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PubMed2002

Proteolysis of bovine beta-lactoglobulin during thermal treatment in subdenaturing conditions highlights some structural features of the temperature-modified protein and yields fragments with low immunoreactivity.

Bovine beta-lactoglobulin was hydrolyzed with trypsin or chymotrypsin in the course of heat treatment at 55, 60 and 65 degrees C at neutral pH. At these temperatures beta-lactoglobulin undergoes significant but reversible structural changes. In the conditions used in the present study, beta-lactoglobulin was virtually insensitive to proteolysis by either enzyme at room temperature, but underwent extensive proteolysis when either protease was present during the heat treatment. High-temperature proteolysis occurs in a progressive manner. Mass spectrometry analysis of some large-sized breakdown intermediates formed in the early steps of hydrolysis indicated that both enzymes effectively hydrolyzed some regions of beta-lactoglobulin that were transiently exposed during the physical treatments and that were not accessible in the native protein. The immunochemical properties of the products of beta-lactoglobulin hydrolysis were assessed by using various beta-lactoglobulin-specific antibodies, and most epitopic sites were no longer present after attack of the partially unfolded protein by the two proteases.

Animals↗

PubMed1998

Role of free Cys121 in stabilization of bovine beta-lactoglobulin B.

Mixed disulfide derivatives of bovine beta-lactoglobulin (BLG) were studied by circular dichroism (CD), gel-permeation HPLC and high-sensitivity differential scanning calorimetry (HS-DSC). It was shown that modification of Cys121 with mercaptopropionic acid and mercaptoethanol does not affect the secondary structure of BLG, but results instead in tertiary and quaternary structure changes. At neutral pH, the equilibrium dimer<==>monomer of modified beta-lactoglobulin is shifted towards monomeric form. In contrast to native BLG, thermal denaturation of modified beta-lactoglobulin is fully reversible in neutral and acidic pH as demonstrated by CD and HS-DSC measurements. Modification of Cys121 results in a significant decrease of transition temperature (-6 degrees C) and enthalpy (-106 kJ/mol) at pH 2.05 while unfolding heat capacity increment remains unchanged. Thermal unfolding transitions of native and modified beta-lactoglobulin at pH 2.05 are well approximated by a two-state model suggesting that no intermediate states appear after modification. The difference in Gibbs energy of denaturation between native and modified beta-lactoglobulin, 8.5 kJ/mol at 37 degrees C and pH 2.05, does not depend on the nature of the introduced group (charged or neutral). Computer analysis of possible interactions involving Cys121 in a three-dimensional structure of beta-lactoglobulin revealed that the thiol group is too far away from neighboring residues to form side-chain hydrogen bonds. This suggests that the sulfhydryl group of Cys121 may contribute to the maintenance of BLG tertiary structure via water mediated H-bonding.

Animals↗

PubMed1992

A sensitive enzyme-linked immunosorbent assay for determination of bovine beta-lactoglobulin in infant feeding formulas and in human milk.

We developed a sensitive sandwich-type ELISA for measuring low levels of cow's milk (CM) beta-lactoglobulin. Purified anti-beta-lactoglobulin was used as coating antibody and also as second antibody conjugated with alkaline phosphatase. Polyethylene glycol 6000 was added to the incubation buffers to improve sensitivity. The detection limit of the assay was 0.002 microgram/l, which is much better than sensitivities reported for other beta-lactoglobulin assays. The sensitivity was not impaired by the presence of other CM proteins. The recovery from breast milk was 93% and from the diluting buffer 127%. The coefficient of variation within day was 5-15% and between days 10%. One hour after oral intake of milk, beta-lactoglobulin could be detected in the breast milk of three mothers at concentrations of about 1-2 micrograms/l. Widely different concentrations of beta-lactoglobulin were measured in two protein hydrolysates based on CM whey and casein proteins; the observed concentrations were 200 and 0.0056 micrograms beta-lactoglobulin/g dry weight, respectively.

Animals↗

PubMed1991

Increased beta-lactoglobulin absorption during rotavirus enteritis in infants: relationship to sugar permeability.

We studied absorption of the potentially allergenic protein beta-lactoglobulin during acute rotavirus diarrhea in infants and assessed the relationship of this macromolecular absorption with intestinal sugar permeability. After oral rehydration, 38 patients with acute gastroenteritis were given orally a 100-ml solution containing 4 g (11.7 mmol/L) of lactulose and 0.8 g (4.4 mmol/L) of mannitol, and their recovery rate as shown in urine passed during the subsequent 5 h was measured. A blood sample was taken 2 h after a milk feed for ELISA measurement of beta-lactoglobulin in circulating immune complexes. Twelve nondiarrhea patients were studied after an overnight fast as controls. Immune complexes containing beta-lactoglobulin were found in the serum of all, but the levels [median (range)] were significantly higher in patients with rotavirus diarrhea [686 (36-4352)] than in nondiarrhea patients [165 (0-2594)]; p = 0.007. The mean (95% confidence interval) lactulose/mannitol urinary recovery ratios were increased in patients with acute diarrhea [0.19 (0.10, 0.30)] compared to nondiarrhea patients [0.01 (0.005, 0.02)]; p = 0.0001. Thus, a significant correlation between beta-lactoglobulin absorption and sugar permeability was found; Spearman's rank correlation coefficient = 0.42, p = 0.004. This correlation was not, however, direct but was due to an inverse relationship between urinary recovery of mannitol and serum beta-lactoglobulin immune complexes. These results indicate that rotavirus gastroenteritis is associated with enhanced beta-lactoglobulin absorption and elevated lactulose/mannitol permeability test results, but these represent different phenomena.

Antigen-Antibody Complex↗

PubMed1985

Homology between the primary structures of beta-lactoglobulins and human retinol-binding protein: evidence for a similar biological function?

Two types of beta-lactoglobulins were identified and isolated from horse colostrum: beta-1g I and beta-1g II. The amino-acid sequence of some tryptic peptides from the new monomeric beta-lactoglobulin II was determined and aligned to the other beta-lactoglobulins of known sequence and to the human plasma retinol-binding protein. The comparison of the primary structures of beta-lactoglobulins and human retinol-binding protein shows an unexpectedly high homology of 25%. We found 37 identities among 149 possible homologous residues. Among them is a tryptophan residue at position 19 of beta-lg which might represent the binding site of beta-ionone. These data suggest a common origin of beta-lactoglobulin and human retinol-binding protein and imply that beta-lactoglobulins may be involved in the metabolism of retinol.

Amino Acid Sequence↗

PubMed1989

Isolation and complete primary sequence of a new ovine wild-type beta-lactoglobulin C.

A new wild type of beta-lactoglobulin has been identified in the milk of sheep. It has been designated as ovine beta-lactoglobulin C. Its primary structure has been determined by direct protein microsequencing of intact protein and RP-HPLC-derived tryptic peptides. The new beta-lactoglobulin C is a subtype of ovine beta-lactoglobulin A with a single exchange Arg-Gln at position 148. This exchange may influence polymerisation of beta-lactoglobulin since in the crystal structure of orthorhombic bovine beta-lactoglobulin, residues 145-150 constitute a short beta-sheet region involved in dimer formation by pairing of dyad-related strands.

Amino Acid Sequence↗

PubMed1990

Covalent structure of the minor monomeric beta-lactoglobulin II component from donkey milk.

The complete primary structure of the minor beta-lactoglobulin II component from donkey milk is presented. It has been established by amino-acid sequencing and mass-spectrometry analysis of intact protein and peptides obtained after enzymatic and chemical cleavages. The molecular mass and the pI of the protein are calculated to be 18,261 Da and 4.5 respectively. Despite the close structural similarity of the donkey and horse major beta-lactoglobulin I components, their minor beta-lactoglobulin II components show substantial differences in sequence. Most observed exchanges are clustered at residues 78-106 where only 6 amino-acid residues are conserved. The primary structure of donkey beta-lactoglobulin II reveals some unusual features of minor beta-lactoglobulins II and gives new light to the evolution of beta-lactoglobulins and other lipocalins involved in retinol binding or reproductive functions.

Amino Acid Sequence↗

PubMed2002

Scale-up of native beta-lactoglobulin affinity separation process.

Affinity separation of beta-lactoglobulin in its native form with all-trans-retinal immobilized on calcium bio-silicate was scaled up and applied to separate it from industrial sweet whey. Three different methods of mixing the modified calcium bio-silicate and whey for the interaction between all-trans-retinal and beta-lactoglobulin were tried at pilot scale. The three methods used were 1) a column packed with calcium bio-silicate, 2) a stirred tank, and 3) a fluidized bed column of calcium bio-silicate particles. Adsorption and desorption of beta-lactoglobulin were carried out at pH 5.1 and 7.0, using 0.01 and 0.1 M phosphate buffers, respectively. The phosphate buffer containing desorbed beta-lactoglobulin was concentrated 20 times using ultrafiltration and then freeze-dried. The packed column, stirred tank, and fluidized bed column produced beta-lactoglobulin with purity of 80, >95, and >95%, and recovery of 0.65, 2.88, and 2.88 g per kilogram of calcium bio-silicate, respectively. The comparative poor purity and recovery of beta-lactoglobulin in the case of the packed column was attributed to insufficient contact between the passing fluids and the calcium bio-silicate during adsorption, desorption, and intermittent washing. The fluidized bed column method, with a gentle mixing action, was considered the best suited for further scale up to the industrial level.

Adsorption↗

PubMed2006

Use of chitosan for selective removal of beta-lactoglobulin from whey.

A method is described for selective removal of undenatured beta-lactoglobulin from cheese whey based on interactions between whey proteins and chitosan. Whey was previously clarified at pH 4.5 with addition of chitosan (25 mg/100 mL), and selective removal of beta-lactoglobulin was studied in the pH interval 4.6 to 6.5. Addition of chitosan caused selective precipitation of beta-lactoglobulin that increased with pH. The content of beta-lactoglobulin in whey decreased as the amount of chitosan added was increased. At pH 6.2, addition of 1.9 to 3.0 mg/mL of chitosan led to complete removal of beta-lactoglobulin, whereas at least 80% of the rest of whey proteins remained in solution. The production of cheese whey without beta-lactoglobulin could help to expand the applications of dairy by-products in food processing, and to isolate hypoallergenic whey protein concentrates.

Cheese↗

PubMed1997

Long-term consumption of whey hydrolysate formula by lactating women reduces the transfer of beta-lactoglobulin into human milk.

Food antigens transferred into breast milk sometimes cause an allergic reaction in exclusively breast-fed infants. This study will show whether the intake of a whey hydrolysate formula for lactating women (MOM HA) can reduce the appearance of food antigens in breast milk. Lactating women in the MOM group (n = 12) consumed MOM HA as a substitute for cow's milk and those in the COW group (n = 13) consumed cow's milk for more than 4 months. After the ingestion of 200 mL of MOM HA and cow's milk by the women in the MOM and COW groups, respectively, the first breast milk samples were obtained and beta-lactoglobulin was measured using enzyme-linked immunosorbent assay. The number of subjects with detectable beta-lactoglobulin (> 0.1 ng/mL) in the MOM group was two (17%), which was significantly less than that in the COW group (11 subjects, 85%, p < 0.01). The level of beta-lactoglobulin was also lower in the MOM group than the COW group (p < 0.01). Subsequently, the women in the MOM group consumed cow's milk and those in the COW group consumed MOM HA for one week; then a second sampling was performed. beta-Lactoglobulin was detected in three (25%) and 8 subjects (62%) in the MOM and COW groups, respectively. The level of beta-lactoglobulin was still lower in the MOM group (p < 0.05). The consumption of whey hydrolysate formula by lactating women over a considerable time reduces the transfer of beta-lactoglobulin into their breast milk, and the low level can be maintained even after inadvertent ingestion of cow's milk.

Animals↗

PubMed1990

Expression of recombinant bovine beta-lactoglobulin in Escherichia coli.

Bovine beta-lactoglobulin A was expressed in Escherichia coli in its mature form. The gene was constructed using a cDNA clone which coded for amino acid residues Leu-11 to Ile-162 and a synthetic oligonucleotide coding for the initial 10 amino acids preceded by a translational start. The met-beta-lactoglobulin was expressed using a tac promoter vector, pTTQ18, and accounted for approximately 15% of the total cellular protein. The recombinant met-beta-lactoglobulin migrated with the same molecular weight as native beta-lactoglobulin A on SDS-PAGE. The majority of the met-beta-lactoglobulin produced was found in an insoluble form but could be solubilized using guanidine-HCl. The renatured preparation was greater than 80% pure and migrated similarly to purified beta-lactoglobulin A under nondenaturing conditions.

Amino Acid Sequence↗

PubMed1999

Adsorption Dynamics of alpha-Lactalbumin and beta-Lactoglobulin at Air-Water Interfaces.

Dynamics of adsorption of 14C radiolabeled beta-lactoglobulin and alpha-lactalbumin at the air-water interface was investigated through the measurement of surface pressure (pi) and surface concentration (Gamma) via a radiotracer technique. Adsorption was diffusion controlled at short times, the rates of increase of pi and Gamma being lower at longer times because of an energy barrier. At low concentrations, an apparent time lag was observed in the evolution of pi for beta-lactoglobulin but not for alpha-lactalbumin which was shown to be due to the nonlinear nature of the pi-Gamma relationship for the former. The area per molecule of an adsorbed beta-lactoglobulin during the dynamics of adsorption was smaller than that for spread monolayer since beta-lactoglobulin was not fully unfolded during adsorption. For alpha-lactalbumin, however, no such difference in the molecular areas for adsorbed and spread monolayer was observed indicating thereby that alpha-lactalbumin unfolded much more rapidly than beta-lactoglobulin. Evolution of Gamma for alpha-lactalbumin was found to occur in two steps possibly due to the change in the orientation of the adsorbed protein from a side-on to an end-on orientation. A previously developed mechanistic model (G. Narsimhan and F. Uraizee, Biotechnology Prog. 8, 187 (1992)) was improved to account for the presence of hydrophobic patches on the surface of the protein molecule as well as an adsorbed protein layer at the air-water interface. The model predictions agreed quite well with the experimental evolution of Gamma for beta-lactoglobulin and alpha-lactalbumin. The model calculations seem to indicate that alpha-lactalbumin changes its orientation at the air-water interface from side-on to other orientations at higher surface concentrations. Copyright 1999 Academic Press.

Journal Article↗

PubMed1993

Reversible effects of medium dielectric constant on structural transformation of beta-lactoglobulin and its retinol binding.

The secondary structure transformation of beta-lactoglobulin from a predominantly beta-structure into a predominantly alpha-helical one, under the influence of solvent polarity changes is reversible. Independent of the alcohol used--methanol, ethanol, or 2-propanol--the midpoints of the observed structural transformation occur around dielectric constant epsilon approximately 60. The structural change destroying the hydrophobic core formed by the beta-barrel structure leads, at room temperature, to the dissociation of the retinol/beta-lactoglobulin complex in the neighborhood of dielectric constant epsilon approximately 50. However, when the dielectric constant of the medium is raised back to epsilon approximately 70 by the decrease of the temperature, both the refolding of BLG into a beta-structure and the reassociation of the retinol/beta-lactoglobulin complex are observed. The esterification of beta-lactoglobulin carboxyl groups has two effects: on the one hand it accelerates the beta-strand<==>alpha-helix transition induced by alcohols. On the other hand, the esterification of beta-lactoglobulin strengthens its interaction with retinol as it may be deduced from the smaller apparent dissociation constant of retinol/methylated beta-lactoglobulin complex. The binding of retinol to modified or unmodified beta-lactoglobulin has no influence (stabilizing or destabilizing) on the folding changes induced by alcohol.

Alcohols↗

PubMed2005

Selective separation of beta-lactoglobulin from sweet whey using CGAs generated from the cationic surfactant CTAB.

The selective separation of whey proteins was studied using colloidal gas aphrons generated from the cationic surfactant cetyl trimethyl ammonium bromide (CTAB). From the titration curves obtained by zeta potential measurements of individual whey proteins, it was expected to selectively adsorb the major whey proteins, i.e., bovine serum albumin, alpha-lactalbumin, and beta-lactoglobulin to the aphrons and elute the remaining proteins (lactoferrin and lactoperoxidase) in the liquid phase. A number of process parameters including pH, ionic strength, and mass ratio of surfactant to protein (M(CTAB)/M(TP)) were varied in order to evaluate their effect on protein separation. Under optimum conditions (2 mmol/l CTAB, M(CTAB)/M(TP) = 0.26-0.35, pH 8, and ionic strength = 0.018 mol/l), 80-90% beta-lactoglobulin was removed from the liquid phase as a precipitate, while about 75% lactoferrin and lactoperoxidase, 80% bovine serum albumin, 95% immunoglobulin, and 65% alpha-lactalbumin were recovered in the liquid fraction. Mechanistic studies using zeta potential measurements and fluorescence spectroscopy proved that electrostatic interactions modulate only partially the selectivity of protein separation, as proteins with similar surface charges do not separate to the same extent between the two phases. The selectivity of recovery of beta-lactoglobulin probably occurs in two steps: the first being the selective interaction of the protein with opposite-charged surfactant molecules by means of electrostatic interactions, which leads to denaturation of the protein and subsequent formation and precipitation of the CTAB-beta-lactoglobulin complex. This is followed by the separation of CTAB-beta-lactoglobulin aggregates from the bulk liquid by flotation in the aphron phase. In this way, CGAs act as carriers which facilitate the removal of protein precipitate.

Animals↗

PubMed2004

Purification and physicochemical characterization of ovine beta-lactoglobulin and alpha-lactalbumin.

Ovine whey proteins were fractionated and studied by using different analytical techniques. Anion-exchange chromatography and reversed-phase high-performance liquid chromatography (HPLC) showed the presence of two fractions of beta-lactoglobulin but only one of alpha-lactalbumin. Gel permeation and sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis allowed the calculation of the apparent molecular mass of each component, while HPLC coupled to electrospray ionisation-mass spectrometry (ESI-MS) technique, giving the exact molecular masses, demonstrated the presence of two variants A and B of ovine beta-lactoglobulin. Amino acid compositions of the two variants of beta-lactoglobulin differed only in their His and Tyr contents. Circular dichroism spectroscopy profiles showed pH conformation changes of each component. The thermograms of the different whey protein components showed a higher heat resistance of beta-lactoglobulin A compared to beta-lactoglobulin B at pH 2, and indicated high instability of ovine alpha-lactalbumin at this pH.

Amino Acids↗

PubMed1996

Hormonal influences on beta-lactoglobulin transgene expression inferred from chromatin structure.

The major milk whey protein of ruminants is beta-lactoglobulin. Transgenic mice which carry genomic fragments of ovine beta-lactoglobulin express the transgene at high levels in the mammary gland. Using DNaseI as a probe for transcription complex formation in chromatin, the temporal induction pattern of beta-lactoglobulin in transgenic mice has been addressed and compared to the known hormonal profiles during pregnancy. Prior to the 9th day of pregnancy no obvious hypersensitivity to DNaseI digestion at the beta-lactoglobulin promoter was evident. From the 9th day of pregnancy through to lactation, the beta-lactoglobulin promoter displays DNaseI hypersensitivity. These results support the hypothesis that placental lactogens are the major lactogenic influence from mid-pregnancy to parturition.

Animals↗

PubMed1998

Chromatin structures of goat and sheep beta-lactoglobulin gene differ.

Different levels of the major milk protein beta-lactoglobulin are found in evolutionarily related ruminant species: with sheep milk containing as much as three times the concentration in goat milk. In an attempt to understand why these differences exist, we have characterised, using DNaseI as a probe of structure, the chromatin surrounding the goat beta-lactoglobulin promoter and compared it to that of the sheep homologue. The goat gene displays a mammary-specific chromatin pattern, which is reformed on expressing goat beta-lactoglobulin transgenes. This implies that this chromatin structure is sequence dependent and suggests that it plays a role in regulating beta-lactoglobulin gene expression. This pattern differs from that seen on the ovine beta-lactoglobulin gene in lactating sheep mammary chromatin. Thus, even between highly related species, the transcriptional mechanisms regulating activity of a gene can differ.

Animals↗

PubMed1996

The burst-phase intermediate in the refolding of beta-lactoglobulin studied by stopped-flow circular dichroism and absorption spectroscopy.

The kinetics of the guanidine hydrochloride-induced unfolding and refolding of bovine beta-lactoglobulin, a predominantly beta-sheet protein in the native state, have been studied by stopped-flow circular dichroism and absorption measurements at pH 3.2 and 4.5 degrees C. The refolding reaction was a complex process composed of different kinetic phases, while the unfolding was a single-phase reaction. Most notably, a burst-phase intermediate of refolding, which was formed during the dead time of stopped-flow measurements (approximately 18 ms), showed more intense ellipticity signals in the peptide region below 240 nm than the native state, yielding overshoot behavior in the refolding curves. We have investigated the spectral properties and structural stability of the burst-phase intermediate and also the structural properties in the unfolded state in 4.0 M guanidine hydrochloride of the protein and its disulfide-cleaved derivative. The main conclusions are: (1) the more intense ellipticity of the intermediate in the peptide region arises from formation of non-native alpha-helical structure in the intermediate, apparently suggesting that the folding of beta-lactoglobulin is not represented by a simple sequential mechanism. (2) The burst-phase intermediate has, however, a number of properties in common with the folding intermediates or with the molten globule states of other globular proteins whose folding reactions are known to be represented by the sequential model. These properties include: the presence of the secondary structure without the specific tertiary structure; formation of a hydrophobic core; broad unfolding transition of the intermediate; and rapidity of formation of the intermediate. The burst-phase intermediate of beta-lactoglobulin is thus classified as the same species as the molten globule state. (3) The circular dichroism spectra of beta-lactoglobulin and its disulfide-cleaved derivative in 4.0 M guanidine hydrochloride suggests the presence of the residual beta-structure in the unfolded state and the stabilization of the beta-structure by disulfide bonds. Thus; if this residual beta-structure is part of the native beta-structure and forms a folding initiation site, the folding reaction of beta-lactoglobulin may not necessarily be inconsistent with the sequential model. The non-native alpha-helices in the burst-phase intermediate may be formed in an immature part of the protein molecule because of the local alpha-helical propensity in this part.

Circular Dichroism↗