Molecular self-assembling and the fluorescence enhancement in morin-Al3+-cetyltrimethylammonium bromide-protein system

Fluorescence enhancement effect of the morin-Al3+-cetyltrimethylammonium bromide (CTAB)-bovine serum albumin (BSA) system is reported here and the interaction mechanism is studied using fluorescence, resonance light scattering (RLS), absorption spectroscopy, Far-UV circular dichroism (CD) spectrum and small angle X-ray scattering (SAXS) measurement. It is considered that protein can bind with Al3+, morin and CTAB through self-assembling function with electrostatic attraction, hydrogen bond, hydrophobic interaction and Vander Waal force etc, and forms a supermolecular association with multilayer structure, in which morin-Al3+ is clamped between BSA and CTAB. In this system, the fluorescence enhancement of morin originates from both intermolecular energy transfer between BSA and morin, and the hydrophobic microenvironment provided by BSA and CTAB. Whereas Al3+ plays a key role for the enhancement of energy transfer efficiency because it provides an efficient channel for the energy transfer between BSA and morin.     

Improvement of the acridine orange-protein-surfactant system for protein estimation based on aromatic ring stacking effect of sodium dodecyl benzene sulphonate.

The fluorescence of acridine orange (AO) is greatly quenched by the anionic surfactant sodium dodecyl benzene sulphonate (SDBS), but when protein is added into the AO-SDBS system, the fluorescence intensity of the latter is enhanced again. Based on this, a new fluorimetric method of determination of protein was developed. Under optimum conditions, the enhanced intensity of fluorescence is in proportion to the concentration of protein, such as bovine serum albumin (BSA), human serum albumin (HSA) and egg albumin (EA), over a wide range with detection limits at the 10(-9) g/mL level. This method has been satisfactorily used for the determination of protein in samples. We compared results using 280 nm and 490 nm excitation wavelengths and the mechanism of the assay.     

Fluorescence enhancement monitoring of pyrromethene laser dyes by metallic Ag nanoparticles

Fluorescence enhancement monitoring of pyrromethene laser dyes using their complexation with Ag nanoparticles (Ag NPs) was studied. The size of the prepared Ag NPs was determined by transmission electron spectroscopy and UV/Vis absorption spectroscopy. Mie theory was also used to confirm the size of NPs theoretically. The effect of different nanoparticle concentrations on the optical properties of 1 × 10^‐4 M PM dyes shows that 40%of Ag NPs concentration (40%C Ag NPs) in complex is the optimum concentration. Also, the effects of different concentrations of PM dyes in a complex was measured. Emission enhancement factors were calculated for all samples. Fluorescence enhancement efficiencies depended on the input pumping energy of a Nd‐YAG laser (wavelength 532 nm and 8 ns pulse duration) were reported and showed the lowest energy (28 and 32 mJ) in the case of PM567 and PM597, respectively. Copyright © 2014 John Wiley & Sons, Ltd.     

Relaxation data in NMR structure determination: model calculations for the lysozyme-Gd3+ complex

The effect of including paramagnetic relaxation data as additional restraints in the determination of protein tertiary structures from NMR data has been explored by a systematic series of model calculations. The system used for testing the method was the 2.0 A resolution tetragonal crystal structure of hen egg white lysozyme (129 amino acid residues) and structures were generated using a version of the hybrid "distance geometry-dynamic simulated annealing" procedure. A limited set of 769 NOEs was used as restraints in all the calculations; the strengths of these were categorized into three classes on the basis of distances observed in the crystal structure. The values of 50 phi angles were also restrained on the basis of amide-alpha coupling constants calculated from the X-ray structure. Five sets of 12 structures were determined using differing sets of paramagnetic relaxation data as restraints additional to those involving the NOE and coupling constant data. The paramagnetic relaxation data were modeled on the basis of the distances of defined protons from the crystallographic binding site of Gd3+ in lysozyme. Analysis of the results showed that the relaxation data significantly improved the correspondence between the set of generated structures and the crystal structure, and that the more well defined the relaxation data, the more significant the improvement in the quality of the structures. The results suggest that the inclusion of paramagnetic relaxation restraints could be of significant value for the experimental determination of protein structures from NMR data.     

Study on the fluorescent enhancement effect in terbium-gadolinium-protein-sodium dodecyl benzene sulfonate system and its application on sensitive detection of protein at nanogram level

The co-luminescence effect in a terbium-gadolinium-protein-sodium dodecyl benzene sulfonate (SDBS) system is reported here. Based on it, the sensitive quantitative analysis of protein at nanogram levels is established. The co-luminescence mechanism is studied using fluorescence, resonance light scattering (RLS), absorption spectroscopy and NMR measurement. It is considered that protein could be unfolded by SDBS, then a efficacious intramolecular fluorescent energy transfer occurs from unfolded protein to rare earth ions through SDBS acting as a "transfer bridge" to enhance the emission fluorescence of Tb3+ in this ternary complex of Tb-SDBS-BSA, where energy transfer from protein to SDBS by aromatic ring stacking is the most important step. Cooperating with the intramolecular energy transfer above is the intermolecular energy transfer between the simultaneous existing complexes of both Tb3+ and Gd3+. The fluorescence quantum yield is increased by an energy-insulating sheath, which is considered to be another reason for the resulting enhancement of the fluorescence. F?rster theory is used to calculate the distribution of enhancing factors and has led to a greater understanding of the mechanisms of energy transfer.     

Resonance light‐scattering enhancement effect of the protein–Y3+–TTA–SLS system and its analytical application

In this paper, a sensitive resonance light scattering (RLS) method for the determination of protein is reported. In the Tris–HCl (pH 7.50) buffer, protein enhanced the RLS intensity of the Y³⁺–2‐thenoyltrifluoroacetone (TTA)–sodium dodecyl sulphate (SLS) system. The enhanced RLS intensities were in proportion to the concentrations of proteins in the range 8.0 × 10^?9–1.0 × 10^?5 g/mL for BSA, 1.0 × 10^–8–1.0 × 10^?5 g/mL for HSA and 1.0 × 10^–8–1.0 × 10^?6 g/mL for EA, and their detection limits were 5.0, 5.4 and 6.7 ng/mL, respectively. Actual samples were satisfactorily determined. The interaction mechanism was also studied. Copyright © 2008 John Wiley & Sons, Ltd.     

Using Co-Cultures Expressing Fluorescence Resonance Energy Transfer Based Protein Biosensors to Simultaneously Image Caspase-3 and Ca<Superscript>2+</Superscript> Signaling

Fluorescence resonance energy transfer (FRET)-based protein biosensors allow the spatial and temporal imaging of signaling events in living cells. However, the simultaneous correlation of multiple events of a signaling pathway is hindered by the spectral cross-talk between fluorescent proteins. Here, we show, for signaling pathways that progress synchronously, multiple events can be correlated by using co-cultures expressing different FRET-based protein biosensors. As a demonstration, we investigated the simultaneous caspase-3 and Ca²⁺ signaling events involved in cell death of COS-7 cells induced by 10 mM H₂O₂. Interestingly, this H₂O₂ stimulus induced synchronous caspase-3 activation and Ca²⁺ signaling. In parallel to caspase-3 activation, cytosolic Ca²⁺ concentration, [Ca²⁺]_c, gradually rises to its peak and then slowly drops. As cell shrinkage and rounding ensues, [Ca²⁺]_c again gradually rises to its peak and then reaches a plateau. These observations reveal the relative timing and location of these signaling events in cell death induced by this stimulus of H₂O₂. Finally, our approach offers an exciting opportunity for spatial and temporal imaging of multiple events in a signaling pathway in living cells.     

Synthesis and photoluminescence enhancement of the LiLa(MoO4)2:Sm3+ red phosphors by co-doping with Bi3+

The LiLa(MoO₄)₂:Sm³⁺ and LiLa(MoO₄)₂:Sm³⁺,Bi³⁺ phosphors were prepared using the citric-acid-fueled combustion method and the influence of concentrations of Bi³⁺ dopant on LiLa(MoO₄)₂:Sm³⁺ red luminescence was investigated. The LiLa(MoO₄)₂:Sm³⁺ and LiLa(MoO₄)₂:Sm³⁺,Bi³⁺ samples matched well with the scheelite structure and I4₁/a space group and did not detect structural changes. Under an excitation of 403 nm, the prepared LiLa(MoO₄)₂:Sm³⁺,Bi³⁺ phosphor was excited and produced orange-red emission. When compared with the LiLa(MoO₄)₂:Sm³⁺ phosphor, the LiLa(MoO₄)₂:Sm³⁺,Bi³⁺ phosphor exhibited enhanced fluorescence intensity because the Bi³⁺ dopant ions are doped as a sensitizer. The optimal doping concentrations of Sm³⁺ and Bi³⁺ were 5 and 1 mol%, respectively. Furthermore, the energy transfer from Bi³⁺ to Sm³⁺ is effective (³P₁ → ⁴K_11/2). Subsequently, the electrons in an unstable excited state were transferred to a stable ground state (⁴G_5/2 → ⁶H_5/2, ⁶H_7/2, ⁶H_9/2). The Commission Internationale de L'Eclairage (CIE) chromaticity coordinates of the optimized LiLa(MoO₄)₂:Sm³⁺,Bi³⁺ phosphor were situated in the orange-red region. The luminescence of the LiLa(MoO₄)₂:Sm³⁺,Bi³⁺ phosphor generated under near-ultraviolet (UV) irradiation could be used to produce a warm white light, indicating its possible applications in white light-emitting diodes.     

Fluorescence lifetime and rotational correlation time of bovine serum albumin-sodium dodecyl sulfate complex labeled with 1-dimethylaminonaphthalene-5-sulfonyl chloride: Effect of disulfide bridges in the protein on these fluorescence parameters

A fluorescent dye, 1-dimethylaminonaphthalene-5-sulfonyl chloride, was used to label bovine serum albumin (BSA), intact and disulfide bridges-cleaved. The fluorescence lifetime and fluorescence anisotropy of the adducts in sodium dodecyl sulfate (SDS) solutions were studied by the nanosecond fluorescence depolarization method. The volume of equivalent sphere (V _e) was calculated to be 2.1×10^–19 cm³ for BSA with the intact disulfide bridges from the rotational correlation time. The value ofV _e was 4.4×10^–19 cm³ for the disulfide bridges-cleaved BSA. With an increase in SDS concentration, the rotational correlation time of the intact BSA became longer, while that of the disulfide bridges-cleaved BSA became shorter. This suggests that upon the binding of SDS, the total volume of the intact BSA increases while the expanded state of the protein, caused by the cleavage of the disulfide bridges, becomes compact.     

Fluorescence enhancement of rare earth Tb(III) by Tm(III) in benzyl benzoylmethyl sulphoxide complexes

A series of rare earth complexes [(Tb_xTm_y)L₅(ClO₄)₂](ClO₄)·3H₂O (x:y = 1.000:0.000, 0.999:0.001, 0.995:0.005, 0.990:0.010, 0.950:0.050, 0.900:0.100, 0.800:0.200, 0.700:0.300; L = C₆H₅CH₂SOCH₂COC₆H₅) (Tb(III) luminescence ion; Tm(III) doped inert ion) were synthesized and characterized by elemental analysis, infrared spectra (IR) and ¹H‐NMR. The photophysical properties of these complexes were studied in detail using ultraviolet absorption spectra, fluorescent spectra and lifetimes. The fluorescence spectra of complexes indicated that the fluorescence emission intensity was significantly enhanced by Tm(III). The complexes showed the best luminescence properties when the mole ratio Tb(III):Tm(III) was 0.990:0.010. The fluorescence intensity could be increased to 390%. Additionally, phosphorescence spectra and the luminescence mechanisms are discussed. Copyright © 2011 John Wiley & Sons, Ltd.     

Fe3+对雨生红球藻(Haematococcus pluvialis)生长及荧光特性的影响

为探求适宜雨生红球藻CG-06株生长的Fe³⁺浓度和Fe³⁺对藻细胞荧光特性的影响,以BBM为基础培养基,选用EDTA-FeNa(Ⅲ)为Fe³⁺源,设置0、1.79、8.95、17.9、35.8、71.6μmol·L^-16种Fe³⁺浓度梯度,实验测定藻的生长并分析不同Fe³⁺对藻细胞叶绿素荧光和77 K低温荧光等的影响。结果表明:适合雨生红球藻CG-06株生长的Fe³⁺浓度为8.95μmol·L^-1,Fe³⁺浓度较高时,雨生红球藻的生长受到抑制,Fe³⁺浓度低于1·79μmol.L^-1将产生低铁限制。W alz-PAM测定数据显示,在铁不足或高铁抑制条件下,雨生红球藻光系统Ⅱ活性明显下降,开放态的反应中心数目减少,光合作用受到抑制。77 K低温荧光光谱显示,在铁不足或高铁抑制条件下,710 nm荧光峰降低,684 nm和694 nm荧光峰相对增强,说明能量在两个光系统分配上发生变化,能量更多的分配给光系统Ⅱ,限制了光系统Ⅰ的活性。在高铁抑制条件下,CP₄₇蛋白荧光峰降低,CP₄₃蛋白荧光峰增强,推测存在D₁蛋白降解。     

The fluorescence quenching of Ru(bipy)32+: an application for the determination of bilirubin in biological samples

A simple, sensitive and efficient fluorescence method has been established for the quantitative analysis of bilirubin. The fluorometric determination method was based on the kinetic quenching of ruthenium(II) fluorescence. The quenching effect may be due to the complexation reaction of bilirubin with ruthenium(II). Therefore, the effects of ruthenium concentrations and different surfactants have been studied. Under the optimized experimental parameters, the fluorescence intensity decreased proportionally with the bilirubin concentration and linearity was established in the range of 3.3 × 10⁻⁷ to 3.0 × 10⁻⁴ M bilirubin. The detection limit calculated from the calibration graph was found to be 5.2 × 10⁻⁸ M. The relative standard deviation (RSD) of 10 consecutive measurements of 8.0 × 10⁻⁶ M bilirubin was 3.0%, while the recoveries of bilirubin in both human serum and urine samples were obtained in the range 94.0–99.5%. The interference study shows that the developed fluorescence based technique is fast, easy to carry out and shows negligible interference. The developed technique was successfully applied for the analysis of bilirubin in human urine and serum samples. All the experimental results and quality parameters confirmed the sensitivity and reproducibility of the proposed technique for bilirubin determination in human urine and serum samples .     

Factors determining the reconstructive denaturation of proteins in sodium dodecyl sulfate solutions. Further circular dichroism studies on structural reorganization of seven proteins

The factors determining the onset and extent of reconstructive denaturation of proteins were considered by comparing circular dichroism (CD) data of seven proteins and previously published findings. The effects of sodium dodecyl sulfate (SDS) on the conformation of the following proteins were tested: lysozyme, the mitogens fromPhytolacca americana (fractions Pa2 and Pa4), lectin fromWistaria floribunda, ovine lutropin, a Bence Jones protein, and histone H2B. While the helix content of lysozyme was raised by SDS slightly, in the Bence Jones protein andW. floribunda lectin it increased from near zero to about 25–30%. In histone H2B the helix content was raised by SDS even to about 48%. However, no clear indication of helix formation could be observed in the mitogens and lutropin, even at low pH or 2.0–2.5. The tertiary structure of the proteins was perturbed by SDS. It was concluded that the reorganization of secondary structure of the proteins was favored by the following factors: (1) presence of helicogenic amino acid sequences in the protein, (2) availability of positively charged sites of the basic amino acids for interactions with the dodecyl ion, (3) absence of a large surplus of negatively charged sites on the surface of protein, and (4) absence of extensive disulfide cross-linking within the macromolecule. Both hydrophobic and electrostatic interactions occur in reconstructive denaturation, and the newly formed helices are stabilized by hydrophobic shielding by the alkyl chains of the alkyl sulfate.     

Investigation of the interactions of quercetin and morin with trypsin

The interactions of quercetin and morin with trypsin were investigated by UV–vis absorption, fluorescence, synchronous fluorescence and three‐dimensional fluorescence spectra techniques under physiological pH 7.40. Quercetin and morin effectively quenched the intrinsic fluorescence of trypsin via static quenching. The process of binding quercetin and morin on trypsin was a spontaneous molecular interaction procedure. The binding constants and thermodynamic parameters at two different temperatures, the binding locality and the binding power were obtained. The conformation of trypsin was discussed by synchronous and three‐dimensional fluorescence techniques. Copyright © 2009 John Wiley & Sons, Ltd.     

Properties of the iron--sulphur proteins of the benzene dioxygenase system from Pseudomonas putida.

A purification procedure was developed to stabilize the iron-sulphur proteins of the benzene dioxygenase system from Pseudomonas putida. The intermediate electron-carrying protein has a mol. wt. of 12300 and possesses one (2Fe--2S) cluster, whereas the terminal dioxygenase has a mol.wt. of 215300 and possesses two (2Fe--2S) clusters. The order and stoicheiometry of electron transfer and of the whole system are described.     

Kinetics of protein agglomeration. A nephelometric method for the determination of total protein in biological samples

The kinetics of agglomeration of proteins precipitating in a viscous solution was measured by light scattering. The resulting transitory maximum was linearly proportional to the mass of protein over three orders of magnitude. The change in scattering intensity is described as a change in scattering symmetry due to a continuous in particle size.This method is both fast (minutes) and sensitive (30 ng protein) and is independent of the chemical composition of the different protein species and is barely influenced by size and shape of the proteins.By solubilising the protein in an alkaline dedecyl sulfate solution this method can be applied to all types of biological samples (e.g. tissue homogenates proteins) and also to all types of biological preparations containing detergents as well as urea, sucrose, salts and lipids.     

Fluorescence and the structure of proteins. XXI. Fluorescence of aminotyrosyl residues in peptides and helical proteins.

1. Five peptides containing tyrosine were converted to the 3-aminotyrosyl peptides by nitration with tetranitromethane and subseuqent reduction of the nitro groups to amino groups. The fluorescence of these aminotyrosyl residues was found to be quite similar to that of 3-aminotyrosine and it is concluded that the fluorescence is not sensitive to incorporation of the amino acid into the peptide chain. 2. Fluorescence of 3-aminotyrosine derivatives was sensitive, however, to the nature of the solvent; as the dielectric constant decreased, fluorescence was enhanced ten fold and the emission maximum shifted from the 350-370 nm value in aqueous solution to 320 nm. It is predicted that similar differences might be expected for exposed and buried aminotyrosyl residues in a protein. 3. Exposed tyrosyl residues on the helical protein tropomyosin and a helical segment of paramyosin were aminated in part (39% and 34% of the total tyrosyl residues, respectively). The fluorescence of the aminated tyrosyl residues on these proteins was similar to that of the aminotyrosyl peptides in an aqueous medium. Although the fluorescence efficiency of an aminotyrosyl residue was much lower than that of a tyrosyl residue, it was easy to distinguish the fluorescence of the aminotyrosyl residues (350-355 nm) on the protein from that arising from unmodified tyrosyl residues (305 nm).     

Rocket and crossed immunoelectrophoresis of proteins solubilized with sodium dodecyl sulfate

A method for the immunoelectrophoretic analysis of both hydrophilic and hydrophobic proteins from whole-cell extracts solubilized with 2% (w/v) sodium dodecyl sulfate (SDS) is described. For rocket immunoelectrophoresis, Triton X-100 is added to the sample before electrophoresis to sequester non-protein-bound SDS, and polyethylene glycol (PEG) is added to the antibody gel to enhance precipitin formation. With the optimal ratio of Triton X-100 to PEG, the quantitative determination of 5 ng of protein is possible. The SDS-solubilized sample can also be analyzed by crossed immunoelectrophoresis using SDS-polyacrylamide gels in the first dimension and antibody-containing agarose gels in the second. The best results are obtained when intermediate gels without nonionic detergents are used and when ionic detergents are omitted from the cathodal gel. Precipitin peaks of high quality, reproducibility, and without artifacts are obtained using antibody concentrations 5- to 50-fold lower than with other crossed-immunoelectrophoresis procedures.     

The impact of window functions on NMR-based paramagnetic relaxation enhancement measurements in membrane proteins

Though challenging, solution NMR spectroscopy allows fundamental interrogation of the structure and dynamics of membrane proteins. One major technical hurdle in studies of helical membrane proteins by NMR is the difficulty of obtaining sufficient long range NOEs to determine tertiary structure. For this reason, long range distance information is sometimes sought through measurement of paramagnetic relaxation enhancements (PRE) of NMR nuclei as a function of distance from an introduced paramagnetic probe. Current PRE interpretation is based on the assumption of Lorentzian resonance lineshapes. However, in order to optimize spectral resolution, modern multidimensional NMR spectra are almost always subjected to resolution-enhancement, leading to distortions in the Lorentizian peak shape. Here it is shown that when PREs are derived using peak intensities (i.e., peak height) and linewidths from both real and simulated spectra that were produced using a wide range of apodization/window functions, that there is little variation in the distances determined (< 1 Å at the extremes). This indicates that the high degree of resolution enhancement required to obtain well-resolved spectra from helical membrane proteins is compatible with the use of PRE data as a source of distance restraints. While these conclusions are particularly important for helical membrane proteins, they are generally applicable to all PRE measurements made using resolution-enhanced data.     

The dependence of radiation enhancement effect on the concentration of gold nanoparticles exposed to low- and high-LET radiations

We examined the dependence of hydroxyl radical production on the concentration of 15 nm citrate-capped AuNPs and dose using coumarin-3-carboxylic acid in phosphate buffered saline (PBS), and investigated the radiosensitisation of different concentration AuNPs on human cervix carcinoma HeLa cells through clonogenic survival assay for X-rays and carbon ions. The enhancement factor of AuNPs for hydroxyl radical production reached a maximum 3.66 for X-rays at the concentration of 0.1 μg/mL while the maximum was 5.52 for carbon ions in presence of 1.0 μg/mL AuNPs in PBS. At 50% survival level, the sensitizer enhancement ratios of X-rays and carbon ions varied from 1.14 to 2.88 and from 1.27 to 1.44, respectively, when cells were co-cultured with 1.5–15.0 μg/mL AuNPs. Our data indicate AuNPs showed radiosensitisation in terms of hydroxyl radical production and cell killing for low- and high-LET radiations. The concentration of AuNPs in PBS and cells played an important role in radiosensitizing effect. Based on the fact-the AuNPs in PBS could improve the production of hydroxyl radical and no accumulation of cells in the G₂/M phase was observed, we deduce that the increment of hydroxyl radical production with AuNPs provided a mechanism for radiosensitisation.