Comparison of the results of thermal denaturation of β-lactoglobulin obtained by DSC and UV-spectroscopy

The thermal denaturation of β-lactoglobulin in the presence of urea and alkylurea solutions were measured. In the presence of a high concentration of urea this protein shows not only heat but also cold denaturation. For studying the effect of temperature two methods were used, differential scanning calorimetry (DSC) and UV-spectroscopy. DSC provides direct model-independent determination of the transition enthalpy in comparison with UV-spectroscopy, which gives only apparent or van't Hoff enthalpy of transition. The UV-melting curves were analyzed on the basis of a two-state approximation. The apparent standard enthalpies of thermal denaturation, ΔH _app. ^o , were compared with calorimetric ones.     

Thermodynamic denaturation of β-lactoglobulin in the presence of cetylpyridinium chloride

In this work, we determined the stability parameters of bovine β-lactoglobulin, variant A, (BLG-A), in relation to their transition curves induced by cetylpyridinium chloride (CPC) as a cationic surfactant. The experiments took place over the temperature range of 298 K to 358 K. For each transition curve at any specific temperature, the conventional method of analysis, which assumes a linear concentration dependence of the pre- and post-transition base lines, gave the most realistic values for ΔG_D(H₂O). Results show that the minimum value of ΔG_D(H₂O) occurs at T = 328 K. Using the Gibbs–Helmholtz equation, the values of enthalpy, ΔH_D, and entropy, ΔS_D, of denaturation have been calculated considering temperature dependence of ΔG_D at any specified concentration of CPC. The values of 12.05 kJ · mol⁻¹, 18.54 kJ · mol⁻¹, and 18.32 J · mol⁻¹ · K⁻¹, were obtained for ΔG_D(H₂O), ΔH_D(H₂O), and ΔS_D(H₂O), respectively. The results show that the enthalpy term dominates the entropy term.     

A differential scanning calorimetric study of β-lactoglobulin and vitamin D3 complexes

In the study, ??-lactoglobulin and vitamin D₃ complexes were obtained through spray drying at inlet temperature of 120 or 150?°C. Additionally, complexes with lactose were synthesised. Modulated differential scanning calorimetry (MDSC) was used in order to explain the glass transitional behaviour of spray dried ??-lactoglobulin?Cvitamin D₃ and ??-lactoglobulin?Cvitamin D₃?Clactose complexes and the influence of applied spray drying conditions on calorimetric parameters. The glass transition temperatures of the powders in this study ranged from 112.93 to 112.99?°C (T _g onset), from 118.42 to 119.20?°C (T _g midpoint) and from 122.07 to 125.08?°C (T _g endpoint). The present study has shown that the values of glass transition temperatures at a _w?=?0 did not differ significantly for studied samples obtained in a form of spray-dried powders, despite of various process conditions applied. The different values of heat capacity changes can be related to the various vitamin D₃ content in tested samples.     

Hydration and dehydration processes of β-cyclodextrin: A raman spectroscopic study

A linear relationship between the integrated intensity of the Raman OH stretching band (I_OH) and ambient humidity in equilibrium with a sample of a crystalline -Cyclodextrin (CD) hydrate, for humidities between 15% and 100%, was obtained. In addition, hydration and dehydration processes were monitored by measuring I_OH as a function of time. For the normally hydrated crystal, both hydration and dehydration processes present essentially continuous and similar variations with hysteresis for times shorter than 20 min.     

Isolation of ovine β-lactoglobulin genetic variants by chromatofocusing: Heterogeneity of β-lactoglobulin A

Summary Chromatofocusing was applied to the isolation of ovine -Lactoglubulin (-Lg) genetic variants. Ovine -Lg A and B, which are known to differ only by a single amino-acid substitution, were separated. Moreover, one component differing from A variant in isoelectric point and relative molecular mass has been isolated in samples containing ovine -Lg A. Although elution of -Lgs occurred far from their normal isoelectric points, chromatofocusing is a powerful technique effective for both analytical and preparative scale.     

Quantum chemical study of the host-guest inclusion complexes of the local anaesthetic drugs, procaine hydrochloride and butacaine hydrochloride, with α- and β-cyclodextrins

Quantum mechanical (QM) calculations were carried out in order to study the host-guest inclusion complexes of procaine hydrochloride (Pro-H) and butacaine hydrochloride (But-H) with α- and β-cyclodextrins (α- and β-CDs) by PM3 and AM1 methods. The systems were studied by a 1:1 (α-CD/Pro-H, α-CD/But-H, β-CD/Pro-H, and β-CD/But-H) stoichiometric ratio. In this work we calculated the energy of complex formation in vacuo, and this investigation was carried out on the basis of the host-guest approach. The stabilization energy results for the 1:1 host-guest inclusion complexes indicate that the β-CD/Pro-H complex is more stable than the α-CD/Pro-H complex. Furthermore, stabilization energy for the 1:1 inclusion complex of α-CD with But-H is lower than that for the 1:1 inclusion complex of β-CD with But-H. The calculation results show that all complexation processes for the four complexes are exothermic. Enthalpy changes for the α-CD/But-H and β-CD/Pro-H host-guest inclusion complexes are more negative than those for the other ones. ΔG ^o values for both the β-CD/Pro-H and α-CD/But-H complexes are negative. Correspondence: S. M. Hashemianzadeh, Department of Physical Chemistry, College of Chemistry, Iran University of Science and Technology, Tehran, Iran.     

A comparative study of the structure and properties of β-galactosidases

A comparative study of β-galactosidase amino acid sequences of E. coli and another four out of 11 microorganisms selected at the first stage was performed. It was shown that the functional amino acid residues in the catalytic domain and the ligand environment of the magnesium cation for all five sequences are identical. The mechanism of the catalytic action of E. coli and K. lactis β-galactosidases was investigated by the method of nucleophilic competition. It was shown that the mechanism of the effects of nucleophilic agents is kinetically identical both enzymes: the presence of methanol or butanediols affects the stage of degalactosylation; the presence of magnesium cations promotes the activity of both β-galactosidases; and the mechanisms of the thermal inactivation of E. coli and K. lactis β-galactosidases are different.     

Theoretical study of the inclusion processes of octopamine with β-cyclodextrin: PM6, ONIOM,and NBO analysis

The inclusion process involving β-cyclodextrin with octopamine (OA) was investigated by using PM6, HF/3-21G*, B3LYP/6-31G (d), WB97XD/6-31G (d) methods and several combination of ONIOM2 hybrid calculations. The obtained results clearly indicate the orientation in which the guest molecule penetrates into the hydrophobic cavity of β-CD with the aromatic ring is energetically preferred. The structures show the presence of several intermolecular hydrogen bond interactions that were studied on the basis of natural bonding orbital (NBO) analysis, employed to quantify donor–acceptor interactions between host and guest molecules. A study of these complexes in solution was carried out using the CPCM model to examine the influence of solvation on the stability of the octopamine/β-CD complex.     

Characterization of β-cyclodextrin inclusion complex with procaine hydrochloride by 1H NMR and ITC

The inclusion of local anesthetic drug procaine hydrochloride by β-cyclodextrin was investigated by 1D and 2D proton NMR spectroscopy and isothermal titration calorimetry (ITC) at 298 K. The stoichiometry of the complex was determinate by the method of continuous variation, using the chemical induced shift of both host and guest protons. The association constant K, of the obtained complex was calculated and found to be 293.17 M^?1. Rotating frame NOE spectroscopy, was used to ascertain the solution geometry of the host–guest complex. The result reveals that the procaine molecule penetrates into the β-cyclodextrin cavity with the aromatic ring. The energetics of complexation process is investigated by ITC technique. The analysis indicates that the complexation of procaine by β-CD is an exothermic process and show that both enthalpy and entropy contribute to the binding process. The obtained value for the association constant is in good agreement with that obtained from NMR.     

Synthesis and study of cesium-containing phosphates with a β-tridymite structure

Complex phosphates CsMg_{1 ? x} M _x PO₄ (M = Mn, Co, Cu, Zn), containing cesium and metals in the oxidation state +2, have been synthesized, and their structure and thermal behavior have been studied. Continuous solid solutions (0 ?? x ?? 1) of the ??-tridymite structure type are formed in the CsMg_{1 ? x} Mn _x PO₄, CsMg_{1 ? x} Co _x PO₄, and CsMg_{1 ? x} Zn _x PO₄ systems, whereas limited solid solutions (0 ?? x ?? 0.4) are formed in the CsMg_{1 ? x} Cu _x PO₄ system. Based on DTA data, phase transitions have been revealed in the cobalt-, copper-, and zinc-containing phosphates, and the orthorhombic or monoclinic crystal system has been identified. Unit cell parameters of the solid solutions have been calculated. Thermal expansion of the CsMPO₄ phosphates has been studied.     

A quartz crystal microbalance study of β-cyclodextrin self assembly on gold and complexation of immobilized β-cyclodextrin with adamantane derivatives

Quartz crystal microbalance (QCM) was used to study the self-assembly of per-6-thio-β-cyclodextrin (t₇-βCD) on gold surfaces, and the subsequent inclusion interactions of immobilized βCD with adamantane-poly(ethylene glycol) (5,000 MW, AD-PEG), 1-adamantanecarboxylic acid (AD-C) and 1-adamantylamine (AD-A). From a 50 μM solution of t₇-βCD in 60:40 DMSO:H₂O, a t₇-βCD layer was formed on gold with surface density of 71.7 ± 2.7 pmol/cm², corresponding to 80 ± 3% of close-packed monolayer coverage. Gold sensors with immobilized t₇-βCD were then exposed alternately to six different concentrations of AD-PEG, 500 μM AD-C or 500 μM AD-A aqueous solutions for association, and water for dissociation. Association of AD-PEG conformed to a Langmuir isotherm, with a best fit equilibrium constant K = 125,000 ± 18,000 M⁻¹. For AD-C and AD-A, association (k _a ) and dissociation (k _d ) rate constants were extracted from kinetic profiles by fitting to the Langmuir model, and equilibrium constants were calculated. The parameters for AD-C were found to be: k _a = 100 ± 5 M⁻¹ s⁻¹, k _d = 110 (±18) × 10⁻⁴ s⁻¹, and K = 9,400 ± 1,700 M⁻¹. For AD-A, k _a = 58 ± 6 M⁻¹ s⁻¹, k _d = 154 (±7) × 10⁻⁴ s⁻¹, and K = 3,800 ± 400 M⁻¹. The results demonstrate the utility of QCM as a tool for studying small molecule surface adsorption and guest–host interactions on surfaces. More specifically, the kinetic and thermodynamic data of AD-C, AD-A, and AD-PEG inclusion with immobilized t₇-βCD form a basis for further surface association studies of AD-X conjugates to advance surface sensory and coupling applications.     

DSC and NMR Study on the Inclusion Complex of Lappaconitine with β—Cyclodextrin

The inclusion complex of lappaconitine(Lap) with β-cyclodextrin (β-CD) has been studied by the differential scanning calorimetry (DSC) and ^1H-NMR,2D-NMR spectroscopy. The structure of the complex has been deduced.     

A thermodynamic study of the inclusion processes of α- andβ-cyclodextrins with the acid forms of methyl orange and methyl yellow

The effect of temperature on the UV-visible spectra of the acid forms of methyl orange and methyl yellow in the presence of - and-cyclodextrins was analysed in terms of 1 : 1 inclusion processes. Three inclusion processes were considered for each azo dye/cyclodextrin system. The processes are the apparent inclusion process, the ammonium inclusion process, and the azonium inclusion process. The values of H ⁰ and S ⁰ are reported for each process. The inclusion complexes of the ammonium tautomer are more stable than those of the azonium tautomer and -cyclodextrin inclusion complexes are more stable than those of-cyclodextrin. The inclusion processes resulted in a shift in the position of the tautomeric equilibrium of an azo dye. A significant color fading was observed in -cyclodextrin/methyl yellow solutions. No measurable changes in the absorbances were observed in the case of azo dye solutions containing -cyclodextrin.     

A study of the tautomerism of β-dicarbonyl compounds with special emphasis on curcuminoids

Six β-diketones related to curcumin and curcumin itself have been studied by ¹³C NMR spectroscopy in chloroform in order to determine the equilibrium constant between the two keto/enol tautomers. In order to do this GIAO/B3LYP/6-31G^∗∗ calculations of absolute shieldings (σ, ppm) were carried out. To establish relationships between σ and experimental chemical shifts (δ, ppm), three simple β-diketones (acetylacetone, dibenzoylmethane and benzoylacetone) have been studied. The preference for different groups to be conjugated with the CO has been determined.     

Theoretical study of β- and γ-cyclodextrin complexes with ferrocene-containing azoles

The interaction between cyclodextrins (β- and γ-CD) and ferrocenyl azoles (i.e., pyrazole ferrocenes (I, III–V) and benzimidazole ferrocenes (VI, VII)), along with 1-ferrocenylethanol (II), each in the form of (R)- and (S)-enantiomers, in forming inclusion complexes is studied for the first time using detailed quantum chemical calculations. Compounds are calculated in terms of the density functional theory (DFT), using the Becke–Lee–Yang–Parr (B3LYP) approach in the 6-31G* basis sets. For the considered CD complexes with enantiomers of I–VII, structures in which a guest partially enters a host cavity from the side of the heterocyclic substituent (pyrazole or benzimidazole) are found to be energetically advantageous. It is shown that for successful resolution of (R,S)-enantiomers on chiral phases containing cyclodextrins, we must consider the interaction between outer hydroxyl groups on the CD cone’s surface, in addition to the correspondence of geometric dimensions. The calculated data correlate well with the data from the chromatographic separation of guest enantiomers on cyclodextrin sorbents.     

Stabilization mechanism of oil-in-water emulsions by β-lactoglobulin and gum arabic

Natural biopolymer stabilized oil-in-water emulsions were formulated using β-lactoglobulin (β-lg), gum arabic (GA), and β-lg:GA solutions as an alternative to synthetic surfactants. Emulsions using these biopolymers and their complexes were formulated varying the biopolymer total concentration, the protein-to-polysaccharide ratio, and the emulsification protocol. This work showed that whereas β-lg enabled the formulation of emulsions at concentration as low as 0.5 (w/w)%, GA allowed to obtain emulsions at concentrations equal to or higher than 2.5 (w/w)%. In order to improve emulsion stability, β-lg and GA were complexed through strong attractive electrostatic interactions. GA solution had to be added to previously prepared β-lg emulsions in order to obtain stable emulsions. Interfacial tension and interfacial rheological measurements allowed a better understanding of the possible stabilizing mechanism. β-lg and GA both induced a very effective decrease in interfacial tension and showed interfacial elastic behaviour. In the mixed system, β-lg adsorbed at the interface and GA electrostatically bound to it, leading to the formation of a bi-layer stabilized emulsion. However, emulsion stability was not improved compared to β-lg stabilized emulsion, probably due to depletion or bridging flocculation.     

Effect of urea on the β-hairpin conformational ensemble and protein denaturation mechanism

Despite the daily use of urea to influence protein folding and stability, the molecular mechanism with which urea acts is still not well understood. Here the use of combined parallel tempering and metadynamics simulation allows us to study the free-energy landscape associated with the folding/unfolding of β-hairpin GB1 equilibrium in 8 M urea and pure water. The nature of the unfolded state in both solutions has been analyzed: in urea solution the addition of denaturants acts to expand the denatured state, while in pure water solution the unfolded state is noticeably more compact. For what concerns the mechanism by which urea acts as a denaturant, a preferential direct interaction between urea molecules and protein backbone has been found. However, the bias toward urea solvation is largest at intermediate values of the gyration radius.     

A correction function to determine the β-fusion heat in a mixture of α- and β-PP

Blends of known amounts of α- and β-PP crystals were prepared from pure α- and pure β-PPsamples. Their fusion behaviours were studied by differential scanning calorimetry (DSC).The fusion heats of the α- and β-crystals were approximated from the DSC curves and compared with those calculated on the basis of the compositions of the blends. A correction function was found which improved the accuracy of the respective fusion heats significantly from the DSC analysis. The correction function can be used to determine the respective fusion heats of a PP sample which contains an unknown mixture of the α- and β-crystals. This revised version was published online in July 2006 with corrections to the Cover Date.     

Complexes of α-, β-, and γ-cyclodextrins with nitrophenols: A theoretical study of the structure and energy

Detailed quantum mechanical calculations of the interaction of cyclodextrin (α-, β-, and γ-CD) with 4-nitrophenol (I), 4-nitro-2,6-dimethylphenol (II), 4-nitro-3,5-dimethylphenol (III), and their anions (IVVI) with the formation of intercalation complexes are carried out for the first time. The calculations of the compounds are performed within the density functional theory by the hybrid Becke–Lee–Yang–Parr (B3LYP) method with LanL2DZ basis sets. For the α-CD+III and α-CD+VI complexes it is shown that a nitrophenol molecule of III and a nitrophenolate anion of VI are not contained in the α-CD torus, which agrees with the experimental equilibrium constants. It is found that the calculated equilibrium constants of the formation of guest–host complexes with phenolate anions are much larger than those of neutral molecules. The most stable CD complexes with nitrophenols and their anions should be expected for γ-CD. The β-CD complexes when the guest enters into the host cavity are formed only with compounds I, V, and VI.     

A new sample preparation method compatible with capillary electrophoresis and laser-induced fluorescence for improving detection of low levels of β-lactoglobulin in infant foods

β-Lactoglobulin (βLG) is the main allergenic protein in cow's milk and can cause allergy even when present at very low concentration. The aim of this work is to develop an innovative sample preparation method fully compatible with capillary electrophoresis and laser-induced fluorescence detection for improving the sensitivity when analyzing βLG. Different types of baby food were on purpose contaminated with diverse dairy desserts and submitted to thermal treatment to simulate potential contamination at production. Sample preparation prior to CE analysis was performed by the classical extraction method and by the innovative one, and the results were compared. Analysis was performed by capillary electrophoresis with laser-induced fluorescence detection. The innovative method permitted to detect contaminations as low as 1 part of yoghurt in 10 000 parts of baby food.