Interaction of albumins and heparinoids investigated by affinity capillary electrophoresis and free flow electrophoresis

A fast and precise affinity capillary electrophoresis (ACE) method has been applied to investigate the interactions between two serum albumins (HSA and BSA) and heparinoids. Furthermore, different free flow electrophoresis methods were developed to separate the species which appears owing to interaction of albumins with pentosan polysulfate sodium (PPS) under different experimental conditions. For ACE experiments, the normalized mobility ratios (∆R/R_f), which provided information about the binding strength and the overall charge of the protein‐ligand complex, were used to evaluate the binding affinities. ACE experiments were performed at two different temperatures (23 and 37°C). Both BSA and HSA interact more strongly with PPS than with unfractionated and low molecular weight heparins. For PPS, the interactions can already be observed at low mg/L concentrations (3 mg/L), and saturation is already obtained at approximately 20 mg/L. Unfractionated heparin showed almost no interactions with BSA at 23°C, but weak interactions at 37°C at higher heparin concentrations. The additional signals also appeared at higher concentrations at 37°C. Nevertheless, in most cases the binding data were similar at both temperatures. Furthermore, HSA showed a characteristic splitting in two peaks especially after interacting with PPS, which is probably attributable to the formation of two species or conformational change of HSA after interacting with PPS. The free flow electrophoresis methods have confirmed and completed the ACE experiments.     

Effects of Temperature and Common Ions on Binding of Puerarin to BSA

The effects of temperature and common ions on binding of puerarin to bovine serum albumin (BSA) are investigated. The binding constants (K _a) between puerarin and BSA are 1.13×10⁴ L⋅mol⁻¹ (20 °C) and 1.54×10⁴ L⋅mol⁻¹ (30 °C), and the number of binding sites (n) is (0.95±0.02). However, at a higher temperature (40 °C) the stability of the puerarin–BSA system decreases, which results in a lower binding constant (1.58×10³ L⋅mol⁻¹) and number of binding sites (n=0.73) of the puerarin–BSA system. However, the presence of Cu²⁺ and Fe³⁺ ions increases the binding constants and the number of binding sites in the puerarin–BSA complex.     

Characterization of lidocaine:hydroxypropyl-β-cyclodextrin inclusion complex

An inclusion complex was prepared between the local anesthetic lidocaine (LDC) and hydroxypropyl-β-CD (HP-β-CD). The complex was characterized by thermal analysis (differential scanning calorimetry, DSC), UV absorption and high-pressure liquid chromatography (HPLC). DSC results were indicative of complexation, due to the loss of the characteristic endothermic peak of LDC (77 °C). Phase-solubility diagrams allowed the determination of the association constant between LDC and HP-β-CD (35.7 ± 4.7 M⁻¹). The rate of LDC release decreased after complexation and thermodynamic parameters from the HPLC studies (ΔG° = −2.65 kJ/mol) revealed that a stable complex was formed.     

Development of a UHPLC-MS/MS method for the measurement of chlortetracycline degradation in swine manure

An ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was developed capable of simultaneously measuring chlortetracycline (CTC), epi-chlortetracycline, and isochlortetracycline (ICTC), as well as other structurally related tetracyclines in swine manure. A simple sample preparation was used consisting of extraction, dilution, centrifugation, and ultrafiltration. The concentrations of analyte were calculated using d₆-tetracycline as an internal standard in the matrix-matched standard curve. A solvent gradient resolved the compounds in 3.5 min with an additional 1.5 min of re-equilibration allowing the analyses of a large number of samples in a short period of time. MS/MS was used as the detection method giving analyte confirmation in addition to a large dynamic range and low detection limit. The UHPLC-MS/MS method successfully resolved multiple degradation products of CTC from the complex manure matrix. The method detection limits ranged from 1.9 pg/μL for CTC to 7.3 pg/μL for ICTC, and the calibration curve was linear from 1 to 10,000 pg/μL. The method was tested by measuring CTC and its degradation products as a function of time in incurred swine manure that had been incubated at three different temperatures (22 °C, 38 °C, and 55 °C). CTC concentration at 22 °C decreased 44% after 25 days; greater percentage decreases were observed when the manure was stored at elevated temperatures (96% and 98% for 38 °C and 55 °C, respectively). The concentration of the microbiologically inactivate isomer, ICTC, increased over the incubation period. At 22 °C, ICTC continued to increase through 25 days of incubation; at 38 °C, ICTC concentration plateaued on day 14 while at 55 °C ICTC concentration plateaued on day 7, with concentration increases of 198%, 374%, and 282% for 22 °C, 38 °C, and 55 °C, respectively.     

Study by DTA/TG of the formation of calcium aluminate obtained from an aluminium hazardous waste

A Spanish hazardous waste from tertiary aluminium industry was used as a raw material for the synthesis of calcium aluminate. An amorphous precursor was obtained by a hydrothermal method at different values of pH. The transformation of the precursor in a crystalline aluminate was followed by TG/DTA up to 1300 °C. At temperatures between 719 and 744 °C, the precursors evolve towards the formation of C₁₂A₇ which becomes CA at circa 1016 °C. Mass spectrometry coupled to thermal analyser allowed the identification of the decomposition products.     

Eudragit-coated albumin nanospheres carrying inclusion complexes for oral administration of indomethacin

Oral administration of indomethacin (IN) as well as drugs with low aqueous solubility usually results in poor absorption and bioavailability. The aim of this study was to prepare enteric-coated bovine serum albumin (BSA) nanospheres carrying cyclodextrin complex for IN delivery. Inclusion complex composed of IN and ??-cyclodextrin (CD) was prepared by spray-drying. Indomethacin alone and its inclusion complex were incorporated into albumin nanospheres using a coacervation method followed by thermal cross-linking. Then nanosphere suspensions were spray-dried. The inclusion complex and the nanospheres were characterized by FT-IR spectroscopy and DSC analysis. Phase-solubility diagrams and stability constants were determined at pH 2.0 and 7.4 and at different temperatures (10, 25 and 37 °C). Swelling ability of nanospheres were evaluated as well as the in vitro release behaviour at pH 2.0 and 7.4. The nanospheres were coated with Eudragit^® L-100 (EudL) or S-100 (EudS) using spray-drying to give protection in the stomach. The results showed that IN solubility can be increased by complexation with ??-CD or protein/drug interaction with albumin nanospheres. The inclusion complex loaded into BSA nanospheres provided a zero order drug release kinetic. The coating process with EudL and EudS allowed to obtain a negligible release at acidic pH without limiting drug availability at pH 7.4.     

In vitro evaluation of potential complexation between bovine insulin and bovine serum albumin

The objective of this study was to examine the possible binding of bovine insulin (BI) with bovine serum albumin (BSA) to form a new potential diabetogenic irreversible complex protein. Several preparations of BSA and BI were prepared. Both capillary electrophoresis and spectrophotometric analysis were undertaken to test the possibility of complexation between BI and BSA. HPLC was used to test whether the potential complex of BI and BSA is reversible or irreversible. The optimum deviation between the real and calculated absorbances was observed at a BI/BSA ratio of 2. Moreover, the migration time of BI decreased substantially with increasing ratio of BI to BSA until it became almost constant at equal molar ratio of BI/BSA. While the majority of the 2:1 BI–BSA sample detached during the HPLC analysis, which confirms the reversible character of BI–BSA binding, the HPLC chromatogram also emphasizes the formation of an irreversible complexation between the two proteins. This study provides evidence of the formation of reversible and irreversible new BI–BSA complexes under physiological conditions. This highlights the importance of examining the possible diabetogenicity of BI–BSA complex in genetically susceptible people. Copyright © 2013 John Wiley & Sons, Ltd.     

Synthesis,characterization, and evaluation of enzymatically degradable poly(N‐isopropylacrylamide‐co‐acrylic acid) hydrogels for colon‐specific drug delivery

The enzymatically degradable poly(N‐isopropylacrylamide‐co‐acrylic acid) hydrogels were prepared using 4,4^′‐bis(methacryloylamino)azobenzene (BMAAB) as the crosslinker. It was found that the incorporated N‐isopropylacrylamide (NIPAAm) monomer did not change the enzymatic degradation of hydrogel, but remarkably enhanced the loading of protein drug. The hydrogels exhibited a phase transition temperature between 4°C (refrigerator temperature) and 37°C (human body temperature). Bovine serum albumin (BSA) as a model drug was loaded into the hydrogels by soaking the gels in a pH 7.4 buffer solution at 4°C, where the hydrogel was in a swollen status. The high swelling of hydrogels at 4°C enhanced the loading of BSA (loading capability, ca. 144.5 mg BSA/g gel). The drug was released gradually in the pH 7.4 buffer solution at 37°C, where the hydrogel was in a shrunken state. In contrast, the enzymatic degradation of hydrogels resulted in complete release of BSA in pH 7.4 buffer solution containing the cecal suspension at 37°C (cumulative release: ca. 100 mg BSA/g gel after 4 days). Copyright © 2008 John Wiley & Sons, Ltd.     

Effect of Bovine Serum Albumin (BSA) on Enzymatic Cellulose Hydrolysis

Bovine serum albumin (BSA) was added to filter paper during the hydrolysis of cellulase. Adding BSA before the addition of the cellulase enhances enzyme activity in the solution, thereby increasing the conversion rate of cellulose. After 48 h of BSA treatment, the BSA adsorption quantities are 3.3, 4.6, 7.8, 17.2, and 28.3 mg/g substrate, each with different initial BSA concentration treatments at 50 °C; in addition, more cellulase was adsorbed onto the filter paper at 50 °C compared with 35 °C. After 48 h of hydrolysis, the free-enzyme activity could not be measured without the BSA treatment, whereas the remaining activity of the filter paper activity was approximately 41 % when treated with 1.0 mg/mL BSA. Even after 96 h of hydrolysis, 25 % still remained. Meanwhile, after 48 h of incubation without substrate, the remaining enzyme activities were increased 20.7 % (from 43.7 to 52.7 %) and 94.8 % (from 23.3 to 45.5 %) at 35 and 50 °C, respectively. Moreover, the effect of the BSA was more obvious at 35 °C compared with 50 °C. When using 15 filter paper cellulase units per gram substrate cellulase loading at 50 °C, the cellulose conversion was increased from 75 % (without BSA treatment) to ≥90 % when using BSA dosages between 0.1 and 1.5 mg/mL. Overall, these results suggest that there are promising strategies for BSA treatment in the reduction of enzyme requirements during the hydrolysis of cellulose.     

A comprehensive study on the inclusion mechanism of benzophenone into supramolecular nanoassemblies prepared using two water-soluble associative polymers

In this study, the entrapment of benzophenone (BZ) into supramolecular nanoassemblies prepared by mixing two water-soluble associative polymers (i.e. polymerized β-CD (pβ–CD) and dextran grafted with lauryl-side chains (MD)) has been investigated by using isothermal titration microcalorimetry (ITC) and molecular modeling. ITC experiments have been performed at various temperatures (4 °C (277 K), 25 °C (298 K), and 37 °C (310 K)) to evaluate the interaction of BZ with pβ–CD in comparison with β-CD. The inclusion complexation for both β-CD/BZ and pβ–CD/BZ interactions was entropy-driven (|ΔH| < |TΔS|) when the temperature of the experiment was low (4 °C) and enthalpy-driven (|ΔH| > |TΔS|) with minor entropic contribution when the temperature was increased (25 and 37 °C). Using all the thermodynamic data obtained for β-CD/BZ and pβ–CD/BZ interactions when the temperature of the experiment was varied, the \Updelta H   =   f(T\Updelta S ) \Updelta H\; = \;f(T\Updelta S ) plot was perfectly linear, which reflected an enthalpy–entropy compensation process. Finally, the combination of ITC data with molecular modeling provided consistent information in regard to the location of MD side chains and BZ inside the cyclodextrin cavity, as well as concerning the stability of the nanoassemblies loaded with BZ.     

Complexation study and spectrofluorometric determination of the binding constant for diquat and p-sulfonatocalix[4]arene

The interactions between diquat (DQ) and p-sulfonatocalix[4]arene (C4S) were studied in an aqueous solution as a function of the ionic strength. Evidence for the formation of a complex between DQ and C4S was obtained using fluorescence measurements, while the stoichiometry of the complex was confirmed as 1:1 for DQ/C4S using UV–vis spectroscopy. The ionic strength had no influence on the stoichiometry of the complex, but exerted a significant influence on the complexation constant, K_c, decreasing with an increase in the ionic strength. The thermodynamic complexation constant, K_c′, was computed as 5.25±1.11×10⁷ using the extended Debye–Hückel law. The rate constants for the heterogeneous electron transfer for the reduction of DQ at an electrode surface were evaluated as 0.150±0.010 cm s^?1 in the absence of C4S and 0.065±0.010 cm s^?1 when C4S was added to the solution in a 1:1 ratio.     

Temperature effect on the recovery of SO2-Poisoned GC/Nano-Pt electrode towards oxygen reduction

The SO₂ poisoning of Pt nanoparticle (n-Pt) modified glassy carbon (GC/n-Pt) electrode and the recovery of its activity for the oxygen reduction reaction (ORR) were studied using cyclic voltammetry at ambient (25 °C) and elevated (70 °C) temperatures. Recovery of the GC/n-Pt electrode by cycling the potential within the ORR range (1.0 to 0.2 V (standard hydrogen electrode)) in 0.1 M H₂SO₄ was not effective at 25 °C, but at 70 °C the onset potential of the ORR was almost the same as that at the fresh GC/n-Pt electrode. For the two different temperatures used here, the recovery on cycling the potential between 0.4 and 1.7 V was efficient. However, the number of cycles and the amount of charge required for the recovery at 70 °C were the smallest, which is of great interest for the proton exchange membrane fuel cell performance. The recovery using such a wide potential range at 70 °C resulted in an enhancement of the electrocatalytic activity of the GC/n-Pt electrode over a non-poisoned (bare) GC/n-Pt electrode.     

Thermodynamic study of complex formation between dibenzo-18-crown-6 and UO2 2+ cation in different non-aqueous binary solutions

In the present work the complexation process between UO₂ ²⁺ cation and the macrocyclic ligand, dibenzo-18-crown-6 (DB18C6) was studied in ethylacetate–dimethylformamide (EtOAc/DMF), ethylacetate–acetonitrile (EtOAc/AN), and ethylacetate–tetrahydrofuran (EtOAc/THF) and ethylacetate–propylencarbonate (EtOAc/PC) binary solutions at different temperatures using the conductometric method. The results show that the stoichiometry of the (DB18C6 . UO₂)²⁺ complex in all binary mixed solvents is 1:1. A non-linear behavior was observed for changes of log K_f of this complex versus the composition of the binary mixed solvents. The stability constant of (DB18C6 . UO₂)²⁺ complex in various neat solvents at 25 °C decreases in order: THF > EtOAc > PC > AN > DMF, and in the binary solvents at 25 °C is: THF–EtOAc > PC–EtOAc > DMF–EtOAc ≈ AN–EtOAc. The values of thermodynamic quantities (?H°_c, ?S°_c) for formation of this complex in the different binary solutions were obtained from temperature dependence of its stability constant and the results show that the thermodynamics of complexation reaction between UO₂ ²⁺ cation and DB18C6 is affected strongly by the nature and composition of the mixed solvents.     

The influence of precursor temperature on strontium sulphide doped silver for optoelectronic application

This research aims at the study of strontium sulphide doped silver using 0.1 mol of strontium chloride hexahydrate (SrCl2.6H2O), Thioacetamide (C₂H₅NS), and 0.01 mol of silver nitrate (AgNO3) as the cationic, anionic, and dopant concentrations via electrochemical deposition technique. The film had a strong peak at (111) and (211) which corresponds to 2theta values of 26.69° and 51.77° for undoped SrS and doped SrS respectively, and a flawless crystalline peak with a cubic phase that is indexed at orientations (111), (112), (200), and (211). SrS/Ag of deposited different precursor temperatures (room, 35, 40, and 45)^o correspond to 2theta values of 26.69°, 33.79°, 37.60°, and 51.77° respectively. The crystal lattice is shown by the rise in peak intensity with higher 2theta degree values; the appearance of an unindexed peak is caused by the substrate utilized for the deposition. Clove-like material with precipitate is visible in the SrS material's micrograph; the big nano grain on the surface of the substrate exhibits photon absorption but lacks any signs of pinholes. At the introduction of dopant and heating the precursor at 35 °C, 40 °C, and 45 °C there is a drastic change in the micrograph of the films, for the films at 35 °C the nanoparticle clave together with a melted wax with a sharp large white precipitate which is very visible on the surface of the film and the material deposited at 40 °C and 45 °C there is no visible precipitate on the film which show that as the precursor temperature increases it eliminate lattice strain and improve the photovoltaic properties of the deposited material. The energy band gap of strontium sulphide (SrS) and strontium sulphide doped silver (SrS/Ag) at different precursor temperatures of 35 °C, 40 °C, 45 °C is 1.50–2.35 eV.     

Novel water based cerium acetate precursor solution for the deposition of epitaxial cerium oxide films as HTSC buffers

A novel water based precursor solution using ethylenediaminetetraacetic acid (H₄EDTA) as a complexant and acetic acid and ethylenediamine (EDA) as additional components to obtain CeO₂ buffer layers on Ni (5%W) tapes is described in detail. The influence of complexation behavior in the formation of transparent and homogenous sols and gels by the combination of cerium acetate, acetic acid and H₄EDTA has been studied. The optimal growth conditions for cerium oxide were Ar-5% H₂ gas processing atmosphere, solution concentration levels of 0.2–0.4 M, a dwell time of 60 min at 900 °C and 5–30 min at 1,050 °C. X-ray diffraction, SEM, spectroscopic ellipsometry and pole figures were used to characterize the CeO₂ films. Highly textured CeO₂ layers were obtained.     

Temperature‐responsive “tadpole‐shaped” protein–polymer hybrids and their self‐assembly behavior

The temperature‐responsive poly (N, N‐diethylacrylamide) (pDEAAm) with narrower molecular weight distribution was prepared by the atom transfer radical polymerization and characterized by ¹HNMR and gel permeation chromatography. The temperature‐responsive “tadpole‐shaped” BSA–pDEAAm hybrids were fabricated via a free Cys‐34 residue of bovine serum albumin (BSA) site specifically binding to the end group disulfide bonds of pDEAAm and characterized by native‐polyacrylamide gel electrophoresis (Native‐PAGE) and matrix‐assisted laser desorption/ionization time of flight mass spectrometry. Their temperature‐responsive behaviors were measured by ultraviolet‐visible spectra (UV‐Vis). The lower critical solution temperature (LCST) of the pDEAAm was identified as 28°C, and the LCST of BSA–pDEAAm hybrids was identified as 31°C. The morphologies of BSA–pDEAAm hybrids self‐assembled in the aqueous solutions with two different temperatures at 25 °C and 40°C were investigated by transmission electron microscopy. Below the LCST of BSA–pDEAAm hybrids, the separate spherical nanoparticles were observed. In contrast, bundles and clusters were observed above the LCST of BSA–pDEAAm hybrids. The results suggested that the self‐assembly morphology of BSA–pDEAAm hybrids depended upon the pDEAAm block in BSA–pDEAAm hybrids, and the morphology transitions were effected by the LCST of BSA–pDEAAm hybrids. It would be expected to be used in biomedicine and materials science. Copyright © 2016 John Wiley & Sons, Ltd.     

Effect of temperature on self-assembly of an ionic tetrapeptide

The self-aggregation of the ionic tetrapeptide RWDW (R = arginine, W = tryptophan, D = aspartic acid) was studied at three temperatures (15, 25 and 35 °C) by different experimental techniques such as atomic force microscopy (AFM), isothermal titration calorimetry (ITC) and differential scanning calorimetry (DSC). AFM was used to investigate the morphology of the aggregates; the AFM images at 15 °C showed the presence of a dense network of entangled fibres, while at 35 °C the peptide assembled into sparse globular and fibrillar structures. Moreover, the calorimetric experiments showed that in all cases the disaggregation process is endothermic and dependent on the investigated temperature. Both the enthalpy of disaggregation and the cac change with temperature. In particular, at 35 °C, we obtained the lower enthalpy of disaggregation and higher cac, showing that the disaggregation process is favoured at high temperature. The DSC scans strengthen the hypothesis that the RWDW aggregation is a rather complex phenomenon.     

Structural evolution of turbostratic carbon: Implications in H2 storage

Structural evolution of turbostratic carbon samples as a function of annealing temperature has been investigated in detail using small angle X-ray scattering (SAXS), solid state nuclear magnetic resonance (NMR) and Raman spectroscopic techniques. From these studies, it is established that, samples heated at lower temperatures (700 °C and 800 °C) consist carbon particles with rough surfaces forming structure of surface fractal in nature. Whereas the sample heated at higher temperature (900 °C) consists of larger clusters with nearly smooth surface as well as smaller size particles forming dense mass fractal structure. For this sample, solid state NMR and Raman Spectroscopic studies indicate an increased extent of overlapping of 2p_z orbital of carbon atoms due to improved long range ordering and clustering. Hydrogen adsorption studies further substantiated that energetically more homogeneous surface exists for particles of 900 °C heated sample as compared to those of 700 °C and 800 °C heated samples. A highest hydrogen storage capacity of 0.152 H/M has been observed at 123 K and 45 bar pressure for the sample heated at 900 °C.     

Curie-point pyrolysis–gas chromatography–mass spectroscopic analysis of theabrownins from fermented Zijuan tea

Zijuan tea theabrownins (ZTTBs) was extracted from a type of fermented Zijuan tea and separated into fractions according to molecular weight. The extract was found to contain predominantly two fractions: <3.5 kDa and >100 kDa. These two fractions were analyzed for chemical composition, structural characteristics by Curie-point pyrolysis–gas chromatography–mass spectroscopy (CP-Py–GC/MS). The affects of pyrolysis temperature on pyrolytic products were also investigated. The fraction >100 kDa produced 50 GC/MS peaks during pyrolysis at 280 °C, 70 peaks at 386 °C, and 134 peaks at 485 °C. Fourteen of the products formed at 280 °C, 12 of those formed at 386 °C, and 21 of those formed at 485 °C were identified with match qualities of greater than 80%. The fraction <3.5 kDa gave 51 peaks during pyrolysis at 280 °C, 99 peaks at 386 °C, and 257 peaks at 485 °C. Six products formed at 280 °C, four products formed at 386 °C, and 61 products formed at 485 °C were identified with match qualities of greater than 80%. Pyrolysis temperatures of 485 °C and 386 °C were found suitable for the two fractions respectively. CP-Py–GC/MS revealed that, the fraction >100 kDa mainly consisted of phenolic pigments, esters, proteins, and polysaccharides, while the fraction <3.5 kDa contained no polysaccharide. CP-Py–GC/MS is an effective tool for the composition difference and structural characteristics of ZTTBs as well as other complex macromolecular plant pigments.     

In vitro release modulation and conformational stabilization of a model protein using swellable polyamidoamine nanosponges of β-cyclodextrin

New swellable cyclodextrin-based poly(amidoamine) nanosponges, named PAA-NS10 and PAA-NS11, were synthesized by crosslinking β-cyclodextrin with either 2,2-bisacrylamidoacetic acid or with polyamidoamine segments deriving from 2,2-bisacrylamidoacetic acid and 2-methylpiperazine, respectively. Water uptake studies showed a tremendous swelling capacity of both nanosponges, forming hydrogels. Time dependent swelling experiments in various aqueous media showed that the nanosponge hydrogels were stable over a period of at least 72 h maintaining their integrity. Thermal analysis showed that the two nanosponges were stable up to 250 and 300 °C, respectively. Both PAA-NS10 and PAA-NS11 were converted to aqueous nanosuspensions using the High Pressure Homogenization technique. Bovine serum albumin (BSA) was used as model protein to study the encapsulating capacity of these new β-cyclodextrin-based PAA-nanosponges. High protein complexation capacity was observed, as confirmed by UV spectroscopy. BSA encapsulation efficiency was greater than 90% on w/w basis for both nanosponges. In vitro BSA release studies were carried out showing a prolonged release of albumin from the two swollen BSA loaded β-CD PAA-NS over a period of 24 h.