A cucurbit[6]uril analogue: host properties monitored by fluorescence spectroscopy

This paper describes the host properties of a new cucurbit[6]uril analogue, studied by fluorescence and 1H NMR spectroscopy. This host has an elongated cavity with oval-shaped portals. It is intrinsically fluorescent, and more importantly, this fluorescence is sensitive to guest encapsulation, allowing for the study of the inclusion of nonfluorescent guests by fluorescence spectroscopy. In the case of benzene as guest, significant enhancement of the cucurbit[6]uril analogue host fluorescence was observed upon addition of benzene; this allowed for the determination of the binding constant for 1:1 host-guest complexation, yielding a value of K = 6900 +/- 1100 M(-1). This complexation was also studied by 1H NMR, yielding a similar value of K = 8980 +/- 500 M(-1). The binding of a much larger guest, the dye Nile Red, was also studied, but in this case using guest fluorescence. Significant suppression of the Nile Red fluorescence was observed upon 1:1 complexation with the cucurbit[6]uril analogue, with an extremely large binding constant of 8.2 +/- 0.5 x 10(6) M(-1), indicating a very strong host-guest interaction and an excellent size and shape match. In both cases, binding was much stronger than in the case of the same guests with cucurbit[6]uril itself, and in the case of Nile Red, binding was also much stronger than with modified beta- or gamma-cyclodextrins. This is partly a result of the partial aromatic nature of the host walls, which allow for pi-pi interactions not possible in cucurbiturils or cyclodextrins. The ability to study its inclusion complexes using either host or guest fluorescence, and the very high binding constants observed, illustrates the versatility and potential usefulness of this new host compound.     

Probing the interaction between fluorophores and DNA nucleotides by fluorescence correlation spectroscopy and fluorescence quenching

We have investigated the association interactions between the fluorescent dyes TAMRA, Cy3B and Alexa-546 and the DNA deoxynucleoside monophosphates by means of fluorescence quenching and fluorescence correlation spectroscopy (FCS). The interactions of Cy3B and TAMRA with the nucleotides produce a decrease in the apparent diffusion coefficient of the dyes, which result in a shift toward longer times in the FCS autocorrelation decays. Our results with Cy3B demonstrate the existence of Cy3B-nucleotide interactions that do not affect the fluorescence intensity or lifetime of the dye significantly. The same is true for TAMRA in the presence of dAMP, dCMP and dTMP. In contrast, the diffusion coefficient of Alexa 546 remains practically unchanged even at high concentrations of nucleotide. These results demonstrate that interactions between this dye and the four dNMPs are not significant. The presence of the negatively charged sulfonates and the bulky chlorine atoms in the phenyl group of Alexa 546 possibly prevent strong interactions that are otherwise possible for TAMRA. The characterization of dye-DNA interactions is important in biophysical research because they play an important role in the interpretation of energy transfer experiments, and because they can potentially affect the structure and dynamics of the DNA.     

Extended para-hydrogenation monitored by NMR spectroscopy

A system that provides a sustained hyperpolarized (1)H NMR signal in an aqueous medium is reported. The enhanced signal lasts much longer than typical (1)H T(1) values, uncovering new possibilities for implementing hyperpolarized (1)H NMR/MRI experiments or performing kinetics studies that would not otherwise be detectable.     

荧光光谱法研究辛硫磷与牛血清白蛋白的相互作用

用荧光光谱法研究了在生理pH条件下杀虫剂辛硫磷与牛血清白蛋白(BSA)的相互作用. 结果表明: 辛硫磷对BSA的荧光有较强的猝灭作用, 该猝灭属于静态猝灭. 根据猝灭结果求得了不同温度下辛硫磷与牛血清白蛋白结合作用的结合位点数、结合常数及反应热力学参数, 并据此确定它们之间主要的相互作用力为疏水作用力. 用同步荧光光谱法探讨了辛硫磷对BSA构象的影响.     

Beta-amyloid oligomerisation monitored by intrinsic tyrosine fluorescence

Aggregation of the peptide beta-amyloid is known to be associated with Alzheimer's disease. According to recent findings the most neurotoxic aggregates are the oligomers formed in the initial stages of the aggregation process. Here we use beta-amyloid's (Aβ's) intrinsic fluorophore tyrosine to probe the earliest peptide-to-peptide stages of aggregation, a region often merely labelled as a time lag, because negligible changes are observed by the commonly used probe ThT. Using spectrally resolved fluorescence decay time techniques and analysis we demonstrate how the distribution of 3 rotamer conformations of the single tyrosine in Aβ tracks the aggregation across the time lag and beyond according to the initial peptide concentration. At low Aβ concentrations (≤5 μM), negligible aggregation is observed and this is mirrored by little change in the fluorescence decay parameters, providing a useful baseline for comparison. At higher concentrations (≈50 μM), and contrary to what is generally accepted from ThT studies the rate of aggregation can be described by an exponential growth to a plateau in terms of the relative contributions of two of the three rotamers, with a characteristic aggregation time of ≈33 h.     

荧光光谱法研究克仑特罗与蛋白质的结合作用

应用荧光光谱法研究了水溶液中盐酸克仑特罗与牛血清白蛋白分子间的结合反应 ,测定了结合常数 (K =2 .84× 1 0 3 L mol)和结合位点数 (n =5 .65)。依据F ster非辐射能量转移理论 ,确定了授体 受体间的结合距离 (r=1 .69nm)和能量转移效率 ,采用同步荧光技术考察了盐酸克仑特罗对牛血清白蛋白构象的影响。利用盐酸克仑特罗对蛋白质荧光猝灭 ,对作用机理做了初步探讨     

Study of the interaction between N-confused porphyrin and bovine serum albumin by fluorescence spectroscopy

The fluorescence and ultraviolet spectroscopy were explored to study the interaction between N-confused porphyrins (NCP) and bovine serum albumin (BSA) under imitated physiological condition. The experimental results indicated that the fluorescence quenching mechanism between BSA and NCP was static quenching procedure at low NCP concentration at 293 and 305 K or a combined quenching (static and dynamic) procedure at higher NCP concentration at 305 K. The binding constants, binding sites and the corresponding thermodynamic parameters ΔH, ΔS, and ΔG were calculated at different temperatures. The comparison of binding potency of the three NCP to BSA showed that the substituting groups in benzene ring could enhance the binding affinity. From the thermodynamic parameters, we concluded that the action force was mainly hydrophobic interaction. The binding distances between NCP and BSA were calculated using F?rster non-radiation energy transfer theory. In addition, the effect of NCP on the conformation of BSA was analyzed using synchronous fluorescence spectroscopy.     

同步荧光法研究胺菊酯与牛血清白蛋白的相互作用

血清白蛋白是血浆中含量最丰富的蛋白,能够与许多内源性、外源性化合物相结合.药物小分子进入生物体后,通过与血清白蛋白相互作用,贮存运输至受体部位,进而发生药效作用.     

Penetration of pyrene and its derivatives into polystyrene latex particles as studied by fluorescence spectroscopy

Small hydrophobic pores (channels) in polystyrene latex particles were found by investigating the penetration of pyrene using fluorescence techniques.     

Silica hydrogel formation and aging monitored by pyrene-based fluorescence probes

Fluorescent probes pyrene (Py), di(1-pyrenylmethyl)ether (DiPyM) and newly synthesized 2,3-bis-[4-(1-pyrenemethoxy)methylphenyl]butane (DiPyS) were used to monitor the formation and aging of silica hydrogel prepared from poly(glyceryl silicate) (PGS) sol. The fluorescence emission spectra of these probes are sensitive to their environment and this feature is utilized for monitoring the evolution of silica hydrogels prepared in this work. The polarity of hydrogel matrix during sol–gel transition assessed by all three pyrene probes decreases in the first stage of hydrogel formation, for about 2 h, followed by a gradual increase in polarity and reaching the constant level after 24 h for at least 2 weeks. The process of crosslinking was assessed by DiPyM and DiPyS. These fluorescent probes possess two pyrene structures, which ability to form a dynamic intramolecular excimer can be used to monitor the degree of hydrogel crosslinking with time. These data support the polarity measurements that the hydrogel network is predominatly formed within the first 2 h, stabilized within the 24 h, and that there is a minor increase in the network density for about 10 days until reaching the constant level. In addition, utilizing the second-order diffraction of scattered excitation light may also be used to obtain an adequate information about the silica hydrogel evolution. In summary, this paper demonstrates that pyrene-type fluorescent probes represent simple and precise tool for characterization of formation and aging of the silica hydrogels.     

Exploring the interaction between milrinone drug and lysozyme by fluorescence spectroscopy and differential scanning calorimetry

Journal of Thermal Analysis and Calorimetry - Exploring the mechanism of the interaction between milrinone drug and lysozyme is important for us to understand the transportation in vivo, metabolism...     

Conformational changes monitored by fluorescence study on reconstituted hemoglobins

Intrinsic steady state fluorescence measurements were performed on a series of reconstituted metal ion and hybrid hemoglobins (Hbs). At 296 nm excitation, the spectrum exhibits a broad and asymmetric feature in the case of copper and nickel reconstituted hemoglobins. Deconvolution of the fluorescence bands clearly reveals the existence of two definite peaks. A similar trend was also observed for hybrid hemoglobins (CuNi, NiCu, CuFe-CO, and NiFe-CO). A guassian fit of the fluorescence bands in these proteins again yields two prominent peaks, which are assigned as due to two different tryptophan (Trp) environments. A relative ratio of the amplitudes of these peaks indicates the percentage of T-character in these molecules. This is in support to our previous findings by other spectroscopic studies on the same molecules. These studies therefore, suggest the presence of two different environments of a tryptophan thereby revealing structural heterogeneity among the subunits.     

Side-chain dynamics of an alpha-helical polypeptide monitored by fluorescence

The "blob" model, developed to analyze the fluorescence decays of polymers randomly labeled with pyrene, has been applied to a series of pyrene-labeled poly(glutamic acid)s (PyPGA) in DMF and carbonated buffer solutions at pH 9. Poly(glutamic acid) (PGA) exists in the ionized form in the buffer solutions as poly(sodium glutamate) (PGNa). PGA adopts an alpha-helical conformation in DMF, whereas in aqueous solution PGNa is a random coil. Fluorescence, UV-vis absorption, and circular dichroism measurements indicate that in our studies pyrene pendants attached themselves along PGA in a clustered manner. Simulations were carried out to establish that the geometry of the PGA alpha-helix induces the high level of pyrene clustering. Since the level of pyrene clustering decreased with lower pyrene content, information about naked PGA was retrieved by extrapolating the trends obtained by fluorescence to zero pyrene content. Analysis of the fluorescence decays demonstrated that during its lifetime an excited pyrene probes a 32 amino acid section of the PGA alpha-helix. This result was supported by molecular mechanics optimizations. This study establishes that the blob model, originally used to monitor the encounters between pyrenes attached randomly onto a polymer adopting a random coil conformation, can also be applied to study the dynamics of the side chains of structured proteins. Since the blob model helps in monitoring the encounters between amino acids in the initial state (i.e., random coil) and in the final state (i.e., structured protein) of the folding pathway of a protein, it could be applicable to the study of protein folding.     

Study on the interaction of methylene blue with cyclodextrin derivatives by absorption and fluorescence spectroscopy

The ability of beta-cyclodextrin (beta-CD), hydroxypropyl-beta-cyclodextrin (HP-beta-CD), and carboxymethyl-beta-cyclodextrin (CM-beta-CD) to break the aggregate of the methylene blue (MB) and to form 1:1 inclusion complexes has been studied by absorption and fluorescence spectroscopy. Experimental conditions including concentrations of various cyclodextrins (beta-CD, HP-beta-CD and CM-beta-CD) and media acidity were investigated for the inclusion formation in detail. The formation constants are calculated by using steady-state fluorimetry, from which the inclusion capacity of different cyclodextrins (CDs) is compared. The results suggest that the charged beta-cyclodextrin (CM-beta-CD) is more suitable for inclusion of the cationic dye MB than the neutral beta-cyclodextrins (beta-CD, HP-beta-CD) at pH>5. A mechanism is proposed which is consistent with the stronger binding of MB with CM-beta-CD compared with the other CDs at pH>5.     

Investigation of the interaction between human hemoglobin and five antioxidants by fluorescence spectroscopy and molecular modeling

L-ascorbic acid, α-tocopherol, procyanidin B3, β-carotene, and astaxanthin are five classic dietary antioxidants. In this study, the interaction between the five antioxidants and human hemoglobin (HHb) was investigated by fluorescence spectroscopy and molecular modeling. The quenching mechanisms of HHb by the five antioxidants are all static quenching. The downward curvature of the Stern–Volmer plots for HHb–procyanidin B3 system at higher concentrations of procyanidin B3 come from the reason for the variation in the number of accessible tryptophan (Trp) residues toward HHb. The upward curvature of the Stern–Volmer plots for HHb–β-carotene system at higher concentrations of β-carotene predominantly by the “sphere of action” quenching mechanism. The binding constants of HHb with the five antioxidants are in the following order as: astaxanthin > L-ascorbic acid > β-carotene > α-tocopherol > procyanidin B3 at 298 K. The binding processes of the five antioxidants to HHb are all entropy process. Thermodynamic analysis and molecular modeling suggest that the hydrophobic forces are the main interaction force in the binding of the five antioxidants to HHb and hydrogen bond interactions between HHb and L-ascorbic acid/α-tocopherol/procyanidin B3/astaxanthin should be also considered. The fluorescence experimental results are in agreement with the results obtained by molecular modeling study.     

荧光光谱法研究亚甲蓝与三种芳香族氨基酸的相互作用

本文运用荧光光谱法研究亚甲蓝(MB)分别与色氨酸(Trp)、酪氨酸(Tyr)和苯丙氨酸(Phe)三种芳香族氨基酸的相互作用。在pH 7.4 Tris-HCl缓冲溶液中,MB引起上述三种氨基酸明显的荧光猝灭现象,最大荧光猝灭波长分别位于346、303和282nm。其荧光猝灭值(F0-F)在一定范围内与MB成正比,用于测定MB具有高灵敏度。通过Stern-Volmer作图及温度的影响,表明MB与三种芳香族氨基酸之间以摩尔比1∶1形成基态复合物,均产生显著的静态猝灭,相互作用力较强,其中MB主要通过疏水作用与Trp结合,与Tyr和Phe之间的结合过程以静电作用力为主。     

荧光猝灭法研究胆红素与牛血清白蛋白的相互作用

在模拟生理条件下,利用荧光猝灭法研究了胆红素(BR)和牛血清白蛋白(BSA) 的相互作用.结果表明胆红素对BSA有较强的荧光猝灭作用,两者形成了新的复合物,属于静态荧光猝灭,发生了分子内的非辐射能量转移.计算了不同温度下的结合位点数n,结合常数KA,以及对应的热力学参数ΔG,ΔH和ΔS.根据Foster非辐射能量转移理论确定了胆红素和BSA间的结合距离r.此外,利用同步荧光光谱,分析了胆红素对牛血清白蛋白构象的影响.     

Proteins in action monitored by time-resolved FTIR spectroscopy.

In the post genome era proteins coming into the focus of life sciences. X-ray structure analysis and NMR spectroscopy are established methods to determine the geometry of proteins. In order to determine the molecular reaction mechanism of proteins, time-resolved FTIR (trFTIR) difference spectroscopy emerges as a valuable tool. In this Minireview we describe the trFTIR difference spectroscopy and show its application on the light-driven proton pump bacteriorhodopsin (bR), the photosynthetic reaction center and the GTPase Ras, which is crucial in signal transduction. The main principles of the technique are presented, including a summary of triggering techniques, scan modes and analysis.     

甲基紫与牛血清白蛋白作用的荧光光谱研究

在pH 7.40的Tris-HC1缓冲溶液体系中,采用荧光光谱法和同步荧光光谱法研究了甲基紫(MV)与牛血清白蛋白(BSA)相互作用.结果表明MV与BSA相互作用两者存在一个结合位点,结合常数(KA)为7.628×103 L/mol,MV与BSA主要发生疏水作用,反应是一个吸热、熵增的自发过程.