Proteins Α-lactalbumin Research Articles

The Formation of an Anti-Cancer Complex Under Simulated Gastric Conditions

A potent anti-cancer complex has previously been formed from two major components of milk. Human/bovine α-lactalbumin made lethal to tumour cells (H/BAMLET) is a protein–fatty acid complex that has been produced using the whey protein α-lactalbumin (α-LA) and the fatty acid oleic acid (OA). It was shown that it possesses selective anti-tumour and anti-microbial activity, which was first identified in acidic fractions of human breast milk. The aim of this study was to determine whether the two components would form a bioactive complex during simulated gastric (GI) transit. Results showed that a complex consisting of α-LA and OA is formed as the protein unfolds under acidic conditions and subsequently refolds upon pH increase. Analysis of this complex using Nuclear Magnetic Resonance and Fourier Transform Infra-Red (FTIR) spectroscopies estimated a stoichiometry of 4.1 and 4.4 oleic acids per mole of protein, respectively. FTIR and fluorescence spectroscopies showed that the structure was similar to that of BAMLET. Cytotoxicity testing against cancer cell line U937 cells showed that the complex had an LC50 value of 14.08 μM compared to 9.15 μM for BAMLET. These findings suggest that a BAMLET-like complex may be formed under the tested in vitro GI conditions.

Synthesis and Characterization of Various Protein <i>α</i>-Lactalbumin Nanotubes Structures by Chemical Hydrolysis Method

New water-based nanofluids including unparalleled milk protein α-lactalbumin hollow nano-bio-tubes using low cost, available and advanced partial chemical hydrolysis strategy in bottom-up nano-assembly have been employed in this work. The aqueous sol-gel chemistry in nanotechnology which we selected for this goal offers new fabrication as interesting smart protein nanotubes. The kinds of nanometer sized tubular structures such as waved, helically coiled, bent, bamboo-shaped, bead-like and branched single-walled protein nanotubes (SWPNTs) with a range of 3 - 8 nm in outer diameters were produced by this method. Complete characterization for natural produced nanotubes including SEM, TEM images, G bond and D bond in Raman spectroscopy, XRD patterns, DLS (Dynamic Light Scattering) and FTIR analysis were evaluated which they are most significant experiments in synthesized protein nanotubes soluble in clear water nanofluids and stabilization of transparent nanofluids was proved within more than one year after preparation. Various necessary ligand ion salts such as Mn2+, Zn2+ and Ca2+ or mixtures as bridge makers and producing biological self-assembly hollow SWPNTs were performed and we focused on new chemical technology under specific acidic hydrolysis method not conventional enzymatic proteolysis and applying surfactants, pH reagent, Tris-HCl buffer, polar solvent which could be produced by β-sheet stacked hydrolysed protein α-lactalbumin mechanism under appropriate conditions to achieving high efficiency new protein nanotubes skeleton. They can be promising materials applied in food science, diet nutrition, nanomedicine, nano-biotechnology and surgery.

Producing the Controlled Hybrid Lattice of Natural Protein Nanotubes by Chemical Hydrolysis for Nanomedicine

This work was instigated by the fact that sol-gel chemistry provides a relatively simple way to incorporate recognition species in a stable host green environment. In this new strategy, which represents a low cost example of bottom-up nanoassembly, chemistry art is entering the field of nanobiotechnology in fabrication and control of an expanded homogeneous length of separate single-walled and ordered, helical lattice-like open-ended natural protein nanotubes (PNTs) and also creative novel bio-nanohybrids. A new type of protein nanohybrids containg nanotubes-nanorods, nanotubes-nanofibers, nanotubes core-shell nanofibers were prepared using an electrostatic self-assembly method with the aid of chemical partial hydrolysis of milk protein α-lactalbumin (sol-gel technique) at a suitable pH value for the first time. They can have long helically coiled length and are promising for high capacity drug loading and applying in nanomedicine as organ transplantation in human body and implant material, because of their improved stability and unique mechanical and lattice thermal resistivity properties. In this study, various valuable ligand or binding sites such as distinct Mn+2, Ca+2or Zn+2 cations were used for incorporated into protein nanostructures as the self-assembly essential stimulant motor. It was found that the designed nanobioproducts could retain and stabilize as very clear and transparent green aqueous nanobiofluids during two years.

Electrostatic interactions play an essential role in the binding of oleic acid with α‐lactalbumin in the HAMLET‐like complex: A study using charge‐specific chemical modifications

Human α-lactalbumin made lethal to tumor cells (HAMLET) and its analogs are partially unfolded protein-oleic acid (OA) complexes that exhibit selective tumoricidal activity normally absent in the native protein itself. To understand the nature of the interaction between protein and OA moieties, charge-specific chemical modifications of lysine side chains involving citraconylation, acetylation, and guanidination were employed and the biophysical and biological properties were probed. Upon converting the original positively-charged lysine residues to negatively-charged citraconyl or neutral acetyl groups, the binding of OA to protein was eliminated, as were any cytotoxic activities towards osteosarcoma cells. Retention of the positive charges by converting lysine residues to homoarginine groups (guanidination); however, yielded unchanged binding of OA to protein and identical tumoricidal activity to that displayed by the wild-type α-lactalbumin-oleic acid complex. With the addition of OA, the wild-type and guanidinated α-lactalbumin proteins underwent substantial conformational changes, such as partial unfolding, loss of tertiary structure, but retention of secondary structure. In contrast, no significant conformational changes were observed in the citraconylated and acetylated α-lactalbumins, most likely because of the absence of OA binding. These results suggest that electrostatic interactions between the positively-charged basic groups on α-lactalbumin and the negatively-charged carboxylate groups on OA molecules play an essential role in the binding of OA to α-lactalbumin and that these interactions appear to be as important as hydrophobic interactions.

Global and local indices for characterizing biomolecular flexibility and rigidity

Understanding flexibility and rigidity characteristics of biomolecules is a prerequisite for understanding biomolecular structural stability and function. Computational methods have been implemented that directly characterize biomolecular flexibility and rigidity by constraint network analysis. For deriving maximal advantage from these analyses, their results need to be linked to biologically relevant characteristics of a structure. Such links are provided by global and local measures ("indices") of biomolecular flexibility and rigidity. To date, more than 14 indices are available with sometimes overlapping or only vague definitions. We present concise definitions of these indices, analyze the relation between, and the scope and limitations of them, and compare their informative value. For this, we probe the structural stability of the calcium binding protein α-lactalbumin as a showcase, both in the "ground state" and after perturbing the system by changing the network topology. In addition, we introduce three indices for the first time that extend the application domain of flexibility and rigidity analyses. The results allow us to provide guidelines for future studies suggesting which of these indices could best be used for analyzing, understanding, and quantifying structural features that are important for biomolecular stability and function. Finally, we make suggestions for proper index notations in future studies to prevent the misinterpretation and to facilitate the comparison of results obtained from flexibility and rigidity analyses.

Qualitative and quantitative characterization of the arsenic‐binding behaviour of sulfur‐containing peptides and proteins by the coupling of reversed phase liquid chromatography to electrospray ionization mass spectrometry

Phenylarsenic-substituted cysteine-containing peptides and proteins were completely differentiated from their unbound original forms by the coupling of reversed phase liquid chromatography with electrospray ionization mass spectrometry. The analysis of biomolecules possessing structure-stabilizing disulfide bridges after reduction provides new insights into requirements concerning the accessibility of cysteine residues for reducing agents as well as for arsenic compounds in a spatial protein structure. Complementary binding studies performed using direct ESI-MS without chromatographic coupling in different solvent systems demonstrated that more than one binding site were activated for aprotinin and lysozyme in denaturing solvents because of a stronger defolding. From the intensities of the different charge states occurring in the mass spectra as well as from the LC elution behaviour, it can be deduced that the folding state of the arsenic-bound protein species resembles the native, oxidized conformation. In contrast, although the milk protein α-lactalbumin has several disulfide bridges, only one phenylarsenic moiety was bound under strongly denaturing conditions. Because of the charge state distribution in the ESI mass spectra, a conformational change to a molten globule structure is assumed. For the second considered milk protein ß-lactoglobulin, a noncovalent interaction with phenylarsine oxide was detected. In general, smaller apparent binding constants for the condensation reactions of the biomolecules with phenylarsine oxide leading to covalent arsenic-sulfur bindings were determined from direct injection ESI-MS measurements than from LC-ESI-MS coupling. The following order of binding affinities for one phenylarsenic group can be assumed from both ESI-MS and LC-ESI-MS: nonapeptide vasopressin > nonapeptide vasotocin > lysozyme > aprotinin > α-lactalbumin > thioredoxin. Kinetic investigations by LC-ESI-MS yielded a partial reaction order of 2 for vasopressin, Lys and α-lactalbumin and corresponding half-lives of 0.93, 2.56 and 123.5 min, respectively.

Statistical Analysis of Ion Mobility Spectrometry. II. Adaptively Biased Methods and Shape Correlations

Following a recent effort [J. Am. Soc. Mass Spectrom. 23, 386-396 (2012)], we continue to explore computational methodologies for generating molecular conformations to support collisional cross sections suggested by ion mobility measurements. Here, adaptively biased molecular dynamics (ABMD) simulations are used to sample the configuration space and to achieve flat-histogram sampling along the reaction coordinates of the first two moments of the gyration tensor. The method is tested and compared with replica-exchange simulations on triply-protonated bradykinin and on a larger 25-residue peptide. It is found to have a much higher efficiency for producing large sets of conformations in a broad range of diffusion cross-sections, whereas it does not compete with conventional replica-exchange molecular dynamics in locating the lowest-energy structure. Nevertheless, the broad sampling obtained from the ABMD method allows to quantitatively correlate the diffusion cross-section Ω with other geometric order parameters that have simpler interpretation. The strong correlations found between the diffusion cross-section and the radius of gyration, the surface area and the volume of the convex hull suggest an optimal template for accurately mimicking the variations of Ω in a broad range of conformations, using only geometrical information and doing so at a very moderate computational cost. The existence of such a correlation is confirmed on the much larger protein α-lactalbumin.

Structure and antioxidant activity of Maillard reaction products from α-lactalbumin and β-lactoglobulin with ribose in an aqueous model system

Maillard reaction products (MRPs) were prepared from aqueous model mixtures containing 3% (w/w) ribose and 3% (w/w) of the dairy proteins α-lactalbumin (α-LA) or β-lactoglobulin (β-LG), heated at 95 °C, for up to 5 h. The pH of MRPs decreased significantly during heat treatment of α-LA-Ribose and β-LG-Ribose mixtures from 8.4 to 5.3. The amino group content in MRPs, derived from the α-LA-Ribose and β-LG-Ribose model system, was decreased noticeably during the first hour and did not change thereafter. The loss of free ribose in MRPs was higher for β-LG-Ribose than for α-LA-Ribose. During the Maillard reaction, the concentration of native and non-native α-LA, or β-LG, decreased and the formation of aggregates was observed. Fluorescence intensity of the β-LG-Ribose MRPs reached maximum within 1 h, compared to 2 h for α-LA-Ribose MRPs. Meanwhile, modification of the UV/vis absorption spectra for α-LA and β-LG was mainly due to a condensation reaction with ribose. Dynamic light scattering showed a significant increase in the particle size of the MRPs. Size exclusion chromatography of MRPs revealed the production of both high and low molecular weight material. Electrophoresis of MRPs indicated polymerization of α-LA and β-LG monomers via inter-molecular disulfide bridge, but also via other covelant bonds. MRPs from α-LA-Ribose and β-LG-Ribose exhibited increased antioxidant activities, therefore theses MRPs may be used as natural antioxidants in food products.

Synthesis of Biopolymeric α-Lactalbumin Protein Nanoparticles and Nanospheres as Green Nanofluids Using in Drug Delivery and Food Technology

New nanofluids carrier containing fine homogeneous whey protein α-lactalbumin spherical nanoparticles and nanospheres have been synthesized by three methods in this research. Biochemical enzymatic catalysis by V8- protease, chemical hydrolysis (sol-gel assisted self assembly method) using cationic templates and metal ions ligand forming, and also by direct acidic titration method with applying various ethylene glycol derivatives containg alcohol solvents. Repeatability and stability of synthetic nanoparticles and nanospheres milk protein structures in clear and crystalline green water-based nanofluids was confirmed by dynamic light scattering (DLS) analysis and TEM images (2-5nm) even after a one year period of preparation. Fabrication of non-toxic nanosolutions-nanofluids including nonionic and cationic surfactants, cosurfactants and suitable solvents, besides proper incubation temperatures (37-50°C), adjusted aging times and doped of divalent ions–bridging (Ca2+, Mn2+ and Zn2+ ) between specific amino acids and carboxyl groups, are so vital factors in kinetically controlled self-assembly phenomenon of the natural protein building blocks to form high resistant and homogeneous nanoparticles and nanospheres in transparent and clear green nanofluids. Such nanofluids can have many applications in food and pharmaceutical industries for example in formulate foods with high nutritional value in oral and digestive (ingestion) routes, and drug delivery. Interesting textures of SEM images have also shown these new nanobiostructures of natural milk protein morphologies.

Transcript abundance of hormone receptors, mammalian target of rapamycin pathway-related kinases, insulin-like growth factor I, and milk proteins in porcine mammary tissue1

Prolactin, glucocorticoids, and insulin are commonly used to induce milk protein synthesis in bovine mammary cell cultures. In addition, administration of GH increases milk yield in dairy cows, likely via the mammalian target of rapamycin (mTOR) pathway and IGF-I synthesis. As such, the hypothesis of this study was that mRNA abundance of hormone receptors, mammalian target of mTOR pathway-related kinases, IGF-I, and milk protein-encoding genes increases in the porcine mammary gland in response to greater lactation demand. Selected genes included those encoding for receptors of GH (GHR), insulin (INSR), glucocorticoid (NR3C1), prolactin (PRLR), IGF-I (IGF-I), mTOR (FRAP1), and p70S6 kinases (RPS6KB1), and the milk proteins α-lactalbumin (LALBA) and β-casein (CSN2). Mammary tissue was biopsied from 4 sows on d 110 of gestation (prepartum), d 5 and 17 of lactation, and d 5 after weaning (postweaning), and gene expression was quantified by reverse-transcription quantitative PCR. Compared with prepartum, d 5 of lactation increased (P < 0.001) NR3C1, tended to increase (P = 0.06) GHR, and decreased (P < 0.001) PRLR mRNA abundance. Compared with d 5 of lactation, d 17 of lactation increased PRLR (P < 0.001) and decreased GHR (P < 0.01). Expression of INR and FRAP1 did not differ when comparing either prepartum or d 17 of lactation with d 5 of lactation. Compared with d 17 of lactation, postweaning decreased (P < 0.001) PRLR, did not affect INSR, and increased both IGF-I and GHR (P < 0.05) mRNA abundance. From prepartum to d 17 of lactation, NR3C1 mRNA abundance was positively correlated with CSN2 (r = 0.85; P < 0.001) and LALBA mRNA abundance (r = 0.79; P = 0.002), whereas mRNA abundance of GHR tended to be positively correlated with that of IGF-I (r = 0.46; P = 0.06). In conclusion, expression of the genes NR3C1, PRLR, GHR, and IGF-I changed in the porcine mammary gland during the prepartum to postweaning periods, but only NR3C1 mRNA abundance was positively correlated with expression of CSN2 and LALBA.

Transcript abundance of amino acid transporters, β-casein, and α-lactalbumin in mammary tissue of periparturient, lactating, and postweaned sows

The objective of these experiments was to test the hypothesis that transcript abundance of cationic AA transporter- and milk protein-encoding genes increase in the porcine mammary gland in response to higher lactation demand. Genes of interest included those encoding for the milk proteins α-lactalbumin (α-LA) and β-casein (β-CN; LALBA and CSN2, respectively), and AA transporter b0,+AT, y+LAT1, y+LAT2, ATB0,+, CAT-1, and CAT-2b (SLC7A9, SLC7A7, SLC7A6, SLC6A14, SLC7A1, and SLC7A2, respectively). Mammary tissue was biopsied from 4 sows on d 110 of gestation (prepartum), on d 2 (early postpartum), on d 5 (early), and d 17 (peak) of lactation, and on d 5 after weaning (postweaning), and mRNA of target genes quantified by reverse transcription quantitative PCR. Compared with prepartum, CAT-1, ATB0,+, y+LAT2, β-CN, and α-LA mRNA abundance was higher at early lactation, whereas compared with early lactation, only CAT-1 and α-LA mRNA abundance was higher at peak lactation. The CAT-2b, y+LAT1, and b0,+AT mRNA abundance did not differ when comparing either prepartum or peak lactation to early lactation. Compared with peak lactation, postweaning mRNA abundance of CAT-1, ATB0,+, α-LA, and β-CN decreased, y+LAT2, CAT-2b, and b0,+AT remained unchanged, and y+LAT1 increased. The mRNA abundance of y+LAT2 increased from early postpartum to early lactation, and remained unchanged for CAT-1, ATB0,+, α-LA, and β-CN. From prepartum to peak lactation, the mRNA abundance of CAT-1, y+LAT2, and ATB0,+ was positively correlated with that of β-CN and α-LA. In conclusion, the expression of genes encoding for y+LAT1, CAT-2b, and b0,+AT remained unchanged in porcine mammary glands over prepartum to peak lactation period, whereas expression of genes encoding for CAT-1, ATB0,+, and y+LAT2 was upregulated and positively correlated to expression of genes encoding for the mammary synthesized milk proteins β-CN and α-LA.

Comparison of the Chaperoning Action of Glycerol and β-Casein on Aggregation of Proteins in the Presence of Crowding Agent

β-Casein is one of the major components of the milk micelles of most mammals and has been shown to exhibit in vitro chaperone-like activity. Glycerol is a chemical chaperone belonging to the polyol family, which increases protein stability and inhibits protein aggregation. These prompted us to compare the chaperone-like activity of β-casein and glycerol. In this study, the effect of β-casein and glycerol on folding of the target proteins (ovotransferrin, insulin and α-lactalbumin) in the presence of dextran, as a macromolecular crowding agent, is examined using visible absorption spectroscopy, intrinsic fluorescence spectroscopy, 1-anilino-8-naphthalene sulfonic acid fluorescence binding and near CD spectroscopy. In the presence of dextran, the rate and extent of aggregation of target proteins was enhanced and β-casein was less effective in preventing the aggregation and precipitation of target proteins. These data support the hypothesis that β-casein interacts more effectively with slowly aggregating rather than rapidly aggregating target proteins. It is proposed that dextran-induced changes to protein conformation and the rate of intermolecular association are in a kinetic competition with the chaperoning action of β-casein; however our results demonstrated the higher activity of glycerol, as a chemical chaperone, than β-casein on the folding of target proteins, especially in the presence of dextran. This is likely due to the stabilizing effect of glycerol on protein structure and environment. The implications for the in vivo functions of β-casein and glycerol, based on their exhibiting such in vitro chaperone-like activities, are discussed.

Comparison of the metabolic effects of milk-derived α-lactalbumin and amino acids mixture with equal composition in diet-induced obese mice

The effects of milk-derived whey protein α-lactalbumin (α-lac) and amino acid mixture with equal amino acid profile (AA) on weight loss and weight regain in a model of diet-induced obesity were compared. Obesity was induced in C57BL/6J mice with a high-fat diet. Energy restriction was performed for 50 days on casein (calcium 0.8%) and two different high-calcium (1.8%) whey protein-based (α-lac + Ca and AA + Ca) diets, after which the animals were fed with the same diets ad libitum for additional 50 days. The mice on α-lac and AA lost significantly more weight and body fat than mice on casein diet ( P < 0.05). There was no difference between α-lac and AA diets. Ad libitum feeding after weight loss resulted in weight regain and fat accumulation in all groups. Our findings indicate that the anti-obesity effects of α-lac during energy restriction can be reproduced by amino acid mixture with equal amino acid profile.

MALDI-TOF MS Characterization of Glycation Products of Whey Proteins in a Glucose/Galactose Model System and Lactose-free Milk

The major modifications induced by thermal treatment of whey proteins α-lactalbumin (α-La) and β-lactoglobulin (β-Lg) in a model system mimicking lactose-free milk (L(-) sugar mix) were investigated by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS). The analysis of the intact α-La revealed species with up to 7 and 14 adducts from lactose and sugar mix, respectively, whereas for β-Lg 3 and up to 5 sugar moieties were observed in the case of lactose and sugar mix experiments, respectively. A partial enzymatic hydrolysis with endoproteinase AspN prior to mass spectrometric analysis allowed the detection of further modifications and their localization in the amino acid sequence. Using α-cyano-4-chlorocinnamic acid as MALDI matrix, it could be shown that heating α-La and β-Lg with glucose or galactose led to the modification of lysine residues that are not glycated by lactose. The higher glycation degree of whey proteins in a lactose-free milk system relative to normal milk with lactose reflects the higher reactivity of monosaccharides compared to the parent disaccharide. Finally, the analysis of the whey extract of a commercial lactose-free milk sample revealed that the two whey proteins were present as three main forms (native, single, and double hexose adducts).

Single-Walled Natural Protein Nanotubes Soluble in Polar Solvent

ABSTRACT Nano-biotechnology has the ability to unravel nature's secrets, yielding a deeper understanding of how she effortlessly produces intricate structures from simple building blocks. Attempts are made to mimic the natural bottom-up fabrication with the aim of creating new and varied milk protein nanotubes structures with valuable uses. A novel design and synthesis of various α-lactalbumin protein nanotubes by chemical hydrolysis (sol-gel method) and biochemical enzymatic hydrolysis as fabricated. Using different organic surfactants (directing agents) as suitable templates in order to form various remarkable structures such as beaded protein nanotubes, Y-junction, bamboo-shaped, perpendicular and horizontal hollow, sweep-like network and interesting morphologies including nanoemulsions and green nanofluids for application in food and pharmaceutical industries is the essential goal of this research. Several surfactants (for example, nonionic acetylacetone [acac], citric acid, urea, triethanolamine [TEA...

A prophylactic vaccine for breast cancer? Why not?

In a recent issue of Breast Cancer Research, Watson and Gusterson wrote a viewpoint article [1] about our strategy for developing a prophylactic breast cancer vaccine [2]. Th e authors base much of their view on several misconceptions and misrepresentations that we wish to clarify. We did not immunize mice with an antibody as the authors claim in their abstract. We immunized mice with a single injection of whole recombinant mouse α-lactalbumin protein, and this single immunization provided highly signifi cant protection and therapy against the growth of tumors in several widely used mouse models for breast cancer. Th e authors indicate that our ‘approach was based on the hypothesis that removal of an organ will provide protection against cancer developing in that organ’ [1]. Th e notion that excised organs have decreased risk for developing tumors is obviously simplistic and misrepresents our actual hypothesis stated in the fi rst sentence of our article: ‘a full-strength autoimmune attack suffi cient to induce organ-specifi c failure may provide protection and therapy against tumors derived from the targeted organ’ [2].

Fast Proton Titration Scheme for Multiscale Modeling of Protein Solutions.

Proton exchange between titratable amino acid residues and the surrounding solution gives rise to exciting electric processes in proteins. We present a proton titration scheme for studying acid-base equilibria in Metropolis Monte Carlo simulations where salt is treated at the Debye-Hückel level. The method, rooted in the Kirkwood model of impenetrable spheres, is applied on the three milk proteins α-lactalbumin, β-lactoglobulin, and lactoferrin, for which we investigate the net-charge, molecular dipole moment, and charge capacitance. Over a wide range of pH and salt conditions, excellent agreement is found with more elaborate simulations where salt is explicitly included. The implicit salt scheme is orders of magnitude faster than the explicit analog and allows for transparent interpretation of physical mechanisms. It is shown how the method can be expanded to multiscale modeling of aqueous salt solutions of many biomolecules with nonstatic charge distributions. Important examples are protein-protein aggregation, protein-polyelectrolyte complexation, and protein-membrane association.

Partitioning of α-lactalbumin and β-lactoglobulin in whey protein concentrate/hydroxypropylmethylcellulose aqueous two-phase systems

Whey protein concentrate (WPC) was fractionated by using hydroxypropylmethylcellulose (HPMC) at pH 6.5. Incompatible mixtures with different proportions of HPMC and WPC were prepared. After phase separation, the protein concentration in both phases was determined by the Kjeldahl method and the proportion of each protein by SDS-PAGE combined with image analysis. The results show that the low molecular weight proteins α-lactalbumin (α-lac) and β-lactoglobulin (β-lg) were retained in high proportion in the upper phase (about 90% compared to 64% of WPC). The most efficient condition to fractionate β-lg and α-lac was the phase separation of an incompatible mixed system with a high initial concentration of WPC and a low initial concentration of HPMC i.e., WPC 20%, wt/wt/HPMC 0.5%, w/w. It can be concluded that the thermodynamic incompatibility which arises from mixing WPC with HPMC could be used as a method for fractionation of whey proteins.

Effects of fermentation by lactic acid bacteria on the antigenicity of bovine whey proteins

The main whey proteins alpha-lactalbumin (alpha-LA) and beta-lactoglobulin (beta-LG) are considered as the major allergens in cow's milk. Microbial fermentation can produce some proteolytic enzymes, which can induce the degradation of milk protein allergens. In this study, the effects of fermentation by lactic acid bacteria on the antigenicity of alpha-LA and beta-LG were investigated using indirect competitive ELISA. Meanwhile, the proteolysis of milk proteins was detected by TNBS assay and SDS-PAGE electrophoresis. Fermentation by lactic acid bacteria could significantly reduce the antigenicity of alpha-LA and beta-LG in skim milk. Combined strains of Lactobacillus helveticus and Streptococcus thermophilus were the most effective in reducing the antigenicity of both whey proteins. In addition, alpha-LA and beta-LG antigenicity decreased to a lower value at 6 h of fermentation and at 0.5 d of cold storage by fermentation with the combined strains. The results of TNBS assay and SDS-PAGE electrophoresis showed that lactic acid bacteria strains used in this study hydrolysed whey proteins only to a limited extent. The fermentation with lactic acid bacteria is an effective way to reduce whey proteins antigenicity.

Experimentally Approaching the Solvent-Accessible Surface Area of a Protein: Insights into the Acid Molten Globule of Bovine α-Lactalbumin

Each conformational state of a protein is inextricably related to a defined extent of solvent exposure that plays a key role in protein folding and protein interactions. However, accurate measurement of the solvent-accessible surface area (ASA) is difficult for any state other than the native (N) state. We address this fundamental physicochemical parameter through a new experimental approach based on the reaction of the photochemical reagent diazirine (DZN) with the polypeptide chain. By virtue of its size, DZN is a reasonable molecular mimic of aqueous solvent. Here, we structurally characterize nonnative states of the paradigmatic protein α-lactalbumin. Covalent tagging resulting from unspecific methylene (:CH2) reaction allows one to obtain a global estimate of ASA and to map out solvent accessibility along the amino acid sequence. By its mild apolar nature, DZN also reveals a hydrophobic phase in the acid-stabilized state of α-lactalbumin, in which there is clustering of core residues accessible to the solvent. In a fashion reminiscent of the N state, this acid-stabilized state also exhibits local regions where increased :CH2 labeling indicates its nonhomogenous nature, likely pointing to the existence of packing defects. By contrast, the virtual absence of a defined long-range organization brings about a featureless labeling pattern for the unfolded state. Overall, :CH2 labeling emerges as a fruitful technique that is able to quantify the ASA of the polypeptide chain, thus probing conformational features such as the outer exposed surface and inner cavities, as well as revealing the existence of noncompact apolar phases in nonnative states.