Thermodiffusion of aqueous solutions of various potassium salts.

Thermophoresis or thermodiffusion has become an important tool to monitor protein-ligand binding as it is very sensitive to the nature of solute-water interactions. However, the microscopic mechanisms underlying thermodiffusion in protein systems are poorly understood at this time. One reason is the difficulty to separate the effects of the protein system of interest from the effects of buffers that are added to stabilize the proteins. Due to the buffers, typical protein solutions form multicomponent mixtures with several kinds of salt. To achieve a more fundamental understanding of thermodiffusion of proteins, it is therefore necessary to investigate solutions of buffer salts. For this work, the thermodiffusion of aqueous potassium salt solutions has been studied systematically. We use thermal diffusion forced Rayleigh scattering experiments in a temperature range from 15 °C to 45 °C to investigate the thermodiffusive properties of aqueous solutions of five potassium salts: potassium chloride, potassium bromide, potassium thiocyanate, potassium acetate, and potassium carbonate in a molality range between 1 mol/kg and 5 mol/kg. We compare the thermophoretic results with those obtained for non-ionic solutes and discuss the thermophoresis of the salts in the context of ion-specific solvation according to the Hofmeister series.

Tofogliflozin Salt Cocrystals with Sodium Acetate and Potassium Acetate.

We investigated the salt cocrystals formed by tofogliflozin with sodium acetate and potassium acetate by determining the crystal structures of the salt cocrystals and characterizing the solid states. The salt cocrystal screening using the slurry method and the liquid-assisted grinding method resulted in the formation of tofogliflozin-sodium acetate 1 : 1 and tofogliflozin-potassium acetate 1 : 1 salt cocrystals. Single-crystal X-ray diffraction revealed that, although each salt cocrystal belongs to a different space group, both of the salt cocrystals have almost similar structural features, including the conformation of tofogliflozin molecules, the coordination to Na+/K+ ions, and hydrogen bonds. The salt cocrystals exhibited extreme hygroscopicity with deliquescence, which is also a property of sodium acetate and potassium acetate. In addition, tofogliflozin-potassium acetate salt cocrystal had two polymorphs, which were enantiotropically related.

Improved conventional and microwave-assisted silylation protocols for simultaneous gas chromatographic determination of tocopherols and sterols: Method development and multi-response optimization.

This paper reports on improved conventional thermal silylation (CTS) and microwave-assisted silylation (MAS) methods for simultaneous determination of tocopherols and sterols by gas chromatography. Reaction parameters in each of the methods developed were systematically optimized using a full factorial design followed by a central composite design. Initially, experimental conditions for CTS were optimized using a block heater. Further, a rapid MAS was developed and optimized. To understand microwave heating mechanisms, MAS was optimized by two distinct modes of microwave heating: temperature-controlled MAS and power-controlled MAS, using dedicated instruments where reaction temperature and microwave power level were controlled and monitored online. Developed methods: were compared with routine overnight derivatization. On a comprehensive level, while both CTS and MAS were found to be efficient derivatization techniques, MAS significantly reduced the reaction time. The optimal derivatization temperature and time for CTS found to be 55°C and 54min, while it was 87°C and 1.2min for temperature-controlled MAS. Further, a microwave power of 300W and a derivatization time 0.5min found to be optimal for power-controlled MAS. The use of an appropriate derivatization solvent, such as pyridine, was found to be critical for the successful determination. Catalysts, like potassium acetate and 4-dimethylaminopyridine, enhanced the efficiency slightly. The developed methods showed excellent analytical performance in terms of linearity, accuracy and precision.

Identification of RNase-resistant RNAs in Saccharomyces cerevisiae extracts: Separation from chromosomal DNA by selective precipitation.

High-quality chromosomal DNA is a requirement for many biochemical and molecular biological techniques. To isolate cellular DNA, standard protocols typically lyse cells and separate nucleic acids from other biological molecules using a combination of chemical and physical methods. After a standard chemical-based protocol to isolate chromosomal DNA from Saccharomyces cerevisiae and then treatment with RNase A to degrade RNA, two RNase-resistant bands persisted when analyzed using gel electrophoresis. Interestingly, such resistant bands did not appear in preparations of Escherichia coli bacterial DNA after RNase treatment. Several enzymatic, chemical, and physical methods were employed in an effort to remove the resistant RNAs, including use of multiple RNases and alcohol precipitation, base hydrolysis, and chromatographic methods. These experiments resulted in the development of a new method for isolation of S. cerevisiae chromosomal DNA. This method utilizes selective precipitation of DNA in the presence of a potassium acetate/isopropanol mixture and produces high yields of chromosomal DNA without detectable contaminating RNAs.

Water-mediated potassium acetate intercalation in kaolinite as revealed by molecular simulation.

Molecular simulations are suitable tools to study the adsorption and intercalation of molecules in clays. In this work, a recently proposed thermodynamically consistent force field for inorganic compounds (INTERFACE, Heinz H, Lin TJ, Mishra RK, Emami FS (2013) Langmuir 29:1754-1765), which enables accurate simulations of inorganic-organic interfaces, was tested for a two-sheet type clay mineral. All-atom NpT molecular dynamics simulations were used to describe the characteristics (basal spacing, loading, molecular orientation) of some intercalate complexes of kaolinite with potassium acetate and the results were compared with the available experimental data. The most probable structural configurations of the kaolinite/potassium acetate intercalate complexes were determined from the simulations. Our examinations confirmed some supposed (single- or double-layered) arrangements of guest molecules. The need of interlayer water in the intercalate complex, which can be produced by the basic synthesis procedure in air atmosphere, was verified.

Functionalization of an sp(3) C-H bond via a redox-neutral domino reaction: diastereoselective synthesis of hexahydropyrrolo[2,1-b]oxazoles.

A diastereoselective synthesis of pyrrolidinooxazolidines was achieved by a metal-free, base-promoted reaction of pyrrolidine and aromatic aldehydes under microwave irradiation. The rare functionalization of an sp(3) C-H bond probably results from an in situ generated azomethine ylide that undergoes cycloaddition with aldehydes.

Structure and substrate-induced conformational changes of the secondary citrate/sodium symporter CitS revealed by electron crystallography.

The secondary Na+/citrate symporter CitS of Klebsiella pneumoniae is the best-characterized member of the 2-hydroxycarboxylate transporter family. The recent projection structure gave insight into its overall structural organization. Here, we present the three-dimensional map of dimeric CitS obtained with electron crystallography. Each monomer has 13 a-helical transmembrane segments; six are organized in a distal helix cluster and seven in the central dimer interface domain. Based on structural analyses and comparison to VcINDY, we propose a molecular model for CitS, assign the helices, and demonstrate the internal structural symmetry. We also present projections of CitS in several conformational states induced by the presence and absence of sodium and citrate as substrates. Citrate binding induces a defined movement of a helices within the distal helical cluster. Based on this, we propose a substrate translocation site and conformational changes that are in agreement with the transport model of ''alternating access''.

CKC: isolation of nucleic acids from a diversity of plants using CTAB and silica columns.

To assay for viruses in plant samples, we required a method for nucleic acid isolation that is rapid, simple, and applicable to the widest possible variety of plants. A protocol for isolation of total nucleic acid (TNA) was developed by combining common CTAB methods with silica spin columns. We report data on TNA purity and RNA quality from over 30 plant genera representing 25 families. Measurements showed that RNA is of high quality, and one-step RT-PCR was successfully performed on all samples. The protocol can be completed in less than 2 h.

The effect of a mild base on curcumin in methanol and ethanol.

Steady-state and time-resolved emission techniques were employed to study the effect of acetate, a mild base, on the luminescence of curcumin in methanol and ethanol. We found that the steady-state emission intensity as well as the average fluorescence decay time are reduced by a factor of 5 when the acetate concentration is raised to about 1.8 M. We attribute this large effect to an excited-state proton transfer (ESPT) from the acidic groups of curcumin to the acetate anion. We analyze the experimental data in terms of an ESPT reaction occurring between a photoacid and a base.

Stable isotope analysis of organic carbon in small (µg C) samples and dissolved organic matter using a GasBench preparation device.

The stable isotopes of organic matter can provide valuable information on carbon cycling dynamics, microbial metabolisms, and past climates. Since bulk measurements may mask dynamic changes to critical portions of the organic pool, researchers are increasingly isolating individual compounds for isotopic analysis. The amount of carbon isolated is frequently small, requiring specialized equipment for its analysis. We present a simple and accurate method to measure the δ(13)C values of µg-amounts of organic compounds and dissolved organic matter in freshwaters using wet oxidation and a GasBench II preparation device. Samples containing 3 µg C can be analyzed with a precision of <0.4‰. For samples containing 1.2 µg C, the precision is <0.8‰. The blank is estimated to be ~0.2 µg C. The accuracy of the method is demonstrated for a wide range of compounds including those that are difficult to oxidize such as humic acid and phthalic acid. The δ(13)C values of DOC from river and riparian ground water determined by this method are comparable with those determined with an elemental analyzer on freeze-dried samples of DOC. The low detection limit and the ease with which it can be combined with isolation techniques such as liquid chromatography make this technique attractive for the off-line analysis of organic compounds, and open new possibilities for the development of methodologies for compound-specific carbon isotope analysis of complex mixtures separated by HPLC.

Sorption of water vapor, hydration, and viscosity of carboxymethylhydroxypropyl guar, diutan, and xanthan gums, and their molecular association with and without salts (NaCl, CaCl2, HCOOK, CH3COONa, (NH4)2SO4 and MgSO4) in aqueous solution.

Gums are routinely used in food industry, pharmacy and oil recovery process. In these uses, the hydrocolloids very often encounter interactions with salts at moderate to high temperature. Since they are normally employed in the form of solution and gel, their viscous or fluidity properties need detailed investigation. In the present work, properties such as water vapor adsorption of finely powdered carboxymethylhydroxypropyl derivatized guar (CMHPG) as well as xanthan (Xn) and diutan (Dn) gums, their hydration in solution, their viscosity behaviors, and salt effects on fluidity have been studied. The concentration domains for the existence of free and associated molecules in the studied solutions have been assessed from the viscosity results. The gums have been found to bind a fair amount of water from the vapor phase with them. In solution, they can interact and arrest a large amount of water in their folded configuration. Intrinsic viscosities of the gums in aqueous medium declined in the presence of salts. The activation energies for their viscous flow were moderate and comparable, and were dependent on their concentrations. From the power law relation and viscosity master curve behavior mostly two critical association states of the macromolecular dispersions were envisaged.

Purification of total DNA extracted from activated sludge.

Purification of the total DNA extracted from activated sludge samples was studied. The effects of extraction buffers and lysis treatments (lysozyme, sodium dodecyl sulfate (SDS), sonication, mechanical mill and thermal shock) on yield and purity of the total DNA extracted from activated sludge were investigated. It was found that SDS and mechanical mill were the most effective ways for cell lysis, and both gave the highest DNA yields, while by SDS and thermal shock, the purest DNA extract could be obtained. The combination of SDS with other lysis treatment, such as sonication and thermal shock, could apparently increase the DNA yields but also result in severe shearing. For the purification of the crude DNA extract, polyvinyl polypyrrolidone was used for the removal of humic contaminants. Cetyltrimethyl ammonium bromide, potassium acetate and phenol/chloroform were used to remove proteins and polysaccharides from crude DNA. Crude DNA was further purified by isopropanol precipitation. Thus, a suitable protocol was proposed for DNA extraction, yielding about 49.9 mg (total DNA)/g volatile suspended solids, and the DNA extracts were successfully used in PCR amplifications for 16S rDNA and 16S rDNA V3 region. The PCR products of 16S rDNA V3 region allowed the DGGE analysis (denatured gradient gel electrophoresis) to be possible.

The kinesin family member BimC contains a second microtubule binding region attached to the N terminus of the motor domain.

The kinesin family member BimC has a highly positively charged domain of approximately 70 amino acids at the N terminus of the motor domain. Motor domain constructs of BimC were prepared with and without this extra domain to determine its influence. The level of microtubules needed for half saturation of the ATPase of BimC motor domain constructs is reduced by approximately 7000-fold at low ionic strength upon addition of this extra N-terminal extension. Although the change in microtubule affinity is less at higher salt, addition of the N-terminal domain still produces a 20-fold increase in affinity for microtubules in 200 mm potassium acetate. A fusion protein of the N-terminal domain and thioredoxin binds tightly to MTs at low salt, consistent with the increased affinity of motor domain constructs (which contain the N-terminal domain) being due to the additional binding of the N-terminal domain to the microtubule. Hydrodynamic analysis indicates that the N-terminal extension is in a highly extended conformation, suggesting that it may be intrinsically disordered. Fusion of the N-terminal extension of BimC onto the motor domain of conventional kinesin produces a similar large increase in microtubule affinity without significant reduction in kcat or velocity in an in vitro motility assay, suggesting that the N-terminal extension can act in a modular manner to increase the microtubule affinity of kinesin motor domains without a decrease in velocity.

A DRIFT spectroscopic study of potassium acetate intercalated mechanochemically activated kaolinite.

Kaolinite has been mechanochemically activated by dry grinding for periods of time up to 10 h. The kaolinite was then intercalated with potassium acetate and the changes in the structure followed by DRIFT spectroscopy. Intercalation of the kaolinite with potassium acetate is difficult and only the layers, which remain hydrogen bonded, are intercalated. The mechanochemical activation of the kaolinite may be followed by the loss of intensity of the hydroxyl-stretching vibrations. The intensity of the 3695 and 3619 cm(-1) bands reach a minimum after 10 h of grinding. The observation of a band at 3602 cm(-1) is indicative of the intercalation of the kaolinite with potassium acetate. The degree of intercalation decreases with mechanochemical treatment. The effect of exposure of the intercalated mechanochemically activated kaolinite to moist air results in de-intercalation. The effect of the mechanochemical treatment is loss of layer stacking, which prevents the intercalation of the kaolinite.

Transport and degradation of propyleneglycol and potassium acetate in the unsaturated zone.

De-icing chemicals used during the winter season are potential pollutants for the groundwater underneath the new main airport of Norway. Several field experiments examining the transport and degradation of propyleneglycol (PG), potassium acetate (KAc) and non-reactive tracers were performed in a lysimeter trench under natural snowmelting conditions. Chemicals were applied underneath the snow cover and the transport in a heterogeneous coarse sandy soil was examined by extracting soil water from 30 or 40 suction cups placed at five depths between 0.4 and 2.4 m depth. Transport and degradation was analysed by spatial moment calculations. The de-icing chemicals showed the same basic displacement as chemically inactive tracers, an initial fast transport during the melting period followed by a period of stagnation throughout the summer season. PG seemed to be displaced to greater depths compared to non-reactive tracer after the first application. However, computer simulations of transport and degradation in a heterogeneous unsaturated soil showed that decreasing degradation constants with depth can generate a downward movement of the centre of mass without any flow occurring in the system. Potassium acetate showed some adsorption, with calculated retardation factors of approximately 1.3 and 1.2. The degradation rate constant for PG was calculated to be 0.015 day-1 in 1994 and increased to 0.047 day-1 in the second application made in 1995. The degradation rate constant for acetate was estimated to be 0.02 day-1. Increased manganese concentrations seem to be a good indicator of degradation of PG and Ac.

Raman spectroscopy of potassium acetate-intercalated kaolinites at liquid nitrogen temperature.

Raman microscopy has been used to study low and high defect kaolinites and their potassium acetate intercalated complexes at 298 and 77 K. Raman spectroscopy shows significant differences in the spectra of the hydroxyl-stretching region of the two types of kaolinites, which is also reflected in the spectroscopy of the hydroxyl-stretching region of the intercalation complexes. Additional bands to the normally observed kaolinite hydroxyl stretching frequencies are observed for the low and high defect kaolinites at 3605 and 3602 cm(-1) at 298 K. Upon cooling to liquid nitrogen temperature, these bands are observed at 3607 and 3604 cm(-1), thus indicating a weakening of the hydrogen bond formed between the inner surface hydroxyls and the acetate ion. Upon cooling to liquid nitrogen temperature, the frequency of the inner hydroxyls shifted to lower frequencies. Collection of Raman spectra at liquid nitrogen temperature did not give better band separation compared to the room temperature spectra as the bands increased in width and shifted closer together.

Relationship between hexamerization and ssDNA binding affinity in the uvsY recombination protein of bacteriophage T4.

In bacteriophage T4, homologous genetic recombination events are catalyzed by a presynaptic filament containing stoichiometric quantities of the T4 uvsX recombinase bound cooperatively to single-stranded DNA (ssDNA). The formation of this filament requires the displacement of cooperatively bound gp32 (the T4 ssDNA-binding protein) from the ssDNA, a thermodynamically unfavorable reaction. This displacement is mediated by the T4 uvsY protein (15.8 kDa, 137 amino acids), which interacts with both uvsX- and gp32-ssDNA complexes and modulates their properties. Previously, we showed that uvsY exists as a hexamer under physiological conditions and that uvsY hexamers bind noncooperatively but with high affinity to ssDNA. We also showed that a fusion protein containing the N-terminal 101 amino acid residues of uvsY lacks interactions with uvsX and gp32 but retains both weak ssDNA-binding activity and a residual ability to stimulate uvsX-catalyzed recombination functions. Here, we present quantitative data on the oligomeric structure and ssDNA-binding properties of a closely related fusion protein designated uvsY. Sedimentation velocity and equilibrium results establish that uvsY, unlike native uvsY, behaves as a monomer in solution (M(app) = 14.2 kDa, = 2.1). Like native uvsY, uvsY binds noncooperatively to an etheno-DNA (epsilonDNA) lattice with a binding site size of 4 nucleotides/monomer; however at physiological ionic strength, the association constant for uvsY-epsilonDNA is decreased 10(4)-fold relative to native uvsY. Nevertheless, the magnitude of the salt effect on the association constant (K) is essentially unchanged between uvsY and uvsY, indicating that disruption of the C-terminus does not disrupt the electrostatic ssDNA-binding determinants found within each protomer of uvsY. Instead, the large difference in ssDNA-binding affinities reflects the loss of hexamerization ability by uvsY, suggesting that a form of intrahexamer synergism or cooperativity between binding sites within the uvsY hexamer leads to its high observed affinity for ssDNA.