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PubMed2006

Self-assembly of monoglycerides in beta-lactoglobulin adsorbed films at the air-water interface. Structural, topographical, and rheological consequences.

In this work, we have analyzed the structural, topographical, and surface dilatational characteristics of pure beta-lactoglobulin adsorbed films and the effect of the self-assembly of monoglycerides (monopalmitin or monoolein) in beta-lactoglobulin films at the air-water interface. Measurements were performed in a single device that incorporates a Wilhelmy-type film balance, Brewster angle microscopy, and interfacial dilatational rheology. The structural and topographical characteristics of beta-lactoglobulin adsorbed and spread films are similar. However, the surface dilatational modulus of beta-lactoglobulin films shows a complex behavior depending on film formation. The self-assembly of monoglyceride in a beta-lactoglobulin adsorbed film has an effect on the structural, topographical, and dilatational properties of the mixed films, depending on the interfacial composition and the surface pressure (pi). At low pi, a mixed film of monoglyceride and beta-lactoglobulin may exist. At high pi (after the collapse of beta-lactoglobulin), the mixed films are dominated by monoglyceride molecules. However, the small amounts of collapsed beta-lactoglobulin have a significant effect on the surface dilatational properties of the mixed films. Protein displacement by monoglyceride is higher for monopalmitin than for monoolein. However, some degree of interaction exists between proteins and monoglycerides, and these interactions are more evident in adsorbed films than in spread films.

Adsorption↗

PubMed1995

Characterisation of a second, apparently inactive, copy of the bovine beta-lactoglobulin gene.

A bovine cosmid clone was isolated which contains the previously characterised beta-lactoglobulin gene and, in addition, a related sequence which appears to be a beta-lactoglobulin pseudogene. The total length of the pseudogene, as determined by DNA sequencing, is 4.8 kb, similar to that of beta-lactoglobulin. Both genes are in the same orientation and are separated by approximately 14 kb intergenic sequence. Although most of introns I-V are extremely divergent, the exon sequences are clearly related, exons I-V exhibiting nucleotide sequence similarities in the range 60-87%. Exons VI and VII, together with the final intron, comprise a region of sequence extending over 730 bp, which displays 92.5% identity to the corresponding beta-lactoglobulin sequence. It is suggested that this is the result of a recent gene conversion event involving conversion of the pseudogene by the authentic beta-lactoglobulin gene. Identification of the new lactoglobulin sequence as a pseudogene is based on the occurrence of a stop codon in exon V. Comparison of the inferred translation product encoded by the pseudogene before its mutational inactivation, with the sequences of equine and feline beta-lactoglobulins I and II, indicates that the bovine pseudogene is more closely related to these type-II lactoglobulin sequences than to the type-I sequences.

Amino Acid Sequence↗

PubMed1986

Bovine beta-lactoglobulin in the human milk. A longitudinal study during the whole lactation period.

Human milk samples (n = 232) collected during the whole lactation period from 25 healthy, Swedish mothers were analyzed by radioimmunologic method for content of bovine beta-lactoglobulin. Detectable amounts (5-800 micrograms/l) were found in 93 of 232 milk samples (40%). Six mothers had no detectable beta-lactoglobulin in their breast milk on any occasion. Two mothers had measurable beta-lactoglobulin in all their milk samples. No correlation was found between daily cow's milk intake and concentration of beta-lactoglobulin in the milk samples. Six mothers with allergic symptoms such as asthma, hay-fever, eczema all had detectable amounts of beta-lactoglobulin in their milk. Of 19 mothers without allergy, 13 had detectable amounts. This difference did not show statistical significance. The presence of symptoms in the infant such as diarrhoea, vomiting, colic, exanthema was significantly correlated to high levels of beta-lactoglobulin in the milk. Bovine beta-lactoglobulin was also detected in 7 of 13 serum samples. The two mothers with detectable beta-lactoglobulin in all milk samples had the highest serum values, and their infants suffered from gastro-intestinal symptoms, weight decline and exanthema.

Animals↗

PubMed1996

Reduction of ferrylmyoglobin by beta-lactoglobulin.

Reduction of iron (IV) in ferrylmyoglobin in the presence of beta-lactoglobulin in aqueous solution is the result of two parallel reactions: (i) a so-called autoreduction, and (ii) reduction by beta-lactoglobulin in a second-order-reaction resulting in bityrosine formation in beta- lactoglobulin. In the pH-region investigated (5.4-7.4), the rate of reduction increased for both reactions with decreasing pH. The second order-reaction had for non-denatured beta-lactoglobulin the activation parameters: delta H* = 45 kJ.mol-1 and delta S not equal to = -93 J.mol-1.K-1 at pH = 7.0 and ionic strength 0.16 (NaCl). Reduction of ferrylmyoglobin by beta-lactoglobulin denatured by heat (86 degrees C for 3 min) or by hydrostatic pressure (300 MPa for 15 min) resulted in formation of higher molecular weight species as detected by size-exclusion chromatography and by SDS-PAGE. No molecular weight changes were observed for reduction of ferrylmyoglobin by native beta-lactoglobulin. Detection of bityrosine in the native beta-lactoglobulin fraction after oxidation with ferrylmyoglobin indicated intra-molecular bityrosine formation. In heat-denatured beta-lactoglobulin bityrosine formation could be of intra-molecular and/or of inter-molecular origin, the latter being confirmed by size-exclusion chromatography.

Hydrogen Peroxide↗

PubMed1990

[Total IgE, specific antibodies to cow's milk proteins, beta-lactoglobulin and eczema in one month infants].

Total IgE, specific IgE.IgA.IgG.IgM antibodies to whole cow's milk and beta-lactoglobulin were measured in 32 term and 23 premature infants. 1) The term infants who developed eczema till one month had significantly high specific IgG titers to whole cow's milk and beta-lactoglobulin in cord blood serum. It is concluded that specific IgG antibody to whole cow's milk and beta-lactoglobulin in cord blood serum have predictive value for the development of eczema till one month. 2) Some of mixed feeding premature infants produced specific IgE.IgA.IgG.IgM antibodies to whole cow's milk and beta-lactoglobulin till one month. These infants produced various kind of specific antibodies to whole cow's milk and beta-lactoglobulin. 3) The premature infants who developed eczema at one month had significantly high specific IgA.IgG titers to cow's milk and high specific IgA titers to beta-lactoglobulin in serum at one month. These infants had a tendency to show high total IgE value and high specific IgM titers to cow's milk and high specific IgG.IgM titers to beta-lactoglobulin. It is concluded that specific antibodies to whole cow's milk and beta-lactoglobulin are responsible for the development of eczema in one month infants.

Eczema↗

PubMed1992

Binding of benzo(a)pyrene, ellipticine, and cis-parinaric acid to beta-lactoglobulin: influence of protein modifications.

The binding of benzo(a)pyrene, ellipticine, and cis-parinaric acid to native, esterified, and alkylated beta-lactoglobulin was followed by enhancement of the ligand fluorescence. Three studied ligands bind to native or modified beta-lactoglobulin in apparent molar ratios varying between 1/8 and 2/1, with apparent dissociation constants in the range of 10(-8) M for ligand/beta-lactoglobulin complexes. The studied, chemically modified beta-lactoglobulin derivatives display higher binding affinities for all studied ligands, cis-parinaric acid excluded. The reductive alkylation of epsilon-NH2 lysyl residues of beta-lactoglobulin increases the apparent molar ratios of benzo(a)pyrene and cis-parinaric acid, and decreases it for ellipticine. The esterified and native beta-lactoglobulin complexed to the investigated ligands display similar stoichiometries. Dynamic light scattering study of ligand-beta-lactoglobulin complexes in solution shows the formation of aggregates: the apparent hydrodynamic radius value of beta-lactoglobulin dimer (3.4 nm) reaches 49, 46, and 74 nm upon addition and binding of benzo(a)pyrene, ellipticine, and cis-parinaric acid, respectively.

Alkylation↗

PubMed2006

Observation of salt-induced beta-lactoglobulin aggregation using sedimentation field-flow fractionation.

Sedimentation field-flow fractionation (SdFFF) was applied in order to characterize particle sizes of beta-lactoglobulin aggregates induced by Ca2+ or Zn2+. Aggregation induced by Zn2+ was faster than that induced by Ca2+. Effects of Zn2+ and beta-lactoglobulin concentrations, as well as contact time, on the aggregation of beta-lactoglobulin were examined. All factors exhibited a combined effect on the size of aggregates, whereby larger aggregates were obtained at increased concentrations of Zn2+ and beta-lactoglobulin. At fixed concentrations of 2% (w/v) beta-lactoglobulin and 10 mM Zn2+, the particle size of the aggregates increased from 0.19 microm (at 15 min) to 0.38 microm (at 2880 min). Further, a hyphenated technique of SdFFF and inductively coupled plasma-optical emission spectrometry (ICP-OES) was used to examine whether intermolecular ionic bridges take part in salt-induced beta-lactoglobulin aggregation. With SdFFF-ICP-OES, protein-cation-protein cross-linkages were observed for beta-lactoglobulin aggregation induced by Zn2+, but not for that induced by Ca2+.

Calcium↗

PubMed1995

Uptake and passage of beta-lactoglobulin, palmitic acid and retinol across the Caco-2 monolayer.

Caco-2 cell line grown on collagen coated polycarbonate membranes in bicameral chambers has been used to study the effect of the binding of palmitic acid or retinol on the uptake and passage of iodinated beta-lactoglobulin and albumin across cell monolayers. The percentage of beta-lactoglobulin transported through the monolayer was higher than that of albumin, about 50% and 30% of the total protein after 24 h of incubation, respectively. In all cases, less than 1% of protein was retained intracellularly. No differences were found in the uptake and transport of beta-lactoglobulin or albumin in the presence or absence of ligands. Furthermore, uptake and passage across Caco-2 monolayer of retinol or palmitic acid added either bound to beta-lactoglobulin or to albumin have been compared. The percentage of retinol found in the lower chamber was about 35% of the total retinol after 24 h of incubation for both proteins. However, the amount of retinol associated to cells was higher when it was added bound to beta-lactoglobulin than to albumin, about 26% and 10%, respectively. This fact suggests that the metabolic processing of retinol by Caco-2 cells is the rate-limiting step for retinol transport. The percentage of palmitic acid that crossed the monolayer was about 7%, remaining approx. 90% in the cells for beta-lactoglobulin and albumin. These data support the hypothesis that palmitic acid internalized by Caco-2 cells is mainly destined to serve the structural and energy needs. These results show evidence of retinol and palmitic uptake by Caco-2 cells when beta-lactoglobulin or albumin are the donors, and indicate that the type of binding protein does not affect the transport of both ligands through Caco-2 monolayer.

Biological Transport↗

PubMed1994

High-pressure effects on beta-lactoglobulin interactions with ligands studied by fluorescence.

The effects of pressure (0.1 MPa to 400 MPa) on intrinsic fluorescence of beta-lactoglobulin and on its binding of retinol and cis-parinaric acid have been studied at neutral and acid pHs. In neutral pH, fluorescence emission spectra of beta-lactoglobulin tryptophanes are characterized by an irreversible 14 nm red-shift indicating pressure-induced folding changes. The intensity of the fluorescence of retinol in beta-lactoglobulin-retinol complex is enhanced by a pressure increase up to 150 MPa. It decreases at higher pressures and disappears altogether at 300 MPa. beta-Lactoglobulin-retinol complex does not reassociate after decompression at neutral pH. At acid pH condition, the fluorescence quenching by pressure of beta-lactoglobulin tryptophans is coupled with a 2 nm spectral shift and is fully reversible demonstrating almost complete restoration of globulin folding. The evolution of retinol fluorescence in beta-lactoglobulin-retinol complex is also entirely reversible between 0.1 MPa and 400 MPa and the complex never dissociates in the studied pressure range. beta-lactoglobulin-cis-parinaric acid complexes at neutral and acid pH values dissociate irreversibly at 200 MPa and 350 MPa, respectively.

Binding Sites↗

PubMed1995

Impact of esterification on the folding and the susceptibility to peptic proteolysis of beta-lactoglobulin.

beta-Lactoglobulin was esterified and the differences between unmodified and ethylated beta-lactoglobulin were studied by microcalorimetry, circular dichroism and limited proteolysis. Microcalorimetric studies and circular dichroic spectra in aromatic regions revealed changes of esterified beta-lactoglobulin tertiary structure compared with native beta-lactoglobulin conformation in aqueous media. These changes are characteristic of molten globule state. While beta-lactoglobulin is resistant to peptic hydrolysis in aqueous and physiological conditions, a study of peptic action on esterified (ethylated, approximately 40% of the carboxyl groups substituted) beta-lactoglobulin in aqueous conditions showed that it is hydrolysed rapidly by this enzyme. The main part of the obtained peptic peptides has been purified and identified. Their analysis shows that 22 new sites of pepsin cleavage are induced by esterification of beta-lactoglobulin. Fourteen cleavage sites are pepsin specific and their unveiling is due to imposed tertiary structure changes. Eight of the observed new cleavage targets are entirely atypical containing either one or two distal dicarboxylic acid moieties. Apparently, the ethylation of beta- and/or gamma-carboxylates removing charges and grafting hydrophobic ethyl groups adapts substituted dicarboxylic amino-acid side chains for the recognition by pepsin.

Amino Acid Sequence↗

PubMed2004

Improved purification of beta-lactoglobulin from acid whey by means of ceramic hydroxyapatite chromatography with sodium fluoride as a displacer.

The successful separation of beta-lactoglobulin from other bovine whey proteins was performed by ceramic hydroxyapatite chromatography with a fluoride ion gradient in phosphate buffer as displacement agent. The method was applied to acid whey originating from milk of healthy as well as of mastitic cows. beta-Lactoglobulin was completely eluted in one peak at a fluoride concentration of about 0.6 mol/l. The purity of beta-lactoglobulin in this fraction was at least 96% if whey from healthy milk was processed. Co-eluted contaminants are traces of immunoglobulin G, serum albumin and lactoferrin. In case of mastitic whey the proportion of beta-lactoglobulin is diminished as the amounts of immunglobulin G, serum albumin and lactoferrin are increased within this fraction. Size exclusion chromatography on Superdex 75 pg effectively removed contaminants resulting in a purity for beta-lactoglobulin from normal whey of approximately 99%. The yield of beta-lactoglobulin from physiological whey was 50-55% referring to the fraction highly enriched with beta-lactoglobulin by hydroxyapatite chromatography. In case of mastitic milk the higher amounts of contaminants were also removed successfully by size exclusion chromatography.

Chromatography, Liquid↗

PubMed1997

Charge-dependent insertion of beta-lactoglobulin into monoglyceride monolayers.

The interactions between beta-lactoglobulin and 1-monostearoyl-glycerol were studied in order to gain insight into protein-gel-phase monoglyceride interactions. Using a monomolecular layer at the air-water interface, we determined the insertion of beta-lactoglobulin into the monoglycerides under different conditions of protein and surface charge by varying the pH and/or incorporating charged amphiphiles into the monolayer, respectively, and using subphases with either a low or high ionic strength. The interactions were quantified by determining the binding of 14C-labeled beta-lactoglobulin to the monolayer. Our results show the importance of electrostatics for binding of beta-lactoglobulin to condensed monoglycerides. Moreover, electrostatic interactions were found to be important for specific insertion of beta-lactoglobulin into the monolayer. A negatively charged surface in particular allowed positively charged beta-lactoglobulin to insert in a surface charge density-dependent manner, even at surface pressures as high as 36 mN/m, whereas under other conditions, the limiting insertion pressure was 32 mN/m. The rheological properties of the monolayer were not affected by the interactions with beta-lactoglobulin.

Animals↗

PubMed1996

Effects of physicochemical factors on the secondary structure of beta-lactoglobulin.

Fourier transform infrared spectroscopy and differential scanning calorimetry were used as complementary techniques to study changes in the secondary structure of beta-lactoglobulin under various physicochemical conditions. The effects of pH (3-9), NaCl (0-2 M), and lactose, glucose and sucrose (100-500 g/l) in the temperature range 25-100 degrees C on the conformation sensitive amide I band in the i.r. spectrum of beta-lactoglobulin in D2O solution were examined. The 1692 cm-1 band in the amide I band profile had not been definitively assigned in previous studies of the i.r. spectrum of beta-lactoglobulin. The decrease in this band at ambient temperature with time or upon mild heating was attributed to slow H-D exchange, indicating that it was due to a structure buried deep within the protein. The disappearance of the 1692 cm-1 band on heating was accompanied by the appearance of two bands at 1684 and 1629 cm-1, assigned to beta-sheets. The 1692 cm-1 band was therefore attributed to a beta-type structure. beta-Lactoglobulin showed maximum thermal stability at pH 3 and was easily denatured at pH 9. On denaturation, the protein unfolded into more extensive random coil structures at pH 9 than at pH 3. After 10 h at pH 9 (25 degrees C), beta-lactoglobulin was partly denatured. Heating to 60-80 degrees C generally resulted in the loss of secondary structure. At all pH values studied, two new bands at 1618 and 1684 cm-1, characteristic of intermolecular beta-sheet structure and associated with aggregation, were observed after the initial denaturation. Differential scanning calorimetry studies indicated that the thermal stability of beta-lactoglobulin was enhanced in the presence of sugars. The Fourier transform i.r. results obtained provide evidence that sugars promoted the unfolding of beta-lactoglobulin via multiple transition pathways leading to a transition state resisting aggregation.

Calorimetry, Differential Scanning↗

PubMed1999

Differential expression of bovine beta-lactoglobulin A and B promoter variants in transiently transfected HC11 cells.

Quantification of beta-lactoglobulin A and B in the milk of heterozygous animals has revealed a differential content of these two variants. Nucleotide sequence analysis of the first 733 bp of the bovine beta-lactoglobulin promoter has indicated the presence of ten polymorphic sites, nine of them being allele A and B specific mutations. To study the differential expression of these alleles, constructs containing 753 bp long fragments of the bovine beta-lactoglobulin A and B associated promoters were used in transient transfection experiments in HC11 cells. A differential transcription activity directed by the A and B specific promoters was consistently observed. The relative expression levels were 57% for beta-lactoglobulin A and 43% for beta-lactoglobulin B promoters. An allele-specific mutation has been reported to have a differential binding affinity to the activator protein-2 between the beta-lactoglobulin A and B promoters. However, experiments in HC11 cells where the activator protein-2 binding sites were mutated in both beta-lactoglobulin A and B promoters showed no major differences in activity between the mutated and native promoters.

Alleles↗

PubMed2004

Effect of sulfated polysaccharides on heat-induced structural changes in beta-lactoglobulin.

The mechanism that leads to a decreased aggregation of beta-lactoglobulin in the presence of dextran sulfate and lambda-carrageenan was investigated by assessing changes in the denaturation thermodynamics and protein structure. Differential scanning calorimetry results showed that the denaturation temperature (Tp) was about 4.6 degrees C higher in the presence of dextran sulfate, as compared with beta-lactoglobulin alone, whereas in the presence of lambda-carrageenan the difference in Tp was about 1.2 degrees C. Changes in protein structure studies using near-UV circular dichroism (CD) provided support for the calorimetric results. The transition midpoint (Tm) for denaturation of beta-lactoglobulin was about 5 degrees C higher in the presence of dextran sulfate than that found with beta-lactoglobulin alone and about 2 degrees C in the presence of lambda-carrageenan. Thermal modifications of the tertiary structure of beta-lactoglobulin were irreversible at temperatures above 67 degrees C; the addition of dextran sulfate reduced the extent of such modifications. Far-UV CD studies indicated that the addition of dextran sulfate or lambda-carrageenan did not affect secondary structure changes of beta-lactoglobulin upon heating. These studies indicate that dextran sulfate and lambda-carrageenan can enhance the stability of beta-lactoglobulin and thereby inhibit heat denaturation and aggregation.

Carrageenan↗

PubMed2000

Thermal unfolding and refolding of beta-lactoglobulin. An intrinsic andextrinsic fluorescence study.

The conformational features of beta-lactoglobulin, refolded by cooling from a thermally perturbed state, has been characterized by intrinsic and extrinsic fluorescence measurements on the protein. It is found that even at 85-90 degrees C, beta-lactoglobulin does not completely lose its folded structure. The unfolding and refolding of beta-lactoglobulin as observed through intrinsic tryptophan fluorescence is nearly reversible because the native beta-lactoglobulin and its refolded form, following heating and cooling, show nearly identical tryptophan fluorescence properties. However, the fluorescence properties of an extrinsic probe 1-anilino 8-naphthalene sulfonic acid (ANS) for the native and refolded forms are quite different from each other. Significant increase in fluorescence intensity and blue shifts in emission maxima of ANS bound to refolded beta-lactoglobulin is observed compared to that of the native form. Our results indicate that beta-lactoglobulin, refolded after heating to above 70 degrees C, has deep hydrophobic pockets which can be accessed by ANS. These pockets are either nonexistent or inaccessible to ANS in native beta-lactoglobulin. The opening of the central cavity collapses at pH close to the isoelectric pH of the protein. This indicates that electrostatic repulsion is necessary to keep this access open.

Animals↗

PubMed2003

EF loop conformational change triggers ligand binding in beta-lactoglobulins.

Beta-lactoglobulins, belonging to the lipocalin family, are a widely studied group of proteins, characterized by the ability to solubilize and transport hydrophobic ligands, especially fatty acids. Despite many reports, the mechanism of ligand binding and the functional role of these proteins is still unclear, and many contradicting concepts are often encountered in the literature. In the present paper the comparative analysis of the binding properties of beta-lactoglobulins has been performed using sequence-derived information, structure-based electrostatic calculations, docking simulations, and NMR experiments. Our results reveal for the first time the mechanism of beta-lactoglobulin ligand binding, which is completely determined by the opening-closing of EF loop, triggered by Glu89 protonation. The alkaline shift observed for Glu89 pKa in porcine beta-lactoglobulin (pKa 9.7) with respect to the bovine species (pKa 5.5) depends upon the interplay of electrostatic effects of few nearby key residues. Porcine protein is therefore able to bind fatty acids provided that the appropriate pH solution conditions are met (pH > 8.6), where the EF loop conformational change can take place. The unusually high pH of binding detected for porcine beta-lactoglobulin seems to be functional to lipases activity. Theoretical pKa calculations extended to representative beta-lactoglobulins allowed the identification of key residues involved in structurally and functionally important electrostatic interactions. The results presented here provide a strong indication that the described conformational change is a common feature of all beta-lactoglobulins.

Amino Acid Sequence↗

PubMed1993

Transfer of cow's milk beta-lactoglobulin to human serum after a milk load: a pilot study.

A sensitive and specific enzyme linked immunosorbent assay (ELISA) was developed for the quantification of cow's milk beta-lactoglobulin in human serum. The assay had a sensitivity of 80 pg/ml (2 standard deviations from zero) and did not cross react significantly with any other tested milk proteins. The absorption of cow's milk beta-lactoglobulin and beta-lactoglobulin immunoglobulin G antibody levels after a milk load was investigated in eight healthy non-allergic individuals. There was a significant variation in the circulating beta-lactoglobulin antigen levels during the six hour period after feeding (p < 0.01) for the combined data. A biphasic absorption pattern was seen in all subjects except one subject who had undetectable levels. No significant variation with time was observed for the beta-lactoglobulin IgG antibody levels and no correlation was found between the beta-lactoglobulin antigen levels and the beta-lactoglobulin IgG antibody levels. This pilot study offered information on the quantity of immunogenic cows' milk protein transferred to the blood after a milk load in non-allergic healthy adults and confirmed a biphasic profile of antigen transfer. The sensitivity of the assay used has shown that transfer of food antigen does occur in the majority of healthy nonallergic subjects.

Adult↗