Fibrinogen-β-Estradiol Binding Studied by Fluorescence Spectroscopy: Denaturation and pH Effects

Fibrinogen is a blood plasma protein that plays a crucial role in hemostasis. It is known that erythrocyte aggregation increases in the presence of fibrinogen, and that β-estradiol decreases erythrocyte aggregation with a constant fibrinogen concentration. In this work, we have used intrinsic tryptophan fluorescence to obtain information on the conformational changes of fibrinogen upon the recently proposed interaction with β-estradiol. To evaluate the effect on the conformational changes during fibrinogen-β-estradiol binding, fluorescence experiments were performed using guanidine hydrochloride (0–6 M) as denaturant, at different pH values. The results obtained for pH 6.5 and 8.0 showed no effect during the binding. The main differences were observed between pH 4.2 and 7.4, in the absence and in the presence of two different denaturant concentrations (1 and 5 M). A red shift of the fluorescence emission from 344 to 354 nm is observed when denaturant concentration is above 3 M for all studied pH values. This phenomenon may be explained by the loss of compact structure of the protein in the presence of denaturant, with tryptophan residues exposure to the aqueous environment and alteration of fibrinogen-β-estradiol binding. These results demonstrate that the binding sites of fibrinogen are strongly dependent on the conformational state of the protein.     

Fluorescence lifetime study of ECFP/EYFP labeled MBP in Förster resonance energy transfer

This paper studies the conformational change of the binding protein by a fluorescence lifetime method. As a model protein, maltose binding protein (MBP) where enhanced cyan protein (ECFP) and enhanced yellow fluorescent protein (EYFP) were genetically fused to act as a donor and an acceptor in Förster resonance energy transfer (FRET) was used. The ECFP and the EYFP were linked to the C-terminal and N-terminal regions of the MBP, respectively. In order to investigate the conformational change of the MBP, the lifetime of the ECFP, which acts as a donor in the ECFP:MBP:EYFP fusion protein, was analyzed during the FRET process. We observed that two lifetime components exist when the ECFP is linked to the MBP and that the lifetime of the ECFP is shortened when ECFP:MBP:EYFP protein undergoes a conformational change as a result of the maltose binding. In addition, we observed that the lifetime of the donor is gradually shorter in the ECFP:MBP:EYFP fusion protein as the maltose concentration increases. By a lifetime analysis and simulation study, we found that the participant rate of the ECFP:MBP:EYFP protein in FRET is the main cause of the donor lifetime shortening in relation to the increase of the maltose concentration.     

Luminescent spectral characteristics of eosin in solutions of human serum albumin when denatured by treatment with sodium dodecyl sulfate

From analysis of the fluorescence spectra of eosin molecules in a solution with human serum albumin (HSA), we have obtained information about the dynamics of protein conformational rearrangements during denaturing of the protein when treated with sodium dodecyl sulfate (SDS) for different pH values of the solution. We hypothesize that HSA denaturing in the presence of SDS occurs in two stages: the first stage is loosening of the protein globules, and the second stage is complete unfolding of the protein molecules. We have shown that denaturating of the protein in the presence of SDS passes through both stages for a solution pH below the isoelectric point of the albumin, while the denaturing stops in the first stage for a solution pH above the isoelectric point of the albumin. __________ Translated from Zhurnal Prikladnoi Spektroskopii, Vol. 73, No. 5, pp. 661–665, September–October, 2006.     

Exploration of twisted intramolecular charge transfer fluorescence properties of trans-2-[4-(dimethylamino)styryl]benzothiazole to characterize the protein–surfactant aggregates

The characterization of aggregates of an anionic surfactant, sodium dodecyl sulphate (SDS) with bovine serum albumin (BSA) in various regions of binding isotherm of SDS to BSA with increasing concentration of the former have been done by exploring the twisted intramolecular charge transfer (TICT) fluorescence properties of a probe, trans-2-[4-(dimethylamino)styryl] benzothiazole (DMASBT). The TICT fluorescence, steady-state fluorescence anisotropy and time-resolved fluorescence of DMASBT, and the fluorescence resonance energy transfer (FRET) study reveal the characteristics of the native protein as well as the protein–surfactant aggregates viz., micropolarity, microviscosity, locations of probe, denaturation of protein in various regions of binding isotherm, and also the validation of necklace-bead model. The changes in the polarity and the viscosity of the microenvironment around the probe from one binding region of SDS to other have been reflected in the highly sensitive fluorescence properties of DMASBT. The study of FRET between the DMASBT and the tryptophan residue (Trp) of BSA has identified the locations of the probe molecule in the native protein as well as that in various BSA–SDS aggregates. The energy transfer efficiency decreases, whereas the distance between the DMASBT and the Trp residue increases with increasing concentration of SDS. The significant change in the conformations of protein molecules during the non-cooperative binding region of SDS is evidenced by the fluorescence anisotropic behavior of DMASBT in the same region.     

Fluorescent Probing of Protein Bovine Serum Albumin Stability and Denaturation Using Polarity Sensitive Spectral Response of a Charge Transfer Probe

The polarity sensitive photo-induced intra-molecular charge transfer (ICT) fluorescence probe (E)-3-(4-methylamino-phenyl)-acrylic acid ethyl ester (MAPAEE) has been used to study the model protein Bovine Serum Albumin (BSA) in its native and thermal and urea induced denatured states. The interaction between BSA and the regular surfactant Sodium Dodecyl Sulphate (SDS) as well as the biologically relevant steroid-based amphiphile Sodium Deoxycholate (NaDC) has also been very keenly followed using this ICT probe. The variation of micellar properties of both SDS and NaDC with increasing ionic strengths and in presence of the chaotrope urea has also been well documemted by the same probe. Steady-state spectroscopy, FRET, and fluorescence anisotropy measurements have been used to gain better insight into these processes and the molecule MAPAEE to be a full-bodied fluorescent probe for studying such intricate biological systems, their properties and interactions.     

低盐和高盐环境下α-Synuclein构象的~(19)F NMR研究

天然无结构的突触核蛋白α-synuclein(AS)与帕金森病密切相关.最近研究发现低盐与高盐环境下AS纤维化的速率不同,所形成的纤维结构,细胞毒性与传染性也不一样,但盐效应对AS聚集及纤维结构影响的具体分子机制仍不清楚.该文通过生物标记方法在AS的酪氨酸芳香环上引入19F标记的探针,利用19F核磁共振(NMR)方法研究了低盐与高盐环境下AS的构象差异,发现19F NMR对天然无结构蛋白构象变化非常灵敏,AS在低盐中的构象比较紧密,而在高盐下比较松散,这种在溶液中起始的构象差异可能是导致最终AS纤维结构与生物效应不同的原因.     

A Fluorescence and Fluorescence Probe Study of Benzonaphthyridines

A series of benzo[b][1,8]naphthyridines has been synthesized by Friedl?nder condensation of 2-aminoquinoline-3-carbaldehyde 1 (o-aminoaldehyde) with alicyclic ketones in basic medium. Benzonaphthyridines branched with various side-chains and substituents are prepared with the aim of being investigated as a good fluorescent material. Electronic absorption and fluorescence properties of some representative benzonaphthyridines in organic solvents, water-dioxane, and SDS, CTAB and Triton-X-100 micelles have been examined. The linear correlation between solvent polarity and fluorescence properties is observed. This study may provide new directions for the development of fluorescence probes as reporters of microenvironments of organized assemblies.     

丝素蛋白在甲醇-水混合溶剂中构象变化的光谱学研究

应用圆二色谱、内源荧光和外源荧光探针研究了丝素蛋白在甲醇-水混合溶剂中的构象变化及机理。结果显示,在浓度低于30%(V/V)的甲醇-水混合溶剂中,处于无规卷曲状态的丝素蛋白可以通过疏水相互作用形成小的疏水区域,随着甲醇浓度的增加,丝素蛋白发生由无规卷曲向β-折叠的构象转变,削弱了疏水侧链的相互作用。分析表明,丝素蛋白的构象变化与溶剂体系的微观结构密切相关,其稳定性主要由肽键单元与混合溶剂中的分子簇之间的相互作用决定。低浓度的甲醇-水混合溶剂保持水固有的氢键结构,对丝素蛋白肽键单元的溶剂化和丝素蛋白构象的影响较小,而随着甲醇浓度的增加,溶剂结构出现由四面体结构的水分子簇向链状结构的甲醇分子簇的转变,丝素蛋白通过形成分子内氢键减少肽键单元与溶剂分子的接触,从而引发丝素蛋白的构象转变。     

A thermodynamic and photophysical study of the effects of thiourea on the micellar properties of aqueous sodium dodecyl sulphate solution

The micellar properties of sodium dodecyl sulphate (SDS) in aqueous thiourea solutions were investigated with the aim of studying the effect of thiourea on these properties. The critical micelle concentrations (cmc) and the degree of dissociation of the micelle were determined by the conductometric method over the temperature range 298–323 K for different concentrations of thiourea. The cmc values of the surfactant in the presence of thiourea were obtained by following the change in the relative intensities of vibrational fine structure of the pyrene fluorescence spectra. The aggregation numbers were determined by employing the static quenching fluorescence method. The cmc values in the presence of varying electrolyte concentrations (NaCl) were obtained with 8-anilinonaphthalene 1-sulphonate (ANS) as a fluorescence probe, and from these values the degree of dissociation was calculated. The mass action model was applied in the present study to obtain various thermodynamic parameters of micellization. All these properties were compared with the micellar properties of an aqueous urea/SDS system, and it was found that thiourea is a better demicellization agent.     

Spectrophotometric and spectrofluorimetric investigation of different equilibria of a recently synthesized Schiff base with the aid of chemometric methods

In this study ground and excited states acidic dissociation constants of a recently synthesized Schiff base was obtained in a DMF:water mixture of 30:70 (v/v) using absorption and fluorescent spectra of the Schiff base in different pH values with the aid of chemometric methods. In addition, the fluorescent of the two kinds of tautomers of this Schiff base was investigated and the rate of tautomerization was obtained using rank annihilation factor analysis (RAFA). The effect of different kinds of surfactants such as sodium dodecyl sulfate (SDS), cetyltrimethylammonium bromide (CTAB) and Triton X-100 on fluorescence spectrum of the Schiff base in a DMF:water mixture of 2:98 (v/v) was investigated. CTAB increased the fluorescence intensity of the Schiff base while SDS and Triton X-100 had no significant effect on it. β-Cyclodextrin increased the fluorescence intensity of the Schiff base. Also the sensing behavior of this Schiff base toward metal ions was studied in DMF and ethanol by fluorescence spectroscopy. The Schiff base showed prominent fluorescent signal in the presence of Zn²⁺, whereas other metal ions failed to induce response and ground-state dissociation constant of the complex was determined by direct fluorimetric titration as a function of Zn²⁺ concentration.     

Structural Transition in the Micellar Assembly: A Fluorescence Study

Structural transition can be induced in charged micelles by increasing the ionic strength of the medium. Thus, spherical micelles of sodium dodecyl sulfate (SDS) that exist in water at concentrations higher than the critical micelle concentration assume an elongated rod-like structure in the presence of an increased electrolyte concentration. This is known as sphere-to-rod transition. In this paper, we characterize the change in organization and dynamics that is accompanied by the salt-induced sphere-to-rod transition in SDS micelles using wavelength-selective fluorescence and other steady-state and time-resolved fluorescence parameters. Since the change in micelle organization during such structural transition may not be limited to one region of the micelle, we have selectively introduced fluorophores in two distinct regions of the micelle. We used two probes, N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (NBD-PE) and 25-[N-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-methyl]amino]-27-norcholesterol (NBD-cholesterol), for monitoring the two regions of the micelle. NBD-PE monitors the interfacial region of the micellar assembly, while NBD-cholesterol acts as a reporter for the deeper regions of the micellar interior. Our results show that wavelength-selective fluorescence, in combination with other fluorescence parameters, offers a powerful way to monitor structural transitions induced in charged micelles. These results could be significant to changes in membrane morphology observed under certain physiological conditions.     

EB荧光探针法研究多粘菌素B与DNA的作用方式

本文以溴化乙锭（EB）为荧光探针，对多粘菌素B（Polymyxin B sulfate)与DNA的作用机制进行了研究。对荧光光谱法、紫外光谱法、荧光偏振法、热变性等方面的研究结果表明：在pH=7.4的溶液中PB与DNA双螺旋碱基对之间存在嵌插作用，在一定离子强度下该嵌插作用会减弱。此外，还存在着非特性的静电作用。     

Fluorescence of tryptophan in the denaturation of human serum albumin under the action of sodium dodecyl sulfate

Tryptophan fluorescence of human serum albumin (HSA) when sodium dodecyl sulfate (SDS) was added to a solution of the protein was studied at various pH values, which allowed us to get an idea about the mechanism of the denaturation of HSA under the action of SDS. The two-stage quenching of tryptophan fluorescence of HSA as the concentration of SDS increased was evidence of the two-stage character of denaturation. The first stage involved loosening of protein globules, and the second one was the complete uncoiling of the amino acid protein chain. At solution pH higher than the isoelectric point of the protein (pI 4.7), denaturation stopped at the first stage. At pH values below pI of the protein, the denaturation of the protein under the action of SDS was more effective and deep (involved both stages).     

Complexation of pyrene and anthracene with human blood plasma

We have studied the interaction between polycyclic aromatic hydrocarbons (pyrene and anthracene) with human serum albumin (HSA) and human blood plasma. We have shown that the increase in the fluorescence intensity and the decrease in the polarity index of pyrene on going from an aqueous solution to a pH 7.4 buffer solution of HSA suggests that polycyclic aromatic hydrocarbons are localized in the hydrophobic microphase of the proteins. The increase in the fluorescence intensity for anthracene and pyrene, and also the decrease in the polarity index of pyrene on going from HSA to blood plasma is connected with the fact that polycyclic aromatic hydrocarbons can bind both to plasma proteins and to plasma lipids. When sodium dodecyl sulfate (SDS) is added to the blood plasma in a concentration greater than the critical micelle concentration, we observe an increase in the fluorescence intensity and the polarity index of pyrene. We hypothesize that this is connected with localization of pyrene near the interface between the hydrophobic and hydrophilic phases of the protein-SDS system. We have established that SDS leads to a change in the structure of blood plasma proteins and promotes escape of polycyclic aromatic hydrocarbons from the protein globules. __________ Translated from Zhurnal Prikladnoi Spektroskopii, Vol. 75, No. 3, pp. 379–382, May–June, 2008.     

Polymer-doped ferroelectric liquid crystal: UV absorbance,fluorescence and electro-optical study

In this study, UV absorption, fluorescence and electro-optical study have been performed for pure ferroelectric liquid crystal (FLC) and its three different concentrations, i.e. 1%, 3% and 5% of fluorescent polymer in the pure FLC. We observed that there is higher value of UV absorption for fluorescent polymer-doped system in comparison to the pure system. We have found excitation wavelength, emission wavelength and quantum yield for all the mixtures. Quantum yield shows the probability of excited state being deactivated by fluorescence rather than non-radiative mechanism. The higher value of spontaneous polarization and faster response has been observed for fluorescent polymer-doped system compared to the pure FLC system. The electro-optical study also suggests that there exists an optimum concentration of fluorescent polymer for which we have found highest spontaneous polarization and least response time.     

十二烷基硫酸钠(SDS)增强若丹明6G水溶液激光激发荧光谱研究

报道了若丹明6G水溶液添加不同浓度的表面活性剂十二烷基硫酸钠(SDS)时激光激发染料的变化,发现较低的掺入量导致R6G荧光减弱,适量SDS的加入使荧光增强,在5×10-5 mol·L-1的R6G水溶液中,加入6×10-2 mol·L-1 SDS,荧光增强因子达到3.1。当R6G浓度为1×10-4 mol·L-1时,加入2×10-2 mol·L-1,染料激光阈值显著降低。测量了不同浓度的R6G溶液的吸收光谱及加入不同浓度SDS后的荧光谱,分析了不同SDS加入量下R6G荧光减弱及增强的物理机制。     

Propidium Iodide and PicoGreen as Dyes for the DNA Fluorescence Correlation Spectroscopy Measurements

Many experimental designs, in which nucleic acid conformational changes are of interest, require reliable fluorescence labeling. The appropriate fluorescence probe should have suitable optical properties and, more importantly, should not interfere with the investigated processes. In order to avoid chemical modifications the fluorescence label needs to be associated with nucleic acid via weak non-covalent interactions. There are a number of fluorescent probes that change their fluorescent properties (i.e. their quantum yield and/or spectral characteristics) upon association with nucleic acid. Such probes are frequently used to detect, visualize and follow processes involving nucleic acid and its conformational changes. In order to obtain reliable data regarding macromolecule or aggregate topology a detailed knowledge of probe–nucleic acid interactions on the molecular level is needed. In this paper we show that the association of propidium iodide with DNA alters its conformation and that it selectively labels plasmid fragments and/or its subpopulations in a concentration-dependent meaner. Another dye, PicoGreen, exhibits better properties. It labels nucleic acid uniformly and without any concentration-dependent artifacts.     

铽离子发光探针研究pH诱导的植物钙调素构象变化

利用脱钙钙调素(apo-CaM),Tb.CaM和Ca.CaM系统的荧光光谱及酪氨酸-铽离子(Tyr-Tb3 )敏化荧光光谱,研究了pH诱导钙调素构象的改变.随着pH值的降低,apo-CaM的荧光强度下降并出现蓝移,Ca.CaM系统的荧光强度较Tb.CaM下降显著,Tyr-Tb3 敏化荧光光谱也有相当程度的降低.对荧光强度的变化与钙调素分子结构和构象的关系和pH诱导钙调素构象改变的机理作了详细的解释,H 可通过与Ca2 和Tb3 产生竞争结合,影响金属离子与钙调素的结合作用,还可以通过正电相斥或与肽链上带负电荷原子相结合而使CaM分子的极性增强,改变CaM表面的疏水性,降低CaM的活性.文章对pH诱导钙调素构象改变在胞外钙调素信号转导机制中的重要意义进行了阐述.     

Label-free tungsten disulfide quantum dots as a fluorescent sensing platform for highly efficient detection of copper(Ⅱ) ions

A fluorescent probe for the sensitive and selective determination of copper ion(Cu~(2+)) is presented. It is based on the use of tungsten disulfide quantum dots(WS_2 QDs) which is independent of the p H of solution and emits strong blue fluorescence. Copper ions could cause aggregation of the WS_2 QDs and lead to fluorescence quenching of WS_2 QDs. The change of fluorescence intensity is proportional to the concentration of Cu~(2+), and the limit of detection is 0.4 μM. The fluorescent probe is highly selective for Cu~(2+) over some potentially interfering ions. These results indicate that WS_2 QDs,as a fluorescent sensing platform, can meet the selective requirements for biomedical and environmental application.     

Surfactant fluorescence in the study of aggregation and clouding

The intrinsic fluorescence of Triton X-114 and Igepal CO-630 was used to monitor the aggregation behavior of micellar solutions of these surfactants. The response to changes in surfactant concentration, increases in temperature up to and beyond the cloud point, and addition of an ionic surfactant (SDS) was monitored. The intrinsic fluorescence was used to measure aggregate anisotropy as a function of SDS concentration and temperature. Relative aggregate abundance showed a minimum at the CMC, confirming the existence of premicellar assemblies. Structural differences in the hydrophobic portions of the two nonionic surfactants led to vastly different packing in their aggregates. The addition of SDS produced smaller, more closely packed micelles.