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At least 487 records · Page 27Linked to original sources
PubMed2005

Effect of cationic surfactants on the interaction between sodium perfluorooctanoate and beta-lactoglobulin.

The interaction between the fluorocarbon surfactant, sodium perfluorooctanoate (SPFO), and beta-lactoglobulin (BLG) was studied. In particular, the effects of cationic surfactants, such as alkyltriethylammonium bromide (C(n)NE, n=8, 10, 12), on SPFO-BLG interaction were examined. It was shown that the anionic fluorocarbon surfactant, SPFO, was a strong denaturant of BLG. The ability of SPFO to denature BLG could be weakened by the addition of C(n)NE. The effect of C(n)NE on SPFO-BLG interaction was related to the hydrocarbon chain length of C(n)NE, and also the molar ratio of the added C(n)NE to the SPFO in SPFO-BLG solutions ([C(n)NE]/[SPFO]). Our findings might provide a way to design surfactant systems that are less denaturing to proteins or tailor the ability of surfactant to denature proteins through the appropriate mixing with other surfactants.

Caprylates↗
PubMed2005

Adsorption behaviour of lactoferrin in oil-in-water emulsions as influenced by interactions with beta-lactoglobulin.

Oil-in-water emulsions (pH 7.0 or pH 3.0) containing 30 wt% soya oil and various concentrations of lactoferrin were made in a two-stage valve homogenizer. The average droplet size (d32), the surface protein coverage (mg/m2) and composition, and the zeta-potential of the emulsions were determined. The value of d32 decreased with increasing lactoferrin concentration up to 1%, and then was almost independent of lactoferrin concentration beyond 1% at both pH 7.0 and pH 3.0. The surface protein coverage of the emulsions made at pH 7.0 increased almost linearly with increasing lactoferrin concentration from 0.3 to 3%, but increased only slightly in emulsions made at pH 3.0 at lactoferrin concentrations >1%. The surface protein coverage of the emulsions made at pH 3.0 was lower than that of the emulsions made at pH 7.0 at a given protein concentration. The emulsion droplets had a strong positive charge at both pH 7.0 and pH 3.0, indicating that stable cationic emulsion droplets could be formed by lactoferrin alone. When emulsions were formed with a mixture of lactoferrin and beta-lactoglobulin (beta-lg) (1:1 by weight), the charge of the emulsion droplets was neutralized at pH 7.0 suggesting the formation of electrostatic complexes between the two proteins. The composition of the droplet surface layer showed that both proteins were adsorbed, presumably as complexes, from the aqueous phase at pH 7.0 in equal proportions, whereas competitive adsorption occurred between lactoferrin and beta-lg at pH 3.0. At this pH, beta-lg was adsorbed in preference to lactoferrin at low protein concentrations (1%), whereas lactoferrin appeared to be adsorbed in preference to beta-lg at high protein concentrations.

Adsorption↗
PubMed2005

Microstructure of beta-lactoglobulin-stabilized emulsions containing non-ionic surfactant and excess free protein: influence of heating.

The influence of the non-ionic surfactant Tween 20 on the microstructure of beta-lactoglobulin-stabilized emulsions with substantial excess free protein present was investigated via confocal microscopy. The separate distributions of oil droplets and protein were determined using two different fluorescent dyes. In the emulsion at ambient temperature the excess protein and protein-coated oil droplets were associated together in a reversibly flocculated state. The pore-size distribution of the initial flocculated emulsion was found to depend on the surfactant/protein ratio R, and at higher values of R the system became more inhomogeneous due to areas of local phase separation. Evidence for competitive displacement of protein from the oil-water interface by surfactant was obtained only on heating (from 25 to 85 degrees C) during the process of formation of a heat-set emulsion gel. By measuring fluorescence intensities of the protein dye inside and outside of the oil-droplet-rich areas, we have been able to quantify the evolving protein distribution during the thermal processing. The results are discussed in relation to previous work on the competitive adsorption of proteins and surfactants in emulsions and the effect of emulsion droplets on the rheology of heat-set protein gels.

Emulsions↗
PubMed2006

Complex coacervation between beta-lactoglobulin and Acacia gum: a nucleation and growth mechanism.

Complex coacervation between proteins and polysaccharides is a demixing process mainly driven by electrostatic interactions. During this process many structural transitions occur, involving the formation of soluble complexes, aggregated complexes, and coacervates. The dynamic mechanism of complexation/coacervation was studied on beta-lactoglobulin (BLG)/Acacia gum (AG) mixed dispersions (0.1 wt% total concentration; BLG:AG ratio of 2:1) using small angle static light scattering (SALS). Acidification of BLG/AG dispersions was induced by dissolution of 0.11 wt% glucono-delta-lactone, allowing in situ SALS measurements. Time evolution of turbidity, scattered light intensity at 46 degrees scattering angle (I46) or slope of scattering functions at high q range revealed the existence of six pH-induced structural transitions. During BLG/AG complexation and before coacervation took place, scattering profiles displayed a monotonic decrease of I(q) as a function of q. A correlation peak in the scattering functions was only observed when coacervates appeared in the system. The wave vector q(max) corresponding to the maximum in scattered intensity first shifted toward larger q values, indicating an increasing number of coacervates, then shifted toward smaller q values, as a consequence of the system coarsening. The power laws q(max) approximately t(-alpha) and I(max) approximately t(-beta) gave values of 1.9 and 9.2, respectively, values much larger than those expected for intermediate and late stages of spinodal decomposition. From these results, it was concluded that complex coacervation between BLG and AG was a nucleation and growth type process. In addition, the temporal evolution of I46 followed power laws with two different exponents. First exponent corresponding to BLG/AG complexation was 3.0+/-0.3 and indicated a diffusion-controlled growth mechanism. Second exponent corresponding to the initiation of phase separation to the coacervation process was 6.5+/-0.3 and revealed an interfacially-controlled growth mechanism.

Gum Arabic↗
PubMed2006

Interactions of beta-lactoglobulin with sodium decylsulfonate, decyltriethylammonium bromide, and their mixtures.

The interactions of beta-lactoglobulin (BLG) with anionic surfactant sodium decylsulfonate (C10SO3), cationic surfactant decyltriethylammonium bromide (C10NE), and the mixtures of cationic-anionic surfactants (C10NE-C10SO3) were investigated by circular dichroism (CD) and fluorescence methods. At pH 7.0, C10NE and the C10NE-rich surfactant mixtures of C10NE-C10SO3 could form precipitates with BLG, while C10SO3, equimolar mixtures of C10NE-C10SO3, or C10SO3-rich mixtures of C10NE-C10SO3 form homogeneous solutions with BLG. CD observed that both C10NE and C10SO3 could change the BLG structure. The effects of the mixtures of C10NE-C10SO3 on BLG structure depended on the ratio of C10NE to C10SO3. The C10NE-rich or the C10SO3-rich mixtures of C10NE-C10SO3 could significantly affect BLG structure, while the equimolar mixtures of C10NE-C10SO3 exhibited weaker interaction with BLG. Fluorescence measurements showed that both C10NE and C10SO3 could induce the enhancement of fluorescence of BLG, and C10NE enhanced the BLG fluorescence more than C10SO3 did. The effect of the mixtures of C10NE-C10SO3 on the fluorescence of BLG became stronger with the increase of the molar fraction of C10NE in C10NE-C10SO3 mixtures.

Aspartic Acid↗
PubMed2005

Helical and expanded conformation of equine beta-lactoglobulin in the cold-denatured state.

The thermal unfolding transition of equine beta-lactoglobulin (ELG) was investigated by circular dichroism (CD) over a temperature range of -15 degrees C to 85 degrees C. In the presence of 2 M urea, a cooperative unfolding transition was observed both with increasing and decreasing temperature. The CD spectrum indicated that the heat and cold-denatured states of ELG have substantial secondary structures but lack persistent tertiary packing of the side-chains. In order to clarify the relation between the heat or cold-denatured state and the acid-denatured (A) state characterized previously, we have attempted to observe the temperature dependence of the CD spectrum at pH 1.5. The CD spectrum in the heat-denatured state is similar to that in the A state. The CD spectrum in the A state does not change cooperatively with increasing temperature. These results indicate that the heat-denatured state and the A state are the same structural state. On the other hand, the CD intensity at acid pH cooperatively increased with decreasing temperature. The CD spectrum at low temperature and acid pH is consistent with that in the cold-denatured state. Therefore, the cold-denatured state is distinguished from the heat-denatured state or the A state, and ELG assumes a larger amount of non-native alpha-helices in the cold-denatured state. Small angle X-ray scattering and analytical ultracentrifugation have indicated that ELG assumes an expanded chain-like conformation in the cold-denatured state in contrast to the compact globular conformation in the A state. The relation between the molecular size and the helical content in the partially folded states is discussed.

Animals↗
PubMed2005

Dynamics and mechanism of the Tanford transition of bovine beta-lactoglobulin studied using heteronuclear NMR spectroscopy.

The Tanford transition is a conformational change of bovine beta-lactoglobulin (betaLG) occurring at around pH 7, identified originally on the basis of optical rotatory dispersion and the accessibility of a thiol group. X-ray analysis has suggested that a conformational change to the EF-loop is responsible for the Tanford transition, with the loop closing the hydrophobic cavity of the beta-barrel of the betaLG molecule below pH 7 and flipping to open the cavity above pH 7. To clarify the dynamics of this conformational change, NMR measurements were made at neutral pH. Since severe signal broadening due to monomer-dimer equilibrium prevented NMR measurements of wild-type betaLG at neutral pH, we searched for optimal sample conditions, finding that a disulfide bond-linked dimer of the mutant A34C gives an HSQC spectrum without signal broadening. The HSQC and CD spectra indicated that in overall conformation A34C is similar to wild-type betaLG, suggesting that the A34C dimer is a good model with which to study the structure and dynamics of the wild-type at neutral pH. The pH-dependent HSQC signal changes and Lipari-Szabo type relaxation analyses of the A34C dimer revealed that the conformational change to the EF-loop occurs above pH 7. We observed two types of motions in the EF-loop region; relatively fast (micro- to milliseconds) and slow (milliseconds or slower) conformational exchanges of the residues located in the hinge and top of the EF-loop regions, respectively. Furthermore, the GH-loop adjacent to the EF-loop exhibited conformational change at a pH slightly lower than that at which the EF-loop motions occurred. From these observations, we propose a three-step mechanism of conformational change in the EF-loop leading to the Tanford transition, in which the GH-loop conformational change, the cleavage of the hydrogen bonds at the hinge, and the flip of the EF-loop occur sequentially.

Animals↗
PubMed2006

Structure of bovine beta-lactoglobulin (variant A) at very low ionic strength.

Bovine beta-lactoglobulin (BLG) is a globular protein of uncertain physiological function and a member of the lipocalin superfamily of proteins. Here, we present the X-ray structure at 3.0 angstroms of BLG (variant A) from an orthorhombic (P2(1)2(1)2(1)) pseudo-tetragonal crystal form that suffers from pseudo-merohedral twinning (final R(working) = 0.224, R(free) = 0.265). Crystals were grown by dialysis against ultra-purified water (i.e., at very low ionic strength), at pH approximately 5.2 (approximately pI), conditions vastly different from all other BLG structures determined previously. This allows critical assessment of the BLG structure and of the influence that pH, ionic strength, and crystal packing may have on the molecular structure of BLG. The pH-sensitive EF loop is found in the closed conformation characteristic of BLG at pH less than 7 and moderate to high ionic strength. Although the hydrophobic pocket appears to be empty, the presence of highly disordered water molecules cannot be excluded. The dimer interface and the hydrophobic pocket (calyx) are preserved. However, the orientation of the subunits in the dimer varies considerably with crystal form. Structure is deposited with PDB ID 2akq.

Animals↗
PubMed2002

Role of calcium as trigger in thermal beta-lactoglobulin aggregation.

Divalent calcium ions have been suggested to be involved in intermolecular protein-Ca2+-protein cross-linking, intramolecular electrostatic shielding, or ion-induced protein conformational changes as a trigger for protein aggregation at elevated temperatures. To address the first two phenomena in the case of beta-lactoglobulin, a combination of chemical protein modification, calcium-binding, and aggregation studies was used, while the structural integrity of the modified proteins was maintained. Although increasing the number of carboxylates on the protein by succinylation results in improved calcium-binding, calcium appears to be less effective in inducing protein aggregation. In fact, the larger the number of carboxylates, the higher the concentration of calcium that is required to trigger the aggregation. Lowering the number of negative charges on the protein surface via methylation of carboxylates reduces calcium-binding properties, but calcium-induced aggregation at low concentration is improved. Monovalent sodium ions cannot take over the specific role of calcium. The relation between net surface charge and number of calcium ions bound required to trigger the aggregation suggests that calcium needs to bind site specific to carboxylates with a threshold affinity. Subsequent site-specific screening of surface charges results in protein aggregation, driven by the partial unfolding of the protein at elevated temperatures, which is then facilitated by the absence of electrostatic repulsion.

Animals↗
PubMed2002

Retinoic acid binding properties of the lipocalin member beta-lactoglobulin studied by circular dichroism, electronic absorption spectroscopy and molecular modeling methods.

Interaction between the Vitamin A derivative all-trans retinoic acid and the lipocalin member bovine beta-lactoglobulin (BLG) was studied by circular dichroism (CD) and electronic absorption spectroscopy at different pH values. In neutral and alkaline solutions achiral retinoic acid forms a non-covalent complex with the protein as indicated by the appearance of a negative Cotton effect around 347 nm associated to the narrowed and red shifted pi-pi(*) absorption band of the ligand. The induced optical activity is attributed to the helical distortion of the conjugated chain caused by the chiral protein binding environment. As the disappearing CD activity showed in the course of CD-pH titration experiment, retinoic acid molecules dissociate from BLG upon acidification but this release is completely reversible as proved by the reconstitution of the CD and absorption spectra after setting the pH back to neutral. This unique behavior of the complex is explained by the conformational change of BLG (Tanford transition) which involves a movement of the EF loop at the entrance of the central cavity from open to closed conformation in the course of pH lowering. From these results it was inferred that retinoic acid binds within the hydrophobic calyx of the beta-barrel.

Animals↗
PubMed2000

Characterization and isolation of intermediates in beta-lactoglobulin heat aggregation at high pH.

The early stages of heat induced aggregation at 67.5 degrees C of beta-lactoglobulin were studied by combined static light scattering and size exclusion chromatography. At all conditions studied (pH 8.7 without salt and pH 6.7 with or without 60 mM NaCl) we observe metastable heat-modified dimers, trimers, and tetramers. These oligomers reach a maximum in concentration at about the time when large aggregates (1000-4000 kg/mol) appear, after which they decline in concentration. By isolating the oligomers it was demonstrated that they rapidly form aggregates upon heating in the absence of monomeric protein, showing that these species are central to the aggregation process. To our knowledge this is the first time that intermediates in protein aggregation have been isolated. At all stages of aggregation the dominant oligomer was the heat-modified dimer. Whereas the heat-modified oligomers are formed at a higher rate at pH 8.7 than at pH 6.7, the opposite is the case for the formation of aggregates from the metastable oligomers indicating cross-linking via disulfide bridges for the oligomers and noncovalent interaction in the formation of the aggregates. The data suggest that an aggregate nucleus is formed from four oligomers. For protein concentrations of 10 or 20 g/l a heat-modified monomer can be observed until about the time when the maximum in concentration appears of the heat-modified dimer. The disappearance of this heat-modified monomer correlates to the formation of dimers (trimers and tetramers).

Dimerization↗
PubMed1979

Absorption spectra for the complexes formed from vitamin-A and beta-lactoglobulin.

The interactions between vitamin-A and beta-lactoglobulin have been investigated. We have found that two different complexes can be formed: one involving vitamin-A, and one involving a derivative of vitamin-A that most probably has a retro-beta-ionylidene structure. Room temperature absorption spectra for these complexes in phosphate buffer at pH 7.50 are reported and discussed.

Animals↗
PubMed1998

Pressure-induced subunit dissociation and unfolding of dimeric beta-lactoglobulin.

Effects of hydrostatic pressure on dimeric beta-lactoglobulin A (beta-Lg) were investigated. Application of pressures of up to 3.5 kbar induced a significant red shift ( approximately 11 nm) and a 60% increase in intrinsic fluorescence emission of beta-Lg. These changes were very similar to those induced by guanidine hydrochloride, which caused subunit dissociation and unfolding of beta-Lg. A large hysteresis in the recovery of fluorescence parameters was observed upon decompression of beta-Lg. Pressure-induced dissociation and unfolding were not fully reversible, because of the formation of a nonnative intersubunit disulfide bond that hampered correct refolding of the dimer. Comparison between pressure dissociation/unfolding at 3 degrees C and 23 degrees C revealed a marked destabilization of beta-Lg at low temperature. The stability of beta-Lg toward pressure was significantly enhanced by 1 M NaCl, but not by glycerol (up to 20% v/v). These observations suggest that salt stabilization was not related to a general cosolvent effect, but may reflect charge screening. Interestingly, pressure-induced dissociation/unfolding was completely independent of beta-Lg concentration, in apparent violation of the law of mass action. Possible causes for this anomalous behavior are discussed.

Animals↗
PubMed1999

Two-phase induction of the nonnative alpha-helical form of beta-lactoglobulin in the presence of trifluoroethanol.

The trifluoroethanol-dependent induction of the nonnative alpha-helical form of beta-lactoglubulin has been studied by circular dichroism spectroscopy. Data analysis is performed by factor analysis and multivariate curve resolution. An intermediate form in the induction of the alpha-helical form of the beta-lactoglobulin has been identified at low TFE concentration. By application of an alternating least-squares algorithm, the CD spectrum corresponding to the intermediate form has been resolved. The deconvolution of this CD spectrum shows a secondary structure content more in agreement with the one predicted from the amino acid sequence than the secondary structure of the helical form obtained at higher TFE concentrations. The additional alpha-helical content of the form present at higher TFE concentrations could be due to nonspecific interaction of TFE with the polypeptide chain.

Algorithms↗
PubMed2001

Refolding of beta-lactoglobulin studied by stopped-flow circular dichroism at subzero temperatures.

Refolding of bovine beta-lactoglobulin was studied by stopped-flow circular dichroism at subzero temperatures. In ethylene glycol 45%-buffer 55% at -15 degrees C, the isomerization rate from the kinetic intermediate rich in alpha-helix to the native state is approximately 300-fold slower than that at 4 degrees C in the absence of ethylene glycol, whereas the initial folding is completed within the dead time of the stopped-flow apparatus (10 ms). At -28 degrees C, we observed at least three phases; the fastest process, accompanied by an increase of alpha-helix content, is completed within the dead time of the stopped-flow apparatus (10 ms), the second phase, accompanied by an increase of alpha-helix content with the rate of 2 s(-1), and the third phase, accompanied by a decrease of alpha-helix content. This last phase, corresponding to the isomerization process at -15 degrees C described above, was so slow that we could not monitor any changes within 4 h. Based on the findings above, we propose that rapid alpha-helix formation and their concurrent collapse are common even in proteins rich in beta-structure in their native forms.

Circular Dichroism↗
PubMed2002

Induced chirality upon binding of cis-parinaric acid to bovine beta-lactoglobulin: spectroscopic characterization of the complex.

Binding of the polyunsaturated cis-parinaric acid to bovine beta-lactoglobulin (BLG) was studied by circular dichroism (CD), electronic absorption spectroscopy and mass spectrometry methods. Upon protein binding, the UV absorption band of parinaric acid is red shifted by ca. 5 nm, showing hypochromism and reduced vibrational fine structure, suggesting that the ligand binds as a monomer in non-planar geometry. In the CD spectra measured at pH 7.36 and 8.5 a strong, negative Cotton band appears centered at 310 nm (Delta epsilon = -25 M(-1) cm(-1)) corresponding to the long-wavelength absorption band of cis-parinaric acid. The source of this induced optical activity is the helical distortion of the polyene chromophore caused by the chiral protein environment. From CD spectral data the value of the association constant was calculated to be 4.7 x 10(5) M(-1) at pH 7.36. CD and mass spectrometry measurements showed that parinaric acid binds weakly to BLG in acidic solution, though small peaks at mass 18,559 and 18,645 can be obtained in the reconstructed electrospray mass spectrum; these correspond to the binding of parinaric acid in 1:1 stoichiometry to both monomer variants of BLG B and A. The hydrophobic interior cavity of BLG was assigned as the primary binding site of cis-parinaric acid.

Animals↗
PubMed2003

A new ligand for an old lipocalin: induced circular dichroism spectra reveal binding of bilirubin to bovine beta-lactoglobulin.

This study reports that bilirubin-bovine beta-lactoglobulin (BLG) complexes exhibit very characteristic induced circular dichroism (CD) spectra in the visible absorption region. Due to intramolecular chiral exciton coupling between the dipyrrinone chromophores, the long-wavelength negative and short-wavelength positive CD bands clearly prove that a single bilirubin molecule binds to BLG in a left-handed helical conformation (in pH 7.4 phosphate buffer Deltaepsilon(min) is -54 M(-1) cm(-1) at 467 nm and Deltaepsilon(max) is +48.5 M(-1) cm(-1) at 412 nm). The very low aqueous solubility and strong tendency of bilirubin molecules to aggregate around pH 7.4 meant that much more intense CD bands were measured at alkaline pH values owing to the increasing solubility of the ligand. Vanishing CD activity obtained upon titration of the complex with palmitic acid known to bind in the hydrophobic cavity of BLG indicates bilirubin to be bound at the open end mouth of the beta-barrel. Reversible changes of the induced CD spectrum due to acidic pH shift of the sample solution lead to the same conclusion.

Animals↗
PubMed1997

Identification of a novel angiotensin-I-converting enzyme inhibitory peptide corresponding to a tryptic fragment of bovine beta-lactoglobulin.

The angiotensin-I-converting enzyme (ACE) inhibitory activity of a tryptic digest of bovine beta-lactoglobulin (beta-lg) was investigated. Intact beta-lg essentially did not inhibit ACE while the tryptic digest gave an 84.3% inhibition of ACE. Peptide material eluting between 20 and 25% acetonitrile during C18 solid-phase extraction of the beta-lg tryptic digest inhibited ACE by 93.6%. This solid-phase extraction fraction was shown by mass spectroscopy to contain beta-lg f(142-148). This peptide had an ACE IC50 value of 42.6 micromol/l. The peptide was resistant to further digestion with pepsin and was hydrolysed to a very low extent with chymotrypsin. The contribution of specific amino acid residues within the peptide to ACE inhibitory activity and the potential application of this peptide as a nutraceutical is discussed.

Amino Acid Sequence↗