Spectrofluorimetric determination of nebivolol by using boron doped carbon dots as fluorescence probe.
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•Boron-doped carbon dots were synthesized and characterized by various ...techniques.•The quantum yield and other optical features of the prepared carbon dots were identified.•A new and selective spectrofluorimetric method is developed for the determination of nebivolol.•The method was applied for the determination of nebivolol in tablets, spiked human plasma, and content uniformity testing.
Recently, nanomaterials have attracted a lot of attention due to their potential as effective fluorescent nano-sensor probes. They were distinguishing substitutes for other luminescent techniques, such as fluorescent dyes and luminous derivatization, because of their affordability, environmental friendliness, and special photocatalytic properties. In the suggested work, a straightforward method was used to create boron and nitrogen carbon dots (B@CDs) with a good quantum yield value of 31.15 % utilizing boric acid and di-sodium EDTA. For the purpose of characterizing QDs, a variety of instruments were employed, such as transmission electron microscopy, fluorescence spectroscopy, X-ray FTIR, and UV–VIS spectroscopy. Nebivolol (NEB) is a cardiovascular medication used globally to treat congestive heart failure and hypertension, is in the meantime. For this reason, a brand-new, environmentally friendly analytical technique was created to determine the amount of human plasma, uniformity test, and commercial nebivolol (NEB) tablets. After gradually adding NEB, the response of B@CQDs was enhanced at 438 nm (excitation at 371 nm). The calibration graph ranged between 20 and 500 ng mL−1 with a quantification limit (LOQ) of 2.50 ng mL−1 and a detection limit (LOD) of 0.82 ng mL−1.
Enzymes are attractive tools for synthetic applications. To be viable for industrial use, enzymes need sufficient stability towards the desired reaction conditions such as high substrate and ...cosolvent concentration, non‐neutral pH and elevated temperatures. Thermal stability is an attractive feature not only because it allows for protein purification by thermal treatment and higher process temperatures but also due to the associated higher stability against other destabilising factors. Therefore, high‐throughput screening (HTS) methods are desirable for the identification of thermostable biocatalysts by discovery from nature or by protein engineering but current methods have low throughput and require time‐demanding purification of protein samples. We found that nanoscale differential scanning fluorimetry (nanoDSF) is a valuable tool to rapidly and reliably determine melting points of native proteins. To avoid intrinsic problems posed by crude protein extracts, hypotonic extraction of overexpressed protein from bacterial host cells resulted in higher sample quality and accurate manual determination of several hundred melting temperatures per day. We have probed the use of nanoDSF for HTS of a phylogenetically diverse aldolase library to identify novel thermostable enzymes from metagenomic sources and for the rapid measurements of variants from saturation mutagenesis. The feasibility of nanoDSF for the screening of synthetic reaction conditions was proved by studies of cosolvent tolerance, which showed protein melting temperature to decrease linearly with increasing cosolvent concentration for all combinations of six enzymes and eight water‐miscible cosolvents investigated, and of substrate affinity, which showed stabilisation of hexokinase by sugars in the absence of ATP cofactor.
Enzymes
Alcohol dehydrogenase (NADP+) (EC 1.1.1.2), transketolase (EC 2.2.1.1), hexokinase (EC 2.7.1.1), 2‐deoxyribose‐5‐phosphate aldolase (EC 4.1.2.4), fructose‐6‐phosphate aldolase (EC 4.1.2.n).
nanoDSF is a valuable tool to rapidly determine thermal stability of native proteins in crude protein extracts. It can be used for high‐throughput screening of enzyme libraries from metagenomic sources or from saturation mutagenesis, for rapid screening of tolerance for water‐miscible organic cosolvent and of substrate affinity, for example, sugar binding by hexokinase even in the absence of ATP.
•A comprehensive evaluation on various aspects of uranium in groundwater.•Detection and determination of elemental and isotopic concentrations of uranium in water.•Currently available remediation ...technologies for the removal of uranium from water.
Uranium concentration/contamination in groundwater is currently a subject of concern all over the world due to related severe health problems to humans, as groundwater is the main drinking water source in rural and urban India and also in several parts of the world. Uranium concentration in groundwater in shallow aquifers in various states such as Punjab, Rajasthan, Karnataka Telangana, and Madhya Pradesh of India varies from 0 to 1443 ng/ml exceeding the permissible levels by WHO for drinking water (30 ng/ml), at several places. Very high concentrations ranging up to 1400 ng/ml were reported in some areas in other countries such as Canada, the USA, Mongolia, Burundi, Zambia, Nigeria, South Korea, Pakistan, Jordon, Afghanistan, China, and Myanmar. Various natural aspects which influence the uranium concentration in groundwater such as bedrock geology, water chemistry, and redox conditions, and anthropogenic sources such as mining activities (uranium, coal, and phosphate rock), nuclear activities, agricultural practices of using phosphate fertilizers, and prevalence of excessive nitrate in some areas, are described with examples. Some of the important analytical techniques for the precise and accurate determination of elemental and isotopic concentrations of uranium in water samples, such as LED fluorimetry, Raman spectroscopy, inductively coupled plasma mass spectrometry (ICP-MS), high-resolution ICP-MS (HR-ICP-MS), and multi-collector ICP-MS (MC-ICP-MS), are described. A number of advancements have taken place in remediation technologies for the removal of uranium in drinking water using different physical, chemical, and biological methods including rainwater harvesting. Various mitigation strategies for the effective removal of uranium from water during treatment, such as bioremediation using biochars from different sources, nanoparticle technology, and adsorption by magnesium (Mg)-iron (Fe)-based hydrotalcite-like compounds (MF-HT), are described in detail.
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A new fluorescein-based chemodosimeter (II) for Hg2+ ion was designed and synthesized, and it displayed excellent selective and sensitive toward Hg2+ ion over other commonly metal ions in aqueous ...media. II was a colorless, non-fluorescent compound. Upon addition of Hg2+ to the solution of II, the thiosemicarbazide moiety of II would undergo an irreversible desulfurization reaction to form its corresponding oxadiazole (IV), a colorful and fluorescent product. During this process, the spirocyclic ring of II was opened, causing instantaneous development of visible color and strong fluorescence emission in the range of 500-600nm. Based on the above mechanism, a fluorogenic Hg2+-selective chemodosimeter was developed. The fluorescence increase is linearly with Hg2+ concentration up to 1.0 is a subset of molL-1 with a detection limit of 8.5X10-10molL-1 (3 sigma ). Compared with the rhodamine-type chemodosimeter, II is more stable in aqueous media and exhibits higher sensitivity toward Hg2+. The findings suggest that II will serve as a practical chemodosimeter for rapid detection of Hg2+ concentrations in realistic media.
Lipopolysaccharide (LPS) synthesis in Gram‐negative bacteria is completed at the outer leaflet of the inner membrane (IM). Following synthesis, seven LPS transport (Lpt) proteins facilitate the ...movement of LPS to the outer membrane (OM), an essential process that if disrupted at any stage has lethal effects on bacterial viability. LptB2FG, the IM component of the Lpt bridge system, is a type VI ABC transporter that provides the driving force for LPS extraction from the IM and subsequent transport across a stable protein bridge to the outer leaflet of the OM. LptC is a periplasmic protein anchored to the IM by a single transmembrane (TM) helix intercalating within the lateral gate formed by LptF TM5 and LptG TM1. LptC facilitates the hand‐off of LPS from LptB2FG to the periplasmic protein LptA and has been shown to regulate the ATPase activity of LptB2FG. Here, using an engineered chromosomal knockout system in Escherichia coli to assess the effects of LptC mutations in vivo, we identified six partial loss of function LptC mutations in the first unbiased alanine screen of this essential protein. To investigate the functional effects of these mutations, nanoDSF (differential scanning fluorimetry) and site‐directed spin labeling (SDSL) electron paramagnetic resonance (EPR) spectroscopy in combination with an in vitro ATPase assay show that specific residues in the TM helix of LptC destabilize the LptB2FGC complex and regulate the ATPase activity of LptB.
The characterization of protein stability is essential for understanding the functions of proteins. Hydroxysteroid dehydrogenase is involved in the biosynthesis of steroid hormones and the ...detoxification of xenobiotic carbonyl compounds. However, the stability of hydroxysteroid dehydrogenases has not yet been characterized in detail. Here, we determined the changes in Gibbs free energy, enthalpy, entropy, and heat capacity of unfolding for 3α‐hydroxysteroid dehydrogenase/carbonyl reductase (3α‐HSD/CR) by varying the pH and urea concentration through differential scanning fluorimetry and presented pH‐dependent protein stability as a function of temperature. 3α‐HSD/CR shows the maximum stability of 30.79 kJ mol−1 at 26.4°C, pH 7.6 and decreases to 7.74 kJ mol−1 at 25.7°C, pH 4.5. The change of heat capacity of 30.25 ± 1.38 kJ mol−1 K−1 is obtained from the enthalpy of denaturation as a function of melting temperature at varied pH. Two proton uptakes are linked to protein unfolding from residues with differential pKa of 4.0 and 6.5 in the native and denatured states, respectively. The large positive heat capacity change indicated that hydrophobic interactions played an important role in the folding of 3α‐HSD/CR. These studies reveal the mechanism of protein unfolding in HSD and provide a convenient method to extract thermodynamic parameters for characterizing protein stability using differential scanning fluorimetry.
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The structural maintenance of therapeutic proteins during formulation and/or storage is a critical aspect, particularly for multi-domain and/or multimeric proteins which usually ...exhibit intrinsic structural dynamics leading to aggregation with concomitant loss-of-function. Protein freeze-drying is a widely used technique to preserve protein structure and function during storage. To minimize chemical/physical stresses occurring during this process, protein stabilizers are usually included, their effect being strongly dependent on the target protein. Therefore, they should be screened for on a time-consuming case-by-case basis. Herein, differential scanning fluorimetry (DSF) and isothermal denaturation fluorimetry (ITDF) were employed to screen, among different classes of freeze-drying additives, for the most effective stabilizer of the model protein human phenylalanine hydroxylase (hPAH). Correlation studies among retrieved DSF and ITDF parameters with recovered enzyme amount and activity indicated ITDF as the most appropriate screening method. Biochemical and biophysical characterization of hPAH freeze-dried with ITDF-selected stabilizers and a long-term storage study (12 months, 5 ± 3 °C) showed that the selected compounds prevented protein aggregation and preserved hPAH structural and functional properties throughout time storage. Our results provide a solid basis towards the choice of ITDF as a high-throughput screening step for the identification of protein freeze-drying protectors.
All biological processes rely on the formation of protein–ligand, protein–peptide and protein–protein complexes. Studying the affinity, kinetics and thermodynamics of binding between these pairs is ...critical for understanding basic cellular mechanisms. Many different technologies have been designed for probing interactions between biomolecules, each based on measuring different signals (fluorescence, heat, thermophoresis, scattering and interference, among others). Evaluation of the data from binding experiments and their fitting is an essential step towards the quantification of binding affinities. Here, user‐friendly online tools to analyze biophysical data from steady‐state fluorescence spectroscopy, microscale thermophoresis and differential scanning fluorimetry experiments are presented. The modules of the data‐analysis platform (https://spc.embl‐hamburg.de/) contain classical thermodynamic models and clear user guidelines for the determination of equilibrium dissociation constants (Kd) and thermal unfolding parameters such as melting temperatures (Tm).
eSPC is an online tool to analyze biophysical data from fluorescence, microscale thermophoresis and differential scanning fluorimetry experiments. The modules from the data‐analysis platform contain classical thermodynamic models and clear user guidelines for the determination of dissociation constants (Kd) and thermal unfolding parameters such as melting temperatures (Tm).
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