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PubMed2001

Structural and kinetic characterization of early folding events in beta-lactoglobulin.

We have defined the structural and dynamic properties of an early folding intermediate of beta-lactoglobulin known to contain non-native alpha-helical structure. The folding of beta-lactoglobulin was monitored over the 100 micros--10 s time range using ultrarapid mixing techniques in conjunction with fluorescence detection and hydrogen exchange labeling probed by heteronuclear NMR. An initial increase in Trp fluorescence with a time constant of 140 micros is attributed to formation of a partially helical compact state. Within 2 ms of refolding, well protected amide protons indicative of stable hydrogen bonded structure were found only in a domain comprising beta-strands F, G and H, and the main alpha-helix, which was thus identified as the folding core of beta-lactoglobulin. At the same time, weak protection (up to approximately 10-fold) of amide protons in a segment spanning residues 12--21 is consistent with formation of marginally stable non-native alpha-helices near the N-terminus. Our results indicate that efficient folding, despite some local non-native structural preferences, is insured by the rapid formation of a native-like alpha/beta core domain.

Animals↗
PubMed1996

Non-native alpha-helical intermediate in the refolding of beta-lactoglobulin, a predominantly beta-sheet protein.

It is generally assumed that folding intermediates contain partially formed native-like secondary structures. However, if we consider the fact that the conformational stability of the intermediate state is simpler than that of the native state, it would be expected that the secondary structures in a folding intermediate would not necessarily be similar to those of the native state. beta-Lactoglobulin is a predominantly beta-sheet protein, although it has a markedly high intrinsic preference for alpha-helical structure. We have studied the refolding kinetics of bovine beta-lactoglobulin using stopped-flow circular dichroism and find that a partly alpha-helical intermediate accumulates transiently before formation of the native beta-sheets. The present results suggest that the folding reaction of beta-lactoglobulin follows a non-hierarchical mechanism, in which non-native alpha-helical structures play important roles.

Animals↗
PubMed1976

The whey proteins of the milk of red deer (Cervus elaphus L.). A homologue of bovine beta-lactoglobulin.

1. The whey proteins from the milk of red deer are compared with those of cattle. Gel chromatography and electrophoresis showed a close similarity between the whey proteins of the two species in the size, mobility and relative amounts of the main constituents and in the changes in their relative amounts with time after parturition. 2. The major constituent of the deer whey was isolated. It appeared to be homologous with bovine beta-lactoglobulin and had the following properties: m=-5.2X10(-9)m2-s-1-V-1 at 4 degrees C and pH 8.6; pI=5.17; S020, w =2.89S; v=0.748 ml/g; E1g/dl 1cm= 9.12 at 278 nm; deltan/c=1.794 X 10(-3)dl/g at 579 nm (all at 20 degrees C except m). Its molecular weight was that of a dimer with a subunit weight of 18 000. 3. Amino acid analyses of this protein, adjusted to lysine = 15 residues showed that it contains one more residue of aspartic acid, alanine and methionine and one less glutamic acid residue and two less leucine residues than bovine beta-lactoglobulin A. 4. On starch-gel electrophoresis at pH 8.2, this protein migrated at the same rate as bovine beta-lactoglobulin B, although its isoelectric point is close to that of the bovine A variant. Milk from three out of 27 hinds examined showed a variant. This migrated in starch gel at the same rate as the bovine A variant but had a more acid pI = 5.02. 5. The two species whose milk whey proteins are compared represent two different families of ruminants. The similarities found support the view that the milk whey proteins of the bovids are probably typical of the suborder as a whole.

Amino Acids↗
PubMed1988

Complete amino acid sequence of human placental protein 14: a progesterone-regulated uterine protein homologous to beta-lactoglobulins.

Placental protein 14 (PP14), also known as progestagen-dependent endometrial protein and pregnancy-associated endometrial alpha 2-globulin, is synthesized by the human secretory endometrium and decidua. We have isolated from a human decidual cDNA library clones corresponding to PP14 and deduced its entire amino acid sequence. PP14 contains 180 amino acids, 18 of which correspond to a putative signal peptide. The predicted molecular weight of the pre-PP14 is 20,555 and that of the mature protein is 18,787. PP14 is encoded by a 1-kilobase-pair mRNA that is expressed in human secretory endometrium and decidua but not in postmenopausal endometrium, placenta, liver, kidney, and adrenals. The 162-residue-long sequence of PP14 is highly homologous to beta-lactoglobulins, with a 53.4% identity with the amino acid sequence of horse beta-lactoglobulin I. The four cysteinyl residues (positions 66, 106, 119, and 160) responsible for intramolecular disulfide bridges in beta-lactoglobulins are all conserved in PP14. Southern blot analysis of human DNA suggested that PP14 gene sequences encompass some 20 kilobase pairs of the human genomic DNA.

Amino Acid Sequence↗
PubMed1996

Biopolymer-surfactant interaction: 4. Kinetics of binding of cetyltrimethyl ammonium bromide with gelatin, hemoglobin, beta-lactoglobulin and lysozyme.

The binding of CTAB with the proteins, gelatin, hemoglobin, beta-lactoglobulin and lysozyme follow first order kinetics and occurs either in two or three distinct stages. The number of stages depends on the overall configuration of the biopolymers. The denatured protein, gelatin has shown three-stage kinetics under all conditions, whereas the native proteins, hemoglobin, beta-lactoglobulin and lysozyme have exhibited two stage kinetics. Heat treated lysozyme in 8 mol dm-3 urea medium has also shown a two-stage kinetics. On the basis of non interacting binding sites on the proteins and independent sequential binding, the rates of reaction have been observed to increase with temperature and follow the trend k1 >> k2 > k3. The interaction of CTA+ with the proteins is both electrostatic and hydrophobic. Hemoglobin has shown maximum reaction rate whereas, beta-lactoglobulin has shown a minimum. The activation parameters for the kinetic process have exhibited almost non-variant delta G++ and delta H++ < T delta S++. The formation of activation complex in the Eyring model is entropy controlled so also the overall kinetics. An isokinetic entropy-enthalpy compensation phenomenon has been observed for the respective kinetic stages.

Biopolymers↗
PubMed1996

Gastrojejunal kinetics and the digestion of [15N]beta-lactoglobulin and casein in humans: the influence of the nature and quantity of the protein.

The evolution and luminal effects of different quantities of casein and beta-lactoglobulin were investigated in the upper jejunum of 35 volunteers who ingested 400 mL water with either beta-lactoglobulin or casein in either low or high doses (72.6 mmol N, Lbetalg; 71.7 mmol N, LCas; 368.2 mmol N, Hbetalg; 386.8 mmol N, HCas). The flow rate of the liquid effluents as well as the nitrogen movements were measured and the exogenous ([15N]) and endogenous nitrogen fractions analyzed in the upper jejunum after milk protein ingestion. The basal jejunal liquid flow rate (mL/min) was 3.88+/-1.84 and peaked in the 0-20 min period for water (9.92+/-3.72) and Lbetalg (7.27+/-3.08) and during the 20-40 min period for LCas (5.69+/-2.49), HCas (6.32+/-1.85), and Hbetalg (6.11+/-2.31). One hour after water, LCas, Lbetalg, Hbetalg, and HCas ingestion, 100%, 95%, 85%, 71%, but only 38% of the liquid phase of the meal were passed through the jejunum, respectively. The flow rate of the endogenous nitrogen was 12.93+/-5.22 mmol N/h before meal ingestion; remained unchanged after water, LCas or Hbetalg ingestion; but increased significantly (P<0.05) after Lbetalg and HCas ingestion. The net disappearance of exogenous nitrogen in the upper jejunum 240 min after HCas, Lbetalg, LCas and Hbetalg ingestion was 82.6+/-9.5%, 61.6+/-9.6%, 58.4+/-14.7%, and 44.7%+/-24.4%, respectively. The exogenous fraction of protein nitrogen recovered in the upper intestinal lumen represented 43.3% of the ingested Hbetalg nitrogen, but only 4.9% of the ingested HCas nitrogen. In conclusion, casein and beta-lactoglobulin present differences in both the intestinal kinetics of amino acid delivery and in the nature of the products in the intestinal lumen. These differences have to be taken into account from both nutritional and physiologic points of view for the utilization of these proteins in humans.

Adult↗
PubMed1999

Insertion of a casein kinase recognition sequence induces phosphorylation of ovine beta-lactoglobulin in transgenic mice.

We have shown that the cellular mechanisms of the mammary gland can be used to produce a phosphorylated form of a normally unphosphorylated milk protein. This was achieved by the insertion of a beta-casein DNA sequence coding for a group of mammary gland casein kinase recognition sites into ovine beta-lactoglobulin. Transgenic mice carrying this modified gene were generated and lactating females were shown to produce a novel beta-lactoglobulin in their milk. The infrared spectrum, reactivity to antiphosphoserine antibody and reduction of electrophoretic mobility on treatment with alkaline phosphatase showed that the novel protein recovered from the milk whey (serum) was phosphorylated and molecular mass determination by mass spectrometry was consistent with the phosphorylation of one or two residues. A similar level of phosphorylation was measured by quantitative infrared spectroscopy. Centrifugation of the milk to pellet the casein micelles showed that most of the phosphorylated beta-lactoglobulin was in the whey and hence not incorporated into casein micelles.

Amino Acid Sequence↗
PubMed2001

Salt-dependent monomer-dimer equilibrium of bovine beta-lactoglobulin at pH 3.

Although bovine beta-lactoglobulin assumes a monomeric native structure at pH 3 in the absence of salt, the addition of salts stabilizes the dimer. Thermodynamics of the monomer-dimer equilibrium dependent on the salt concentration were studied by sedimentation equilibrium. The addition of NaCl, KCl, or guanidine hydrochloride below 1 M stabilized the dimer in a similar manner. On the other hand, NaClO(4) was more effective than other salts by about 20-fold, suggesting that anion binding is responsible for the salt-induced dimer formation, as observed for acid-unfolded proteins. The addition of guanidine hydrochloride at 5 M dissociated the dimer into monomers because of the denaturation of protein structure. In the presence of either NaCl or NaClO(4), the dimerization constant decreased with an increase in temperature, indicating that the enthalpy change (DeltaH(D)) of dimer formation is negative. The heat effect of the dimer formation was directly measured with an isothermal titration calorimeter by titrating the monomeric beta-lactoglobulin at pH 3.0 with NaClO(4). The net heat effects after subtraction of the heat of salt dilution, corresponding to DeltaH(D), were negative, and were consistent with those obtained by the sedimentation equilibrium. From the dependence of dimerization constant on temperature measured by sedimentation equilibrium, we estimated the DeltaH(D) value at 20 degrees C and the heat capacity change (DeltaC(p)) of dimer formation. In both NaCl and NaClO(4), the obtained DeltaC(p) value was negative, indicating the dominant role of burial of the hydrophobic surfaces upon dimer formation. The observed DeltaC(p) values were consistent with the calculated value from the X-ray dimeric structure using a method of accessible surface area. These results indicated that monomer-dimer equilibrium of beta-lactoglobulin at pH 3 is determined by a subtle balance of hydrophobic and electrostatic effects, which are modulated by the addition of salts or by changes in temperature.

Animals↗
PubMed1984

Preparation of maltosyl, beta-cyclodextrinyl, glucosaminyl, and glucosamineoctaosyl derivatives of beta-lactoglobulin.

beta-Lactoglobulin was modified to various degrees with maltose or beta-cyclodextrin using the cyclic carbonate method and with glucosamine or glucosamine-octaose using the carbodiimide method. Up to 65% of the amino or the carboxyl groups of b-LG were glycosylated using the two methods, respectively. Up to 32 maltose residues were coupled to b-LG using the cyclic carbonate method while up to 16.6 glucosamine residues were coupled to b-LG using the carbodiimide method. This resulted because more than one disaccharide was attached to each group of b-LG that was modified. Even so, the coupling efficiency of the active compound was higher for the carbodiimide derivative than for the cyclic carbonate derivative. The electrophoretic analyses of the glycosylated proteins indicated that a heterogeneous population of protein molecules was formed during modification. The electrophoretic mobility of maltosyl-beta-lactoglobulin derivatives was essentially unchanged while the mobility of glucosaminyl-beta-lactoglobulin derivatives decreased as the extent of modification increased. The glycosylated proteins were suitable for studying their structural and functional properties.

Animals↗
PubMed1988

Structural stability of beta-lactoglobulin in the presence of kosmotropic salts. A kinetic and thermodynamic study.

The thiol group of beta-lactoglobulin reacted very sluggishly with dithio-bis-nitro-benzoic acid as compared to that of glutathione at pH 6.85. The pKapp value of the thiol group of the protein was 9.35. In the presence of 3 M urea, the thiol group reacted completely with dithio-bis-nitrobenzoic acid at pH 6.85. Heating (from 50 degrees to 80 degrees) increased the exposure of the thiol by dissociating the dimer unit. From the pseudo-first order rate constants of heat-exposure of thiol, thermodynamic activation parameters, delta G++, delta H++, and delta S++, for the heat-dissociation of beta-lactoglobulin dimer were estimated to be 23,290 cal/mol, 31,160 cal/mol, and 22.9 e.u. (at 70 degrees), respectively. Addition of kosmotropic salts, chloride, tartrate, sulfate, phosphate, and citrate (0.2 M) decreased the heat-induced exposure of the thiol group (at 70 degrees), probably by decreasing the dissociation of the dimer at pH 6.85. The relative change in free energy of activation for the dissociation of the dimer, delta(delta G++dimer), in the presence of the salts was positive, suggesting that these additives increase the stability of the dimer against heat. These salts also increased the conformational stability of beta-lactoglobulin as revealed by an increase in -delta(delta G0conf) values in their presence. Both delta(delta G++dimer) and -delta(delta G0conf) values followed the order, chloride less than tartrate less than sulfate less than phosphate less than citrate. These salts seem to manifest their structure-stabilizing effect by increasing both inter- and intramolecular hydrophobic interactions via changes in structure of water.

Chlorides↗
PubMed1995

Peptic proteolysis of esterified beta-casein and beta-lactoglobulin.

Moderate esterification induces slight secondary structure changes in two major milk proteins, beta-lactoglobulin and beta-casein. Esterification of beta-lactoglobulin prompts its tertiary structure 'melting', opening it to peptic cleavage. Twenty-two new cleavage sites were characterised in beta-lactoglobulin and five in beta-casein. Some of them are due to esterification-improved peptide bond accessibility, some to the bias of pepsin specificity by glutamate and aspartate esters. The resulting fragmentation yields original and partially amphiphilic peptide populations.

Amino Acid Sequence↗
PubMed1985

Dietary bovine beta-lactoglobulin is transferred to human milk.

Human milk from 38 mothers was analysed by radioimmunological method for content of bovine beta-lactoglobulin. Detectable amounts (5-33 micrograms/l) of immunoactive beta-lactoglobulin were found in 18 human milk samples. Milk from 3 mothers, whose infants suffered from infantile colic contained high amounts of beta-lactoglobulin (32, 18 and 14 micrograms/l respectively). With the mothers on a cow's milk free diet the contents fell to non-detectable amounts in two mothers and to 6 micrograms/l in the third. All three infants became free from colic.

Animals↗
PubMed1994

Modulation of beta-lactoglobulin transport in rabbit ileum.

This study was undertaken to investigate the mechanisms of beta-lactoglobulin transport and its regulation in rabbit intestinal mucosa. beta-Lactoglobulin, the major bovine milk whey protein, was slightly degraded by ileal brush-border membranes, indicating the presence of phosphoramidon-insensitive neutral endoproteases. Modulation of 14C-radiolabeled beta-lactoglobulin transport across rabbit ileum mounted in Grass diffusion cells was studied in Ringer solution in the presence and absence of (in mM) 25 glucose, 10 galactose, and 0.5 colchicine. The transport was also measured in Dulbecco's modified Eagle's medium as a more "balanced" medium. The transport of the degraded products and of [3H]polyethylene glycol-4000, which was used as an extracellular marker, was enhanced by addition of glucose, presumably indicating passage through the tight junctions. The transepithelial passage of intact protein was estimated to be 10-20% by trichloroacetic acid precipitation and high-performance liquid chromatography, thus indicating the effective transport of the intact protein. The passage of the intact protein was inhibited by both colchicine, a microtubule-depolymerizing agent, and galactose by 50 and 30%, respectively. Galactose-like glycoconjugates, known to be present on the membrane, might be involved in the interaction between the protein and the brush border.

Animals↗
PubMed1984

Sidedness of the reaction to beta-lactoglobulin in sensitised colonic epithelia.

The short circuit current reaction to beta-lactoglobulin shown by the epithelial lining of the colon from guinea-pigs fed with cows' milk is elicited only when the challenge is applied to the basolateral side of the tissue. However if the apical surface of the epithelium is subjected to controlled lesioning with ultraviolet irradiation then apical challenge with beta-lactoglobulin becomes effective. A similar situation ensues when preparations are aged in vitro at room temperature in oxygenated Krebs-Henseleit solution. Although irradiation causes insignificant changes in the electrical resistance of the tissue, the evidence suggests that it allows beta-lactoglobulin to penetrate the epithelium from the apical side. If the model is applicable to clinical conditions of food allergic disease then it appears that concomitant changes in epithelial permeability as well as the development of sensitivity are required.

Animals↗
PubMed1985

Investigation of the allergenicity of beta-lactoglobulin and its cleavage fragments.

Fragments of beta-lactoglobulin were produced by proteolytic cleavage with trypsin, and chemical cleavage with cyanogen bromide, followed by gel filtration on Sephadex G-50. The antigenicity and allergenicity of the products were studied, before and after reductive cleavage by treatment with 2-mercaptoethanol. The former was determined by inhibition ELISA using IgG anti-beta-lactoglobulin raised in rabbits, whilst the latter was determined by inhibition ELISA and mast cell challenge, using respectively the sera and peritoneal cells of rats experimentally sensitised to beta-lactoglobulin. The findings raise interesting points about the structural basis of allergenicity in relation to antigenicity.

Allergens↗
PubMed1979

The amino acid sequence of goat beta-lactoglobulin.

The isolation of beta-lactoglobulin from milk of the goat is described. The purified protein was checked for purity and has been characterized by its gross composition and end groups. The native or the modified protein was then degraded by tryptic and cyanogen bromide cleavage. The cleavage products were isolated and sequenced in the sequenator using a Quadrol and propyne program. These data provide the complete sequence of beta-lactoglobulin of the goat. The results are discussed and compared particularly with bovine beta-lactoglobulin components AB. Some biological aspects are described.

Amino Acid Sequence↗
PubMedDate not supplied

The primary structure of the beta-lactoglobulin of the waterbuffalo (Bubalus arnee).

The complete amino acid sequence of the beta-lactoglobuline of the waterbuffalo (Bubalus arnee) was established. The sequence of peptides obtained by cleavage with BNPS-Skatole, CNBr and trypsin were determined automatically by the help of the sequenator. Only two differences were found in the beta lactoglobulin of the waterbuffalo compared with the bovine beta-lactoglobulin B.

Amino Acid Sequence↗
PubMed2001

Mild isolation procedure discloses new protein structural properties of beta-lactoglobulin.

To explore the potentially available functional properties of beta-lactoglobulin in, for example, the processing of food products, it is important to isolate the protein by a procedure that avoids all possible denaturing conditions, such as low pH, high ionic strength, or low or elevated temperatures that could cause the protein to undergo irreversible conformational changes. In this work, a mild isolation protocol for beta-lactoglobulin from bovine milk is presented, applicable to semi large-scale isolations (50 to 200 g). The protein could be isolated with a high efficiency (>80%) and a good purity (>98%). Biochemical characterization of the material demonstrated no lactosylation of the protein, nor the formation of irreversibly associated dimers. Also, no proteose peptones could be detected. The ability of beta-lactoglobulin to undergo conformational changes is studied by far and near-ultraviolet circular dichroism and differential scanning calorimetry. A "global" unfolding of the protein is detected around 72 (tertiary level) and 77 degrees C (secondary level). The dimer-monomer dissociation occurring around 52 degrees C could also be monitored at a secondary structural level. Remarkably, a low temperature transition around 30 degrees C was observed, where approximately 10 beta-stranded residues unfold cooperatively, not been reported previously. This low temperature transition is irreversible at temperatures higher than 35 degrees C or upon freezing the material at -20 degrees C. The addition of 20% glycerol could prevent this irreversible conformational change. The effect of the low temperature transition on the protein's functionality remains to be investigated.

Animals↗