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PubMed1998

Monomeric bovine beta-lactoglobulin adopts a beta-barrel fold at pH 2.

We have determined a crude structure of the apo form of bovine beta-lactoglobulin, a protein of 162 amino acid residues with a molecular mass of 18 kDa, at a low pH on the basis of data collected using only homonuclear 1H NMR spectroscopy. An ensemble of protein conformations was calculated with the distance-geometry algorithm for NMR applications (DYANA). The monomeric protein at low pH adopts a beta-barrel fold, well-superimposable on the structure determined by X-ray crystallography for the dimer at physiological pH. NMR evidence suggests the presence of disordered loop regions and terminal segments. Structural differences between the monomer at pH 2 and the dimer at pH 7, obtained by X-ray crystallography, are discussed, paying particular attention to surface electrostatic properties, in view of the high charge state of the protein at low pH.

Algorithms↗
PubMed1991

Adsorption-desorption isotherm hysteresis of beta-lactoglobulin A with a weakly hydrophobic surface.

Adsorption-desorption isotherms of bovine beta-lactoglobulin A (beta-lact A) on a weakly hydrophobic stationary phase (C1-ether) were measured by frontal analysis. The adsorption isotherms obtained at different pH were found to be dramatically different in shape, column capacity and desorption reversibility. At pH 4.5, an S-shaped adsorption isotherm was observed whereas at pH 6.0 a Langmuir isotherm was found. In addition, the desorption isotherm at pH 6.0 was found to overlap with the adsorption isotherm, and the adsorption-desorption process of beta-lact A under this condition could be characterized by a fully reversible Langmuir model. The desorption isotherm at pH 4.5, however, did not retrace the adsorption isotherm, resulting in hysteresis loops. A higher aggregate (tetramer) of beta-lact A is shown to be in an equilibrium with the beta-lact A protomer (dimer) at pH 4.5 whereas the dimer alone is predominant at pH 6.0. It is further shown that changes in the absorption coefficient between the adsorption and the desorption cycles for the tetramer at pH 4.5 can account for the hysteresis. The results demonstrate that pH can be a sensitive parameter in protein adsorption isotherm behavior and ultimately the behavior of species in preparative-scale chromatography.

Adsorption↗
PubMed1989

Role of association on protein adsorption isotherms. Beta-lactoglobulin A adsorbed on a weakly hydrophobic surface.

This paper explores the role of association on the adsorption isotherms of beta-lactoglobulin A on a weakly hydrophobic stationary phase at 4 degrees C and mobile phases of 0.85 M and 1 M ammonium sulfate, pH 4.5. The isotherms, obtained by frontal analysis, show an S-shape and the corresponding Scatchard plots indicate positive cooperativity. The slopes and intercepts of the Scatchard plots at low solute concentration are analyzed in terms of two species--a promoter and a higher order stronger adsorbing species. An explicit equation of the isotherm is developed based on this model, and this expression is shown to reproduce the isotherm shape using the appropriate derived parameters. It is further shown from this equation that a Langmuir-shaped adsorption isotherm can be obtained if the higher order associate or aggregate binds weaker to the support than the promoter. These results indicate that protein-protein interactions and the formation of associates can play a significant role on the shape of the isotherm and ultimately on the behavior of the species in preparative scale chromatography.

Adsorption↗
PubMed2000

Size-exclusion chromatography--multiangle laser light scattering analysis of beta-lactoglobulin and bovine serum albumin in aqueous solution with added salt.

The solution characteristics of beta-LGB (beta-lactoglobulin) and BSA (bovine serum albumin) are reported as determined by size-exclusion chromatography with on-line multiangle laser light scattering, differential refractive index and UV detection. The order of the three in series placed detectors as well as the interdetector volumes have been carefully pointed out. At concentrations below 2.5 mg/ml and at different values of pH the weight-average molecular mass of both proteins have been obtained. They indicate the appearance of monomers, dimers and higher order multimers. For beta-LGB the growth of self-associates could be observed at the isoelectric point over a period of days. The range of applicability of the method is discussed.

Biopolymers↗
PubMed2002

Epitopic characterization of native bovine beta-lactoglobulin.

Two monoclonal antibodies (mAbs) (mAb 97 and mAb 117) selected from a panel of 52 mAbs directed against beta-lactoglobulin (BLG) have previously been used to develop a two-site enzyme immunometric assay (EIA) specific for the native form of the protein [J. Immunol. Methods 220 (1998) 25]. In the present work, the conformational epitopes recognized by these two mAbs and by the 50 others have been studied. Firstly, an epitope map was drawn using a surface plasmon resonance (SPR) biosensor: the epitopes were organized in a circle of 11 overlapping and 1 nonoverlapping antigenic regions. Secondly, 55 site-directed BLGA mutants were prepared and tested by ELISA and competitive immunoassay to localize these 12 antigenic regions on the protein molecule. Among them, 20 mutants showed a 10- to 7500-fold decrease in relative affinity for the mAbs of one or several neighbouring regions: their circular dichroism (CD) spectra were identical to the spectrum of wild-type (WT) BLGA. At least one mutant was found for each of the 11 overlapping antigenic regions which circled the molecule and for the nonoverlapping one which was localized near the entrance of the calyx. The two mAbs initially chosen were each directed towards very conformation-dependent epitopes and were thus suitable for monitoring native BLG in food products and manufacturing processes. Other mAb pairs could be used to follow the fate of specific regions of the molecule during denaturation or proteolytic digestion.

Animals↗
PubMed1998

Identification of epitopes within beta lactoglobulin recognised by polyclonal antibodies using phage display and PEPSCAN.

Two different epitope mapping techniques were used to identify linear epitopes recognised by polyclonal IgG antibodies from rabbits immunised with bovine beta lactoglobulin (BLG), which is generally regarded as a major allergen in milk. The first, PEPSCAN, was used to investigate the binding of several rabbit polyclonal antisera to sequential overlapping peptides (12-mers) across the sequence of BLG. Each peptide was synthesized on a different polypropylene PIN, and a standard ELISA procedure was used to locate which of these peptides bound the antibodies under investigation. Comparisons of PEPSCANs for antisera from six different rabbits showed that each rabbit recognized a similar set of epitopes within BLG. PEPSCAN analysis also showed that polyclonal antibodies from the mouse recognize a set of epitopes similar to those recognized by the rabbit. The second epitope mapping technique is known as phage display and utilizes libraries of randomized short peptides fused to the coat proteins of filamentous phage as a source of epitopes for analysis. A gene VIII phage display library was used in this study with constrained nonapeptides, which were screened for epitopes recognized by affinity purified rabbit anti-BLG IgG. Immobilised rabbit anti-BLG IgG was screened in two separate experiments, each consisting of three rounds of panning. For each separate experiment, a sensitive phage ELISA was used to screen several hundred single phage clones for binding to anti-BLG IgG immobilised on microtiter plates. As a result, a number of positive phage were identified from the two separate screens of the library (19 different peptides were isolated, which resembled four different regions of BLG). The identified sequences were found to constitute a subset of the linear epitopes recognized by the PEPSCAN technique. The coordinates of the crystal structure of BLG were used to display mapped epitopes on its structure. This study has permitted detailed mapping of the major linear antigenic regions within BLG recognised by IgG antibodies from immunised rabbits and mice.

Amino Acid Sequence↗
PubMed1998

Two-site enzyme immunometric assays for determination of native and denatured beta-lactoglobulin.

Two enzyme immunometric assays suitable for measuring native and denatured beta-lactoglobulin (BLg) have been developed. The assays were performed in 96-well microtitre plates and were based on the use of pairs of monoclonal antibodies specific to either the native form or the reduced and carboxymethylated form of BLg (RCM-BLg). Detection limits of 30 and 200 pg/ml were obtained for the native BLg and the RCM-BLg assay, respectively, with very low or negligible cross-reactivity of the other milk proteins and tryptic fragments of BLg. The validity of the assays in different media such as cow's milk and cow's milk products, saline buffer or serum was supported by recovery experiments. The assays were first applied to the determination of BLg and RCM-BLg in PBS and in raw skimmed milk. The ability of the RCM-BLg assay to detect heat-denatured BLg was confirmed by a kinetic study of BLg heat-denaturation in the two media. During heat treatment, the decrease in the concentration of native BLg was associated with an increase in denatured BLg specifically detected by the RCM-BLg assay. By selecting an appropriate monoclonal antibody which failed to recognize caprine BLg, we were able to establish a modified sandwich immunoassay permitting very sensitive detection of cow's milk in goat's milk.

Animals↗
PubMed2000

Evaluation of a high IgE-responder mouse model of allergy to bovine beta-lactoglobulin (BLG): development of sandwich immunoassays for total and allergen-specific IgE, IgG1 and IgG2a in BLG-sensitized mice.

An animal model of food allergy represents an important tool for studying the mechanisms of induction and repression of an allergic reaction, as well as for the development of an immunotherapy to prevent or minimize such an adverse reaction. IgE and IgG1 (Th2 response) vs. IgG2a (Th1 response) are good markers for the induction of an allergic response in mice. Nevertheless, while the total serum concentrations of these isotypes are easy to measure using classical sandwich immunoassays, this is not the case for allergen-specific isotypes. To develop an animal model of allergy to bovine beta-lactoglobulin (BLG), we set up quantitative assays for total and for allergen-specific IgE, IgG1 and IgG2a. Microtiter plates coated either with anti-isotype antibodies (Abs) or with allergen were used for Ab capture, while anti-isotype Fab' fragments coupled to acetylcholinesterase were used for visualization. These assays of anti-BLG specific Abs are original in two ways. First, assay calibration is performed using anti-BLG specific mAbs, thus allowing good quantification of the different isotypes and subclasses of serum antibodies. Second, the detection of all anti-BLG specific Abs, i.e., those recognizing both the native and denatured forms of the protein, is achieved through indirect coating of BLG using biotin-streptavidin binding. The present assays are quantitative, specific to the isotype (cross-reactivity <0.5%), very sensitive (detection limit in the 10 pg/ml range), and reproducible (coefficient of variation less than 10%). Applied to the humoral response in mice sensitized with BLG adsorbed on alum, these assays proved to be a very useful tool for monitoring high IgE-responder mice following BLG immunization, and for an immunotherapy directed at polarizing the immune response.

Adsorption↗
PubMed2002

The ligand-binding site of bovine beta-lactoglobulin: evidence for a function?

Ever since the fortuitous observation that beta-lactoglobulin (beta-Lg), the major whey protein in the milk of ruminants, bound retinol, the details of the binding have been controversial. beta-Lg is a lipocalin, like plasma retinol-binding protein, so that ligand association was expected to make use of the central cavity in the protein. However, an early crystallographic analysis and some of the more recent solution studies indicated binding elsewhere. We have now determined the crystal structures of the complexes of the trigonal form of beta-Lg at pH 7.5 with bound retinol (R=21.4% for 7329 reflections between 20 and 2.4 A resolution, R(free)=30.6%) and with bound retinoic acid (R=22.7% for 7813 reflections between 20 and 2.34 A resolution, R(free)=29.8%). Both ligands are found to occupy the central calyx in a manner similar to retinol binding in retinol-binding protein. We find no evidence of binding at the putative external binding site in either of these structural analyses. Further, competition between palmitic acid and retinol reveals only palmitate bound to the protein. An explanation is provided for the lack of ligand binding to the orthorhombic crystal form also obtained at pH 7.5. Finally, the possible function of beta-Lg is discussed in the light of its species distribution and similarity to other lipocalins.

Animals↗
PubMed2003

Antinociception induced by beta-lactotensin, a neurotensin agonist peptide derived from beta-lactoglobulin, is mediated by NT2 and D1 receptors.

In this study, we examined the antinociceptive effect of beta-lactotensin, a neurotensin agonist that has been isolated from the chymotrypsin digest of beta-lactoglobulin as an ileum-contracting peptide. Beta-lactotensin showed naloxone-insensitive antinociceptive activity by the tail-pinch test after i.c.v. (200 nmol/mouse) or s.c. (300 mg/kg) administration in ddY mice. Tolerance was not developed to antinociception induced by beta-lactotensin after repeated s.c. administration for 5 days. The antinociceptive activity of beta-lactotensin was blocked by treatment with the neurotensin NT2 receptor antisense ODN, while treatment with the NT1 receptor antisense ODN had no effect. The antinociceptive activity was also blocked by a dopamine D1 receptor antagonist, SCH23390 (1 microg/mouse, i.c.v.), while a D2 receptor antagonist, raclopride (0.5 microg/mouse, i.c.v.), did not block the activity. These results indicate that the antinociceptive activity of beta-lactotensin is mediated by NT2 and D1 receptors.

Analgesics↗
PubMed2000

Interaction of beta-lactoglobulin with phospholipid bilayers: a molecular level elucidation as revealed by infrared spectroscopy.

Fourier transform infrared (FTIR) spectroscopy has been used to study, at a molecular level, the interactions between beta-lactoglobulin (BLG), the most abundant globular protein in milk, and some lipids (sphingomyelin, SM; dimyristoylphosphatidylcholine, DMPC; dipalmytoylphosphatidylcholine, DPPC; dimyristoylphosphatidylserine-sodium salt, DMPS; dipalmitoylphosphatidylserine-sodium salt, DPPS) constituting the milk fat globule membrane (MFGM). The interactions were monitored with respect to alteration in the secondary structure of BLG, as registered by the amide I' band, and phospholipid conformation, as revealed by the acyl chain and carbonyl bands. The results show that neither the conformation nor the thermotropism of neutral bilayers containing DMPC or DPPC is affected by BLG. Reciprocally, the secondary structure and thermal behaviour of pure BLG remain the same in the presence of PC. These results suggest that no interaction occurs between PC and BLG, in agreement with previous studies. However, it is found that BLG interacts with neutral bilayers constituted by milk SM lipids, increasing gauche conformers and thus conformational disorder of the lipid acyl chains. This perturbing effect has been attributed to a partial penetration of BLG into the hydrophobic core of the bilayer, which allows hydrophobic interactions between BLG and SM. Moreover, the fact that SM possesses the same headgroup of PC implies that the head group does not prevent the occurrence of BLG-lipid interactions and other lipid regions can control the binding of BLG to lipids. Furthermore, BLG was found to interact electrostatically with charged bilayers containing PS, leading to a rigidification of the lipid hydrocarbon chains and a dehydration of the interfacial region. This last effect suggests that the protein limits the accessibility of water molecules to the interfacial region of the phospholipids by its presence at the membrane surface.

Animals↗
PubMed1992

Gelation of globular proteins: effect of pH and ionic strength on the critical concentration for gel formation. A simple model and its application to beta-lactoglobulin heat-induced gelation.

The influence of pH (2-9) and ionic strength (0-0.14 M NaCl) on the sol-gel transition of beta-lactoglobulin was investigated in order to determine the critical gel concentration (C0). The concentration necessary to form a gel near the isoelectric pH remains approximately constant (approximately 1% w/v) independently of the ionic strength. At other pH values, the higher the ionic strength is, the lower the protein concentration must be to form a gel. A theoretical model to relate the effect of the intensity and the range of electrostatic interactions on the critical concentration (C0) is proposed and fits reasonably with the experimental results.

Electrochemistry↗
PubMed1998

Effects of pH and salt environment on the association of beta-lactoglobulin revealed by intrinsic fluorescence studies.

The effects of pH, ionic strength and heat on the structure of beta-lactoglobulin (beta-lg) have been investigated by studying the intrinsic tryptophan fluorescence of the protein. Between pH 2 and 9, for sodium chloride concentrations varying from 0.0 to 0.2 M, the position of the fluorescence emission maximum at 20 degrees C remained constant at 328 nm, suggesting that the hydrophobic environment of the fluorophores remained unchanged. The fluorescence intensity increased significantly at pH 2, 7 and 9 on reducing the NaCl concentration of the solutions. The most likely explanation for this, supported by recent light scattering data, is that the presence of NaCl is necessary for beta-lg to dimerize. At the higher NaCl concentrations it was found that dimerization accompanied a reduction in fluorescence intensity. Thus, dissociation appears to reduce the self-quenching of tryptophan residues that occurs within the dimer. The fluorescence of solutions heated below the denaturation temperature reflected the state of association of the protein molecules. Above the denaturation temperature and associated with protein aggregation, an irreversible increase in intrinsic tryptophan fluorescence was observed. We also report what we believe to be the first front-face fluorescence measurements on globular protein gels, showing effects of pH and NaCl concentration.

Animals↗
PubMed1998

Protein stability function relations: beta-lactoglobulin-A sulphydryl group reactivity and its relationship to protein unfolding stability.

The effect of protein stability on the reactivity of the free sulphydryl (SH) group in beta-lactoglobulin-A (beta-LgA) provides a model for the study of protein stability-function relations (PSFR). The free energy change for protein unfolding (delta G(o)) and SH group exposure (delta GSH) were determined from (i) the urea unfolding curve for beta-LgA and (ii) the kinetics of beta-LgA SH/disulphide exchange with 2-pyridine disulphide (2-PDS) in 0-8 M urea (pH 3). Protein unfolding profiles determined from extrinsic fluorescence and SH-group reactivity measurements were not coincident. beta-LgA formed a stable intermediate (X) state in the presence of 4 M urea with delta G(o) = 20 (+/- 0.03) kJ/mol. From the low rate of SH/disulphide exchange in 4 M urea, the SH-group within beta-LgA was efficiently masked within the X-state. SH reactivity increased after beta-LgA was unfolded in 6-8 M urea with, delta GSH = 43(+/- 6.4) kJ/mol. Such results are discussed in terms of possible interrelationships between protein unfolding stability and SH reactivity in beta-LgA.

Drug Stability↗
PubMed1998

Some aspects of beta-lactoglobulin structural properties in solution studied by fluorescence quenching.

The technique of protein fluorescence quenching by acrylamide and sodium nitrite (NO2-) was used to study some structural aspects of beta-lactoglobulin in solution. The degree of exposure and the micro-environments of the two tryptophanyl residues (Trp-19 and Trp-61) present in this ruminant milk protein were sensed, and the influence of the pH and the binding of palmitic acid in their accessibilities were analyzed. The results obtained showed that Trp-19 has an accessibility to the quenchers higher than could be supposed from its structural location. The binding of palmitic acid, on the other hand, increases the accessibility of both tryptophanyl residues, a fact that could be associated with a slight conformational change of the protein.

Acrylamide↗
PubMed1999

Structural investigation of beta-lactoglobulin gelation in ethanol/water solutions.

The aggregation and gelation properties of beta-lactoglobulin (BLG), a globular protein from milk, was studied in hydro-ethanolic solutions (50/50% (v/v)) at room temperature. The phase state diagrams as a function of pH and ethanol concentration showed that a gel structure appeared after a period ranging from 1 min to 1 week depending on the physico-chemical conditions. The aggregation kinetics, studied by infrared spectroscopy and dynamical rheological measurements, highly depended upon the pH; the process being the fastest at pH 7. Alcohol-induced aggregation of BLG was characterized by the formation of intermolecular hydrogen bonded beta-sheet structures. Small angle neutron scattering indicated that the aggregates structures in the final gels were similar at pH 7, 8 and 9. Through the data obtained at the molecular and macroscopic levels, it can be concluded that the kinetics of gelation were pH dependent while the spatial arrangements of the aggregates were similar in the final structures. The heterogeneous structures formed in hydro-ethanolic gels could be analysed in terms of a phase separation, the syneresis being the final visible state.

Ethanol↗
PubMed2000

Effect of pH on the structure and aggregation of human glycodelin A. A comparison with beta-lactoglobulin A.

The effect of pH on the structure of glycodelin A (GdA) and of beta-lactoglobulin A (beta-LgA) has been investigated by means of circular dichroism, steady state fluorescence, synchrotron radiation small angle X-ray scattering (SR-SAXS) and gel permeation chromatography. The comparison between GdA and beta-LgA shows that, at pH 7.0, both proteins are dimers with an extended content of beta-sheet conformation, but pH 2.0 and 9.0 yield a different secondary, tertiary and quaternary structural organisation. Whilst beta-LgA is a monomer, that conserves beta-sheet conformation at pH 2.0 and 9.0, GdA has a stable dimeric structure at alkaline pH, but at pH 2.0 increases its alpha-helix content and it aggregates soon. SR beam has been used to perform SAXS comparative measurements of the two proteins. SR-SAXS data provide the radius of gyration and the radii of the cross-section and of the thickness. GdA aggregation at acid pH has been characterised by calculating the distance distribution function (P(r)). Isoelectric focusing and chromatofocusing data show a different charge distribution on the surfaces of the two proteins, supporting the hypothesis that the presence of oligosaccharides deeply influences the conformational state and the aggregation process of GdA at different pH values. In particular, the presence of sialic acid residues, within the oligosaccharide moiety of the GdA, might be responsible for the differences observed between the two proteins.

Animals↗
PubMed2001

Conformational rearrangement of beta-lactoglobulin upon interaction with an anionic membrane.

Interactions between beta-lactoglobulin (beta-lg) and dimyristoylphosphatidylglycerol (DMPG) bilayers were studied using one- and two-dimensional infrared spectroscopy above (pD 7.4) and below (pD 4.4) the protein's (beta-lg's) isoelectric point (pI=5.2). The aim of the study was threefold: (1) gain a better understanding of beta-lg-phospholipid interaction; (2) provide information relative to the structure of beta-lg as it interacts with membranes; (3) determine whether the conformational modifications of the protein in the presence of lipids are strictly caused by thermal effects or whether they are modulated by the chain-melting phase transition. At pD 7.4, the lipid thermotropism, the acyl-chain order, and the membrane interfacial region were essentially unaffected by the presence of beta-lg, whereas the protein amide I region showed dramatic alterations. The results suggested the predominance of beta-sheets and alpha-helix elements, with a lost of structural integrity. At pD 4.4, beta-lg induced an approximately 2 degrees C downshift of the transition temperature, whereas the conformational order of the lipid chain decreased in the gel phase and increased in the liquid-crystalline phase. The hydration state of the DMPG C==O groups increased in the liquid-crystalline phase. The conformation of beta-lg at pD 4.4 in the presence of DMPG showed similarities with that observed at pD 7.4, but an increase in the alpha-helix content and a reduced thermal stability were noticed. In contrast to the protein alone, beta-lg aggregates in the presence of DMPG at pD 4.4 above 50 degrees C. At both pD values, the charged surface of the membrane seemed to be the main factor for inducing protein conformational changes by altering the intramolecular interactions that stabilize the native structure. However, protein incorporation within the membrane seemed to be involved at pD 4.4. The two-dimensional analysis performed with spectra recorded upon heating showed that spectral intensity changes at pD 4.4 and 7.4 occurred at the same frequencies in the amide I' region. The heat-induced structural changes of beta-lg were not correlated with the conformational modifications of the phospholipids along the phase transition, indicating that the thermal behavior of the protein was not modulated by the lipid chain melting, but rather represented the heat-induced protein rearrangement in the presence of DMPG.

Animals↗