)methylthio]ethane (L), has been investigated for the first time and nicely demonstrated by excitation spectra, a fluorescence lifetime experiment, and (1)H NMR titration. The addition of Ni(2+) to a solution of L (DMSO:water = 1:1, v/v; λ(em) = 345 nm, λ(ex) = 280 nm) quenched its monomer emission, with subsequent enhancement of the excimer intensity (at 430 nm) with an isoemissive point at 381 nm. The fluorescence lifetime of free L (0.3912 ns) is much lower than that of the nickel(2+) complex (1.1329 ns). L could detect Ni(2+) as low as 1 × 10(-6) M with a fairly strong binding constant, 2.0 × 10(4) M(-1). Ni(2+)-contaminated living cells of plant origin could be imaged using a fluorescence microscope.  相似文献   

15.
The mechanism of interaction of vinblastin sulphate (VBS) with bovine serum albumin (BSA) has been reported. Association constant for VBS-BSA binding was found to be 3.146+/-0.06 x 10(4) M(-1). Stern-Volmer analysis of fluorescence quenching data showed that the fraction of fluorophore (protein) accessible to the quencher (drug) was close to unity indicating thereby that both tryptophan residues of BSA are involved in drug-protein interaction. The rate constant for quenching, greater than 10(10) M(-1) S(-1), indicated that the drug-binding site is in close proximity to tryptophan residues of BSA. Binding studies in the presence of an hydrophobic probe, 8-anilino-1-naphthalein-sulphonic acid, sodium salt (ANS) indicated that there is hydrophobic interaction between VBS and probe and they do not share common sites in BSA. Thermodynamic parameters obtained from data at different temperatures showed that the binding of VBS to BSA involves predominant hydrophobic forces. The effects of some additives and paracetamol on binding of VBS-BSA have also been investigated. The CD spectrum of BSA in presence of VBS shows that the binding of VBS leads to change in the helicity of BSA.  相似文献   

16.
Binding of farrerol to lysozyme (LYSO) was investigated at 302, 313 and 318 K at pH 7.4 using spectrophotometric techniques such as fluorescence emission, circular dichroism (CD) and UV absorption. The data obtained from fluorescence quenching experiments showed that farrerol was bound to LYSO and the affinity was enhanced by the addition of farrerol. When the concentration ratio of farrerol to LYSO was higher than 5.4, both the binding constant and the binding stoichiometry went up. Based on the thermodynamic parameters evaluated from the van't Hoff equation, the enthalpy change (deltaH degrees ) and entropy change (deltaS degrees ) were derived to be negative values. They indicated that both van der Waals forces and hydrogen bonds are the major interactions between farrerol and LYSO. A value of 2.67 nm for the average distance r between farrerol (acceptor) and tryptophan residues (Trp) of LYSO (donor) was derived from the fluorescence resonance energy transfer. Besides, the change in the conformation of LYSO was observed, being caused by the interaction with farrerol.  相似文献   

17.
The fluorescence of the single tryptophan (Trp69) of cutinase from Fusarium solani pisi, free in aqueous solution and adsorbed onto the surface of poly(methyl methacrylate) (PMMA) latex particles, was studied at pHs of 4.5 and 8.0. The monodisperse PMMA particles (d=106.0+/-0.1 nm) were coated with a quite compact monolayer of cutinase at both pH values. The Trp decay curve of the folded free cutinase in solution can only be fitted with a sum of four exponentials with lifetimes of 0.05, 0.3-0.4, 2-3, and 6-7 ns, irrespective of pH. The 50 ps lifetime is attributed to the population of Trp residues hydrogen bonded with the Ala32 and strongly quenched by a close disulfide bridge, while the other lifetimes are due to the non-hydrogen-bonded Trp rotamers. The 50 ps Trp lifetime component disappears by temperature melting and upon protein adsorption, owing to the disruption of the Trp-Ala hydrogen bond and the release of the Trp residue from the vicinity of the disulfide bridge. This shows that cutinase adsorption occurs by the region of the protein where the Trp is located, which agrees with the retention of cutinase enzymatic activity by adsorption at basic pH.  相似文献   

18.
The differentiation of proteins commonly found as binding media in paintings is presented based on spectrally resolved and time-resolved laser-induced fluorescence (LIF) and total emission spectroscopy. Proteins from eggs and animal glue were analysed with pulsed laser excitation at 248 nm (KrF excimer) and 355 nm (third harmonic of Nd:YAG) for spectrally resolved measurements, and at 337 nm (N2) and 405 nm (N2 pumped dye laser) for spectrally resolved lifetime measurements and fluorescence lifetime imaging (FLIM). Total emission spectra of binding media are used for the interpretation of LIF spectra. Time-resolved techniques become decisive with excitation at longer wavelengths as fluorescence lifetime permits the discrimination amongst binding media, despite minimal spectral differences; spectrally resolved measurements of fluorescence lifetime have maximum differences between the binding media examined using excitation at 337 nm, with maximum observed fluorescence at 410 nm. FLIM, which measures the average lifetime of the emissions detected, can also differentiate between media, is non-invasive and is potentially advantageous for the analysis of paintings. Figure The fluorescence of solid ox glue extracted from collagen can be visualised in this Total Fluorescence Spectrum; three different peaks from multiple fluorophores are present and allow the discrimination between collagen- and non-collagen proteinaceous binding media found in paintings  相似文献   

19.
Steady-state and time-resolved fluorescence spectroscopic studies have been carried out on Trichosanthes cucumerina seed lectin (TCSL). The fluorescence emission maximum of TCSL in the native state as well as in the presence of 0.1 M lactose is centered around 331 nm, which shifts to 347 nm upon denaturation with 8 M urea, indicating that all the tryptophan residues of this protein in the native state are in a predominantly hydrophobic environment. The exposure and accessibility of the tryptophan residues of TCSL and the effect of ligand binding on them were probed by quenching studies employing two neutral quenchers (acrylamide and succinimide), an anionic quencher (I(-)) and a cationic quencher (Cs(+)). Quenching was highest with acrylamide and succinimide with the latter, which is bulkier, yielding slightly lower quenching values, whereas the extent of quenching obtained with the ionic quenchers, I(-) and Cs(+) was significantly lower. The presence of 0.1 M lactose led to a slight increase in the quenching with acrylamide and iodide, whereas quenching with succinimide and cesium ion was not significantly affected. When TCSL was denatured with 8 M urea, both acrylamide and succinimide yielded upward-curving Stern-Volmer plots, indicating that the quenching mechanism involves both dynamic and static components. Quenching data obtained with I(-) and Cs(+) on the urea-denatured protein suggest that charged residues could be present in close proximity to some of the Trp residues. The Stern-Volmer plots with Cs(+) yielded biphasic quenching profiles, indicating that the Trp residues in TCSL fall into at least two groups that differ considerably in their accessibility and/or environment. In time-resolved fluorescence experiments, the decay curves could be best fit to biexponential patterns, with lifetimes of 1.78 and 4.75 ns for the native protein and 2.15 and 5.14 ns in the presence of 0.1 M lactose.  相似文献   

20.
The mechanism of interaction of vincristine sulphate (VS) and rifampicin (RF) with bovine serum albumin (BSA) has been studied by quenching of BSA fluorescence by RF/VS. The Stern-Volmer plot indicates the presence of a static component in the quenching mechanism. Results also show that both the tryptophan residues of BSA are accessible to VS and RF. The high magnitude of rate constant of quenching indicates that the process of energy transfer occurs by intermolecular interaction and VS/RF-binding site is in close proximity to the tryptophan residues of BSA. Binding studies in the presence of a hydrophobic probe, 8-anilino-1-naphthalene-sulphonic acid sodium salt (ANS) indicate that the VS and RF compete with ANS for hydrophobic sites on the surface of BSA. Small decreases in critical micellar concentrations (CMC) of anionic surfactants in presence of VS/ RF show that the ionic character of VS/RF also contributes to binding. The temperature dependence of the association constant is used to estimate the values of the thermodynamic parameters involved in the interaction of VS/RF with BSA and the results indicate that hydrophobic forces play a significant role in the binding. Circular dichroism studies reveal that the change in helicity of BSA are due to binding of VS/RF to BSA.  相似文献   

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