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Food Research International (Ottawa,... May 2020Interaction between α-lactalbumin (α-LA) and three similar chalconoids was compared using a combination of multi-spectral analysis and molecular docking, and their...
Interaction between α-lactalbumin (α-LA) and three similar chalconoids was compared using a combination of multi-spectral analysis and molecular docking, and their influence on structure and functional properties of α-LA was also investigated. Chalconoids strongly quenched α-LA fluorescence in a static mode and their binding constants to α-LA were declined in the order of hydroxy safflower yellow A (SYA), neohesperidin dihydrochalcone (NHDC) and naringin dihydrochalcone (NGDC). The main interaction forces involved in the binding of SYA, NHDC and NGDC to α-LA included hydrophobic forces and hydrogen bonds. There was non-radiative energy transfer between α-LA and three chalconoids, as indicated by the estimated by Förster's distance. The vicinity of SYA to tryptophan residues of α-LA showed the minimum value. Based on Fourier transform infrared spectroscopy (FTIR) spectra, SYA induced the conversion of more α-LA from α-helix into its β-structures than NHDC and NGDC. Also, although the addition of three chalconoids had no significant effect on the emulsifying activity of α-LA, it slightly improved the emulsion stability of α-LA. In addition, SYA showed the maximum decrease on surface hydrophobicity of α-LA. Antioxidant capacity of SYA was also decreased more than that of NHDC and NGDC after the binding to α-LA. Additionally, docking studies indicated that SYA, NHDC and NGDC bound to the cleft between α-domains and β-domains by three, two and two hydrogen bonds, respectively. Therefore, these findings suggest that there are significant differences among the effects of three similar chalconoids on structure and functionality of α-LA.
Topics: Binding Sites; Chalcone; Chalcones; Emulsions; Flavanones; Hesperidin; Hydrogen Bonding; Hydrophobic and Hydrophilic Interactions; Lactalbumin; Molecular Docking Simulation; Protein Binding; Protein Conformation, alpha-Helical
PubMed: 32247452
DOI: 10.1016/j.foodres.2020.109006 -
Journal of Food Science Apr 2009Some of the factors that affect the rheological characteristics of fibril gels are discussed. Fibrils with nanoscale diameters from beta-lactoglobulin (beta-lg) and... (Comparative Study)
Comparative Study Review
Some of the factors that affect the rheological characteristics of fibril gels are discussed. Fibrils with nanoscale diameters from beta-lactoglobulin (beta-lg) and alpha-lactalbumin (alpha-la) have been used to create gels with different rheological characteristics. Values of the gelation time, t(c), the critical gel concentration, c(0), and the equilibrium value of the storage modulus, G, such as at long gelation times, derived from experimental rheological data, are discussed. Fibrils created from beta-lg using solvent incubation and heating result in gels with different rheological properties, probably because of different microstructures and fibril densities. Partial hydrolysis of alpha-la with a serine proteinase from Bacillus licheniformis results in fibrils that are tubes about 20 nm in diameter. Such a fibril gel from a 10% (w/v) alpha-la solution has a higher modulus than a heat-set gel from a 10% (w/w) beta-lg, pH 2.5 solution; it is suggested that one reason for the higher modulus might be the greater stiffness of alpha-la fibrils. However, the gelation times of alpha-la fibrils are longer than those of beta-lg fibrils.
Topics: Biopolymers; Chemical Phenomena; Food Technology; Fractals; Gels; Hot Temperature; Hydrogen-Ion Concentration; Lactalbumin; Lactoglobulins; Rheology; Solvents
PubMed: 19397731
DOI: 10.1111/j.1750-3841.2009.01098.x -
Journal of Chromatography. B,... Sep 2021In complex food matrices, non-directed reactions between food proteins and secondary plant metabolites (SPM) are conceivable. In this study, the interaction between the...
In complex food matrices, non-directed reactions between food proteins and secondary plant metabolites (SPM) are conceivable. In this study, the interaction between the bioactive metabolite from garden cress (Lepidium sativum) and selected Brassicaceae - benzyl isothiocyanate (BITC) - and the dairy protein α-lactalbumin (α-LA) was investigated. It was focused on monitoring the proteolytic degradation behaviour of unmodified and BITC-modified α-LA with two-dimensional high-performance thin-layer chromatography (2D-HPTLC). The two-dimensional approach of HPTLC offers high resolution in the separation of complex peptide mixtures and might enable differentiation of protein modifications. Based on the specific peptide patterns of native and modified peptides, conclusions can be drawn about differences in protein/peptide polarity, location of a modification, and digestibility. The aim was to characterize tryptically hydrolyzed unmodified and BITC-modified peptides using the 2D method and to investigate the influence of BITC modification of α-LA on polarity and digestibility. To determine the repeatability of peptide separation by 2D-HPTLC, the unmodified and BITC-modified protein hydrolyzates were separated six times. The absolute standard deviations between the retardation factors of the individual peptide spots varied between 0.52 and 4.79 mm for the x-coordinates and between 0.41 and 6.47 mm for the y-coordinates for all three samples. Here, the mean relative standard deviations ranged from 5.80 to 10.4% for the x-coordinates and from 5.91 to 18.3% for the y-coordinates. The results of the tryptic hydrolysis indicated that, depending on the concentration of BITC used, the modification sterically hinders the cleavage sites for the enzyme, resulting in a reduced digestibility. Covalent binding of the hydrophobic BITC altered the digestibility and polarity of the protein, leading to a difference in peptide patterns between the unmodified and modified α-LA. It was concluded that the reaction was undirected, resulting in a mixture of unmodified and modified peptides, and that elongated modified peptides were formed by BITC blocking of trypsin cleavage sites.
Topics: Chromatography, High Pressure Liquid; Chromatography, Thin Layer; Isothiocyanates; Lactalbumin; Peptide Fragments; Trypsin
PubMed: 34536835
DOI: 10.1016/j.jchromb.2021.122937 -
Food Chemistry Dec 2023High internal phase Pickering emulsions (HIPPEs) stabilized by protein nanoparticles have been widely reported, but the use of enzymatic methods for preparing these...
High internal phase Pickering emulsions (HIPPEs) stabilized by protein nanoparticles have been widely reported, but the use of enzymatic methods for preparing these nanoparticles remains underexplored. Our hypothesis is that enzymatically crosslinked α-lactalbumin (ALA) nanoparticles (ALATGs) prepared using transglutaminase will demonstrate improved properties as stabilizers for HIPPEs. In this study, we investigated the physicochemical properties and microstructures of ALATGs, finding that enzymatic crosslinking could be enhanced by removing Ca ions from ALA and preheating the proteins (85 °C, 15 min). The electrical charge, secondary structure, and surface hydrophobicity of ALATGs were found to depend on crosslinking conditions. HIPPEs formed with an ALA concentration of 10 mg/mL and an enzyme activity of 120 U/g exhibited the highest apparent viscosity and mechanical strength, as well as significantly improved loading capacity and photostability for the encapsulated lycopene. Overall, our results support the hypothesis that ALATG-nanoparticles show superior performance as emulsifiers compared to ALA-nanoparticles.
Topics: Lactalbumin; Lycopene; Hydrophobic and Hydrophilic Interactions; Emulsions; Nanoparticles; Transcription Factors; Particle Size
PubMed: 37478605
DOI: 10.1016/j.foodchem.2023.136394 -
Physical Chemistry Chemical Physics :... Sep 2022A molten globule (MG) state is an intermediate state of a protein observed during the unfolding of the native structure. The MG state of the protein is induced by...
A molten globule (MG) state is an intermediate state of a protein observed during the unfolding of the native structure. The MG state of the protein is induced by various denaturing agents (like urea), extreme pH, pressure, and heat. Experiments suggest that the MG state of some proteins is functionally relevant even if there is no well-defined tertiary structure. Earlier experimental and theoretical studies show that the MG state of a protein is dynamic in nature, where conformational states are interconverted on nanosecond time scales. These observations lead us to study and compare the conformational fluctuations of the MG state to those of intrinsic disordered proteins (IDPs). We consider a milk protein, α-lactalbumin (aLA), which shows an MG state at low pH upon removal of the calcium (Ca) ion. We use the constant pH molecular dynamics (CpHMD) simulation to maintain the protonation state of titratable residues at a low pH during the simulation. We use the dihedral principal component analysis, the density based clustering method, and the machine learning technique to identify the conformational fluctuations. We observe metastable states in the MG state. The residues containing the essential coordinates responsible for metastability belong to a stable helix in the crystal structure, but most of them prefer unstructured or bent conformation in the MG state. These residues control the exposure of the putative binding residues for fatty acids. Thus, the MG state of a protein behaves as an intrinsic disorder protein, although the disorder here is induced by external conditions.
Topics: Circular Dichroism; Hydrogen-Ion Concentration; Lactalbumin; Protein Conformation; Protein Denaturation; Protein Folding; Protein Structure, Secondary
PubMed: 36043462
DOI: 10.1039/d2cp02168d -
Journal of Molecular Biology Sep 2015Human α-lactalbumin made lethal to tumor cells (HAMLET) is a tumoricidal complex consisting of human α-lactalbumin and multiple oleic acids (OAs). OA has been shown to...
Human α-lactalbumin made lethal to tumor cells (HAMLET) is a tumoricidal complex consisting of human α-lactalbumin and multiple oleic acids (OAs). OA has been shown to play a key role in the activity of HAMLET and its related complexes, generally known as protein-fatty acid (PFA) complexes. In contrast to what is known about the fate of the protein component of such complexes, information about what happens to OA during their action is still lacking. We monitored the membrane, OA and protein components of bovine α-lactalbumin complexed with OA (BLAOA; a HAMLET-like substance) and how they associate with each other. Using ultracentrifugation, we found that the OA and lipid components follow each other closely. We then firmly identify a transfer of OA from BLAOA to both artificial and erythrocyte membranes, indicating that natural cells respond similarly to BLAOA treatment as artificial membranes. Uncomplexed OA is unable to similarly affect membranes at the conditions tested, even at elevated concentrations. Thus, BLAOA can spontaneously transfer OA to a lipid membrane. After the interaction with the membrane, the protein is likely to have lost most or all of its OA. We suggest a mechanism for passive import of mainly uncomplexed protein into cells, using existing models for OA's effect on membranes. Our results are consistent with a membrane destabilization mediated predominantly by OA insertion being a significant contribution to PFA cytotoxicity.
Topics: Animals; Antineoplastic Agents; Cattle; Cell Membrane Permeability; Erythrocyte Membrane; Humans; Lactalbumin; Models, Molecular; Oleic Acid
PubMed: 26297199
DOI: 10.1016/j.jmb.2015.08.009 -
Journal of the Science of Food and... Aug 2021α-lactalbumin (α-La) is of great interest to the industry as a result of its excellent functional properties and nutritional value. Aqueous two-phase flotation (ATPF)...
BACKGROUND
α-lactalbumin (α-La) is of great interest to the industry as a result of its excellent functional properties and nutritional value. Aqueous two-phase flotation (ATPF) of thermo-sensitive polymer poly (ethylene glycol-ran-propylene glycol) monobutyl ether (UCON) and KH PO was applied to directly separate and purify α-La from milk whey, which was purposed to simplify the production process and reduced cost of production.
RESULTS
The effect of ATPF composition and operating parameters on the flotation efficiency (E) and purity of α-La were investigated. The optimal conditions included 2 min of premixing time, 30 mL min flow velocity and 20 min of flotation time, whereas the composition conditions comprised 35.0 mL 0.18 g mL phosphate solution (containing 10% (cow milk whey/salt solution, v/v) cow milk whey, 50 ppm defoamer and 2 g NaCl) and 5.0 mL of 40% (w/w) UCON solution. Under the optimal conditions, E of α-La was 95.67 ± 1.04% and purity of α-La was 98.78 ± 1.19%. UCON was recovered by a thermally-induced phase separation and reused in next ATPF process without reducing E of α-La. Purified α-La was characterized by several key technologies. The results indicated that α-La in cow milk whey could be directly separated and purified by the ATPF and the purity was satisfactory. Moreover, it was suggested there was no obvious structure difference between the α-La separated by ATPF and the α-La standard.
CONCLUSION
The present study enabled the recycling of UCON, providing an effective, economically viable and environmentally friendly approach for the separation and purification of protein. © 2021 Society of Chemical Industry.
Topics: Animals; Cattle; Chemical Fractionation; Hot Temperature; Hydrogen-Ion Concentration; Lactalbumin; Phosphates; Polymers; Whey
PubMed: 33420726
DOI: 10.1002/jsfa.11055 -
Ultrasonics Sonochemistry Mar 2021α-lactalbumin was modified by ultrasound (US, 20 kHz, 43 ± 3.4 W/cm) pre-treatments (0, 15, 30 and 60 min) and laccase cross-linking of sonicated α-lactalbumin...
α-lactalbumin was modified by ultrasound (US, 20 kHz, 43 ± 3.4 W/cm) pre-treatments (0, 15, 30 and 60 min) and laccase cross-linking of sonicated α-lactalbumin was used to evaluate the physical and oxidative stability of conjugated linoleic acid (CLA) emulsions. The emulsions prepared with laccase cross-linking US-α-lactalbumin (α-lactalbumin treated with US pre-treatment) and US-α-lactalbumin were scrutinized for oxidative and physical stability at room temperature for two weeks of storage. Laccase cross-linking US-α-lactalbumin (Lac-US-α-lactalbumin) revealed improved physical stability in comparison with US-α-lactalbumin, specified by droplet size, structural morphology, adsorbed protein, emulsifying properties and creaming index. SDS-PAGE analysis showed that there was formation of polymers in Lac-US-α-lactalbumin emulsion. Surface hydrophobicity of Lac-US-α-lactalbumin was higher than that of US-α-lactalbumin, and gradually enhanced with the increase of ultrasound time. More importantly, the measurements of peroxide values and conjugated dienes were used to study the oxidative stability of the CLA emulsions. The Lac-US-α-lactalbumin emulsion proved to be reducing the synthesis of fatty acid hydroperoxides and less conjugated dienes compared to the native and US-α-lactalbumin emulsions. This study revealed that the combination of US pre-treatment and laccase cross-linking might be an effective technique for the modification of CLA emulsions.
Topics: Adsorption; Electric Conductivity; Emulsions; Hydrogen-Ion Concentration; Laccase; Lactalbumin; Linoleic Acids, Conjugated; Oils; Oxidation-Reduction; Sonication; Temperature; Water
PubMed: 33125963
DOI: 10.1016/j.ultsonch.2020.105365 -
Current Protein & Peptide Science Jul 2016This is a second part of the three-part article from a series of reviews on the abundance and roles of intrinsic disorder in milk proteins. We continue to describe... (Review)
Review
Disorder in Milk Proteins: α-Lactalbumin. Part B. A Multifunctional Whey Protein Acting as an Oligomeric Molten Globular "Oil Container" in the Anti-Tumorigenic Drugs, Liprotides.
This is a second part of the three-part article from a series of reviews on the abundance and roles of intrinsic disorder in milk proteins. We continue to describe α-lactalbumin, a small globular Ca2+-binding protein, which besides being one of the two components of lactose synthase that catalyzes the final step of the lactose biosynthesis in the lactating mammary gland, possesses a multitude of other functions. In fact, recent studies indicated that some partially folded forms of this protein possess noticeable bactericidal activity and other forms might be related to induction of the apoptosis of tumor cells. In its anti-tumorigenic function, oligomeric α-lactalbumin serves as a founding member of a new family of anticancer drugs termed liprotides (for lipids and partially denatured proteins), where an oligomeric molten globular protein acts as an "oil container" or cargo for the delivery of oleic acid to the cell membranes.
Topics: Amino Acids; Animals; Anti-Bacterial Agents; Antineoplastic Agents; Antiviral Agents; Carrier Proteins; Disease Susceptibility; Humans; Lactalbumin; Lactose; Milk Proteins; Models, Molecular; Protein Binding; Protein Conformation; Protein Multimerization; Structure-Activity Relationship
PubMed: 26916155
DOI: 10.2174/1389203717666151203003151 -
Spectrochimica Acta. Part A, Molecular... Nov 2023The combination of light and photoresponsive compounds provides a peculiar way of regulating biological systems. Azobenzene is a classical organic compound with...
The combination of light and photoresponsive compounds provides a peculiar way of regulating biological systems. Azobenzene is a classical organic compound with photoisomerization properties. Exploring the interactions between azobenzene and proteins can deepen the biochemical applications of the azobenzene compounds. In this paper, the interaction of 4-[(2,6-dimethylphenyl)diazenyl]-3,5-dimethylphenol with alpha-lactalbumin was investigated by UV-Vis absorption spectra, multiple fluorescence spectra, computer simulations, and circular dichroism spectra. Most critically, the interaction differences between proteins and the trans- and cis-isomer of ligands have been analyzed and compared. Results showed that both isomers of ligands were bound to alpha-lactalbumin to form ground state complexes and statically quenched the steady-state fluorescence of alpha-lactalbumin. The van der Waals forces and hydrogen bonding dominated the binding; the difference is that the binding of the cis-isomer to alpha-lactalbumin is more rapidly stabilized, and the binding strength is greater than the trans-isomer. These binding differences were modeled and analyzed by molecular docking and kinetic simulations, and we found that both isomers bind through the hydrophobic aromatic cluster 2 of alpha-lactalbumin. However, the bent structure of the cis-isomer is more closely aligned with the construction of the aromatic cluster and may have contributed to the above differences.
Topics: Lactalbumin; Molecular Docking Simulation; Ligands; Thermodynamics; Computer Simulation; Circular Dichroism
PubMed: 37327501
DOI: 10.1016/j.saa.2023.122965