UV Difference Spectroscopy Research Articles

Formation of thermo-reversible gels from whey proteins after combined thermal and ethanol pretreatment

Τhis study investigates the ability of whey proteins, which were pre-treated with ethanol (0–70%) and subjected tothermal treatment (70 °C for 60 min), to form thermo-reversible gels after removing the alcohol through freeze-drying. The ethanol-free gels were monitored by means of rheology and confocal laser scanning microscopy. Moreover, the role of ethanol during heating of whey proteins was investigated by UV difference spectroscopy. The results showed a retention of the denatured character (30–35%) of whey proteins after removal of ethanol, regardless of whether heating was applied. Intermolecular disulfide connections increased in whey protein solutions pretreated with 30 and 50% w/w ethanol. The determination of free –SH groups demonstrated that their number was increased by heating in the presence of ethanol and after ethanol's removal. Dynamic rheological measurements showed that ethanol-free gels, previously heated with 50% w/w ethanol, formed thermo-reversible gels upon cooling to 5 °C and exhibited significantly higher storage modulus values (G’∼300 Pa) than the counterparts without pretreatment of the proteins with ethanol (G’ = 0.0002 Pa). Generally, the partial retention of the denaturing effect of ethanol on whey proteins after its removal and their ability to form thermo-reversible gels could lead to new applications in the food and pharmaceutical industries.

Differences in the spectral characteristics of dissolved organic matter binding to Cu(II) in wetland soils with moisture gradients

The environmental behavior of heavy metals in soil is significantly regulated by their binding with dissolved organic matter (DOM), which is affected by soil moisture contents. However, the mechanism of this interaction in soils with varying moisture is still not well understood. Using a combination of ultrafiltration, Cu(II) titration, and multispectral (ultraviolet-visible absorption, 3D fluorescence, Fourier transform infrared) analysis techniques, we studied the differences in the spectral characteristics and Cu(II) binding properties of soil dissolved organic matter (DOM) and its different molecular weight (MW) fractions with moisture gradients. We found that the abundance and spectral characters of soil DOM changed with increasing soil moisture, i.e., the increase in abundance while the decrease in aromaticity and humification index. The components of DOM, shown by Fluorescence region-integration (FRI) analysis, also changed, with an increase in the proportion of protein-like substances and a decrease of humic-like and fulvic-like substances. The overall Cu(II) binding potential of soil DOM diminished with increasing soil moisture, as indicated by the fluorescence parallel factor (PARAFAC) analysis. This is aligns with the changes in DOM composition, as the humic-like and fulvic-like fractions exhibited higher Cu(II) binding potential compared to the protein-like fractions. The low MW fraction of the MW-fractionated samples showed a stronger binding potential for Cu(II) compared to the high MW fraction. Finally, the active binding site of Cu(II) in DOM, as revealed by UV-difference spectroscopy and 2D-FTIR-COS analysis, decreased with increasing soil moisture, with the order of preferentially functional groups shifting from OH, NH, and CO to CN and CO. This study emphasizes the impact of moisture variations on the characteristics of DOM and its interaction with Cu(II), providing insight into the environmental fate of heavy metal contaminants in soil in areas with alternating land and water conditions.

Study of ascorbic acid stabilization conditions in fresh juices

For the first time the possibility of using purified pectins to stabilize ascorbic acid (AA) in fresh juices was considered and proved. The purpose of the study was to analyze the possibility of using purified pectins to stabilize AA in fresh juices with a concentration of 1⋅10-6-1⋅10-4g/ml. The work used AA, oxalic acid (OA), as well as beet (BP), citrus (CP) and apple (AP) pectins. There was no interfering effect of pectins on AA determination by differential UV spectroscopy. The specific absorption rate of AA (727) was determined, according to which the AA content in model solutions with auxiliary substances during storage was estimated. The optimal weight ratio AA:AP 1:16 used for blending fresh juices, in which the AA concentration was previously determined by titration with 2.6-dichlorophenolindophenolate sodium, was established. It was revealed that AP contributes to the pH shift to the optimal pH area for AA 4.5-8.0; increase of juice viscosity by 5-148% and storage life by 3.3-59 times. The relative error of definitions did not exceed ±5.6%.

CHARACTERISTICS OF THE REDOX STATE OF THE LIGNIN-CARBOHYDRATE WOOD MATRIX AT THE INITIAL STAGES OF DEVELOPMENT ON THE EXAMPLE OF SCOTS PINE

The studies of wood formation at the initial stages allowed confirming the determining role of the redox transformations of monolignols in the lignification of plant matrix. The change in the composition of the phenolic fraction (conjugated and non-conjugated phenolic structures) of the phenol-quinone redox system of the cell wall was studied using the derivative and differential UV spectroscopy. It was shown that the accumulation of low-molecular phenolic compounds in the initial period of ontogenesis occurs mainly in the "needles" of plants. It was confirmed that the seedlings being removed to the hardening site adapt to the existing temperature regime. It is characterized by lower content of low-molecular phenolic compounds preventing the development of oxidative processes. The plant is exposed to oxidative stress at low temperatures, when the enzymatic (peroxidase activity) system of plant protection is activated. The change in the coniferyl alcohol content as a marker of the lignification is shown. The correlation of changes in peroxidase activity with the total content of phenolic compounds and coniferyl alcohol was shown. The IR spectroscopy method showed a change in the ratio of carbohydrate and lignin components at the initial stages of the development of seedlings of scots pine.

Novel indoleoxazine derivative cyanide ion probe: Detection applications and cell-imaging studies

A colorimetric fluorescence probe 1-nitro-(8-ethylene-11- (14-diethylaminophenyl)-benzoindo oxazine (ZBO) based on indoleoxazine structure was prepared for detection of cyanide ions (CN−). The introduction of UV difference spectroscopy provides a guarantee for the accuracy of CN− detection. Results indicate that ZBO can achieve the desired recognition of CN− with excellent anti-interference performance. The minimum detection concentration of CN− is 0.1 μM. For practical applications, ZBO test strips were prepared for the convenient and qualitative detection of CN−. The low-concentration solution of ZBO can facilitate the qualitative detection of CN− in peach kernel, loquat seed, and cassava samples. In addition, low cytotoxicity and cell imaging experiments suggested that ZBO can be used for detect CN− in living cells.

Warfarin Sodium Stability in Oral Formulations.

Warfarin sodium is a low-dose pharmaceutical blood thinner that exists in two forms: the clathrate form and the amorphous form. In commercially available warfarin sodium oral suspension, the active pharmaceutical ingredient (API) is added in the amorphous state. This study investigates the apparent instability of the commercially available warfarin liquid oral formulation using Raman and IR spectroscopy, X-ray diffraction, differential scanning calorimetry, UV spectroscopy, and optical microscopy. Warfarin, not its sodium salt, was identified as the undissolved solid existing in the suspension. This was found to be due to the dissociation of sodium salt and the protonation of the warfarin ion in the liquid phase, which triggered the crystallization of the sparingly soluble unsalted form. The coexistence of protonated and unprotonated warfarin ions in the supernatant, as detected by Raman and UV spectroscopy, confirmed this assumption. Study of the dissolution of warfarin sodium amorphous salt and crystalline sodium clathrate in the placebo and pure water verified the results. The effect of pH and temperature on warfarin precipitation was also explored.

Structural changes of serum albumin in response to oxidative stress caused byWalker-256 carcinosarcoma growth.

To assess oxidative stress and structural changes of the serum albumin in rats with transplanted Walker-256carcinosarcoma (W256) strains with varying sensitivity to doxorubicin (Dox). The study was performed on female Wistar rats with transplanted W256. On the 9th day after tumor cell transplantation an analysis of peripheral blood, oxidative stress parameters, and structural changes of serum albumin of experimental animals was performed. On the 9th day after W256transplantation a significant increase in the leukocyte counts was observed in the groups of animals with the Dox-resistant and parental (Dox-sensitive) W256tumors compared with the group of the intact animals: up to 14.24 ± 1.92•103/μl and 9.78 ± 1.03•103/μl, vs 8.92 ± 1.04•103/μl, respectively, due to the increase of granulocyte and monocyte counts. The number of lymphocytes was within the normal range. The level of hemoglobin and the erythrocyte counts were also within normal limits, but hematocrit in both groups of animals with tumors somewhat increased against the background of 1.2-fold elevation of the mean erythrocyte volume. In the group of rats with Dox-resistant W256, there was observed a decrease in the plateletcrit by almost 22% and thrombocyte counts- by 28%. Analysis of oxidative stress indices revealed a significant increase in the level of reactive oxygen species, 2-fold increase of malonic dialdehyde level and the degree of oxidative damage of blood plasma proteins, as well as a decrease in the activity of catalase in hemolysates (by 12-15%) in both groups of tumor-bearing rats. With the use of differential scanning calorimetry, UV and fluorescence spectroscopy we have revealed anomalous conformational changes of albumin caused by tumor development: structural rearrangements in the region of its first drug binding site located in the IIA domain, separation of globular parts of albumin molecule, and partial "opening" in a protein molecular three-domain structure resulting a loss of its thermal resistance. The development of transplanted Walker-256carcinosarcoma, especially its Dox-resistant variant, results in severe metabolic intoxication reflected in alteration of hematological parameters, and indices of oxidative stress, as well as architectonic changes of serum albumin.

Relationships between DBP concentrations and differential UV absorbance in full-scale conditions.

Differential UV spectroscopy, defined as the difference in UV absorbance spectra before and after chlorination, has shown great potential to predict disinfection by-product (DBP) concentrations at laboratory scale. However, so far, no results have been reported on the full scale application of differential UV spectroscopy in drinking water treatment facilities. The objectives of this study are to determine if relationships can be developed between differential UV absorbance and DBP concentrations, for both regulated and unregulated DBPs, in a full-scale facility and to determine if these relationships vary throughout the year with variations in raw water quality and treatment conditions. The results show that linear and power relationships between differential UV absorbance and DBP concentrations can be developed (0.62 ≤ R2 ≤ 0.99), although differences between relationships obtained in lab- and full-scale conditions need further investigation. Finally, the relationships obtained are different from one sampling campaign to another, which raises the question of whether it is possible to determine relationships that are stable enough to be used as adequate feedback on DBP concentrations.

A novel form of RNA double helix based on G·U and C·A+ wobble base pairing.

Wobble base pairs are critical in various physiological functions and have been linked to local structural perturbations in double-helical structures of nucleic acids. We report a 1.38-Å resolution crystal structure of an antiparallel octadecamer RNA double helix in overall A conformation, which includes a unique, central stretch of six consecutive wobble base pairs (W helix) with two G·U and four rare C·A+ wobble pairs. Four adenines within the W helix are N1-protonated and wobble-base-paired with the opposing cytosine through two regular hydrogen bonds. Combined with the two G·U pairs, the C·A+ base pairs facilitate formation of a half turn of W-helical RNA flanked by six regular Watson–Crick base pairs in standard A conformation on either side. RNA melting experiments monitored by differential scanning calorimetry, UV and circular dichroism spectroscopy demonstrate that the RNA octadecamer undergoes a pH-induced structural transition which is consistent with the presence of a duplex with C·A+ base pairs at acidic pH. Our crystal structure provides a first glimpse of an RNA double helix based entirely on wobble base pairs with possible applications in RNA or DNA nanotechnology and pH biosensors.

Study of the Ability of Reducing Saccharides to Chemically Transform Lignin

The efficiency of chemical transformations of lignin obtained from Picea excelsa wood under the action of galactose, galacturonic acid and xylose (which can be obtained by enzymatic hydrolysis of hemicelluloses and pectin containing in plant material) was evaluated. The results were compared with use of traditional reducing agent which was borohydride sodium. Using the method of differential UV-spectroscopy was confirmed the increase of a number of phenolic hydroxyl units by Xyl, Gal and GA. The increase of lignin reactivity was controlled to sulfuric acid and to peroxide hydrogen. Similarly to NaBH4, a nucleophilic addition mechanism for the reaction of the reducing saccharides with lignin was revealed. Reduction by NaBH4, Xyl, GA and Gal increased the lignin reactivity to acid solubilisation and to peroxide oxidation.

Near UV difference spectroscopy application for ferulic acid esterases substrate specificity determination

Ferulic acid esterases (FAEs) are promising enzymes for potential application in agricultural, food, paper, and pharmaceutical industries. Discovery and characterization of novel FAEs is still up to date. Depending on their substrate specificity towards four methyl hydroxycinnamates (methyl esters of ferulic, p-coumaric, caffeic, and sinapic acids), FAEs are classified into four subgroups, named A–D types. In this work we investigate practical applicability of near UV difference spectroscopy for FAE’s substrate specificity determination. Difference spectroscopy is demonstrated to be a convenient and easily performed approach, enabling either single point or continuous determination of four methyl hydroxycinnamates hydrolysis rate at a concentration range 0.02–1.0mM. Difference maxima wavelengths and corresponding extinction coefficients are determined for free and methyl esterified ferulic, p-coumaric, caffeic, and sinapic acids.

Influence of Buffer Composition and Calcium Chloride on GdnHCl Denaturation of Bacillus licheniformis α-Amylase

The influence of buffer composition on the conformational stability of native and calciumdepleted Bacillus licheniformis α-amylase (BLA) was investigated against guanidine hydrochloride (GdnHCl) denaturation using circular dichroism, fluorescence and UV-difference spectroscopy. Differential effect of buffer composition on GdnHCl denaturation of BLA was evident from the magnitude of these spectral signals, which followed the order: sodium phosphate > Tris-HCl > HEPES > MOPS. These effects became more pronounced with calcium-depleted BLA. Sephacryl S-200 gel chromatographic results showed significant BLA aggregation in the presence of 6 M GdnHCl.

Monitoring of free phenol content in lignosulfonates by ClO2 titration and UV difference spectroscopy

Abstract Lignosulfonates (LS) are water-soluble polyphenolic macromolecules derived from lignins and common industrial additives in plasticizers and dispersants. A key feature for a more efficient utilization of modified LS is the monitoring of their free phenol (OHphen) content during various reactions as these phenols govern a large part of their chemical reactivity. Against this background, the UV difference spectroscopy (Δεi method) and ClO2 titration have been revisited. Synthetic LS model compounds with a SO3H group on their benzylic position and H, G, and S aromatic rings were synthesized and used to assess the suitability of both the ClO2 titration and the Δεi method on LS. The methodology combining the two analytical methods was then used to determine the molar extinction coefficient of a given LS allowing the calibration of the Δεi method for a convenient and rapid monitoring of OHphen in the course of LS reactions involving the OHphen groups.

Calorimetric and Spectroscopic Analysis of the Thermal Stability of Short Duplex DNA-Containing Sugar and Base-Modified Nucleotides.

Base- and sugar-modified analogs of DNA and RNA are finding ever expanding use in medicine and biotechnology as tools to better tailor structured oligonucleotides by altering their thermal stability, nuclease resistance, base-pairing specificity, antisense activity, or cellular uptake. Proper deployment of these chemical modifications generally requires knowledge of how each affects base-pairing properties and thermal stabilities. Here, we describe in detail how differential scanning calorimetry and UV spectroscopy may be used to quantify the melting thermodynamics of short dsDNA containing chemically modified nucleosides in one or both strands. Insights are provided into why and how the presence of highly stable base pairs containing modified nucleosides can alter the nature of calorimetry or melting spectroscopy data, and how each experiment must therefore be conducted to ensure high-quality melting thermodynamics data are obtained. Strengths and weaknesses of the two methods when applied to chemically modified duplexes are also addressed.

Determination of the Stability Constants of Gold(I) Thiosulfate Complexes by Differential UV Spectroscopy

The stability constants of gold(I) thiosulfate complexes have been determined by differential UV spectroscopy. The aqueous solutions contained 6 × 10−4 mol·dm−3 Na3Au(S2O3)2 and a variable amount of Na2S2O3 to vary the Au:S2O3 ratio over the range 1:5–1:200 and NaClO4 to maintain a constant ionic strength. The measurement of the absorbance of the solutions under investigation has been made in the range 220–300 nm with respect to reference solutions containing all constituents except gold(I) complexes. The stability constant values have been determined to be log10β1 (\( {\text{AuS}}_{2} {\text{O}}_{3}^{ - } \)) = 17.43 (±0.05), log10β2\(({\text{Au}}({{\text{S}}_{2}{\text{O}}_{3}})_{2}^{3-})\) = 23.81 (±0.05) at an ionic strength of the solution of 0.5 mol·dm−3 (NaClO4) and a temperature of 295 K. These values are close to the log10β values which are predicted on the basis of correlation relationships.

Interaction of Zn(2+) ions with single-stranded polyU and polyC in neutral solutions.

Effect of Zn(2+) ions on the conformation of single-stranded polynucleotides polyU and polyC in a wide temperature range at pH 7 was studied by differential UV spectroscopy and by thermal denaturation. The atoms coordinating Zn(2+) ions were determined (O4 and N3 in polyU and N3 in polyC). A three-dimensional phase diagram and its two-dimensional components were constructed for a polyC-Zn(2+) system. The phase diagram revealed a region in which ordered single-stranded structures, stabilized by Zn(2+)-mediated cross-links involving N3 atom of cytosine, are formed. The phase diagram also demonstrated that the behavior of the polyC-Zn(2+) system is similar to the effect of retrograde condensation observed in some binary solutions of simple liquids. A dependence of Zn(2+)-polyC binding constant on the metal ion concentration was obtained. The reason why zinc-induced transition of the sequences with adenine-uracil (AU) base pairs from A-form geometry to a metallized m-form requires higher pH compared to the sequences comprised of guanine-cytosine (GC) base pairs is explained. This information can be useful for the development of possible technological applications based on m-DNA.

Metallization of single-stranded polyI by Zn(2+) ions in neutral solutions.

Metallization of single-stranded polyinosinic acid (polyI) by Zn(2+) ions at pH 7.0 was studied by differential UV spectroscopy at different temperatures. It was found that polyI is metallized at N7 and N1 atoms of hypoxanthine. The concentration dependence of the degree of binding of Zn(2+) ions to both N7 and N1 sites was obtained, and the corresponding binding constants were determined. Metallization of N1 occurs due to Zn(2+) substituting the imino protons and is effective not only at alkaline but also at neutral pH. This makes multistranded polyI-based systems more promising candidates for use in nanoelectronics than natural DNA sequences, metallization of which can be achieved only at alkaline pH.

Effect of electrostatic functional monomers on lysozyme recognition by molecularly imprinted hydrogel

ABSTRACTElectrostatic functional monomers (EFMs) play an important role in noncovalent molecular imprinting due to their formed complexes with the complementary segment(s) of the template molecule. In this work using UV difference spectroscopy, interaction saturations of methacrylic‐acid and 2‐dimethyl‐amino‐ethyl‐methacrylate in complex formation with lysozyme (Lyz) surface was found to occur at molar ratios to Lyz of 400 and 100, respectively. Based on these results and the estimated free to total EFMs ratios, four sets of imprinted/nonimprinted hydrogel samples were synthesized alongside the two sets based on lysozyme surface charges and equal EFMs. Comparisons showed that the highest absorption capacity of 59 mg/g and imprinting effect of 1.58 correspond to samples with EFM/Lyz ratios at saturation and minimum free to total EFM/Lyz ratios, respectively. Minimization of free monomers is hence important in recognition of proteins by avoiding the nonspecific binding. This can have generic application for specific separation of other macromolecules. © 2014 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2015, 132, 41366.

The Different Inhibition Mechanisms of OXA-1 and OXA-24 β-Lactamases Are Determined by the Stability of Active Site Carboxylated Lysine

The catalytic efficiency of class D β-lactamases depends critically on an unusual carboxylated lysine as the general base residue for both the acylation and deacylation steps of the enzyme. Microbiological and biochemical studies on the class D β-lactamases OXA-1 and OXA-24 showed that the two enzymes behave differently when reacting with two 6-methylidene penems (penem 1 and penem 3): the penems are good inhibitors of OXA-1 but act more like substrates for OXA-24. UV difference and Raman spectroscopy revealed that the respective reaction mechanisms are different. The penems form an unusual intermediate, a 1,4-thiazepine derivative in OXA-1, and undergo deacylation followed by the decarboxylation of Lys-70, rendering OXA-1 inactive. This inactivation could not be reversed by the addition of 100 mM NaHCO3. In OXA-24, under mild conditions (enzyme:inhibitor = 1:4), only hydrolyzed products were detected, and the enzyme remained active. However, under harsh conditions (enzyme:inhibitor = 1:2000), OXA-24 was inhibited via decarboxylation of Lys-84; however, the enzyme could be reactivated by the addition of 100 mM NaHCO3. We conclude that OXA-24 not only decarboxylates with difficulty but also recarboxylates with ease; in contrast, OXA-1 decarboxylates easily but recarboxylates with difficulty. Structural analysis of the active site indicates that a crystallographic water molecule may play an important role in carboxylation in OXA-24 (an analogous water molecule is not found in OXA-1), supporting the suggestion that a water molecule in the active site of OXA-24 can lower the energy barrier for carboxylation significantly.

Effect of Zn2+ and temperature on the conformational equilibrium of single-stranded polyA in neutral solutions

Effect of Zn2+ ions on the conformation of polyA in cacodilic buffer at pH 7 was investigated by differential UV spectroscopy (DUV) and by thermal denaturation. The shapes of the DUV spectra and melting curves suggest a transition of polyA into a more ordered “metallized”, possibly double-helical conformation at Zn2+ concentrations above 3×10−5M. A phase diagram of polyA complexes with Zn2+ was constructed for the temperature range from 20°C to 95°C and Zn2+ concentrations between 10−5M and 5×10−4M. It was found that the transition of a single strand into the “metallized” form is possible only if the length of the disordered single-stranded region becomes larger than a certain critical value, ranging between 98% and 78% as the metal concentration increases from 3×10−5 to 5×10−4M.