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PubMed2006

Frontal analysis for characterizing the adsorption-desorption behavior of beta-lactoglobulin on immunoadsorbents.

High-performance frontal affinity chromatography was employed to study the adsorption-desorption kinetics characterizing the retention of beta-lactoglobulin (beta-LG) onto polyclonal anti-beta-lactoglobulin (anti-beta-LG) chromatographic supports. The adsorption and desorption processes were studied by analyzing two different elution fronts separated by a relatively long rinsing step. The method consists in performing two successive frontal injections of the protein. In between, the column was rinsed with a given volume of mobile phase (buffer alone). During this rinsing stage, a partial desorption may occur and a novel amount of protein could be adsorbed in the second frontal injection step. The whole process (first adsorption, possible desorption, and second adsorption) was simulated by a numerical procedure, in which the column was divided into a large number of slices. A model based on bi-Langmuir type kinetics was used to describe the adsorption of the protein on the support. The model assumes a non-uniform adsorbent with two types of binding sites. At equilibrium the adsorption isotherm is of the bi-Langmuir type. A global adsorption effect was considered which includes the effective binding process and the mass transfer resistances due to the transport to the binding site. Therefore, the column capacity and the kinetic parameters of the model (apparent adsorption and desorption rate constants) were determined from the best fit of the first and second adsorption fronts to the experimental ones. The other parameters of the model are the saturation capacities for the adsorption on each type of sites. The equilibrium affinity constants were estimated in a single experiment from the ratio of the apparent adsorption and desorption rate constants. The high values found (around 10(8) M(-1)) reveal a strong interaction of beta-LG with the immunoadsorbent. Kinetic measurements were carried out at different flow rates. Both the apparent adsorption and desorption kinetics were faster at larger flow rates, indicating an important contribution of the mass transfer resistance in the stagnant fluid at the particle boundary. However, as expected, close values were found for the resulting equilibrium constants calculated from the ratio of the apparent adsorption and desorption rate constant determined at various flow rates.

Chromatography, High Pressure Liquid↗
PubMed2005

Influence of environmental stresses on stability of oil-in-water emulsions containing droplets stabilized by beta-lactoglobulin-iota-carrageenan membranes.

An oil-in-water emulsion (5 wt% corn oil, 0.5 wt% beta-lactoglobulin (beta-Lg), 0.1 wt% iota-carrageenan, 5 mM phosphate buffer, pH 6.0) containing anionic droplets stabilized by interfacial membranes comprising of beta-lactoglobulin and iota-carrageenan was produced using a two-stage process. A primary emulsion containing anionic beta-Lg coated droplets was prepared by homogenizing oil and emulsifier solution together using a high-pressure valve homogenizer. A secondary emulsion containing beta-Lg-iota-carrageenan coated droplets was formed by mixing the primary emulsion with an aqueous iota-carrageenan solution. The stability of primary and secondary emulsions to sodium chloride (0-500 mM), calcium chloride (0-12 mM), and thermal processing (30-90 degrees C) were analyzed using zeta-potential, particle size and creaming stability measurements. The secondary emulsion had better stability to droplet aggregation than the primary emulsion at NaCl </=500 mM, CaCl(2) </= 2 mM, and holding temperatures </= 60 degrees C for 20 min. The interfacial engineering technology used in the study could therefore lead to the creation of food emulsions with improved stability to environmental stresses.

Calcium Chloride↗
PubMed2006

In Vitro and in Vivo nucleosome positioning on the ovine beta-lactoglobulin gene are related.

Although positioned nucleosomes are known to play a direct, localised role in regulating access to DNA sequence, they also have the potential, through their long-range distribution, to affect the detailed structure of the higher-order chromatin fibre. To investigate this possibility, we firstly mapped, in vitro, the sequence-dependent positions that the core histone octamer adopts when reconstituted onto DNA containing the ovine beta-lactoglobulin gene. These positioning sites are discussed in terms of their relative affinity for the histone octamer, their locations with respect to the gene sequence and their periodic distribution throughout the gene region. Secondly, we mapped, in vivo, the sites that nucleosomes occupy on the same sequence in liver nuclei, where the gene is transcriptionally inactive. Although the sequence is largely packaged into regularly spaced nucleosomes, reflecting a fibre of uniform higher-order structure, this organisation is disrupted by a number of unusual chromatin structures in a region stretching from the second to the third introns of the gene. A comparison of the in vitro and in vivo nucleosome positioning data shows that they are qualitatively and quantitatively related, suggesting that the structure of the higher-order chromatin fibre containing the beta-lactoglobulin gene is determined, in part, by the long-range organisation of the non-coding sequences within which the gene is embedded.

Animals↗
PubMed2005

Anomalous "unquenching" of the fluorescence decay times of beta-lactoglobulin induced by the known quencher acrylamide.

Picosecond time-resolved fluorescence, together with the addition of quenching agents, was employed to discriminate the fluorescence contributions of the two tryptophans of beta-lactoglobulin (Trp19 and Trp61) to the fluorescence decays of the protein. The fluorescence decays of beta-lactoglobulin at pH 3, 5 and 8 are best fitted using sums of three exponentials and show a dominant contribution (98%) of the components associated with the buried Trp19, which decays according to a double exponential function. The addition of acrylamide (0.05 M) causes an increase of the decay times associated with Trp19. This effect is observed at all pH values studied, but the effect is stronger at pH 3 and pH 5, than at pH 8. The unexpected increase of the decay times of Trp19 and the variation of the respective amplitudes were rationalized in terms of alterations of Trp19 mobility. The hindrance of Trp19 upon acrylamide binding was also monitored and supported by fluorescence anisotropy measurements.

Acrylamide↗
PubMed2003

Stable monomeric intermediate with exposed Cys-119 is formed during heat denaturation of beta-lactoglobulin.

The role of the free sulfhydryl group of beta-lactoglobulin in the formation of a stable non-native monomer during heat-treatment of beta-lactoglobulin solutions was investigated. Two concomitant events occurred at the earlier stage of heating: unfolding of native globular monomer and intramolecular sulfhydryl/disulfide exchange reaction. Thus, two denatured monomeric species were formed: a non-native monomer with exposed Cys-121 (Mcys121) which became reversible after cooling, and a stable non-native monomer with exposed Cys-119 (Mcys119) which exhibited both a larger hydrodynamic conformation than native monomer and low solubility at pH 4.7. The results also show that the formation of these monomeric species throughout heat-induced denaturation of native beta-lg monomers is faster than their subsequent aggregation. A mechanism describing the behavior of beta-lg denaturation/aggregation during heat-treatment under selected conditions (5.8 mg/ml, low ionic strength, pH 6.6, 85 degrees C) is presented.

Animals↗
PubMed2004

Conformational transitions in beta-lactoglobulin induced by cationic amphiphiles: equilibrium studies.

The conformational transition from the native state in water ("beta-state") to a state containing a considerable amount of alpha-helices ("alpha-state") was studied for the protein beta-lactoglobulin (BLG), from bovine milk, in several colloidal solutions containing mixed micelles or spontaneous vesicles. These aggregates were formed in the bicationic system containing the surfactant dodecyltrimethylammonium chloride (DTAC) and the lipid didodecyldimethylammonium bromide (DDAB). The beta-->alpha transition in BLG, investigated by far-ultraviolet circular dichroism spectroscopy, is induced to the same protein alpha-state by pure and mixed DDAB/DTAC micelles or vesicles. This implies a similar interaction mechanism of BLG with DDAB or DTAC, once the colloidal aggregates are formed. In premicelle DTAC solutions, the fraction of alpha-helix is lower and increases with the DTAC concentration. DDAB and DTAC also promote conformational changes in the protein tertiary structure that expose the tryptophans to a less constrained environment. These unfolding transitions were investigated by near-ultraviolet circular dichroism and steady-state fluorescence spectroscopies. In equilibrium conditions, it was found that higher DTAC (and, probably, DDAB) concentrations are needed to induce the beta-->alpha transition than to unfold the protein. beta-Lactoglobulin may therefore be considered as a model for protein-surfactant and protein-lipid interactions.

Animals↗
PubMed1998

Aggregation of beta-lactoglobulin and influence of D2O.

The conformational stability of beta-lactoglobulin increases in D2O over that in H2O. This is concluded from an increase in peak temperature by about 3 degrees C of differential scanning calorimetry (DSC) thermograms and from a decrease in overall aggregation rate. However, effects of pH and salt concentration on the heat-induced aggregation (reaction kinetics, DSC thermograms and aggregate growth) are similar in H2O and D2O. This indicates that the mechanism of heat-induced aggregation of beta-lactoglobulin is not significantly affected by replacement of H2O with D2O.

Calorimetry, Differential Scanning↗
PubMed1998

12-Bromododecanoic acid binds inside the calyx of bovine beta-lactoglobulin.

The X-ray structure of bovine beta-lactoglobulin with the ligand 12-bromododecanoic acid as a model for fatty acids has been determined at a resolution of 2.23 A in the trigonal lattice Z form. The ligand binds inside the calyx, resolving a long-standing controversy as to where fatty-acid like ligands bind. The carboxylate head group lies at the surface of the molecule, and the lid to the calyx is open at the pH of crystallization (pH 7.3), consistent with the conformation observed in ligand-free bovine beta-lactoglobulin in lattice Z at pH 7.1 and pH 8.2.

Animals↗
PubMed1997

Complete structure elucidation of a globular protein by particle beam liquid chromatography-Fourier transform infrared spectrometry and electrospray liquid chromatography-mass spectrometry. Sequence and conformation of beta-lactoglobulin.

The advantages to the use of both mass spectrometry (MS) and Fourier transform infrared spectrometry (FT-IR) in combination for the structural characterization of the tryptic digest of a model globular protein is demonstrated. HPLC has been interfaced to both spectroscopic techniques and has provided a high degree of structural detail for the target protein. beta-Lactoglobulins A and B were digested with trypsin and chromatographed with narrow-bore, reversed-phase HPLC. As determined by LC-FT-IR spectrometry, the conformation of each form of intact beta-lactoglobulin was randomized upon elution. The particle beam and the electrospray LC-MS interfaces enabled the acquisition of spectra for nanogram injection quantities. The primary structures were determined from the accurate molecular mass determinations of the digest fragments. Infrared spectra confirmed the presence of some amino acid functionalities.

Amino Acid Sequence↗
PubMed1997

Multiple peaks in high-performance liquid chromatography of proteins. beta-Lactoglobulins eluted in a hydrophobic interaction chromatography system.

The chromatographic behavior of beta-lactoglobulins when eluted in hydrophobic interaction chromatography systems is studied. By modifying some factors, such as pH and temperature, the relationship between shape of the chromatographic peak and protein structure is shown. At pH 4.5 and low temperature multiple peaks for beta-LG A and beta-LG B are observed and assigned to aggregates. The effects of other parameters, besides pH and temperature, such as volume and concentration of injected sample, contact time between protein and stationary and/or mobile phases, and nature and concentration of mobile phase upon aggregation are studied. Comparison of the chromatographic behavior of both variants of beta-lactoglobulin is made.

Animals↗
PubMed1998

Analysis of the Maillard reaction products of beta-lactoglobulin and lactose in skimmed milk powder by capillary electrophoresis and electrospray mass spectrometry.

When analysed by capillary electrophoresis, certain skimmed milk powders are seen to exhibit additional peaks migrating after the whey protein beta-lactoglobulin. Using a model reaction between beta-lactoglobulin and lactose, and studying the reaction products using electrospray mass spectrometry, it is demonstrated that these protein peaks are almost certainly due to a Maillard reaction between lactose and the epsilon-amino group of lysine. This results in the formation of a series of lactulose-protein conjugates exhibiting throughout molecular mass increments of 324, which is sufficient to allow their separation by capillary electrophoresis.

Dairy Products↗
PubMed1996

Allergy to the heat-labile proteins alpha-lactalbumin and beta-lactoglobulin in mare's milk.

BACKGROUND: Allergy to mare's milk is rare. Recently, however, mare's milk has been recommended for treatment of various ailments by practitioners of "alternative medicine," and it is available in health food stores. OBJECTIVE: We report a case of allergic reaction to mare's milk in a 51-year-old woman who was able to tolerate cow's milk. METHODS: The protein composition of mare's milk was determined by methods based on measurement of nitrogen content. The patient underwent prick and intracutaneous tests with commercially available bovine milk proteins and several mare's milk preparations, including mare's milk granulate and boiled mare's milk. RAST and immunoblotting were also performed. RESULTS: Results of skin testing and RAST with cow's milk were negative but demonstrated an IgE-mediated allergy to mare's milk. Immunoblotting revealed two allergen bands with molecular weights of 16 and 18 kd, most likely representing the whey proteins alpha-lactalbumin and beta-lactoglobulin. The bands disappeared after the mare's milk was boiled, indicating that the proteins are heat-labile. CONCLUSION: The results of this study demonstrate the existence of an IgE-mediated mare's milk allergy caused by low molecular weight heat-labile proteins, most likely alpha-lactalbumin and beta-lactoglobulin, which do not cross-react with the corresponding whey proteins in cow's milk.

Allergens↗
PubMed1997

Induction of systemic immunologic tolerance to beta-lactoglobulin by oral administration of a whey protein hydrolysate.

BACKGROUND: Oral administration of an antigen has been shown to suppress the specific immune response to this antigen. This approach, called oral tolerance, has been demonstrated with intact proteins in animal models for prevention of allergy and autoimmune diseases. OBJECTIVE: The purpose of this study was to determine whether oral tolerance can be induced with protein peptides. Partially hydrolyzed and extensively hydrolyzed cow's milk formulas were compared for their capacity to induce tolerance to cow's milk proteins. METHODS: Five-week-old Sprague-Dawley rats were fed cow's milk formulas ad libitum from day 1 to day 19. All animals were immunized with beta-lactoglobulin and ovalbumin on day 5 and bled on day 19. Sera were analyzed for specific IgE and IgG antibodies by ELISA and for functional IgE response by in vitro mast cell mediator (tritiated serotonin) release. In vivo modulation of intestinal mast cells was analyzed by the specific release of the rat mast cell protease II, and T-cell response was determined by tritiated thymidine incorporation into lymph node lymphocytes. RESULTS: Oral administration of a partially hydrolyzed cow's milk formula suppresses specific serum IgE and IgG anti-beta-lactoglobulin antibodies, as well as mediator release from rat mast cells and T-lymphocyte response. This suppression was shown to be antigen-specific and dose-dependent. An extensively hydrolyzed formula was unable to achieve the induction of such an oral tolerance. CONCLUSION: These results support the view that partially hydrolyzed proteins are able to induce specific oral tolerance, whereas extensively hydrolyzed proteins are not.

Administration, Oral↗
PubMed2000

Heat-induced gelation of globular proteins: part 3. Molecular studies on low pH beta-lactoglobulin gels.

Heat-set gels and aggregates from beta-lactoglobulin (beta-Lg), one of the major globular proteins from milk, have been studied on a molecular distance scale using negative-staining transmission electron microscopy (TEM), wide-angle X-ray diffraction (WAXD), and Fourier transform infrared spectroscopy (FTIR). The microscopy showed long linear aggregates forming in solutions at pH 2 (and sometimes 2.5) after prolonged heating. While there appeared to be no differences in aggregates formed under these conditions in H(2)O as compared with D(2)O, at all other pH and pD values, and in the presence of added salt, much shorter linear aggregates were formed. These became slightly more extended the further the pH was removed from pI. Wide-angle X-ray diffraction (WAXD) showed a diffuse beta-sheet halo at 2&theta;=19 degrees in patterns for both dried native and aggregated protein (irrespective of pH) with only a small change (sharpening) of this feature on heat treatment. Solution FTIR spectra, measured at pD=2, 2.5, 3, and 7, during heating, indicated shoulder development at 1612 cm(-1) in the carbonyl-stretching Amide I region diagnostic of a modest increase in intermolecular beta-sheet. In terms of the shoulder size, no distinctions could be made between acid and neutral aggregate structures. At all pHs, beta-lactoglobulin showed only limited secondary and tertiary structural changes in aggregation, in contrast to previous studies of insulin aggregation, where highly ordered crystalline fibrils were indicated. The current work has implications both in structural studies of food biopolymers and in ongoing studies of pathological protein self-assembly in disease states, such as spongiform encephalopathies.

Animals↗
PubMed1996

Expression, purification and immunochemical characterization of recombinant bovine beta-lactoglobulin, a major cow milk allergen.

The immunological characteristics of a recombinant beta-lactoglobulin were studied using monoclonal antibodies, polyclonal antiserum and sera from allergic patients. Recombinant beta-lactoglobulin (rBLG) was expressed in Escherichia coli strain DH5alpha and purified as described previously [Cho et al. (1994) J. Biol. Chem. 269, 11 102-11 107]. The method has been modified by adding an immunoaffinity purification step. A quantity of 5-10mg of purified rBLG per liter of medium culture can be produced. rBLG shared the same molecular weight as the natural BLG (nBLG) and also possessed at least one intrachain disulfide bridge. In HPLC, rBLG appeared as a single peak, and the purity was estimated to be greater than 95%. All the monoclonal antibodies (mAbs) used in this study recognized different epitopes of the BLG and presented compatible binding. No differences could be detected between rBLG and nBLG when tested in a Western blot with rabbit polyclonal antiserum or with three mAbs that bound preferentially the reduced and S-carboxymethylated form of BLG. In a competitive enzyme immunoassay (EIA) using either a rabbit polyclonal antiserum or four mAbs that recognized conformational epitopes, we could not distinguish between rBLG or nBLG. In direct ELISA using nBLG or rBLG as the immobilized allergen, we measured a similar concentration of specific anti-BLG IgE in five sera from allergic patients. The results of this study indicate that we have obtained a rBLG with biochemical and immunological properties very similar to nBLG.

Allergens↗
PubMed1999

Alcohol-induced denaturation of beta-lactoglobulin: a close correlation to the alcohol-induced alpha-helix formation of melittin.

Alcohols denature the native structure of proteins and induce alpha-helical structure. The potential of alcohols causing such effects varies substantially depending on the alcohol species. With beta-lactoglobulin as a model protein, we compared the effects of various alcohols and observed the additive contribution of each group constituting the alcohol molecules. Whereas the hydrophobic hydrocarbon group promotes the transition according to their size, hydrophilic hydroxyl group suppresses the transition. Halogen groups promote the transition depending on their type and number. It has been known that alcohols induce the alpha-helical structure on the short peptides such as melittin. There is a close correlation between the potentials of alcohol in denaturing beta-lactoglobulin and those in inducing the helical structure in melittin, indicating that the underlying mechanisms of the two phenomena are the same.

Alcohols↗
PubMed1997

Conformational transitions provoked by organic solvents in beta-lactoglobulin: can a molten globule like intermediate be induced by the decrease in dielectric constant?

BACKGROUND: It is known that nonnative states of protein molecules can exist in living cells and can be involved in a number of physiological processes. It has also been established that the membrane surface can be responsible for the partial denaturation of proteins due to negative charges on it. The local decrease in the effective dielectric constant of water near the organic surface has been suggested to be an additional driving force for protein denaturation in the membrane field, but data to confirm this suggestion were lacking. RESULTS: Conformational transitions induced in beta-lactoglobulin by methanol, ethanol, isopropanol, dimethylformamide and dioxane were studied by near and far UV circular dichroism, steady-state tryptophan fluorescence and fluorescence decay of 8-anilinonaphthalene-1-sulfonate (8-ANS). The existence of at least two noncoinciding cooperative transitions has been established in all solvent systems studied. The first of these transitions describes the disruption of rigid tertiary structure in protein molecules, while the second reflects the formation of an expanded helical conformation typical of proteins in concentrated organic solvents. This means that the organic solvents provoke the formation of a denatured intermediate state with pronounced secondary structure and native-like compactness. We show that the positions of maxima in fI versus dielectric constant dependence virtually coincide for all five solvent systems studied. CONCLUSIONS: The decrease in the dielectric constant of the solvent induces in beta-lactoglobulin an equilibrium intermediate state. This state, being denatured, is relatively compact and has pronounced secondary structure and high affinity for the hydrophobic fluorescent probe 8-ANS, i.e. possesses all the properties of the molten globule intermediate state.

Anilino Naphthalenesulfonates↗
PubMed1999

Process steps for the preparation of purified fractions of alpha-lactalbumin and beta-lactoglobulin from whey protein concentrates.

Fractions enriched with alpha-lactalbumin (alpha-la) and beta-lactoglobulin (beta-lg) were produced by a process comprising the following successive steps: clarification-defatting of whey protein concentrate, precipitation of alpha-lactalbumin, separation of soluble beta-lactoglobulin, washing the precipitate, solubilization of the precipitate, concentration and purification of alpha-la. The present study evaluated the performance of the process, firstly on a laboratory scale with acid whey and then on a pilot scale with Gouda cheese whey. In both cases soluble beta-lg was separated from the precipitate using diafiltration or microfiltration and the purities of alpha-la and beta-lg were in the range 52-83 and 85-94% respectively. The purity of the beta-lg fraction was higher using acid whey, which does not contain caseinomacropeptide, than using sweet whey. With the pilot scale plant, the recoveries (6% for alpha-la; 51% for beta-lg) were disappointing, but ways of improving each step in the process are discussed.

Cheese↗