Preparation of water‐soluble CdSe quantum dots and its application for nitrite detection in the anodic electrochemiluminescence

Water‐soluble CdSe quantum dots (QDs) have been prepared by using L‐cysteine as the stabilizer in an aqueous phase under the optimized conditions. The characteristics and shapes of CdSe QDs have been proposed on the basis of UV‐Vis and fluorescence spectra. A rapid analytical method for electrochemiluminescence (ECL) determination of nitrite has been developed on the basis of the quenching effect on anodic ECL emission of CdSe QDs under the optimum experimental conditions. In a neutral system and at a relatively low potential (+0.960 V), the ECL emission of CdSe QDs could be greatly enhanced by sulfite and could be gradually quenched by nitrite at an indium tin oxide (ITO) electrode. The proposed method may allow the measurement of nitrite ranging from 1 μM to 0.5 mM with a correlation coefficient of 0.9956 (n = 10) and a detection limit of 0.2 μM (3σ), and the relative standard deviation for 10 μM nitrite (n = 9) is 1.72 %. The proposed method could be adopted for the sensitive detection of ECL quenchers by using nitrite as a model molecule. Copyright © 2013 John Wiley & Sons, Ltd.     

The investigation of the interaction between orientin and bovine serum albumin by spectroscopic analysis

In this paper, the interaction between orientin and bovine serum albumin (BSA) was examined using fluorescence and absorbance spectroscopy. The analysis of the quenching mechanism was done using Stern–Volmer plots which exhibit upward (positive) deviation. A linear response to orientin was shown in the concentration range between 3 and 50 μM. The experimental results showed the presence of a static quenching process between orientin and BSA. The thermodynamic parameters ΔH, ΔS and ΔG were also calculated and suggested that the hydrophobic and electrostatic interactions played an important role in the interaction between orientin and BSA. Furthermore, the distances between BSA and orientin were determined according to Förster non‐radiation energy transfer theory. In addition, the results of the synchronous fluorescence obtained indicated that the binding of orientin with BSA could affect conformation in BSA. Copyright © 2013 John Wiley & Sons, Ltd.     

ZnS quantum dots‐based fluorescence spectroscopic technique for the detection of quercetin

Water‐soluble ZnS quantum dots (QDs) modified by mercaptoacetic acid (MPA) were used to determinate quercetin in aqueous solutions by a fluorescence spectroscopic technique. The results showed that the fluorescence of the modified ZnS QDs could be quenched by quercetin effectively in physiological buffer solution. The optimum fluorescence intensity was found to be at incubation time 10 min, pH 7.0 and temperature 25°C. Under the optimal conditions, the detection limit of quercetin was 5.71 × 10^‐7 mol/L. Moreover, the quenching mechanism was discussed to be a static quenching procedure, which was proved by the quenching rate constant K_q (1.14 × 10¹³ L/mol/s). Copyright © 2013 John Wiley & Sons, Ltd. Copyright © 2013 John Wiley & Sons, Ltd.     

Differences in heat stability and ligand binding among β-lactoglobulin genetic variants A,B and C using H NMR and fluorescence quenching

The structure of β-lactoglobulin (β-LG) is well characterized, but the exact location of binding sites for retinol and (−)-epigallocatechingallate (EGCG) is still a subject of controversy. Here we report that the genetic β-LG variants A, B and C have different numbers of binding sites for retinol (almost completely incorporated into the calyx), as well as for EGCG (exclusively bound on the surface), and β-LG A with the most binding sites for EGCG, which include Tyr²⁰, Phe¹⁵¹ and His⁵⁹. Upon heat related unfolding, new unspecific binding sites emerge, which are comparable in number and affinity for retinol and for EGCG, and in the three genetic variants A, B and C. The findings of our study provide new insights into the use of β-LG as nanotransporter.     

Stark fluorescence spectroscopy reveals two emitting sites in the dissipative state of FCP antennas

Diatoms are characterized by very efficient photoprotective mechanisms where the excess energy is dissipated as heat in the main antenna system constituted by fucoxanthin–chlorophyll (Chl) protein complexes (FCPs). We performed Stark fluorescence spectroscopy on FCPs in their light-harvesting and energy dissipating states. Our results show that two distinct emitting bands are created upon induction of energy dissipation in FCPa and possibly in FCPb. More specifically one band is characterized by broad red shifted emission above 700 nm and bears strong similarity with a red shifted band that we detected in the dissipative state of the major light-harvesting complex II (LHCII) of plants [26]. We discuss the results in the light of different mechanisms proposed to be responsible for photosynthetic photoprotection.     

Measuring membrane penetration with depth-dependent fluorescence quenching: Distribution analysis is coming of age

Depth-dependent fluorescence quenching by lipid-attached quenchers (e.g., bromine atoms and doxyl groups) is an important tool for determining the penetration of proteins and peptides into lipid bilayers. Extracting quantitative information and accurate calculations of the depth of the fluorophore are complicated by thermal disorder, resulting in broad distributions of the transverse positions of both quenchers and fluorophores. Twenty-one years ago a methodology called distribution analysis (DA) was introduced, based on the emerging view of the complexity of the transverse organization of lipid bilayer structure. The method is aimed at extracting quantitative information on membrane penetration, such as position and width of fluorophore's distribution along the depth coordinate and its exposure to the lipid phase. Here we review recent progress in refining the DA method and illustrate its applications to protein–membrane interactions. We demonstrate how basic assumptions of the DA approach can be validated using molecular dynamics simulations and how the precision of depth determination is improved by applying a new protocol based on a combination of steady-state and time-resolved fluorescence quenching. Using the example of the MPER fragment of the membrane-spanning domain of the HIV-1 gp41 fusion protein, we illustrate how DA applications and computer simulations can be used together to reveal the molecular organization of a protein–membrane complex. This article is part of a Special Issue entitled: Interfacially Active Peptides and Proteins. Guest Editors: William C. Wimley and Kalina Hristova.     

Fluorescence spectroscopy of soluble E. coli SPase I Δ2‐75 reveals conformational changes in response to ligand binding

The bacterial Sec pathway is responsible for the translocation of secretory preproteins. During the later stages of transport, the membrane‐embedded signal peptidase I (SPase I) cleaves the signal peptide from a preprotein. We used tryptophan fluorescence spectroscopy of a soluble, catalytically active E. coli SPase I Δ2‐75 enzyme to study its dynamic conformational changes while in solution and when interacting with lipids and signal peptides. We generated four single Trp SPase I Δ2‐75 mutants, W261, W284, W300, and W310. Based on fluorescence quenching experiments, W300 and W310 were found to be more solvent accessible than W261 and W284 in the absence of ligands. W300 and W310 inserted into lipids, consistent with their location at the enzyme's proposed membrane‐interface region, while the solvent accessibilities of W261, W284, and W300 were modified in the presence of signal peptide, suggesting propagation of structural changes beyond the active site in response to peptide binding. The signal peptide binding affinity for the enzyme was measured via FRET experiments and the K_d determined to be 4.4 μM. The location of the peptide with respect to the enzyme was also established; this positioning is crucial for the peptide to gain access to the enzyme active site as it emerges from the translocon into the membrane bilayer. These studies reveal enzymatic structural changes required for preprotein proteolysis as it interacts with its two key partners, the signal peptide and membrane phospholipids. Proteins 2014; 82:596–606. © 2013 Wiley Periodicals, Inc.     

Wheat-Aegilops biuncialis amphiploids have efficient photosynthesis and biomass production during osmotic stress

Osmotic stress responses of water content, photosynthetic parameters and biomass production were investigated in wheat-Aegilops biuncialis amphiploids and in wheat genotypes to clarify whether they can use to improve the drought tolerance of bread wheat. A decrease in the osmotic pressure of the medium resulted in considerable water loss, stomatal closure and a decreased CO₂ assimilation rate for the wheat genotypes, while the changes in these parameters were moderate for the amphiploids. Maximal assimilation rate was maintained at high level even under severe osmotic stress in the amphiploids, while it decreased substantially in the wheat genotypes. Nevertheless, the effective quantum yield of PS II was higher and the quantum yield of non-photochemical quenching of PS II and PS I was lower for the amphiploids than for the wheat cultivars. Parallel with this, higher cyclic electron flow was detected in wheat than in the amphiploids. The elevated photosynthetic activity of amphiploids under osmotic stress conditions was manifested in higher biomass production by roots and shoots as compared to wheat genotypes. These results indicate that the drought-tolerant traits of Ae. biuncialis can be manifested in the wheat genetic background and these amphiploids are suitable genetic materials for improving drought tolerance of wheat.     

Effects of pre-industrial,current and future [CO2] in traditional and modern wheat genotypes

Wheat is one of the most important cereal food crops in the world today. The productivity and quality of this crop is greatly affected by environmental conditions during grain filling. In this study, we have analyzed two genotypes of durum wheat, Blanqueta and Sula (traditional and a modern wheat respectively) in pre-industrial, current and future [CO₂]. Plant growth and physiological parameters were analyzed during anthesis and grain filling in order to study the capacity of these plants to create new sinks and their role during the process of the acclimation of photosynthesis. It was observed that plants underwent photosynthetic acclimation at pre-industrial and future [CO₂] (up and down-regulation respectively). However, the modern genotype averts the process of down-regulation by creating a new carbon sink (i.e. the spike). Here, we have shown the essential role that the spike plays as a new sink in order to avert the down-regulation of photosynthesis at future [CO₂]. Moreover, we have demonstrated that at future [CO₂] the growth response will depend on the ability of plants to develop new sinks or expand existing ones.