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PubMed1992

An increased level of antibodies to beta-lactoglobulin is a risk determinant for early-onset type 1 (insulin-dependent) diabetes mellitus independent of islet cell antibodies and early introduction of cow's milk.

Using a case-control design we have studied whether antibodies to cow's milk proteins are risk determinants for childhood-onset Type 1 (insulin-dependent) diabetes mellitus independent of early exposure to cow's milk formula and islet cell antibodies. Sera from 116 recent-onset diabetic children and 112 age- and sex-matched control children were analysed for cow's milk protein IgA, IgG and IgM antibodies, beta-lactoglobulin IgA and IgM antibodies and islet cell antibodies. The titres were compared to questionnaire data on duration of breast-feeding and introduction of formula feeding. Most antibody levels tended to be increased among diabetic compared to control children. This was statistically significant for cow's milk protein IgA antibodies (p less than 0.001) and beta-lactoglobulin IgA antibodies (p less than 0.01) as well as for islet cell antibody-positivity which was found among 92% of the diabetic and 3% of control children. The differences in cow's milk protein antibodies as well as beta-lactoglobulin antibodies were more pronounced among children with an early onset of Type 1 diabetes. Breast-feeding duration was significantly inversely related to the log of beta-lactoglobulin IgG (r = -0.16, p = 0.04) and the log of cow's milk protein IgA antibodies (r = -0.17, p less than 0.001). A positive correlation was found between formula feeding and the logarithm of beta-lactoglobulin IgG antibodies (r = 0.22, p = 0.01) and the log of cow's milk protein IgA antibodies (r = 0.16, p = 0.04).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1996

Isolation and rapid sequence characterization of two novel bovine beta-lactoglobulins I and J.

Two novel bovine beta-lactoglobulins I and J have been isolated from bovine milk and characterized by isoelectric focusing. Their primary structure was determined by a very rapid method consisting of a combination of Edman sequencing, mass analysis, and ladder sequencing by mass spectrometry. We found that both new beta-lactoglobulins are of the bovine beta-lactoglobulin B-variant type. beta-lactoglobulin I shows Gly instead of Glu at position 108, whereas beta-lactoglobulin J shows a Pro-to-Leu exchange at position 126.

Amino Acid Sequence↗

PubMed1994

Conformation changes of beta-lactoglobulin: an ATR infrared spectroscopic study of the effect of pH and ethanol.

Fourier transform infrared spectroscopy has been applied to investigate the secondary structural changes of beta-lactoglobulin in water/ethanol mixtures. The studies were carried out at two different pHs and at high protein concentrations. The spectra were recorded using an attenuated total reflection cell. The amide I band of beta-lactoglobulin in water reveals large amounts of intra extended beta-sheet structure. About 20% ethanol, beta-lactoglobulin unfolds and beta-strand formation is observed. alpha-Helices are built up by increasing the ethanol concentration up to 30%. In 50% ethanol, beta-lactoglobulin gels providing the apparent pH are neutral. The secondary structural changes of beta-lactoglobulin were observed on the similarity maps obtained by Principal Component Analysis.

Animals↗

PubMed1998

Temporal profiles of appearance of DNase I hypersensitive sites associated with the ovine beta-lactoglobulin gene differ in sheep and transgenic mice.

The ovine milk protein beta-lactoglobulin is expressed in a distinct temporal pattern during lactogenesis. This expression pattern is reflected in the temporal profile of appearance of DNase I hypersensitive sites (HS) associated with the beta-lactoglobulin gene in the mammary gland. Specifically, HSIV and HSV are present prior to the first major increase in expression, which occurs at mid-pregnancy, while HSI displays the converse profile, being detected after mid-pregnancy and during lactation. The extent of DNase I digestion at HSIII, encompassing the promoter region, reflects the level of beta-lactoglobulin expression. In transgenic mouse mammary chromatin, beta-lactoglobulin transgenes display the same set of DNase I hypersensitive sites as in sheep mammary chromatin. The temporal profile, however, differs from that seen in sheep: notably, HSIV and HSV are detected during lactation. The fact that beta-lactoglobulin transgenes lacking HSIV and HSV are expressed but display a reduced transcription rate per integrated copy is compatible with a functional role for these regions. This suggests that HSIV and HSV may increase the likelihood of high-level transgene expression.

Animals↗

PubMed2005

Structural changes of beta-lactoglobulin during thermal unfolding and refolding--an FT-IR and circular dichroism study.

We have quantitatively characterized by FT-IR spectroscopy the contents of secondary structure of beta-lactoglobulin during thermal unfolding and subsequent refolding. Our data clearly indicate that considerable amount of secondary structure, particularly beta-sheet, still remained intact even at 90 degrees C. Noticeable changes in secondary structure of beta-lactoglobulin were observed only above 70 degrees C. The refolded protein regained, within limits of experimental error, all of the secondary structure lost during thermal unfolding. The data also indicate that the refolding mechanism operating at pH 7.0 and 2.0 are the same. Identical secondary structure of native and refolded beta-lactoglobulin was also indicated by far-UV circular dichroic spectra of the two forms of protein. Near UV circular dichroic spectra of the same two forms showed considerable differences indicating less tertiary structure of refolded beta-lactoglobulin. The combined CD and FT-IR data indicated that refolded form of beta-lactoglobulin could be characterized as a molten globule state as it had native-like secondary structure and compromised tertiary structure.

Circular Dichroism↗

PubMed1980

Spectroscopic characterization of beta-lactoglobulin-retinol complex.

1. The absorption spectrum of retinol when bound to beta-lactoglobulin is vibrationally resolved. The circular dichroism spectrum exhibits the same structure, as does the fluorescence excitation spectrum. 2. Two molecules of retinol are bound per protein dimer, with a binding constant (Kd) of 2 x 10(-8) M. Also, by fluorescence titration it was found that the monomer binds one molecule of retinol with essentially the same Kd. 2. Energy transfer occurs from tryptophan (donor) to retinol (acceptor) with a rate constant, k, of 4.4 x 10(8) s-1. The distance between the centers of mass of the transition is 34 A, corresponding to the energy transfer efficiency of 44%. 4. The fluoresence lifetime of retinol increases dramatically on binding to beta-lactoglobulin, from approx. 2 to approx. 10 ns, as does the fluorescence quantum yield. 5. The retinol binding to beta-lactoglobulin does not show a pH dependence and the binding site is hydrophobic. 6. On the Sephadex G-100 column, retinol is chemically modified to a retro derivative which binds even more strongly to beta-lactoglobulin than does retinol. 7. The beta-lactoglobulin-retinol complex rotates anisotropically in solution with a fast (3 ns) and a slower (12 ns) component. This may be attributed to retinol being found at a flexible region of the protein, where only segmental flexibility is observed, weighted by its proximity to one of the major axis rotational times.

Binding Sites↗

PubMed1995

Separation of beta-lactoglobulin A, B and C variants of bovine whey using capillary zone electrophoresis.

beta-Lactoglobulin is a whey protein that affects milk composition and product functionality and which can be present in up to eight genetic variant forms. A free zone capillary electrophoresis method has been developed to separate and identify the beta-lactoglobulin A, B and C variants. Three buffer systems [borate, 2-(N-morpholino)-ethanesulphonic acid (MES) and bis(2-hydroxyethyl)imino-tris(hydroxymethyl)methane (Bistris)] were examined over a range of pH values and with the addition of the separation buffer modifiers Tween 20 and/or ethanolamine. The most successful combination of these was 50 mM MES at pH 8.0 with the addition of 0.1% Tween 20 which clearly resolved the three variants from both each other and from the other whey proteins even though the MES buffer was acting well outside its pKa range (pH 5.3-7.3). The retention times and identification of the individual variants were verified by spiking with commercially purified beta-lactoglobulin A and B proteins and a beta-lactoglobulin AC whey. The method was then used to phenotype beta-lactoglobulin in a sample population of New Zealand Jersey cows.

Amino Acid Sequence↗

PubMed1988

Characterization of the gene encoding ovine beta-lactoglobulin. Similarity to the genes for retinol binding protein and other secretory proteins.

Beta-lactoglobulin is the major whey protein in the milk of ruminants and is expressed in the mammary gland during pregnancy and lactation. Here we describe the isolation and characterization of genomic clones encoding ovine beta-lactoglobulin. Two very similar but non-identical, types of beta-lactoglobulin clone were obtained. DNA sequence analysis of one of these showed that the gene is 4900 bases long and contains seven exons. It codes for a protein of 180 amino acid residues, containing an 18-residue signal peptide, within exons I to VI; exon VII is non-coding. We show that the genes encoding serum retinol binding protein, major urinary protein, alpha-1-acid glycoprotein and apolipoprotein D have a similar organization of exons and introns to beta-lactoglobulin. In particular, a comparison between beta-lactoglobulin and retinol binding protein shows that both genes encode equivalent elements of three-dimensional protein structure within analogous exons. These proteins are all members of a large, diverse family of secretory proteins, many of which function in binding small hydrophobic molecules.

Amino Acid Sequence↗

PubMed2006

Comparison of the conformational stability of the non-native alpha-helical intermediate of thiol-modified beta-lactoglobulin upon interaction with sodium n-alkyl sulfates at two different pH.

Bovine beta-lactoglobulin assumes a dimeric native conformation at neutral pH, while the conformation at pH 2 is monomeric but still native. beta-lactoglobulin has a free thiol at Cys121, which is buried between the beta-barrel and the C-terminal major or alpha-helix. This thiol group was specifically reacted with DTNB (5,5'-dithiobis(2-nitrobenzoic acid)) at pH 7.5 and 2, producing a modified beta-lactoglobulin containing a mix disulfide bond with 5-thio-2-nitrobenzoic acid (TNB). beta-Lactoglobulin is a predominantly beta-sheet protein, although it has a markedly high intrinsic preference for alpha-helical structure. The formation of non-native alpha-helical intermediate of thiol modified beta-lactoglobulin (TNB-beta-LG) was induced by n-alkyl sulfates including sodium octyl sulfate, SOS; sodium decyl sulfate, SDeS; sodium dodecyl sulfate, SDS; and sodium tetradecyl sulfate, STS at pH 7.5 and 2. The conformation and stability of non-native alpha-helical intermediate (alphaI) state of TNB-beta-LG were studied by circular dichroism (CD), fluorescence and differential scanning calorimetry (DSC) techniques. The effect of n-alkyl sulfates on the structure of alphaI state at both pH was utilized to investigate the contribution of hydrophobic interactions to the stability of alphaI intermediate. The present results suggest that the folding reaction of beta-LG follows a non-hierarchical mechanism and hydrophobic interactions play important roles in stabilizing the native state of beta-LG at pH 2 with more positive charges repulsion than at pH 7.5. Then TNB-beta-LG will become a useful model to analyze the conformation and stability of the intermediate of protein folding.

Animals↗

PubMed2006

Interfacial and foaming properties of sulfydryl-modified bovine beta-lactoglobulin.

The effects of a control blocking of free cystein by N-ethylmaleimide on the interfacial behavior (kinetics of adsorption at the air/water interface, rheology of the interfacial layer) as well as on the foaming properties (density, stability) of beta-lactoglobulin were investigated. Compared to native beta-lactoglobulin (unmodified beta-lactoglobulin), sulfydryl-modified beta-lactoglobulin exhibited higher surface hydrophobicity, adsorbed faster at the air/water interface, had the capability to develop rapidly an interfacial layer with high shear elastic constant but exhibited a considerably lower shear elastic constant plateau value. Moreover, sulfydryl-modified beta-lactoglobulin exhibited better foaming properties especially regarding the short-term foam stability suggesting that the initial rheology of the interfacial film is at least as much important for the general mechanism of foam stabilization as the potential viscoelasticity the interfacial film could reach on aging.

Adsorption↗

PubMed2006

Beta-lactoglobulin-dextran Maillard conjugates: their effect on interfacial thickness and emulsion stability.

The influence of dextran molecular weight on the steric layer thickness and oil-in-water (O/W) emulsion flocculation stability of beta-lactoglobulin-dextran Maillard conjugates was investigated. Maillard conjugates were formed by reacting beta-lactoglobulin with various molecular weight dextrans (Mw = 18.5-440 kDa) under mild conditions (60 degrees C, 76% RH). Purified Maillard conjugates or beta-lactoglobulin were adsorbed onto latex spheres and the thickness of the adsorbed layer measured using photon correlation spectroscopy. The adsorbed layer thickness was 3 nm for beta-lactoglobulin alone. Attachment of dextran increased adsorbed layer thickness to 5 nm for the conjugate with low molecular weight dextran (Mw = 18.5 kDa) and 20 nm for that with high molecular weight dextran (Mw = 440 kDa). Enzymatic digestion of the adsorbed layers with dextranase reduced the layer to a thickness corresponding to that of beta-lactoglobulin alone. This suggests that the protein segment of the Maillard conjugate anchors the emulsifier to the interface. Attachment of dextran, irrespective of its molecular weight (18.5-440 kDa), increased the stability of emulsions against calcium induced flocculation, demonstrating that a low molecular weight dextran is sufficient for imparting high steric stability. The observation that the steric layer size was controlled by the dextran molecular weight, suggests that the results of layer thickness and emulsion stability should be universal across all globular proteins.

Dextrans↗

PubMed1994

Differences in the processes of beta-lactoglobulin cold and heat denaturations.

The changes in beta-lactoglobulin upon cold and heat denaturation were studied by scanning calorimetry, CD, and NMR spectroscopy. It is shown that, in the presence of urea, these processes of beta-lactoglobulin denaturation below and above 308 K are accompanied by different structural and thermodynamic changes. Analysis of the NOE spectra of beta-lactoglobulin shows that changes in the spin diffusion of beta-lactoglobulin after disruption of the unique tertiary structure upon cold denaturation are much more substantial than those upon heat denaturation. In cold denatured beta-lactoglobulin, the network of residual interactions in hydrophobic and hydrophilic regions of the molecules is more extensive than after heat denaturation. This suggests that upon cold- and heat-induced unfolding, the molecule undergoes different structural rearrangements, passing through different denaturation intermediates. From this point of view, cold denaturation can be considered to be a two stage process with a stable intermediate. A similar equilibrium intermediate can be obtained at 35 degrees C in 6.0 M urea solution, where the molecule has no tertiary structure. Cooling or heating of the solution from this temperature leads to unfolding of the intermediate. However, these processes differ in cooperativity, showing noncommensurate sigmoidal-like changes in efficiency of spin diffusion, ellipticity at 222 nm, and partial heat capacity. The disruption with cooling is accompanied by cooperative changes in heat capacity, whereas with heating the heat capacity changes only gradually. Considering the sigmoidal shape of the heat capacity change an extended heat absorption peak, we propose that the intermediate state is stabilized by enthalpic interactions.

Animals↗

PubMed2002

Adsorption kinetics of beta-lactoglobulin on a polyclonal immunochromatographic support.

Beta-Lactoglobulin is one of the main components of whey proteins. Among other reasons, its allergenicity makes its determination in hypoallergenic foods and bio-pharmaceutical products necessary. Immunoaffinity chromatography is a widely accepted technique for purification and analysis of proteins. Knowledge of the apparent kinetics of the adsorption of beta-lactoglobulin onto the anti-beta-lactoglobulin immunochromatographic column is important to optimize the analytical process. High-performance frontal affinity chromatography was used to study the apparent kinetics of the adsorption process. Langmuir and bi-Langmuir kinetic models, assuming one and two kinds of binding sites, respectively, were used to characterize the adsorption kinetics of beta-lactoglobulin B on a polyclonal immunoadsorbent. Very good fits were obtained with the bi-Langmuir model for two different concentrations of beta-lactoglobulin and this allowed us to calculate the apparent adsorption rate constants and the column capacities for both kinds of sites. Experimental results indicate the possibility that the adsorption process is not irreversible. The values of the apparent dissociation rate constants leading to the best fit were estimated and the affinity constants were calculated.

Adsorption↗

PubMed1999

Relationships between conformation of beta-lactoglobulin in solution and gel states as revealed by attenuated total reflection Fourier transform infrared spectroscopy.

Attenuated total reflection Fourier transform infrared spectroscopy (ATR FT-IR) has been used to compare the structure of beta-lactoglobulin, the major component of whey proteins, in solution and in its functional gel state. To induce variation in the conformation of beta-lactoglobulin under a set of gelling conditions, the effect of heating temperature, pH, and high pressure homogenization on the conformation sensitive amide I band in the infrared spectra of both solutions and gels has been investigated. The results showed that gelification process has a pronounced effect upon beta-lactoglobulin secondary structure, leading to the formation of intermolecular hydrogen-bonding beta-sheet structure as evidenced by the appearance of a strong band at 1614 cm(-1) at the expense of other regular structures. These results confirm that this structure may be essential for the formation of a gel network as it was previously shown for other globular proteins. However, this study reveals, for the first time, that there is a close relationship between conformation of beta-lactoglobulin in solution and its capacity to form a gel. Indeed, it is shown that conditions which promote predominance of intermolecular beta-sheet in solution such as pH 4, prevent the formation of gel in conditions used by increasing thermal stability of beta-lactoglobulin. On the basis of these findings, it is suggested that by controlling the extent of intermolecular beta-structure of the protein in solution, it is possible to modify the ability of protein to form a gel and as a consequence to control the properties of gels.

Gels↗

PubMed2003

Isolation of alpha-lactalbumin, beta-lactoglobulin, and bovine serum albumin from cow's milk using gel filtration and anion-exchange chromatography including evaluation of their antigenicity.

The aim of this study was to introduce a simple, reproducible, and less expensive method for isolation of alpha-lactalbumin, beta-lactoglobulin, and bovine serum albumin from cow's milk while retaining their antigenicity. Whey (lactoserum) was obtained by isolating casein from defatted milk using hydrochloric acid. Globulins were then precipitated from whey by half-saturated ammonium sulfate and beta-lactoglobulin was purified further using Sephadex G-50 gel filtration. The proteins in the supernatant were also fractionated using diethylaminoethyl cellulose chromatography in which beta-lactoglobulin was separated from alpha-lactalbumin and bovine serum albumin. The latter two proteins that co-eluted in anion-exchange chromatography were then gently isolated from each other by Sephadex G-50 gel filtration. Pure beta-lactoglobulin was also obtained by anion-exchange chromatography of the ammonium sulfate-precipitated globulins. Using enzyme-linked immunosorbent assay (ELISA), Western blotting, and ELISA inhibition assay, antigenicity of the purified proteins was evaluated. Our results showed high purity and well-preserved antigenicity of alpha-lactalbumin, beta-lactoglobulin, and bovine serum albumin thus purified.

Animals↗

PubMed2003

Reduction of immunoreactivity of bovine beta-lactoglobulin upon combined physical and proteolytic treatment.

Bovine beta-lactoglobulin was hydrolyzed with trypsin or chymotrypsin before, during and after treatment at 600 MPa and pH 6.8 for 10 min at 30, 37 and 44 degrees C. The extent of beta-lactoglobulin hydrolysis under pressure was noticeably higher than at atmospheric pressure, particularly when chymotrypsin was used. Addition of proteases at ambient pressure to previously pressure-treated beta-lactoglobulin gave only a modest increase in proteolysis with respect to the untreated protein. Products of enzyme hydrolysis under pressure were separated by reverse-phase HPLC, and were found to be different from those obtained at atmospheric pressure when chymotrypsin was used. The residual immunochemical reactivity of the products of combined pressure-enzyme treatment was assessed on the unresolved hydrolysates by ELISA tests using polyclonal and monoclonal antibodies, and on individual hydrolytic fractions by Western Blotting using sera of paediatric patients allergic to whey proteins in cow milk. The immunoreactivity of the whole hydrolysates was related to their content of residual intact beta-lactoglobulin, and no immunochemical reactivity was found for all the products of chymotrypsin hydrolysis under pressure. The results indicate that chymotrypsin effectively hydrolysed hydrophobic regions of beta-lactoglobulin that were transiently exposed during the pressure treatments and that were not accessible in the native protein or in the protein that had been previously pressure treated.

Atmospheric Pressure↗

PubMed1995

Effect of sodium dodecyl sulfate and palmitic acid on the equilibrium unfolding of bovine beta-lactoglobulin.

The unfolding of bovine beta-lactoglobulin, a small globular protein that unfolds reversibly at low pH in the presence of urea or guanidine hydrochloride, has been studied at pH 6.72 in phosphate buffer at 21 degrees C. The midpoint urea concentration for the loss of CD intensity at 220 nm, loss of CD intensity at 293 nm, quenching of intrinsic fluorescence, shift in the wavelength of the maximum of the intrinsic fluorescent emission, and loss of fluorescence intensity from 1-anilino-8-naphthalenesulfonate (ANS) (and probably the hydrophobic binding site) was close to 4.4 M. Addition of sodium dodecyl sulfate (SDS) at concentrations less than 100 microM to the beta-lactoglobulin solutions increased the midpoint urea concentration for the CD and intrinsic fluorescence parameters to about 5.8 M. Palmitic acid had a similar effect to that shown by SDS in altering the CD intensity at 293 nm, and both SDS and palmitic acid attained a maximum effect in altering the CD at 293 nm at a 1:1 molar ratio to beta-lactoglobulin. It seems likely that the beta-sheet structure of beta-lactoglobulin breaks down simultaneously with the loss of the hydrophobic binding site and exposure of tryptophan-19 to the external environment, supporting the view that the major hydrophobic binding site of beta-lactoglobulin is closely involved with the beta-sheet core of the protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Anilino Naphthalenesulfonates↗

PubMed2005

Modification of IgE binding to beta-lactoglobulin by fermentation and proteolysis of cow's milk.

The effect of fermentation by Lactobacilli and of proteolytic hydrolysis of whole milk on the IgE binding ability of beta-lactoglobulin was studied using an ELISA inhibition assay. Sera from nine adult milk allergic patients were tested. The individual sera showed a similar inhibition pattern in the changes during fermentation and proteolysis. The degradation of beta-lactoglobulin was studied with liquid chromatography. In general, fermentation with Lactobacilli gave little effect on IgE binding, even though chromatography data showed a gradual degradation of beta-lactoglobulin. Proteolysis with trypsin, however, gave extensive degradation of beta-lactoglobulin and strongly decreased IgE binding. In addition, we measured the inhibition pattern of beta-lactoglobulin in various selected commercially available fermented milk products. These showed an IgE binding capacity similar to that of nonfermented high pasteurized milk.

Adult↗