Modification of protein structures by altering the whey protein profile and heat treatment affects in vitro static digestion of model infant milk formulas.

Heat treatments induce changes in the protein structure in infant milk formulas (IMFs). The present study aims to investigate whether these structural modifications affect protein digestion. Model IMFs (1.3% proteins), with a bovine or a human whey protein profile, were unheated or heated at 67.5 °C or 80 °C to reach 65% of denaturation, resulting in six protein structures. IMFs were submitted to in vitro static gastrointestinal digestion simulating infant conditions. During digestion, laser light scattering was performed to analyze IMF destabilization and SDS-PAGE, OPA assay and cation exchange chromatography were used to monitor proteolysis. Results showed that, during gastric digestion, α-lactalbumin and ß-lactoglobulin were resistant to hydrolysis in a similar manner for all protein structures within IMFs (p > 0.05), while the heat-induced denaturation of lactoferrin significantly increased its susceptibility to hydrolysis. Casein hydrolysis was enhanced when the native casein micelle structure was modified, i.e. partially disintegrated in the presence of lactoferrin or covered by heat-denatured whey proteins. The IMF destabilization at the end of the gastric digestion varied with protein structures, with larger particle size for IMF containing native casein micelles. During intestinal digestion, the kinetics of protein hydrolysis varied with the IMF protein structures, particularly for IMFs containing denatured lactoferrin, exhibiting higher proteolysis degree (67.5 °C and 80 °C vs. unheated) and essential amino acid bioaccessibility (67.5 °C vs. unheated). Overall, the protein structures, generated by modulating the whey protein profile and the heating conditions, impacted the IMF destabilization during the gastric phase and the proteolysis during the entire simulated infant digestion.

Role of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) in ß-lactoglobulin aggregation.

Protein aggregation is a prevalent phenomenon. It is important to study protein aggregation under different solution conditions. In this study, using 1-anilinonaphthalene-8-sulfonic acid (ANS) fluorescence spectra, we investigated the critical micelle concentration (CMC) of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP). We also studied the effects of DOTAP on amyloid aggregation of ß-lactoglobulin using intrinsic fluorescence spectra, circular dichroism spectra, thioflavin T fluorescence, a Congo red binding assay and transmission electron microscopy. We observed that DOTAP had a dual role on ß-lactoglobulin amyloid aggregation. DOTAP inhibited the amyloid aggregation below the CMC, while it had the opposite effect above the CMC. Moreover, the results of transmission electron microscopy showed that spherical aggregates were formed above the CMC. These results led us to conclude that cationic lipids could be used as modulators of protein self-assembly.

Milk whey protein modification by coffee-specific phenolics: effect on structural and functional properties.

A suitable vehicle for integration of bioactive plant constituents is proposed. It involves modification of proteins using phenolics and applying these for protection of labile constituents. It dissects the noncovalent and covalent interactions of ß-lactoglobulin with coffee-specific phenolics. Alkaline and polyphenol oxidase modulated covalent reactions were compared. Tryptic digestion combined with MALDI-TOF-MS provided tentative allocation of the modification type and site in the protein, and an in silico modeling of modified ß-lactoglobulin is proposed. The modification delivers proteins with enhanced antioxidative properties. Changed structural properties and differences in solubility, surface hydrophobicity, and emulsification were observed. The polyphenol oxidase modulated reaction provides a modified ß-lactoglobulin with a high antioxidative power, is thermally more stable, requires less energy to unfold, and, when emulsified with lutein esters, exhibits their higher stability against UV light. Thus, adaptation of this modification provides an innovative approach for functionalizing proteins and their uses in the food industry.

Influence of calcium on ß-lactoglobulin denaturation kinetics: Implications in unfolding and aggregation mechanisms.

Much research dealing with the processing of milk by-products in heat exchangers has noted the key role of calcium in ß-lactoglobulin (ß-LG) fouling behavior. Nevertheless, the manner by which Ca affects ß-LG denaturation has rarely been quantified using reliable kinetic and thermodynamic data. To this end, the influence of Ca on ß-LG denaturation mechanisms in simulated lactoserum concentrates was studied on the laboratory-scale under 100°C by HPLC analysis. The heat-treated solutions were composed of 53.3g/L ß-LG and were enriched in Ca at various concentrations (0, 66, 132, and 264 mg/kg). The kinetic parameters (reaction order, activation energy, and frequency factor) associated with ß-LG denaturation, along with the unfolding and aggregation thermodynamic parameters were deduced from these experiments and discussed with respect to Ca content. We found that the multistage process characterizing ß-LG thermal denaturation is not greatly affected by Ca addition. In fact, the general model subdividing ß-LG denaturation mechanisms in 2 steps, namely, unfolding and aggregation, remained valid for all tested Ca concentrations. The change in the predominant mechanism from unfolding to aggregation was observed at 80°C across the entire Ca concentration range. Moreover, the classical 1.5 reaction order value was unaffected by the presence of Ca. Interpretation of the acquired kinetic data showed that Ca addition led to a significant increase in kinetic rate, and more so in the aggregation temperature range. This indicates that Ca principally catalyzes ß-LG aggregation, by lowering the Coulombian repulsion between the negatively charged ß-LG reactive species, bridging ß-LG proteins, or via an ion-specific conformational change. To a lesser extent, Ca favors ß-LG unfolding, probably by disturbing the noncovalent binding network of native ß-LG. Simultaneously, Ca has a slight protective role on the native and unfolded ß-LG species, as shown by the increase in activation energy with Ca concentration. The calculation of thermodynamic parameters related to ß-LG denaturation confirmed this observation. A threshold effect in Ca influence was noted in this study: no further significant kinetic rate change was observed above 132 mg/kg of Ca; at this concentration, the studied solution was an almost equimolar mixture of ß-LG and Ca. Finally, we simulated the temporal evolution of ß-LG species concentrations at diverse Ca contents at 3 holding temperatures. The simulations were based on the acquired kinetic parameters. This permitted us to highlight the greater effect of Ca on ß-LG denaturation at high Ca content or for short-time heat treatments at temperatures near 100°C, as in heat exchangers.

Ethanol effect on the structure of beta-lactoglobulin b and its ligand binding.

The changes of structure and ligand binding properties of beta-LG B have been studied by fluorescence and circular dichroism spectroscopy in ethanolic solutions. Fluorescence measurements of retinol/beta-LG interactions at 480 nm in various ethanol concentrations show that the maximal fluorescence intensity induced by this interaction between retinol and beta-LG is observed around 20% v/v of ethanol. It is reduced to zero at 40% and 50% of ethanol. These results suggest that there are two distinct structural changes in beta-LG occurring between 20% and 30% and around 40% of ethanol. The first transition, which increases affinity and the apparent number of binding sites for retinol, may be related or similar to the Tanford transition. The strong quenching of retinol emission at 480 nm in 40% of ethanol indicates the radical transformation of beta-LG tertiary structure and the release of retinol. CD spectra at the aromatic region show that secondary and tertiary structures of beta-LG are not significantly affected between 0% and 20% of ethanol. In 30% of ethanol, beta-sheet percentage of beta-LG decreases with respect to native beta-LG (from 55% to 46%). beta-Sheet percentage in beta-LG increases in 40% and 50% alcohol (51% and 53%) relative to 30% of ethanol, which also indicates the strong rearrangement of the secondary structure of beta-LG, while its tertiary structure and beta-LG interactions are radically changed.

The effect of hydrocarbon addition on the deamidation rate in immune whey proteins.

The influence of some hydrocarbons that are often used at different stages of immunobiological preparation's production as stabilizers of biological activity on the dynamics of nonenzymatic deamidation in proteins of immune whey against conditionally pathogenic microorganisms obtained by means of membrane ultrafiltration technology is investigated. Preparations of whey were incubated in 10 per cent solutions of glucose, fructose and sorbitol at the conditions similar to physiological ones (0.9% NaCl, pH 5.5) and temperature of about +4 degrees C and +35 degrees C for 7, 14 and 28 days. A sample dissolved in 0.9% NaCl (pH 5.5) without addition of hydrocarbons was used as a "control preparate". All explored substances brought about the suppressive effect on deamidation rate of asparaginyl residues whereas that of glutaminyl residues, on the contrary, was obviously increased. The possible reasons for these observations are discussed.

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.

A partially unfolded state of equine beta-lactoglobulin at pH 8.7.

The urea-induced unfolding transition of equine beta-lactoglobulin was studied at pH 8.7 using circular dichroism (CD), ultracentrifugation, and gel filtration chromatography. The unfolding transition curves showed that at least one intermediate accumulates at moderate concentrations of urea. Furthermore, analytical ultracentrifugation experiments indicated that the intermediate forms a dimer. Thus, the urea-induced unfolding transition was measured by CD at various protein concentrations and was analyzed by a model assuming the four conformational states (the native, intermediate, dimeric intermediate, and unfolded states). The characteristics of the intermediate are markedly different from those of the intermediate previously observed at pH 4.0 or 1.5. The intermediate at pH 8.7 does not show the intense far-ultraviolet CD suggestive of the nonnative alpha-helix.

Structural damage to proteins caused by free radicals: asessment, protection by antioxidants, and influence of protein binding.

Oxidative damage to proteins results in biological dysfunctions such as perturbed activity in enzymes, transport proteins, and receptors. Here, we investigated structural damage to proteins induced by free radicals. Structural alterations to lysozyme, human serum albumin (HSA) and beta-lactoglobulin A were monitored by capillary zone electrophoresis. Four well-known antioxidants (quercetin, melatonin, Trolox, and chlorogenic acid) were examined for their ability to inhibit protein damage and to bind to these proteins. Melatonin and chlorogenic acid, which did not bind to any of the three proteins under study, showed scavenging and protective activities well correlated with the amount of free radicals generated. Trolox, which bound only to HSA, was a better protector of HSA than of the two other proteins, indicating that its antioxidant capacity is increased by a shielding effect. Finally, quercetin was a good antioxidant in protecting lysozyme and beta-lactoglobulin A, but its binding to HSA resulted in a pro-oxidant effect that accelerated HSA fragmentation. These results demonstrate that binding of an antioxidant to a protein may potentiate protection or damage depending on the properties of the antioxidant.

Effects of guanidine hydrochloride on the proton inventory of proteins: implications on interpretations of protein stability.

The DeltaG degrees (N)(-)(D) value obtained from extrapolation to zero denaturant concentration by the linear extrapolation method (LEM) is commonly interpreted to represent the Gibbs energy difference between native (N) and denatured (D) ensembles at the limit of zero denaturant concentration. For DeltaG degrees (N)(-)(D) to be interpreted solely in terms of N and D, as is common practice, it must be shown to be independent of denaturant concentration. Because DeltaG degrees (N)(-)(D) is often observed to be dependent on the nature of the denaturant, it is necessary to determine the circumstances under which DeltaG degrees (N)(-)(D) can be interpreted as a property solely of the protein. Here, we use proton inventory, a thermodynamic property of both the native and denatured ensembles, to monitor the thermodynamic character of denaturant-dependent aspects of N and D ensembles and the N right arrow over left arrow D transition. Use of a thermodynamic rather than a spectral parameter to monitor denaturation provides insight into the manner in which denaturant affects the meaning of DeltaG degrees (N)(-)(D) and the nature of the N right arrow over left arrow D transition. Three classes of proteins are defined in terms of the thermodynamic behaviors of their N right arrow over left arrow D transition and N and D ensembles. With guanidine hydrochloride as a denaturant, the classification of protein denaturations by these procedures determines when the LEM gives readily interpretable DeltaG degrees (N)(-)(D) values with this denaturant and when it does not.

Intermolecular beta-sheet results from trifluoroethanol-induced nonnative alpha-helical structure in beta-sheet predominant proteins: infrared and circular dichroism spectroscopic study.

2,2,2-Trifluoroethanol (TFE)-induced nonnative alpha-helical structure in peptides and proteins has been extensively studied with circular dichroism (CD) spectroscopy. However, to date, complementary information from infrared (IR) spectroscopy has not been reported. Using both IR and CD spectroscopy, we demonstrate here that the TFE-induced nonnative alpha-helical structure in two beta-sheet-predominant proteins, beta-lactoglobulin and alpha-chymotrypsin, is unstable in comparison with those found in the alpha-helix-predominant proteins myoglobin and cytochrome c under identical conditions. IR spectra showed that, immediately after dissolution of the beta-sheet proteins in 50% (v/v) TFE, a strong amide I band component appears at 1654 cm-1 in H2O and at 1650 cm-1 in D2O, which is ascribed to alpha-helical structure. However, the intensities of the alpha-helical bands decrease as a function of time, concomitant with the appearance of two new band components near 1620 and 1695 cm-1 in H2O and 1612 and 1684 cm-1 in D2O, a typical IR spectral pattern for an intermolecular beta-sheet aggregate. Clear gels begin to develop within 30 min. No similar spectral changes were observed for the alpha-helical proteins. CD spectra suggested initially that the TFE-induced alpha-helix was retained in the gelled state. However, further analysis of the spectra, and Gaussian function modeling with basic spectra, indicated that the apparent alpha-helix signal was actually due to a combination of signals from intermolecular beta-sheet and residual alpha-helix. These results indicate that the TFE-induced nonnative alpha-helix structure in predominantly beta-sheet proteins is unstable and readily converts to an intermolecular beta-sheet aggregate.

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.

JAK2 and STAT5, but not JAK1 and STAT1, are required for prolactin-induced beta-lactoglobulin transcription.

Several different Janus kinases (JAKs) and signal transducers and activation of transcription (STATs) have been implicated in mediating the biological responses induced by PRL, based on their ligand-dependent tyrosine phosphorylation and activation. However, these criteria alone do not prove that a particular JAK or STAT is essential for signal transduction. We have used mutant cell lines defective in JAK1, JAK2, or STAT1 to examine their roles in PRL-dependent signaling. JAK2 is absolutely required for PRL-dependent phosphorylation of the receptor, activation of STATs, and induction of beta-lactoglobulin. Wild type, but not kinase-negative JAK2, restores all responses to PRL in JAK2-defective cells, suggesting that JAK2 function, not merely the protein, is required. In contrast, JAK1, which is phosphorylated in response to PRL, is not required for any of these functions. Although STAT1 homodimers do form in response to PRL, no defect in PRL-dependent signaling is apparent when STAT1 is missing, suggesting that STAT5, which is strongly activated in response to PRL, is primarily responsible for driving the expression of PRL-responsive genes.

The equilibrium intermediate of beta-lactoglobulin with non-native alpha-helical structure.

It is generally considered that intermediates of protein folding contain partially formed native-like secondary structures. In contrast, we recently reported that the kinetic folding intermediate of bovine beta-lactoglobulin contains non-native alpha-helical structures. To understand the mechanism that stabilizes the non-native intermediate, we characterized by circular dichroism (CD) the equilibrium unfolding transition of beta-lactoglobulin induced by guanidine hydrochloride (Gdn-HCl) at pH 2 and 4 degrees C. The unfolding transition measured by near-UV CD preceded the transition measured by far-UV CD, indicating the accumulation of the intermediate state. The far-UV CD spectrum of the intermediate, obtained by global fitting analysis of the CD spectra in the presence of various concentrations of Gdn-HCl, was similar to the burst-phase intermediate observed in the refolding kinetics, and contained non-native alpha-helical structures. Addition of 10% (v/v) 2,2,2-trifluoroethanol (TFE) increased the helical content of the equilibrium intermediate, although the protein still assumed the native structure in the absence of Gdn-HCl. A phase diagram of the conformational states, i.e. the alpha-helical intermediate, unfolded and native states, against the concentration of TFE and Gdn-HCl was constructed. This indicated that, because of the high helical preference of the amino acid sequence of beta-lactoglobulin, the helical region protrudes into the boundary between the native and unfolded states, resulting in non-monotonic accumulation of the helical intermediate upon equilibrium unfolding of the native beta-sheet structure. This is the first observation to indicate that a non-native alpha-helical intermediate accumulates during equilibrium unfolding of a predominantly beta-sheet protein.

Effect of gamma-interferon on binding of gliadin and other food peptides to the human intestinal cell line HT-29.

gamma-Interferon is one of the main cytokines released during activation of intestinal lymphocytes in coeliac patients. The question has never been addressed whether gamma-interferon influences binding of gliadin and other food peptides to human enterocytes. Therefore, the human intestinal epithelial cell line HT-29 was cultured with gliadin, casein, beta-lactoglobulin and ovalbumin, with or without gamma-interferon, and peptide binding to cells was determined by flow cytometry and fluorescence microscopy. gamma-Interferon stimulated gliadin binding by a factor of 4. Binding was saturable with half maximal binding at 0.15 mg/ml. For maximal binding, an incubation of at least 24 h was necessary. gamma-Interferon increased binding of beta-lactoglobulin and casein, too, but inhibited that of ovalbumin. Binding of gliadin was inhibited by the other peptides. Under the conditions of ongoing mucosal inflammatory reactions and release of gamma-interferon, enhanced binding may trigger intestinal lymphocytes, increase secretion of cytokines and thus induce a vicious circle.

Effect of saponin on the transmucosal passage of beta-lactoglobulin across the proximal small intestine of normal and beta-lactoglobulin-sensitised rats.

The ability of saponins and glycoalkaloids to permeabilise the mammalian intestinal barrier has been previously demonstrated in vitro, leading to the hypothesis that membranolytic saponins may facilitate transfer to the tissues of otherwise excluded macromolecules. An enhanced uptake of, for instance, potentially allergenic species from the lumen is one of the factors that may affect the induction of food allergy, and its presentation in already sensitised individuals. In the experiments described here, an increase in the transmucosal uptake of the milk allergen beta-lactoglobulin (beta LG) was assessed in non-sensitised and sensitised Brown Norway rats in the presence of Gypsophila saponin. Isolated jejunal loops were exposed in vivo to either beta LG followed by saponin, saponin followed by beta LG or the two compounds simultaneously. Portal vein blood samples were collected and assayed for beta LG and rat mucosal mast cell protease (RCMP II) activity. Mucosal tissue was also examined histologically and assayed for histamine content. Sham-operated animals, exposed to physiological buffer alone, were included as controls and beta LG measurements corrected for this component which was negligible. No transfer of beta LG occurred in the absence of saponin in non-sensitised rats, whereas a significant enhancement was observed in the presence of saponin. beta LG was detected in the portal circulation of sensitised rats exposed to beta LG alone; however addition of saponin to the intestinal lumen further enhanced this uptake, possibly by an independent mechanism. Histological examination of the mucosal epithelium exposed to saponin revealed damage, especially at the villus tips. Mucosal histamine and serum RCMP II concentrations were consistent with the differences observed between sensitised and non-sensitised animals. It is concluded that exposure to food constituents capable of permeabilising the mucosal epithelium may increase the risk of sensitisation to dietary antigens.

Structural changes of beta-lactoglobulin B induced by urea. Evidence of residual structure.

Several spectroscopic methods have been used to study the structure of beta-lactoglobulin B at pH 2.1 in the presence of 8M urea. Fluorescence and polarization of fluorescence spectroscopy measurements indicate that the two tryptophanyl residues of the protein are exposed to the solvent in the denatured state. CD in the far-UV indicates that the amount of secondary structure in the denatured state is comparable to that found in the native state, whereas the CD spectrum in the near-UV shows that the tertiary structure is not completely disordered. The results of one-dimensional 1H NMR spectroscopy show that some local non-random structure is maintained in the denatured state, but most of the polypeptide chain has an extended non-globular conformation under the conditions of the present experiments. This conclusion is reinforced by the results of two-dimensional 1H NMR conducted on denatured samples of beta-lactoglobulin B. The study of states with intermediate levels of order will aid the understanding of how the native structure of beta-lactoglobulin B is organised during the refolding pathways.

Effect of sodium dodecyl sulfate and palmitic acid on the equilibrium unfolding of bovine beta-lactoglobulin.

The unfolding of bovine beta-lactoglobulin, a small globular protein that unfolds reversibly at low pH in the presence of urea or guanidine hydrochloride, has been studied at pH 6.72 in phosphate buffer at 21 degrees C. The midpoint urea concentration for the loss of CD intensity at 220 nm, loss of CD intensity at 293 nm, quenching of intrinsic fluorescence, shift in the wavelength of the maximum of the intrinsic fluorescent emission, and loss of fluorescence intensity from 1-anilino-8-naphthalenesulfonate (ANS) (and probably the hydrophobic binding site) was close to 4.4 M. Addition of sodium dodecyl sulfate (SDS) at concentrations less than 100 microM to the beta-lactoglobulin solutions increased the midpoint urea concentration for the CD and intrinsic fluorescence parameters to about 5.8 M. Palmitic acid had a similar effect to that shown by SDS in altering the CD intensity at 293 nm, and both SDS and palmitic acid attained a maximum effect in altering the CD at 293 nm at a 1:1 molar ratio to beta-lactoglobulin. It seems likely that the beta-sheet structure of beta-lactoglobulin breaks down simultaneously with the loss of the hydrophobic binding site and exposure of tryptophan-19 to the external environment, supporting the view that the major hydrophobic binding site of beta-lactoglobulin is closely involved with the beta-sheet core of the protein.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

[Experimental study of a new biological preparation for treating E. coli infection and Proteus dysbacteriosis in young infants]. / Eksperimental'noe izuchenie novogo biologicheskogo preparata dlia lecheniia koli-infektsii i proteinogo disbakterioza u detei rannego vozrasta.

The action of a new biological preparation intended for the treatment of E. coli infection and Proteus dysbacteriosis in children has been studied in experiments on mice and suckling rabbits. The protective activity of anti-E. coli and anti-Proteus lactoglobulin has been found to exceed that of normal lactoglobulin 6- to 13-fold. When subjected to the action of proteolytic enzymes of the gastrointestinal tract, the preparation retains its preventive properties on the level of the native preparation, which confirms the possibility of the oral administration of the preparation in clinical practice without protecting it from the proteolytic action of the enzymes of the gastrointestinal tract.