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PubMedDate not supplied

[Effect of carbohydrates and polyols on amide content and protein fragmentation in a lactoglobulin preparation].

Certain carbohydrates and polyols are used at various stages of the production of immunobiological preparations as stabilizers of biological activity, particularly in the production of lactoglobulin (against opportunistic pathogens) using membrane ultrafiltration. This study concerns the effect of these substances on changes in the amide content in proteins of this lactoglobulin. Lactoglobulin was incubated in near-physiological (0.9% NaCl, pH 5.5) 10% solutions of glucose, fructose, and sorbitol at 4 and 35 degrees C for 7, 14, and 28 days. A lactoglobulin solution in 0.9% NaCl, pH 5.5, was used as the control. All substances studied suppressed the reduction of the amide group content of asparagine and, in contrast, increased the rate of amide group removal from glutamine residues in proteins of lactoglobulin preparations.

Amides↗

PubMed1975

Chemistry of allergens. XXII. Isolation and characterization of three new antigens from the dialysates of six successive pepsin hydrolyses of beta-lactoglobulin.

Twelve new antigens previously have been demonstrated in the pepsin digests of milk proteins. The term "new antigen" is defined as an antigen with a specificity distinct from that of the protein from which it was generated. Three new antigens (alpha-, beta-, and D2i) polypeptides and one nonantigenic gamma-polypeptide have been isolated from the dialysates of six successive pepsin hydrolyses of beta-lactoglobulin. The alpha- and beta-polypeptides were 1/5 and D2i was 1/10-1/20 as potent immunogens as precursor beta-lactoglobulin as determined by the Schultz-Dale technique. The minimum observed amounts of new antigen eliciting maximum response of uterine strips in the Schultz-Dale tests were: alpa-D2, 50 ng; beta-D2, 15 ng; beta-D3; 10 ng; and D2i, 1,000 ng. Except for the alpha-polypeptide, the amino acid contents of the polypeptides differed markedly from that of beta-lactoglobulin. The beta-polypeptide has been tentatively identified as a 33 amino acid fragment of beta-lactoglobulin (3,910 daltons). The gamma-polypeptide has been tentatively identified as a 12 amino acid fragment of beta-lactoglobulin (1,372 daltons).

Amino Acids↗

PubMed1995

Tissue-specific, temporally regulated expression mediated by the proximal ovine beta-lactoglobulin promoter in transgenic mice.

The ovine beta-lactoglobulin gene is expressed abundantly in the mammary gland. This study determines whether the ovine beta-lactoglobulin promoter is sufficient for targeting tissue-specific expression in transgenic mice. To address this, the expression profile of an ovine beta-lactoglobulin promoter driven bacterial chloramphenicol acetyltransferase reporter construct was analysed. Comparison of the expression frequency of this hybrid transgene to that of a genomic beta-lactoglobulin transgene indicates that additional sequences, downstream of the promoter, are required for position-independent expression in transgenic mice. Nevertheless, the hybrid transgene was expressed specifically in the mammary gland. Furthermore, the hybrid transgene was expressed in the appropriate temporal pattern during pregnancy and lactation. Thus, the proximal promoter of the ovine beta-lactoglobulin gene contains sufficient sequence information to target expression to the mammary. This construct constitutes the basis for a compact mammary expression vector.

Animals↗

PubMed1994

Probing the retinol-binding site of bovine beta-lactoglobulin.

The retinol-binding site of beta-lactoglobulin has been located by selective modification of amino acid residues which reside in the two putative binding sites. Based upon two separate crystallographic analyses of bovine beta-lactoglobulin, different binding sites for retinol have been proposed: one proposal favors an interior cavity, the other a surface cleft. To discriminate between these two models, we have made four separate site-directed mutations introducing a W19A or a K70M in the interior pocket and a F136A or K141M in the surface pocket. The K70M beta-lactoglobulin exhibited a marked decrease in its binding of retinoic acid compared to the F136A, K141M, and wild-type proteins. Retinyldenepropylamine, a retinyl Schiff base analog of retinol, was synthesized and its absorption spectrum when bound to the wild-type, K70M, and K141M proteins was examined to probe its interaction with the respective lysine residues. The retinylidenepropylamine bound in the K70M beta-lactoglobulin exhibited a kinetic red shift as distinct from the blue shift observed when it is bound to either the K141M or wild-type beta-lactoglobulins. The blue shift indicates protonation of the Schiff base. The resulting tagged peptide was isolated after cyanogen bromide cleavage and found to be the Ala25-Met107 peptide, consistent with the Lys70 being the residue which interacts with the bound retinol. These results support the proposal that retinol binds to an evolutionarily conserved interior cavity rather than the surface pocket.

Amino Acid Sequence↗

PubMed1998

Conformation of beta-Lactoglobulin at an Oil/Water Interface as Determined from Proteolysis and Spectroscopic Methods.

The rates of appearance of tryptic peptides following the hydrolysis of beta-lactoglobulin in solution or adsorbed at the oil/water interface of an emulsion were investigated as a function of time. It was also shown using hydrophobic labeling that the region 15-40 of beta-lactoglobulin was in the oil phase. The fluorescence results suggested that the conformation of beta-lactoglobulin was modified upon adsorption at the oil/water interface and that at least one tryptophan in adsorbed beta-lactoglobulin was in a more hydrophobic environment. The data obtained by circular dichroism in the peptidic region indicated that the adsorbed beta-lactoglobulin was characterized by a higher content in alpha-helix than the protein in solution. Copyright 1998 Academic Press.

Journal Article↗

PubMed1999

Association behavior of native beta-lactoglobulin.

The association behavior of beta-lactoglobulin has been studied by small-angle neutron scattering as a function of protein concentration, temperature, pH, and NaCl concentration of the solution. By indirect Fourier transformation of the spectra, pair-distance distribution functions for the various samples were obtained. These functions provided information on the maximum size, the weight-averaged molecular mass, and the z-averaged radius of gyration of the beta-lactoglobulin particles. At room temperature and pH values below 4 and above 5.2 the protein consists predominantly of monomers and dimers, consistent with literature. In these pH regimes the formation of dimers is favored upon increasing ionic strength and decreasing protein charge (pH values closer to the isoelectric point of the protein). Around pH 4.7, larger oligomeric structures are formed, enhanced by a decrease in temperature and a decrease in ionic strength. beta-Lactoglobulin A associates more strongly than beta-lactoglobulin B. Surprisingly, at pH 6.9 larger structures than dimers seem to be formed at high protein concentrations (> 30 mg mL-1).

Animals↗

PubMed2002

Thermal modifications of structure and co-denaturation of alpha-lactalbumin and beta-lactoglobulin induce changes of solubility and susceptibility to proteases.

Study of heat denaturation of major whey proteins (beta-lactoglobulin or alpha-lactalbumin) either in separated purified forms, or in forms present in fresh industrial whey or in recomposed mixture respecting whey proportions, indicated significant differences in their denaturation depending on pH, temperature of heating, presence or absence of other codenaturation partner, and of existence of a previous thermal pretreatment (industrial whey). alpha-Lactalbumin, usually resistant to tryptic hydrolysis, aggregated after heating at > or = 85 degrees C. After its denaturation, alpha-lactalbumin was susceptible to tryptic hydrolysis probably because of exposure of its previously hidden tryptic cleavage sites (Lys-X and Arg-X bonds). Heating over 85 degrees C of beta-lactoglobulin increased its aggregation and exposure of its peptic cleavage sites. The co-denaturation of alpha-lactalbumin with beta-lactoglobulin increased their aggregation and resulted in complete exposure of beta-lactoglobulin peptic cleavage sites and partial unveiling of alpha-lactalbumin tryptic cleavage sites. The exposure of alpha-lactalbumin tryptic cleavage sites was slightly enhanced when the alpha-lactalbumin/beta-lactoglobulin mixture was heated at pH 7.5. Co-denaturation of fresh whey by heating at 95 degrees C and pH 4.5 and above produced aggregates stabilized mostly by covalent disulfide bonds easily reduced by beta-mercaptoethanol. The aggregates stabilized by covalent bonds other than disulfide arose from a same thermal treatment but performed at pH 3.5. Thermal treatment of whey at pH 7.5 considerably enhanced tryptic and peptic hydrolysis of both major proteins.

Animals↗

PubMed1977

[Alkali treatment of proteins. I. Behavior of sulfhydryl and disulfide groups in alkali-treated beta-lactoglobulin and alpha-lactalbumin].

The alkali treatment of beta-lactoglobulin and alpha-lactalbumin results in the splitting of disulphide bonds in the protein molecules. When a 0.8-10(4) M beta-lactoglobulin solution in a 0.022 N sodium hydroxide solution (pH = 12) is heated at 90 degrees C for 30 min, 0-4 M disulphide groups, 2.2 M sulfhydryl groups and 1.8 M sulphide ions/M dimeric beta-lactoglobulin are detectable of the total of 4 M disulphide groups and 2 M sulfhydryl groups/M dimeric beta-lactoglobulin. The sulphide ions can be determined directly in the form of hydrogen sulphide or by calculating the difference between the values from the amperometric-argentometric titration and those from the method of ELLMAN (reaction with DTNB). The disulphide groups are determined with the aid of DTNB after reduction with sodium borohydride. The sulfhydryl groups obtained by reduction with sodium borohydride re-oxidize, the reaction velocity being of the second order. If the disulphide groups are reduced with sodium borohydride, the argentometric-amperometric determination of the sulfhydryl groups by means of the platinum rotating-disk electrode is disturbed by the presence of boric acid.

Chemical Phenomena↗

PubMed2003

Electrospray mass spectrometric investigation of the binding of cis-parinaric acid to bovine beta-lactoglobulin and study of the ligand-binding site of the protein using limited proteolysis.

The binding property of parinaric acid, a polyunsaturated fatty acid, to bovine beta-lactoglobulin, has been studied by electrospray ionization mass spectrometry. Stable complexation was observed under acidic conditions in a molar ratio of 1:1. Competitive complexation experiments were performed using saturated and unsaturated fatty acid standards with different chain lengths and number of double bonds to study the specificity of the interaction. It can be concluded that formation of the parinaric acid-lactoglobulin complex is preferred even if the molar concentration of the other fatty acids is ten times higher. In cases of specific complex formation the protein must have an active site that is a good acceptor for the ligand molecule. Limited trypsinolysis was performed on the lactoglobulin molecule to identify which part is responsible for the complexation. An intermediate tryptic fragment with molecular mass of 5200 Da was found to have the same ability to bind parinaric acid as the intact protein. This disulfide-bonded residue, [41-70]S-S[149-162], might thus be involved in the specific complexation of parinaric acid to beta-lactoglobulin. This conclusion is consistent with previous information on this binding site.

Amino Acid Sequence↗

PubMed1995

Trifluoroethanol-induced stabilization of the alpha-helical structure of beta-lactoglobulin: implication for non-hierarchical protein folding.

Trifluoroethanol (TFE) is known to stabilize the alpha-helical structure in proteins and their fragments. However, the relationship between the TFE-induced structures and the native structure is not clear. Here we show that beta-lactoglobulin, which consists predominantly of beta-sheets, exhibited a markedly high propensity to form an alpha-helical structure in the presence of TFE, as measured by far-UV circular dichroism. A cooperative transformation from the beta-sheet structure to an alpha-helical structure occurred at a TFE concentration between 10% and 20%. These results were in contrast to a gradual beta-sheet to alpha-helix transition of the constant fragment of the immunoglobulin light chain, which is also a beta-sheet protein. To understand the significance of the high helical propensity of beta-lactoglobulin, we measured the TFE-induced conformational transition of more than 20 proteins of various secondary structural types. Whereas the alpha-helical proteins showed a propensity to form an extensive helical structure in TFE, the helical propensity of proteins with a low helical content in the native state varied. The helical content in TFE was correlated more with the helical content predicted by a secondary structure prediction than with the helical content of the native structure, suggesting that the stability of the helical structure in TFE is determined by local interactions between nearby amino acid residues. Our results suggest that an alpha-helical intermediate can accumulate during the refolding process of beta-lactoglobulin and that a hierarchical model of protein folding is not necessarily true for some beta-sheet proteins including beta-lactoglobulin.

Animals↗

PubMed1981

Porcine beta-lactoglobulin A and C. Occurrence, isolation and chemical properties.

The occurrence of the dominant 'whey' protein in samples of milk from 1180 sows is examined. It exhibits genetic polymorphism with some unusual features. Although immunologically different from bovine beta-lactoglobulin, it is shown by chemical studies of the isolated protein to be a beta-lactoglobulin. Two homozygous genetic variants, designated porcine beta-lactoglobulin A and C, are isolated and their amino acid compositions and peptide maps compared. It is shown that the C variant has +1 His, -1 Gln, and +1 Asp, -1 Glu, with respect to the A variant. These variants, containing ca. 162 residues per molecule, are considered in relationship to porcine beta-lactoglobulins isolated by other workers. The sequence of the first 50 residues is determined and compared with the sequence of the bovine protein. The sequences of ca. 70% of the remaining residues is proposed on the basis of the composition of tryptic peptides and assumed homology.

Amino Acid Sequence↗

PubMed2006

Structural features of transiently modified beta-lactoglobulin relevant to the stable binding of large hydrophobic molecules.

Binding sites for hydrophobic molecules on bovine beta-lactoglobulin, and their susceptibility to temperature, were studied by using various spectroscopic probes. Binding of probes carrying a single fluorophore moiety, a single nitroxide moiety, or both moieties on the same molecule, was followed by EPR and fluorescence. The presence of a fatty acid side chain in the dual probes was found to be required for binding to beta-lactoglobulin. Binding occurred only after the protein was heated at temperatures below the threshold for its irreversible denaturation. Binding became extremely tight and stable upon cooling of the protein-probe mixture. Comparison among the various probes suggests that multiple binding sites for hydrophobes are present in the native protein, and in the partially-and reversibly-modified form of beta-lactoglobulin present in solution at neutral pH and subdenaturing temperatures. Thus, the specificity of hydrophobes binding to beta-lactoglobulin may be modulated by simple physical treatment of the protein.

Animals↗

PubMed1980

A differential scanning calorimetric study of the thermal denaturation of bovine beta-lactoglobulin. Thermal behaviour at temperatures up to 100 degrees C.

The thermal behaviour of beta-lactoglobulin in aqueous solutions was followed by differential scanning calorimetry in the temperature range from 40 to 100 degrees C. From the results the following information is obtained. 1. The DSC thermograms show a large transition peak in the temperature range from 60 to 90 degrees C, which reflects the denaturation (unfolding) of beta-lactoglobulin. 2. From the peak surface an apparent denaturation enthalpy delta H = 230 +/- 15 kJ/mol is calculated at pH 6.5. 3. The temperature of maximum deflection of the DSC curve is dependent on the heating rate. Extrapolation to zero heating rate results in a denaturation temperature of 70.4 +/- 0.5 degrees C at pH 6.7. 4. A kinetic analysis of the DSC curves shows that the denaturation of beta-lactoglobulin is of the first order at temperatures between 65 to 72 degrees C. The apparent activation energy amounts to 343 kJ/mol, calculated according to the method of Kissinger. After comparison of the results with data from the literature, it was concluded that 70 degrees C is a critical temperature for the denaturation of beta-lactoglobulin. Above 70 degrees C the denaturation behaviour is changed, probably because of the starting of the aggregation process. This change is indicated by the transition temperature of the DSC curve at the corresponding heating rate.

Animals↗

PubMed1976

Self-association of beta-lactoglobulin c in acetate buffers.

The self-association of beta-lactoglobulin C at pH 4.65 (23 degrees C) in acetate buffer has been studied at various temperatures, 10, 16, 20 and 25 degrees C, by a series of sedimentation equilibrium experiments. Two different buffers were used. Buffer I with an ionic strength of 0.1 consisted of 0.1M acetic acid and 0.1M sodium acetate; buffer II had 0.1M KCl in addiation so that its ionic strength was 0.2. The variation of the apparent weight average molecular weight, Mwa, with the total solute concentration, c, was characteristic of a self-association. In contrast to the behavior of beta-lactoglobulin A in acetate buffer, the association of beta-lactoglobulin C did not proceed beyond dimer. Furthermore, within the experimental error, the self-association of beta-lactoglobulin C was independent of temperature and ionic strength; all experimental data could be put on the same Mwa (or M1/Mwa) vs. c plot! Several models were used to test the self-association, and a monomer--dimer association with K2 = 2.10 X 10(3) dl/g and BM1 =- 1.2 X 10(-2) dl/g seemed to give a good description of the M1/Mwa vs. c curve.

Acetates↗

PubMed1996

Involvement of a subset of tyrosine kinases and phosphatases in regulation of the beta-lactoglobulin gene promoter by prolactin.

This study used pharmacological intervention to provide support for a role of kinases and phosphatases in prolactin transactivation of a milk protein gene. It was based on transient cotransfection using a rabbit prolactin receptor expression plasmid and a beta-lactoglobulin promoter/CAT reporter construct. In cotransfected CHO cells, herbimycin A and tyrphostin, two tyrosine kinase inhibitors, were able to decrease the CAT response by over 50%, along with tyrosine phosphorylation of cellular proteins, whereas genistein and lavendustine were without effect on lactoglobulin transactivation. Orthovanadate, an inactivator of tyrosine phosphatases, was able to substitute for prolactin in inducing the CAT response. Staurosporine, a non-specific kinase inhibitor, was able, when used at low concentrations (10 nM), to augment the prolactin response strikingly. Threonine/serine kinases do not appear to be involved early in beta-lactoglobulin promoter transactivation, since four C-kinase inhibitors and okadaic acid a threonine/serine phosphatase inhibitor, were without substantive effect. We conclude that specific tyrosine kinases are responsible for most of the signal transduction from the prolactin receptor to the beta-lactoglobulin gene promoter.

Animals↗

PubMed1990

Feline whey proteins: identification, isolation and initial characterization of alpha-lactalbumin, beta-lactoglobulin and lysozyme.

1. Both alpha-lactalbumin and beta-lactoglobulin-like proteins were detected in the whey fraction of feline milk by immunoblotting with rabbit antisera to alpha-lactalbumin and beta-lactoglobulin, respectively. 2. alpha-Lactalbumin was found to occur in both glycosylated and unglycosylated forms in approximately equal concentrations. No polymorphism of feline alpha-lactalbumin was found. 3. Feline beta-lactoglobulin-like proteins produced complex electrophoretic patterns that appear to be determined by three distinct loci. Between two and five genetic variants are expressed by each locus. 4. Lysozyme was detected at levels of approximately 1 mg/ml in skim milk. 5. The identifications of the proteins as alpha-lactalbumin, beta-lactoglobulin and lysozyme were confirmed by determination of N-terminal amino acid sequences.

Amino Acid Sequence↗

PubMed1996

Truncated beta-lactoglobulin transgenes are expressed in the kidney.

The major milk whey protein of ruminants is beta-lactoglobulin. Ovine beta-lactoglobulin-encoding gene expression is restricted to the sheep mammary gland. This report describes the expression profile of a truncated beta-lactoglobulin transgene which, although not expressed in the mammary gland, is expressed in the kidney in the majority of lines generated. The high frequency of ectopic kidney expression may relate to the ability of the larger beta-lactoglobulin transgenes to be expressed in a position-independent manner in the mammary gland of transgenic mice.

Animals↗

PubMed2005

Duplication of Stat5-binding sites within the beta-lactoglobulin promoter compromises transcription in vivo.

Achieving high and reliable levels of expression is essential for biotechnological application of transgene technology. In an attempt to maximise transgene expression, we analysed the effect of adding extra Stat5-binding sites, while maintaining their spatial arrangement, to the beta-lactoglobulin promoter. A 195 bp-fragment comprising the three Stat5-binding sites in the beta-lactoglobulin promoter was duplicated directly upstream, 88 bp or 177 bp upstream from the original site. We assessed the effect that addition of extra Stat5-binding sites had in transcriptional activation and chromatin structuring of the promoter, both in vitro in HC11 cells and in vivo in transgenic animals. Duplication of Stat5-binding sites in the beta-lactoglobulin promoter did favour chromatin remodelling in vivo but unexpectedly did not enhance transcriptional activation of the transgene, despite the higher basal and induced expression levels achieved in vitro in HC11 cells. Furthermore, the well-described positive relationship between beta-lactoglobulin transgene copy number and mammary expression was lost in the new lines. These results indicate that chromatin remodelling upon Stat5 binding is not sufficient to activate transgene expression, denote that strategies to achieve transgenic transcriptional activation are not obvious.

Animals↗