Quenching of Myosin Intrinsic Fluorescence Unravels the Existence of a High Affinity Binding Site for Decavanadate

Decavanadate, one of the aggregated species of vanadate, is a potent inhibitor of several enzymes, including skeletal muscle myosin. However, its putative binding sites in myosin are largely unknown. Titration of the intrinsic fluorescence of myosin, purified from rabbit skeletal muscle, have been carried out in 0.3 M KCl, 5 mM CaCl₂ and 25 mM Tris-HCl (pH 7.0), with 0.1 mg/ml myosin. In the 0-200 M total vanadate concentration range, decavanadate produced approximately 25% quenching of the intrinsic fluorescence of myosin, with an apparent dissociation constant in the micromolar range. This effect was found to be specific of decavanadate, because titration with metavanadate up to 200 M did not produce a significant quenching of the intrinsic fluorescence of myosin. This quenching was accompanied by a parallel decrease of the accessibility of myosin tryptophans to the water-soluble collisional quencher KI, with an apparent dissociation constant also in the micromolar range. It is concluded that the binding of decavanadate to high-affinity sites in myosin produces local conformational change(s) near the tryptophans more accessible to water in the three-dimensional structure of this protein.     

拉曼光谱在血红蛋白结构及功能研究中的应用进展

血红蛋白发挥多种重要生理功能,但对其结构及功能的认识尚不能满足疾病诊治的需求。拉曼光谱在血红蛋白结构及功能研究中具有很大应用潜力,不但可以检测血红蛋白中血红素及其周围分子结构变化,还可以反映血红蛋白反应动力学具体过程。同时,血红蛋白拉曼光谱在疾病状态下异常血红蛋白检测,血氧饱和度定量测定及血液代用品的高铁血红蛋白含量检测中突显优势。本文综述了拉曼光谱在血红蛋白结构及功能领域中的研究,简述了拉曼光谱在一些病变血红蛋白诊断中的研究进展,分析了影响血红蛋白拉曼光谱检测的因素,以促进拉曼光谱技术在血红蛋白结构和功能研究中的应用。     

Simultaneous Determination of Glucose and Choline Based on the Intrinsic Fluorescence of the Enzymes

It has been possible to perform the simultaneous determination of choline and glucose using the intrinsic fluorescence of the corresponding enzyme as an analytical signal. This can be done in two ways. First, for low glucose and choline concentrations (about 0.55 mM and 0.75 μM respectively) two differentiated signals, without mutual interference, are obtained for both analytes in the same measurement. Second, when glucose and choline concentrations are higher, a new model has been designed which permits the concentrations to be accurately determined in samples containing from 0.55 mM to 3.75 mM glucose and from 0.75 μM to 11.0 μM choline; the method has been applied to simultaneous glucose and choline determinations in serum samples with good results. This method gives a better performance than multivariate calibration based on Partial Least Squares Regression. The methodology here shown could be also used for the simultaneous determination of other pairs of analytes. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Fluorescence intensity and anisotropy decays of the intrinsic tryptophan emission of hemoglobin measured with a 10-GHz fluorometer using front-face geometry on a free liquid surface

We measured the intensity and anisotropy decays of the intrinsic tryptophan emission from hemoglobin solutions obtained using a 10-GHz frequency-domain fluorometer and a specially designed cuvette which allows front-face excitation on a free liquid surface. The cuvette eliminates reflections and stray emissions, which become significant for low-intensity fluorescence such as in hemoglobin. Three lifetimes are detectable in the subnanosecond range. The average lifetime of hemoglobin emission is ligand dependent. The measured values of average lifetimes are 91, 174, and 184 ps for deoxy-, oxy-, and carboxyhemoglobin, respectively. Fluorescence anisotropy decays of oxy-, deoxy-, and carbonmonoxyhemoglobin can be fitted with up to three correlation times. When three components are used, the floating initial anisotropyr _o is, in each case, higher than the steady-state anisotropy of tryptophan in vitrified solution. For deoxy hemoglobin it is close to 0.4. The data are consistent with an initial loss of anisotropy from 0.4 to about 0.3 occurring in the first 2 ps.     

经验模态分解法在近红外无创血红蛋白检测中的应用研究

为提高近红外血红蛋白预测模型的稳健性,分别应用Savitzky-Golay平滑、移动窗口平滑以及经验模态分解(EMD)方法对原始光谱进行去噪处理,以提高数据信噪比。采集了81例临床志愿者的手指指端血流容积脉搏波光谱数据,同时获取相应的血红蛋白浓度值临床化验结果。剔除异常样品,确定78例样品为研究对象,建立反向传播神经网络(BP-ANN)定量分析模型并预测。结果表明,经EMD处理后的模型预测效果最优,预测相关系数由0.74提高至0.87,误差均方根由12.85 g·L-1减小至8.08 g·L-1。实验证明应用EMD方法能够获得高信噪比的容积脉搏信号,提高血红蛋白浓度预测模型的准确性,有利于推动近红外无创血红蛋白检测技术的进一步发展。     

Fluorescence Spectrometric Studies on the Binding of an Amino-Functionalized Ionic Liquid to Cytochrome c

The interaction of an amino-functionalized ionic liquid, 1-(2-aminoethyl)-3-butylimidazolium bromide ([NH₂C₂C₄im]Br), with cytochrome c (cyt c) at pH 7.4 was investigated using fluorescence and UV-Vis absorption spectroscopic techniques. From the experimental results, it is found that cyt c has a strong ability to quench the intrinsic fluorescence of [NH₂C₂C₄im]Br and the quenching mechanism is considered as a static quenching process. The binding constants and the number of binding sites (n) were calculated at different temperatures. The thermodynamic parameters such as free energy change (ΔG), enthalpy change (ΔH), and entropy change (ΔS) were calculated by thermodynamic equations. According to the results, the values of ΔG, ΔH, and ΔS are all negative, suggesting that interaction between [NH₂C₂C₄im]Br and cyt c is spontaneous and mainly driven by hydrogen bonding and van der Waals forces.     

Characterization of the Binding of Metoprolol Tartrate and Guaifenesin Drugs to Human Serum Albumin and Human Hemoglobin Proteins by Fluorescence and Circular Dichroism Spectroscopy

The interactions of metoprolol tartrate (MPT) and guaifenesin (GF) drugs with human serum albumin (HSA) and human hemoglobin (HMG) proteins at pH?7.4 were studied by fluorescence and circular dichroism (CD) spectroscopy. Drugs quenched the fluorescence spectra of HSA and HMG proteins through a static quenching mechanism. For each protein-drug system, the values of Stern-Volmer quenching constant, bimolecular quenching constant, binding constant and number of binding site on the protein molecules were determined at 288.15, 298.15, 310.15 and 318.15 K. It was found that the binding constants of HSA-MPT and HSA-GF systems were smaller than those of HMG-MPT and HMG-GF systems. For both drugs, the affinity of HMG was much higher than that of HSA. An increase in temperature caused a negative effect on the binding reactions. The number of binding site on blood proteins for MPT and GF drugs was approximately one. Thermodynamic parameters showed that MPT interacted with HSA through electrostatic attraction forces. However, hydrogen bonds and van der Waals forces were the main interaction forces in the formation of HSA-GF, HMG-MPT and HMG-GF complexes. The binding processes between protein and drug molecules were exothermic and spontaneous owing to negative ?H and ?G values, respectively. The values of binding distance between protein and drug molecules were calculated from Förster resonance energy transfer theory. It was found from CD analysis that the bindings of MPT and GF drugs to HSA and HMG proteins altered the secondary structure of HSA and HMG proteins.     

Steady-State and Time-Resolved Fluorescence Spectroscopic Studies on Interaction of the N-terminal Region with the Hairpin Loop of the Phytocystain Scb

The steady-state and time-resolved fluorescece spectroscopy is one of the most powerful method to detect and analyze subtle conformation change and interaction between peptide elements in protein. Phytocystatin Scb isolated from sunflower seeds includes a single Trp residue at position 85. In an attempt to investigate the interaction of the N-terminal region of Scb with the first and second hairpin loops by fluorescence spectroscopy of Trp residue, two Scb mutants in which single Trp locates at position 52 and 58, respectively, and their N-terminal removed mutants were generated. The N-terminal truncation changed the fluorescence decay kinetics of Trp52 from the triple exponential to double. Furthermore, the time-resolved fluorescence anisotropy residue indicated that the segmental motion of Trp52 was significantly enhanced by its N-terminal truncation. In contrast, Trp58 and Trp85 had little influence. The N-terminal successive truncations of Scb and its mutants resulted in the weaken inhibitors to papain. These results suggested that the N-terminal region of Scb interacts with the peptide segment preceding the first hairpin loop, thereby stabilizing the conformation of the hairpin loop structure.     

A Fluorescence Analysis of ANS Bound to Bovine Serum Albumin: Binding Properties Revisited by Using Energy Transfer

Determination of binding parameters such as the number of ligands and the respective binding constants require a considerable number of experiments to be performed. These involve accurate determination of either free and/or bound ligand concentration irrespective of the measurement technique applied. Then, an appropriate theoretical model is used to fit the experimental data, and to extract the binding parameters. In this work, the interaction between bovine serum albumin (BSA) and 1-anilino-8-naphthalene sulphonate (ANS) is revisited. Using steady state fluorescence spectroscopy, the binding isotherm of BSA/ANS was obtained applying the Halfman-Nishida approach. The binding parameters, site number, and binding site association constants, were determined from the stoichiometric Adair model and Job's plot. The binding parameters obtained were then correlated to the distance of the respective binding site to the tryptophan residues using the energy transfer technique. This approach, that uses both tryptophans independently from each other, is presented as a tool to help understand the binding mechanism of the albumin fluorescent complex. The results show that ANS molecules bind to BSA in up to five different binding sites. Energy transfer from the tryptophan residues to the BSA/ANS complex shows that the four highest affinity binding sites (>10(4) M(-1)) are located at a reasonably close distance (18-27 A) to at least one of two tryptophan residues, while the lowest affinity binding site (approximately 10(4) M(-1)) is located over 34 A away from the both tryptophans.     

荧光光谱法研究萘酚绿B与牛血清白蛋白相互作用特征

在不同温度下,研究了萘酚绿B(NGB)作用于牛血清白蛋白的荧光猝灭光谱、同步荧光光谱、三维荧光光谱和紫外-可见吸收光谱特征。分别用Stern-Volmer方程和Lineweaver-Burk双倒数方程等处理实验数据,证实了在试验浓度和温度范围内,NGB与BSA可相互作用形成复合物, 荧光猝灭作用符合静态猝灭作用特征,作用力主要是疏水作用力和静电作用力;得到了相互作用的相关参数K_LB,ΔHθ,ΔGθ和ΔSθ等的平均值分别为1.411×105 L·mol-1,-5.707 kJ·mol-1,-30.25 kJ·mol-1和79.95 J·K-1,结合位点数为1.258,为研究NGB对蛋白质构象的影响和在生物体内的生物学效应等提供了重要信息。     

二硫键的氧化还原状态对Trx融合赤霉酸诱导富含半胱氨酸蛋白内源荧光及变性过程影响

在采用亲和层析、SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)对原核表达的赤霉酸诱导的富含半胱氨酸蛋白(Trx-GcGASA)进行纯化、鉴定的基础上,运用稳态荧光光谱手段研究了二硫苏糖醇(DTT)、氧化型谷胱甘肽(GSSG)、过氧化氢、盐酸胍(GdnHCl)对Trx-GcGASA内源荧光及变性过程的影响,发现(1)在中性缓冲体系中融合蛋白的内源荧光以305 nm的酪氨酸的荧光发射为主;(2)伴随着二硫键还原,融合蛋白中色氨酸和酪氨酸的相对荧光强度比值从0.7变化至1.8倍左右;(3)经过0.5 mmol.L-1GSSG、5 mmol.L-1过氧化氢处理后,酪氨酸和色氨酸的荧光强度下降约12~21%;(4)无论是否采用1 mmol.L-1DTT处理,6 mol.L-1盐酸胍均不能诱导融合蛋白彻底变性;(5)二硫键的存在与否影响了盐酸胍诱导的变性过程。通过两态模型拟合获得Trx-GcGASA变性过程Gibbs自由能变化ΔG约为3.7 kJ.mol-1。相关工作不仅为深入研究融合伴侣Trx对GcGASA变性热力学、动力学及复性过程影响奠定了基础;同时,也为通过光谱手段获取GcGASA的结构信息提供了基础的数据。     

Determination by Fluorescence Spectroscopy of Cadmium at the Subnanomolar Level: Application to Seawater

This paper reports the development of a molecular fluorescence spectroscopy-based approach for the determination of cadmium in seawater. Anthrylazamacrocycle derivatives—the fluorescence of which is enhanced when chelated to zinc or cadmium—are used as chemosensors. A detection limit of 5 nM has been found at pH 10 for both metals, and spectral shifts allow simultaneous Cd(II)/Zn(II) determination using multiwavelength analysis. While cadmium emission behavior is similar at pH 13, zinc is not detected anymore. This enables the selective detection of cadmium even at a high Zn(II)/Cd(II) ratio. The detection limit is 1 nM. Interferent removal and preconcentration have been developed using a Dowex resin, with a view to determine cadmium in seawater. A global procedure including interferent elimination, cadmium preconcentration (30 fold), and fluorescence detection at pH 13 has been evaluated on certified reference material SLEW-2.     

Synchronous Fluorescence Method to Check Adulteration of Petrol and Diesel by Kerosene

Commercial automotive fuels available on the Indian subcontinent like petrol and diesel are heavily adulterated with kerosene. Kerosene is used as a cooking fuel among the vast rural and urban populations in India. It is heavily subsidized in the local markets; hence, it becomes a cheap and commonly available adulterant. Intelligent mixing of this product with automotive fuels escapes detection. In this work, we report the synchronous fluorescence scan (SFS) technique as a direct tool to both identify and quantify the amount of adulterant present in commercial petrol and diesel mixtures across the city of Pune. The assay reported here promises to be an efficient tool for such detection purposes (kerosene as low as 1% v/v) within a limited geographical area using conditions of calibration with local components.     

Probing the Liquid-State Structure and Dynamics of Aqueous Solutions by Fluorescence Spectroscopy

Time-resolved fluorescence spectroscopy of the solvent-sensitive molecule 1,8-anilinonaphthalene sulfonate (ANS) is used to probe the structure and dynamics of an aqueous methanol solution (mole fraction = 0.5). The intensity decay of ANS in the mixed solvent displays single exponential kinetics under ambient conditions. At low temperature, a simple two-state solvent relaxation model describes the fluorescence decay for ANS in both methanol and the mixed solvent. The temperature dependence of ANS fluorescence in the mixed solvent is attributed to the onset of glassy dynamics in the aqueous component at higher temperature, implying a partial demixing of the water and methanol due to self-association. We discuss the absence of more complicated fluorescence decays in such a heterogeneous solvent system.     

Study of the Interaction of Fangchinoline with Human Serum Albumin by Fluorescence and UV Spectroscopic Method

The interaction of fangchinoline with human serum albumin (HSA) was studied by use of fluorescence quenching spectra, synchronous fluorescence spectra, and ultraviolet spectra. It was shown that fangchinoline has a strong ability to quench the fluorescence of HSA. The Stern‐Volmer curves based on the quenching of the fluorescence of HSA by fangchinoline indicated that the quenching mechanism of fangchinoline on HSA was static quenching and non‐radiation energy transfer. Based on the Förster theory of non‐radiation energy transfer, the binding distances (r) and the binding constants (K _A) between fangchinoline and HSA were found. The thermodynamic parameters obtained revealed that the interaction between fangchinoline and HSA was mainly driven by hydrophobic force. The conformational changes of HSA were investigated by use of synchronous fluorescence. The result indicates that an ionic electrostatic interaction between fangchinoline and HSA could not be excluded.     

Determination of the Constants of Binding of Acridine Dyes with Micelles of Sodium Dodecyl Sulfate Using the Quenching of Delayed Fluorescence by Heavy Atoms

The constants of binding dye molecules with the micelles of sodium dodecyl sulfate are determined using quenching of delayed fluorescence of acridine dyes by sodium iodide in aqueous–micellar solutions. Kinetic equations have been composed that describe the processes of deactivation of the excited states of dyes. By solving these equations at the concentration of the quencher sodium iodide corresponding to the minimum lifetime of triplet states and at the concentration of micelles corresponding to the least value of the delayed fluorescence quenching rate constants, we obtained the constants of binding dyes with micelles equal to 1.3·10⁷, 2.9·10⁷, and 3.1·10⁷ M^–1 for trypaflavine, acridine orange, and acridine yellow, respectively. We calculated the rate constants of quenching of the triplet states of the molecules of dyes by iodide ions (I ^–) that decreased in transition from trypaflavine to acridine orange and acridine yellow.     

血清白蛋白与小分子化合物相互作用的荧光光谱研究

以牛血清白蛋白-Triton X-100和牛血清白蛋白-盐酸西布曲明两个体系为实例,考察了以血清白蛋白和小分子化合物为荧光检测对象所获得的相互作用信息的差异。发现两种方法获得的分子间结合常数差异显著,说明在以血清白蛋白为检测对象的传统荧光光谱法中,以色氨酸基团的荧光光谱所表达的信息来代表整个蛋白分子的相互作用信息是不准确的。文章提出了以荧光小分子化合物为检测对象的改进荧光光谱方法和荧光背景扣除方法,前者能全面表达相互作用过程中分子的整体信息,后者实现了荧光光谱交叠体系的荧光光谱法相互作用分析。     

氢化物-原子荧光光谱法测定岩藻聚糖硫酸酯中As,Hg含量

岩藻聚糖硫酸酯结合大量的重金属,降低了它的生物活性,使之在保健品、药物开发中的应用受到限制。文章采用高压微波消解、氢化物-原子荧光光谱(HG-AFS)法测定了岩藻聚糖硫酸酯中的As,Hg含量,并对仪器工作参数(原子化器温度、灯电流、负高压等)及氢化物发生条件(载流酸度、载气流量、KBH4浓度等)进行了优化。结果表明,脱盐后岩藻聚糖硫酸酯中As和Hg的含量分别为2.78和0.119 mg.kg-1。说明As和Hg是以结合态形式存在于岩藻聚糖硫酸酯中。As,Hg的检出限分别为0.173和0.012 2ng.kg-1,加标回收率分别为93.31%～100.9%,91.21%～106.1%。     

Single Molecule Fluorescence Imaging and Its Application to the Study of DNA Condensation

Single molecule fluorescence imaging incorporated with optical tweezers and a laminar flow cell has been used to monitor the kinetic process of DNA condensation induced by spermidine. It was found that at least two steps were involved in the condensation process of the hydrodynamically-stretched linear DNA; a lag period followed by a rapid collapse of DNA. The lag time increased with the flow speed and the collapse time remained short within the range of the flow speed studied. The effect of salt concentration on the condensation process was examined, and the results suggest that the longer lag time observed in the higher salt buffer probably results from the displacement of bound cations and rearrangement of spermidine on the DNA. The flow-speed dependence of the lag time suggests that a nucleation event at the free end of the DNA, i.e. formation of a loop, may play a vital role in the kinetic process of condensation.     

Determination of Conjugation Efficiency of Antibodies and Proteins to the Superparamagnetic Iron Oxide Nanoparticles by Capillary Electrophoresis with Laser-Induced Fluorescence Detection

The method based on capillary electrophoresis with laser-induced fluorescence detection (CE/LIF) was developed for determination of magnetic iron oxide nanoparticles (hydrodynamic diameters of 100 nm) functionalized with molecules containing primary amino groups. The magnetic nanoparticles with carboxylic or aminopropyl-trimethoxysilane groups at their surface were conjugated to the model proteins (bovine serum albumin, BSA; streptavidin or goat anti-rabbit immunoglobulin G, IgG) using carbodiimide as a zero-length cross-linker.The nanoparticle–protein conjugates (hydrodynamic diameter 163–194 nm) were derivatized with naphthalene-2,3-dicarboxaldehyde reagent and separated by CE/LIF with a helium–cadmium laser (excitation at 442 nm, emission at 488 nm). The separations were carried out by using a fused-silica capillary (effective length 48 cm, inner diameter 75 um) and 100 mM sodium borate buffer (pH 9.2), the potential was 30 kV. The detection limit for BSA-conjugate was 1.3 pg/10 nl, i.e. about 20 amol. The present method provides an efficient and fast tool for sensitive determination of the efficacy of biomolecular functionalization of magnetic nanoparticles. The CE/LIF technique requires only negligible sample volumes for analysis, which is especially suitable for controlling the process of preparation of functionalized nanoparticles with unique properties aimed to be used for diagnostic or therapeutic purposes.