Nuclear Magnetic Resonance Methods Research Articles

Quantitative NMR using water as internal calibrant.

A new quantitative nuclear magnetic resonance (qNMR) method, called qNMRw, using water as the internal calibrant has been developed. Its principles, procedures, calculations, and test results are presented here. It is shown to avoid the difficulties created by moisture present in other reference materials. High precision and accuracy can be achieved with qNMRw. The method can be used for analyzing technical materials, herbicide formulation products, and other types of chemical samples. It can also be used to measure the purity and concentration of materials to be used as quantitation calibrants.

Evaluation of nuclear magnetic resonance spectroscopy for characterisation and quantitation of water-soluble polymers in river water

Water-soluble polymers (WSPs) are commonly used in industrial, commercial, agricultural and pharmaceutical products and their molecular weights and concentrations vary considerably. Methods commonly used in the analysis of WSPs are often for pure products or formulations with only a few other high MW constituents. These methods, like size exclusion chromatography (SEC) or Gel Permeation Chromatography coupled with Mass Spectrometry (MS) can be frustrated by the impact of the necessary separation steps prior to identification and the limitations of MS when identifying and quantifying polymers. To that end, the employment of a Nuclear Magnetic Resonance (NMR) method to identify, characterize and quantify WSPs in the real-world is reported for the first time. Samples were taken from fourteen UK inland river sites, concentrated via air-drying, freeze-drying or vacuum-drying and analyzed using 1D 1H NMR and 2D 1H Diffusion Ordered Spectroscopy (DOSY) NMR analysis. Seven of the river sites showed the presence of polyethylene glycol (PEG) with a range of molecular weights, evidencing the application of these techniques in analysis of WSPs. Soil percolation models evidenced the proof of principle that these techniques can also be used for the detection of polyacrylamide (PAM) and polyacrylic acid (PAA). This work should better enable the evaluation of the biological impact of WSPs on aquatic organisms in future studies.

Comparison of a rapid enzymatic assay with column chromatography and nuclear magnetic resonance methods for free asparagine detection and quantification in wheat

A rapid, reliable, and user-friendly method that does not require complex sample preparation and sophisticated instrumentation will fulfill an emerging demand for free asparagine (ASN) analysis to ascertain acrylamide-related food safety issues in baked cereal products. This study validated a new method for wheat free asparagine analysis by comparing a rapid enzymatic test with two established analytical methods: UPLC and 1H-NMR using asparagine standards and whole-wheat flours with a range of free asparagine concentrations. For method validation, various quality parameters were assessed, including accuracy, precision, and sensitivity. Experimental and time costs associated with the analyses were estimated. The rapid test method had consistent accuracy, excellent precision of 1.0 or less (CV%), good sensitivity (0.0389 AU L mg−1) of free asparagine signal detection, and the least cost for experimental materials and instrument usage. A strong correlation (r = 0.997) between the results generated by the rapid enzymatic test and the UPLC method for free ASN quantification in whole-wheat flour was obtained. Overall, the rapid test method is recommended for ASN analysis in simple laboratory environments or in industrial applications.

Quantifying Liquid-Solid Mass Transfer in a Trickle Bed Using {T}_{2}-{T}_{2} Relaxation Exchange NMR

Measurement of the liquid-solid mass transfer coefficient within a trickle bed (i.e. gas-liquid flow within a packed bed) of porous silica pellets is achieved through the use of {T}_{2}-{T}_{2} relaxation exchange nuclear magnetic resonance (NMR). Compared to many conventional measurement techniques, the NMR method enables measurement of mass transport using pellets of real commercial interest. Mass transfer coefficients measured using the NMR technique over a range of liquid Reynolds number, 0.2 le R{e}_{mathrm{L}}le 1.4, are compared to a number of literature correlations, with values measured using the NMR method falling within the range predicted by the correlations. The results demonstrate the importance of considering both the flow conditions and the type of pellets used to develop mass transport correlations in trickle beds. This novel NMR application may be utilized in the future to screen catalyst pellets in trickle beds for optimal mass transport properties.

Green synthesis of UV absorber (E)-2-(((4-(benzyloxy)phenyl)imino)methyl)phenol by microwave method

In this work, a UV absorber compound (E)-2-(((4-(benzyloxy)phenyl)imino)methyl)phenol was synthesized by microwave method using a two steps protocol. In the first step, α-(4-hydroxyphenylimino)-o-cresol (1) was synthesized from 4-aminophenol and salicylaldehyde by circulating under microwave heating at 60 oC for 8 minutes, the reaction yield reached 82.91 %. In the second step (E)-2-(((4-(benzyloxy)phenyl)imino)methyl)phenol (2) was synthesized from precursor (1) and benzyl bromide by circulating under microwave heating at 80 oC for 3 minutes, the reaction yield reached 25.08 %. The overall yield for the synthesis of UV absorber 2 was 20.79 %. The molecular structures of precursor (1) and product (2) were analysed by high performance liquid chromatography - mass spectrometry (LC/MS) and nuclear magnetic resonance (NMR) methods. The UV absorbance of precursor (1) and product (2) was investigated by UV absorption spectrometry. The obtained results show that product (2) has a higher UV absorption intensity than precursor (1), the UV absorption region extends from 240 nm to 390 nm, the maximum absorption peak is at the wavelength (λmax) of 350 nm. The UV absorber (E)-2-(((4-(benzyloxy)phenyl)imino)methyl)phenol with cyclic structure, low molecular weight, and high UV absorbance has potential application as an UV absorber for greenhouse covering plastic, paint and ink systems.

Fluoride permeation mechanism of the Fluc channel in liposomes revealed by solid-state NMR.

Solid-state nuclear magnetic resonance (ssNMR) methods can probe the motions of membrane proteins in liposomes at the atomic level and propel the understanding of biomolecular processes for which static structures cannot provide a satisfactory description. In this work, we report our study on the fluoride channel Fluc-Ec1 in phospholipid bilayers based on ssNMR and molecular dynamics simulations. Previously unidentified fluoride binding sites in the aqueous vestibules were experimentally verified by 19F-detected ssNMR. One of the two fluoride binding sites in the polar track was identified as a water molecule by 1H-detected ssNMR. Meanwhile, a dynamic hotspot at loop 1 was observed by comparing the spectra of wild-type Fluc-Ec1 in variant buffer conditions or with its mutants. Therefore, we propose that fluoride conduction in the Fluc channel occurs via a "water-mediated knock-on" permeation mechanism and that loop 1 is a key molecular determinant for channel gating.

Characterization of the lysergic acid diethylamide analog, 1-(thiophene-2-carbonyl)-N,N-diethyllysergamide (1T-LSD) from a blotter product.

Recently, lysergic acid diethylamide (LSD) analogs have appeared worldwide as designer drugs. In this study, we identified a distributed LSD analog from a paper-sheet product. Gas chromatography-mass spectrometry (GC-MS), liquid chromatography-photodiode array-mass spectrometry (LC-PDA-MS), and liquid chromatography with hybrid quadrupole time-of-flight mass spectrometry (LC-QTOF-MS) were used to analyze the sheet product. The sheet product claimed to contain 1-(1,2-dimethylcyclobutanoyl)-N,N-diethyllysergamide (1D-LSD). However, an unknown compound was detected in the product together with tryptamine and L-tryptophan methyl ester. This compound was isolated from the sheets and identified as 1-(thiophene-2-carbonyl)-N,N-diethyl-6-methyl-9,10-didehydroergoline-8β-carboxamide (1-thiophenoyl LSD; 1-(2-thienoyl)-LSD, 1T-LSD), using 1H, 13C nuclear magnetic resonance (NMR) spectroscopy and various two-dimensional NMR techniques. 1T-LSD was shown to have the thiophene-2-carbonyl group at the N1 position instead of the 1,2-dimethylcyclobutane-carbonyl group as claimed. The amount of 1T-LSD (free base) in three individual unit from one sheet was determined to be 87-100 μg per unit using a proton-specific quantitative NMR (1H-qNMR) method. Deacylation of 1T-LSD to LSD was also observed to occur in methanol-d4 during NMR analysis. The UV spectrum of 1T-LSD differed from that of other LSD analogs, and the fluorescence sensitivity was much lower. Because of concerns about the future distribution of products containing new LSD analogs, continued monitoring of newly detected compounds in sheet products is encouraged.

Specific Proton-Donor Properties of Glycine Betaine. Metric Parameters and Enthalpy of Noncovalent Interactions in its Dimer, Water Complexes and Crystalline Hydrate.

Trimethylglycine (glycine betaine, GB) is an important organic osmolyte that accumulates in various plant species in response to environmental stresses and has significant potential as a bioactive agent with low environmental impact. It is assumed that the hydration of GB is playing an important role in the protective mechanism. The hydration and aggregation properties of GB have not yet been studied in detail at the atomistic level. In this work, noncovalent interactions in the GB dimer and its complexes with water and crystalline monohydrate are studied. Depending on the object, periodic and non-periodic DFT calculations are used. Particular attention is paid to the metric parameters and enthalpies of intermolecular hydrogen bonds. The identification of noncovalent interactions is carried out by means of the Bader analysis of periodic or non-periodic electron density. The enthalpy of hydrogen bonds is estimated using the Rosenberg formula (PCCP 2 (2000) 2699). The specific proton donor properties of glycine betaine are due to its ability to form intermolecular C-H∙∙∙O bonds with the oxygen atom of a water molecule or the carboxylate group of a neighboring GB. The enthalpy of these bonds can be significantly greater than 10 kJ/mol. The water molecule that forms a hydrogen bond with the carboxylate group of GB also interacts with its CH groups through lone pairs of electrons. The C-H∙∙∙O bonds contribute up to 40% of the total entropy of the GB-water interaction, which is about 45 kJ/mol. The possibility of identifying C-H∙∙∙O bonds by the proton nuclear magnetic resonance method is discussed.

Calculating 13 C NMR chemical shifts of large molecules using the eXtended ONIOM method at high accuracy with a low cost.

Fragmentation-based methods for nuclear magnetic resonance (NMR) chemical shift calculations have become more and more popular in first-principles calculations of large molecules. However, there are many options for a fragmentation-based method to select, such as theoretical methods, fragmentation schemes, the number of levels of theory, etc. It is important to study the optimal combination of the options to achieve a good balance between accuracy and efficiency. Here we investigate different combinations of options used by a fragmentation-based method, the eXtended ONIOM (XO) method, for 13 C chemical shift calculations on a set of organic and biological molecules. We found that: (1) introducing Hartree-Fock exchange into density functional theory (DFT) could reduce the calculation error due to fragmentation in contrast to pure DFT functionals, while a hybrid functional, xOPBE, is generally recommended; (2) fragmentation schemes generated from the molecular tailoring approach (MTA) with small level parameter n, for example, n = 2 and the degree-based fragmentation method (DBFM) with n = 1, are sufficient to achieve satisfactory accuracy; (3) the two-level XO (XO2) NMR calculation is superior to the calculation with only one level of theory, as the second level (i.e., low level) of theory provides a way to well describe the long-range effect. These findings are beneficial to practical applications of fragmentation-based methods for NMR chemical shift calculations of large molecules.

Microscopic Pore Structure Characteristics and Fluid Mobility in Tight Reservoirs: A Case Study of the Chang 7 Member in the Western Xin’anbian Area of the Ordos Basin, China

This research addresses the limited understanding of movable fluid occurrence characteristics in the Chang 7 reservoir by employing mercury injection capillary pressure, constant-rate mercury injections, and nuclear magnetic resonance methods. This study investigates the microscopic pore structure characteristics and movable fluids in the tight reservoir of the western Xin’an region, located in the Ordos Basin. The finding reveals that as permeability decreases, the distribution of the throat radius becomes more concentrated in the low-value area, resulting in a narrow distribution range with high curve peaks. Conversely, with an increasing permeability, the distribution range expands towards the high-value area while the curve peak decreases. This research underscores the significance of the throat radius, especially the main flow throat radius, in constraining the permeability of rock samples. Furthermore, this study highlights a stronger correlation between permeability and movable fluid saturation than porosity. This finding emphasizes the importance of considering movable fluid saturation when assessing reservoir characteristics. Notably, the throat radius plays a crucial role in influencing the occurrence characteristics of movable fluids, with a smaller throat radii posing hindrances to fluid flow in the reservoir. Additionally, the presence of clay minerals in the reservoir leads to pore segmentation and increased fluid flow resistance, ultimately reducing the saturation of movable fluids. I must be understood that these factors are essential for developing and producing reservoirs with similar characteristics. In conclusion, the insights gained from this study hold considerable theoretical value and provide essential references for developing and producing reservoirs with tight characteristics, particularly in the western Xin’an region of the Ordos Basin.

Bioethanol production from alkali-pretreated rice straw: effects on fermentation yield, structural characterization, and ethanol analysis.

Current ethanol production technology has a dire need for efficient conversion of lignocellulosic biomass to fermentable sugars. The conversion requires pretreatment of the biomass, one of the most expensive steps, and thus it is quite necessary to identify the most cost-effective and high-efficiency conversion method. In this study, rice straw (RS) biomass was pretreated using 4% NaOH alkali, soaked for 4 h, and autoclaved for 30min. The structural and morphological changes were examined using Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), and scanning electron microscopy (SEM) analysis in both native and alkali-treated RS. The FTIR analysis revealed that native RS contains a considerable amount of lignin that was removed after the pretreatment process. The XRD pattern of the RS revealed an increasing crystallite size of the pretreated lignocellulosic biomass. The study of SEM clearly showed the distorted structure and surface porosity after the pretreatment process. Enzymatic hydrolysis efficiency was checked by comparing the commercial enzymes and microbial hydrolysis extracted from a fungal isolate. The best-reducing sugar yield obtained was 0.62g/L, achieved at optimized conditions from the commercial enzymes. Fermentation efficiency was checked using the yeast isolate Saccharomyces cerevisiae in both the native and pretreated substrate, and the highest ethanol concentration (21.45%) was achieved using 20% w/v biomass loading, enzyme loading (2:1:1), and fermentation for a week at 30°C and pH 4.5. This concentration was higher than that of the untreated RS (3.67%). The ethanol thus produced was further checked for analysis by the 1H and 13C nuclear magnetic resonance (NMR) methods.

Nuclear Magnetic Resonance Study on Relationship between Phase Behavior and Rotational and Translational Dynamics of an Ionic Liquid with a Plastic Crystal Phase: N-Butyl-N-methylpiperidinium Hexafluorophosphate

Abstract N-butyl-N-methylpiperidinium hexafluorophosphate, [C1C4pip]PF6, is an ionic liquid with a plastic crystal (PC) phase. To investigate the dynamics of the phase transition between crystalline, plastic crystalline, and liquid phases of [C1C4pip]PF6, we measured the temperature dependences of longitudinal and transverse relaxation times (T1 and T2, respectively) for 1H and 19F using low-frequency pulse nuclear magnetic resonance (NMR) methods. T1 and T2 changes are sensitive to the rotational and translational dynamics of ions, respectively. Since H and F atoms are only present in the cation and anion, respectively, the dynamic behaviors of each ionic molecule can be investigated separately. In the phase transition between the PC and liquid phase, the temperature-dependent curves of 1H-T1 were smoothly connected, indicating the same rotational motion for the cation occurred in both phases. The curves of 19F-T1 yielded the same conclusion for the rotational motion of the anion in both phases. The temperature-dependent curves of 1H-T2 and 19F-T2 jumped abruptly from phase to phase, indicating different modes of translational motion in each phase. We observed the appearance of a translationally mobile component in both the PC phase and the crystalline phase. This was concluded to be surface or interfacial melting.

Xanthohumol hinders invasion and cell cycle progression in cancer cells through targeting MMP2, MMP9, FAK and P53 genes in three-dimensional breast and lung cancer cells culture

BackgroundDespite recent advances in the treatment of lung and breast cancer, the mortality with these two types of cancer is high. Xanthohumol (XN) is known as a bioactive compound that shows an anticancer effect on cancer cells. Here, we intended to investigate the anticancer effects of XN on the breast and lung cancer cell lines, using the three-dimensional (3D) cell culture.MethodsXN was isolated from Humulus lupulus using Preparative-Thin Layer Chromatography (P-TLC) method and its authenticity was documented through Fourier Transform Infrared spectroscopy (FT-IR) and Hydrogen Nuclear Magnetic Resonance (H-NMR) methods. The spheroids of the breast (MCF-7) and lung (A549) cancer cell lines were prepared by the Hanging Drop (HD) method. Subsequently, the IC50s of XN were determined using the MTT assay in 2D and 3D cultures. Apoptosis was evaluated by Annexin V/PI flow cytometry and NFκB1/2, BAX, BCL2, and SURVIVIN expressions. Cell cycle progression was determined by P21, and P53 expressions as well as PI flow cytometry assays. Multidrug resistance was investigated through examining the expression of MDR1 and ABCG2. The invasion was examined by MMP2, MMP9, and FAK expression and F-actin labeling with Phalloidin-iFluor.ResultsWhile the IC50s for the XN treatment were 1.9 µM and 4.74 µM in 2D cultures, these values were 12.37 µM and 31.17 µM in 3D cultures of MCF-7 and A549 cells, respectively. XN induced apoptosis in MCF-7 and A549 cell lines. Furthermore, XN treatment reduced cell cycle progression, multidrug resistance, and invasion at the molecular and/or cellular levels.ConclusionsAccording to our results of XN treatment in 3D conditions, this bioactive compound can be introduced as an adjuvant anti-cancer agent for breast and lung cancer.

State and role of water confined in cement and composites modified with metakaolin and additives

Cement as a porous material contains pores and cracks on the whole nanoscale forming an interconnected pore structure. Its interaction with water during the rehydration of cement plays a key role in every application, such as in nuclear waste treatment, where cement binders are commonly used for conditioning liquid radioactive waste. In the prediction of the long-term disposal the knowledge of water mobility and distribution in the formed porous cement matrix is essential.In this study we systematically investigate the effects of metakaolin and several components of the liquid phase (complexing agents, model ions, boric acid, etc.) on the hydrated solid structure by various nuclear magnetic resonance (NMR) methods. The novelty lies in the complex use of such non-conventional techniques that separately provide only partial information of the cement structure; however, the gained results complete each other to offer a new approach in the structural study under several conditions. Water mobility is deduced from NMR T2 relaxation distributions and D2O-H2O exchange diffusion measurements, and the conclusion on structural properties is supported by the macropore structure revealed from NMR cryoporometry and SEM (scanning electron microscopy) images, as well as by following the formation of pores and water domains in the curing process with NMR relaxometry. The results confirm that metakaolin was built into the CSH (calcium silicate hydrate) gel region without altering its mesoporous structure, however, it significantly decreased the capillary pore size and the diffusion coefficient of water. Complexing agent concentration between 2 and 20 g/l and the presence of model ions did not cause significant structural changes, while the rehydration decreased the size of capillary pores due to swelling.

Impact liquefaction mechanism of sandy silt with a change in impact energy

The impact liquefaction of sediments in the path of a landslide would reduce the friction of the sliding surface, leading to an increase in the speed and distance of the landslide. In this study, a self-designed impact-liquefaction test was used to explore the pressure development of soil under the effect of different impact energies. The microscopic mechanisms were unravelled by using nuclear magnetic resonance (NMR) and scanning electron microscopy (SEM) methods. The results show that soil at the middle depth shows a higher porewater pressure than at the top and bottom of the layer. Furthermore, the variation in the porewater pressure in these parts is relatively stable, and the peak pressure linearly increases with impact energy. The collapse of large pores in sandy silt is the primary reason for the generation of high porewater pressures in sandy silt. But when the soils are high-energy impacted, the mesopores and small pores are compressed and blocked, which also stimulates the porewater pressure. The results also demonstrate weak drainage behaviour of sandy silt under conditions of rapid impact. If the impact energy is sufficient, the porewater pressure in sandy silt suppresses dissipation, causing quick liquefaction in the interior of the sandy silt. Supplementary material: Language embellishment proof and SEM image binarization method are available at https://doi.org/10.6084/m9.figshare.c.6655482

Self-diffusion of aromatic compounds in the carbon tetrachloride – acetone binary system. NMR experiment and MD simulation

In this work, we studied the influence of the structure of molecules of aromatic compounds (methylparaben (MP), propylparaben (PP), syringic acid (SA), gallic acid (GA), protocatechualdehide (PAL), p-coumaric acid (p-CA), and caffeic acid (CA)) on their molecular association and self-diffusion in the binary carbon tetrachloride – acetone solvent. The self-diffusion coefficients of PAL, SA, and GA in the binary carbon tetrachloride – acetone‑d6 solvent were measured by the pulsed gradient spin-echo nuclear magnetic resonance (PGSE NMR) method at temperatures of 288, 298, and 308 K. The data obtained at 298 K were also compared with the data on the self-diffusion coefficients of MP, PP, CFA, and p-CA in the binary solvent available in the literature. According to the presented data, the self-diffusion coefficients of all the substances under consideration become greater with an increase in the acetone‑d6 concentration and decrease in the MP > PAL > PP > p-CA > SA > CA > GA series of compounds. Quantitative assessment of the degree of influence of molecular association on solute self-diffusion was carried out within the framework of a method based on the ratio of the self-diffusion coefficient of a solute to the self-diffusion coefficient of its monomers. The study shows that the degree of influence of molecular association of the solute on its self-diffusion is determined by the number and type of active functional groups in the structure of the solute molecules (MP < PP < PAL < SA < p-CA < CA < GA), but does not depend on the composition of the binary solvent. According to the results of molecular dynamics (MD) simulation, this effect correlates with the increase in the degree of self-association of solutes and decrease in the degree of solute – acetone heteroassociation observed when the carbon tetrachloride concentration becomes higher. This effect is also responsible for the similar concentration dependencies of the self-diffusion coefficients of active and inert compounds.

Experimental Study on the Water Content Distribution of Profile Samples and the Improvement of Sampling Detection Methods

To provide reliable input information for the load design and extraction of lunar soil water ice samples, it is necessary to study the water content distribution and water migration of simulated lunar soil water ice samples. On this basis, the temperature field model and the hydrothermal coupling relationship are proposed. The temperature field model was constructed by combining energy conservation and Fourier’s heat transfer law. The coupling relationship was established, and the hydrothermal coupling model was obtained by testing the unfrozen water content using the nuclear magnetic resonance method. Finite element software was used to solve the model numerically, and the water migration rule of the soil water ice samples at different ambient temperatures were analyzed. Thin-wall drilling tests were carried out on the simulated lunar soil water ice samples to obtain water content data for different locations, and the simulation results were verified. Due to the migration effect of the cold end of the water, the closer we tested to the edge of the sample, the higher the water content was. The higher the ambient temperature was, the more pronounced the water migration phenomenon of the whole sample was. These research results provide a basis for sampling scheme design.

Chemical Characteristics and Thermal Oxidative Stability of Novel Cold-Pressed Oil Blends: GC, LF NMR, and DSC Studies.

Plant oils contain a high content of unsaturated fatty acids. Studies of food products have revealed a considerable disproportion in the ratio of ω6 to ω3. This article presents information on the healthful qualities of eight new oil blends that contain a beneficial proportion of ω6 to ω3 fatty acids (5:1), as well as their degradation during heating at 170 and 200 °C. The fatty acid profile was analyzed by gas chromatography (GC), content of polar compounds and polymers of triacylglycerols by liquid chromatography (LC), water content was measured by the Karl Fischer method, and oxidative stability was measured by differential scanning calorimetry (DSC) and low-field nuclear magnetic resonance (LF NMR) methods. The results showed that during heating, the polar fraction content increased in samples heated at both analyzed temperatures compared to unheated oils. This was mainly due to the polymerization of triacylglycerols forming dimers. In some samples that were heated, particularly those heated to 200 °C, trimers were detected, however, even with the changes that were observed, the polar fraction content of the blends did not go beyond the limit. Despite the high content of unsaturated fatty acids, the analyzed blends of oils are characterized by high oxidative stability, confirmed by thermoanalytical and nuclear magnetic resonance methods. The high nutritional value as well as the oxidative stability of the developed oil blends allow them to be used in the production of food, in particular products that ensure an adequate supply of ω3 fatty acids.

Potential of nuclear magnetic resonance for the determination of organochlorine in edible oils

Chloride contaminants are the culprit for the formation of harmful compounds such as the 3-monochloropropane-1,2-diol esters (3-MCPDE) and 2-monochlororpropane-1,3-diol esters (2-MCPDE) during edible oil processing. Understanding the source of these chloride’s donor is important for steps to be taken to mitigate the formation of undesirable chloropropanols in refined edible oils. Nuclear magnetic resonance (NMR) was used to differentiate between the inorganic chloride and organochlorine present in crude palm oil, palm superolein, refined olive oil and refined sunflower oil. A quantitative NMR (qNMR) method was developed to analyse the amount of organochlorine present in the aforementioned edible oils. Excellent regression coefficient (R ≥ 0.9995) and LOQ (0.45 ppm) showed that the method is linear and feasible. Recoveries of the method spanned between 90 % and 94 % for the four types of oils analysed. This showed the potential of qNMR for the determination of organochlorine in edible oils.

Characterization of a transitionally occupied state and thermal unfolding of domain 1.1 of factor of RNA polymerase from Bacillus subtilis.

factors are essential parts of bacterial RNA polymerase (RNAP) as they allow to recognize promotor sequences and initiate transcription. Domain 1.1 of vegetative factors occupies the primary channel of RNAP and also prevents binding of the factor to promoter DNA alone. Here, we show that domain 1.1 of Bacillus subtilis exists in more structurally distinct variants in dynamic equilibrium. The major conformation at room temperature is represented by a previously reported well-folded structure solved by nuclear magnetic resonance (NMR), but 4% of the protein molecules are present in a less thermodynamically favorable state. We show that this population increases with temperature and we predict its significant elevation at higher but still biologically relevant temperatures. We characterized the minor state of the domain 1.1 using specialized methods of NMR. We found that, in contrast to the major state, the detected minor state is partially unfolded. Its propensity to form secondary structure elements is especially decreased for the first and third helices, while the second helix and strand close to the C-terminus are more stable. We also analyzed thermal unfolding of the domain 1.1 and performed functional experiments with full length and its shortened version lacking domain 1.1 ( ). The results revealed that while full length increases transcription activity of RNAP with increasing temperature, transcription with remains constant. In summary, this study reveals conformational dynamics of domain 1.1 and provides a basis for studies of its interaction with RNAP and effects on transcription regulation.