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PubMed1999

Capillary zone electrophoresis with optimized temperature control for studying thermal denaturation of proteins at various pH.

Capillary electrophoresis (CE) was used to analyze the thermal denaturation of bovine beta-lactoglobulin at different pH. This model protein exhibits complex pH- and temperature association/dissociation dependence balances in its quaternary structure. The study was possible after modification and improvement of a capillary electrophoresis apparatus. The improvement allowed both efficient control (temperature fluctuations <0.05 degrees C) and accurate measurement of the temperature (+/- 0.1 degrees C) within the capillary cartridge. CE allowed the thermodynamic parameters of beta-lactoglobulin thermal denaturation to be estimated. The transition temperature, Tm, was determined at acidic, neutral and alkaline pH. Van't Hoff analysis was performed through direct measurement of native and unfolded protein populations in the slow-time regime. This allowed estimation of thermodynamic parameters (deltaH, deltaS, deltaCp). Finally, the stability curve, i.e., the temperature dependence of the free energy change (deltaG) of protein unfolding was drawn. The accuracy of the parameters values compares with parameters obtained by calorimetric measurements. The available parameters and the requirement of minute amount of protein sample are of potential interest in the field of protein engineering and biological pharmaceuticals. Accordingly, CE can be proposed as a convenient tool to study protein stability and denaturation processes.

Animals↗
PubMed2000

Preparative electrochromatography of proteins in various types of porous media.

The performance of commercial and enzymatically modified size-exclusion (SE) gels in electrochromatography was compared for preparative protein separations. Dextran and agarose-based SE gels were subjected to enzymatic digestion under mild conditions. This treatment partially hydrolyzed the gel matrix modifying its pore size distribution. Enzymatic treatment of agarose-based SE gels was found to increase the resolution of the separation. Successful separation of preparative amounts of the A and B forms of beta-lactoglobulin (difference in electrophoretic mobility of 8.5%) was achieved with a high degree of purity using agarose-based SE gels. The four major whey proteins, beta-lactoglobulin, alpha-lactalbumin, BSA and immunoglobulins, were purified from an acid whey preparation. The degree of retention of a protein in electrochromatography followed their free-solution electrophoretic mobility (mu) when the protein was able to enter the gel pores and the ratio of diffusion/mu when the protein was excluded.

Chromatography↗
PubMed1995

Quantitative analysis of major whey proteins by capillary electrophoresis using uncoated capillaries.

A method that allows separation and quantitation of the main whey proteins by capillary electrophoresis using uncoated capillaries is proposed. Separations are performed using 100 mM borate buffer, pH 8.2, containing 30 mM sodium sulfate. The use of high pH and high ionic strength buffer reduces adsorption of proteins on the capillary wall, making their separation possible. Reproducibility of migration times and areas of peaks are improved by optimizing the capillary equilibration protocol and by using an internal standard. Relative standard deviations ranging between 0.74 and 1.03% for migration times and 2.14 to 5.23% for areas of major peaks are obtained. Detection limits equal to or lower than 0.5 mg/100 mL are achieved. Linear relationships of peak area versus concentration have been used to quantitate bovine serum albumin (BSA), alpha-LA (alpha-lactalbumin), beta-LG A (beta-lactoglobulin A) and beta-LG B (beta-lactoglobulin B) in cow's milk subjected to different thermal treatments. Electrophoretic profiles of these milk samples show peaks from other peptides besides those from main proteins. Characteristic patterns for whey from different species are obtained.

Animals↗
PubMed1975

The protein complex of bread dough as an interacting system.

The interactions occurring in the protein complex of wheat dough have been characterized by the extraction and molecular sieving techniques supported with optical methods as well as with some rheological examinations. The effects of wheat albumin, soya globulin and beta-lactoglobulin preparations have been determined. The interactions of dough protein complex with soluble pentosans isolated from rye flour and with lipid fractions of wheat embryo were analysed as well. In most cases the advanced aggregation, leading to formation of high molecular weight product similar to glutenin fraction and the increase of insoluble protein fraction were observed. The albumin, globulin and gluten type proteins participated in the interactions. The beta-lactoglobulin generally caused an intense disaggregation of the dough protein complex. The increase of low molecular weight fraction contents was, however, accompanied with formation of some quantities of the high molecular weight complex.

Albumins↗
PubMed2004

Cow's milk allergens identification by two-dimensional immunoblotting and mass spectrometry.

Cow's milk allergy (CMA) has become a common disease in early childhood, its prevalence ranging from 1.6% to 2.8% among children younger than 2 years of age. The role of different cow's milk protein (CMP) in the pathogenesis of CMA is still controversial. Even if the proteins most frequently and most intensively recognized by immunoglobulin E (IgE) seem to be the most abundant in milk (caseins and beta-lactoglobulin), with an although great variability all milk proteins appear to be potential allergens, even those that are present in trace amounts (i.e., lactoferrin, IgG, and BSA). In this work proteomics techniques have been applied for CMP allergens analysis. Allergens have been identified by immunoblotting following resolution of CMP components by two-dimensional electrophoresis. Sera from 20 milk-allergic subjects, as proven by oral provocation test, CAP-RAST and skin prick test, have been used for cow's milk major allergen identification. Cow's milk proteins and their isoforms were identified by matrix assisted laser desorption/ionization-time of flight (MALDI-TOF)-mass spectrometry. In our group of patients, the prevalence of CMP allergens, i.e., the total number of subjects sensitized to CMP divided by the total number of the subjects enrolled in the study, was: 55% alpha(s1)-casein, 90% alpha(s2)-casein, 15% beta-casein, 50% kappa-casein, 45% beta-lactoglobulin, 45% BSA, 95% IgG-heavy chain, 50% lactoferrin, and 0% alpha-lactalbumin.

Allergens↗
PubMed2002

Immunocytochemical detection of milk proteins in tracheal aspirates of ventilated infants: a pilot study.

In this study, we evaluated immunocytochemical staining for milk proteins (alpha-lactalbumin and beta-lactoglobulin) in tracheal aspirates of mechanically ventilated infants, and assessed whether this staining technique supported a clinical diagnosis of aspiration in infants receiving orogastric feedings. All newborns requiring mechanical ventilation in the neonatal intensive care unit of a major tertiary care hospital were potential subjects for this study. Tracheal aspirates were obtained prior to the introduction of enteral feeding and at various time points thereafter in newborns requiring mechanical ventilation. Cells were obtained and processed for immunocytochemical staining of alpha-lactalbumin and beta-lactoglobulin. In total, 88 specimens recovered from 34 infants were adequate for staining. Alveolar macrophages recovered from most of the infants who were never fed (true negative controls) did not display immunoreactivity for milk proteins: 4/34 or 12% of infants' aspirates demonstrated presence of milk proteins before enteral feeding was commenced. Tracheal aspirates obtained from 12 infants after introduction of enteral feedings appeared to support clinical and radiological findings suggestive of aspiration events, with positive immunostaining on several occasions. These observations support our work in a murine model and demonstrate that immunocytochemical staining of tracheal aspirates for milk proteins may enhance the ability to diagnose pulmonary aspiration. Further studies are needed to define the clinical significance of our findings and the effects of single and repeated aspiration events on respiratory status.

Female↗
PubMed2001

Evaluation of protein modification during anti-viral heat bioprocessing by electrospray ionization mass spectrometry.

During the preparation of therapeutic plasma and recombinant protein biopharmaceuticals heat-treatment is routinely applied as a means of viral inactivation. However, as most proteins denature and aggregate under heat stress, it is necessary to add thermostabilizing excipients to protein formulations destined for anti-viral heat-treatment in order to prevent protein damage. Anti-viral heat-treatment bioprocessing therefore requires that a balance be found between the bioprocessing conditions, virus kill and protein integrity. In this study we have utilized a simple model protein, beta-lactoglobulin, to investigate the relationship between virucidal heat-treatment conditions (protein formulation and temperature) and the type and extent of protein modification in the liquid state. A variety of industrially relevant heat-treatments were undertaken, using formulations that included sucrose as a thermostabilizing excipient. Using liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) we show here that protein modifications do occur with increasingly harsh heat-treatment. The predominant modification under these conditions was protein glycation by either glucose or fructose derived from hydrolyzed sucrose. Advanced glycation end products and additional unidentified products were also present in beta-lactoglobulin protein samples subjected to extended heat-treatment. These findings have implications for the improvement of anti-viral heat-treatment bioprocesses to ensure the safety and efficacy of protein biopharmaceuticals. CopyrightCopyright 2001 John Wiley & Sons, Ltd.

Blood Proteins↗
PubMed1996

Temperature-jump induced fast refolding of cold-unfolded protein.

Transient protein structures with only microsecond live times may be solved at the resolution of single residues by temperature jumping of cold denatured protein [Nölting, B., Golbik, R., and Fersht, A. R. (1995) Proc. Natl. Acad. Sci. USA 92, 10668-10672]. Here it is shown how this method may be extended to the study of intermediates and transition states which are located on the reaction coordinate between the main folding transition state and the native state. When incubating bovine beta-lactoglobulin A under conditions which favour cold unfolding and rapidly refolding the partially cold unfolded protein by a temperature jump from -4 to 2 degrees C, a fast folding kinetics with a rate constant of about 100 s-1 is observed. The amplitude of the relaxation decays following a single exponential function of the time of incubation at low temperature before the T-jump. The rate constant of decay matches the rate constant of the main unfolding transition, showing that early in the cold denaturation reaction, beta-lactoglobulin is kinetically trapped in a partially unfolded intermediate state. Despite the only small fluorescence change, refolding within about 10 ms after a temperature jump involves a considerable degree of solvent exclusion and burial of hydrophobic surface, suggesting a contraction of the molecule.

Animals↗
PubMed1999

Cellular and humoral immunity against cow's milk proteins in type 1 diabetes.

Cow's milk beta-casein has been proposed as a candidate trigger of autoimmunity associated with type 1 diabetes. In this study, cellular and humoral immunity against beta-casein was compared to that against other major cow's milk proteins in patients with recent onset type 1 diabetes and control subjects. T cell responses were found against alpha-casein, beta-casein, beta-lactoglobulin and bovine serum albumin in both patients with type 1 diabetes (stimulation index: 0.2-22.8, n=23) and control subjects (stimulation index: 0.1-18.2, n=22), with no significant differences between groups. Twelve (52%) patients and nine (41%) control subjects had stimulation indices >3 to at least one protein, including 9 (39%) patients and 4 (18%) control subjects against beta-casein, all but one of these also having elevated responses to alpha-casein. The highest responses (stimulation index >9) were against alpha- and beta-casein in some patients and control subjects who had the HLA DR3 allele. Antibody levels against alpha-casein, beta-casein and beta-lactoglobulin were low in both patients (n=59) and control subjects (n=52). Nevertheless, significantly higher IgG binding to both alpha-casein in ELISA (P=0.02) and beta-casein using ELISA (P=0.02) and RIA (P=0.04) was observed in patients aged <15 years compared to control subjects of similar age. No relationship was found between cellular and humoral immunity against individual antigens. These data show that immune responses to cow's milk are not limited to patients with diabetes and not solely against beta-casein.

Adolescent↗
PubMed2002

Removal of proteinaceous soils using hydroxyl radicals generated by the electrolysis of hydrogen peroxide.

We have developed here for the first time a novel method to generate hydroxyl radicals, *OH, by applying slightly negative electric potentials (-0.2--0.8 V vs Ag/AgCl) to the surface of a metal (or metal oxide) that is in contact with hydrogen peroxide solution containing a supporting electrolyte. Namely, *OH radicals were generated at the surface by the electrolysis of hydrogen peroxide according to the equation, H2O2+e- --> *OH+OH-. This method was used to clean a stainless steel fouled with a model protein, beta-lactoglobulin. The *OHs generated at the surface were effective in removing beta-lactoglobulin that had been irreversibly adsorbed, by several minutes of treatment at room temperature (22+/-2 degrees C). The removal rates measured for various concentrations of H2O2 and supporting electrolyte and different potentials were determined exclusively by the electric current.

Animals↗
PubMed1998

Cloning, expression, purification, and immunocharacterization of placental protein-14.

Human placental protein-14 (PP-14), a member of the lipocalin superfamily, shares homology at the level of the primary and secondary structures with bovine beta-lactoglobulin. It is the most prominent endometrial protein synthesized by the glandular cells of endometrium under estrogen priming and progesterone stimulation. The temporal and spatial expression of PP-14 in the female reproductive tract combined with its biological activities ex vivo suggest that this glycoprotein probably plays an essential physiological role in the regulation of fertilization, implantation, and maintenance of pregnancy. We proposed to elucidate the molecular mechanisms involved in the function of this protein. A prerequisite to such investigations on any protein is the availability of sufficient amounts of the same in a homogenous form. Therefore, recombinant DNA technology was employed. The PP-14 cDNA was obtained from the first-trimester endometrial tissue RNA by RT-PCR using unique primers. After confirming the identity of the gene, the protein was expressed in Escherichia coli and purified to homogeneity. The gene was also cloned and expressed in Pichia pastoris to obtain the protein product in a glycosylated form. The recombinant proteins were immunocharacterized using a cross-reactive antibody raised to bovine beta-lactoglobulin. Polyclonal antiserum raised to the E coli expressed PP-14 also bound to the native PP-14 from amniotic fluid suggesting that recombinant PP-14 may be exploited to elucidate functional aspects of the protein.

Amino Acid Sequence↗
PubMed1992

Rapid analysis of whey proteins from different animal species by reversed-phase high-performance liquid chromatography.

This paper explores the possibilities of reversed-phase high performance liquid chromatography (RP-HPLC) for analysing whey proteins from the milk of cows and other animal species. An RP-HPLC method is proposed to separate and quantify bovine whey proteins. Using this method, bovine serum albumin, alpha-lactalbumin, beta-lactoglobulin A and beta-lactoglobulin B were separated in less than 7 min. It is demonstrated that irreversible adsorption of bovine whey proteins occurs on unused columns (i.e. those not previously used to separate proteins). Therefore, prior conditioning of the column with whey proteins is required for valid protein quantification. Conditioning can be achieved by eluting a large amount of at least one of the bovine whey proteins through the unused column. The calibration curve (peak area vs. protein concentration) for the main bovine whey proteins was linear. This method also allowed good separation of caprine and ovine whey proteins and separation of some homologous whey proteins of different animal species. Detection of milk mixtures from different animal species was carried out using this method.

Animals↗
PubMed1990

Cow's milk protein allergy--results of skin-prick test with purified milk proteins.

The diagnosis of milk-protein allergies is difficult. Therefore, the main cow's milk proteins (alpha-lactalbumin, beta-lactoglobulin A + B, alpha s-casein, beta-casein, kappa-casein), purified to the highest available standards, were used for differential diagnosis of allergic individuals by applying skin-prick test. In the case of adults with uncertain medical history, milk proteins rarely caused skin reactions, while distinct skin reactions were observed in 11 of 13 children with strongly suspected milk-protein allergy. In the presented study alpha-lactalbumin and beta-lactoglobulin B were the main allergens, because skin reactions to these proteins were provoked with highest prevalence and intensity. Intensity and prevalence of reactions to alpha- and kappa-casein were significantly less. In two cases skin reactions to alpha-lactalbumin were observed exclusively.

Adolescent↗
PubMed2001

Fluoroalcohol-induced structural changes of proteins: some aspects of cosolvent-protein interactions.

The conformational transitions of bovine beta-lactoglobulin A and phosphoglycerate kinase from yeast induced by hexafluoroisopropanol (HFIP) and trifluoroethanol (TFE) have been studied by dynamic light scattering and circular dichroism spectroscopy in order to elucidate the potential of fluoroalcohols to bring about structural changes of proteins. Moreover, pure fluoroalcohol-water mixed solvents were investigated to prove the relation between cluster formation and the effects on proteins. The results demonstrate that cluster formation is mostly an accompanying phenomenon because important structural changes of the proteins occur well below the critical concentration of fluoroalcohol at which the formation of clusters sets in. According to our light scattering experiments, the remarkable potential of HFIP is a consequence of extensive preferential binding. Surprisingly, preferential binding seems to play a vanishing role in the case of TFE. However, the comparable Stokes radii of both proteins in the highly helical state induced by either HFIP or TFE point to a similar degree of solvation in both mixed solvents. This shows that direct binding or an indirect mechanism must be equally taken into consideration to explain the effects of alcohols on proteins. The existence of a compact helical intermediate with non-native secondary structure on the transition of beta-lactoglobulin A from the native to the highly helical state is clearly demonstrated.

Animals↗
PubMed1997

Antirotaviral activity of milk proteins: lactoferrin prevents rotavirus infection in the enterocyte-like cell line HT-29.

Different milk proteins were analyzed for their inhibitory effect on either rotavirus-mediated agglutination of human erythrocytes or rotavirus infection of the human enterocyte-like cell line HT-29. Proteins investigated were alpha-lactalbumin, beta-lactoglobulin, apo-lactoferrin, and Fe(3+)-lactoferrin, and their antiviral action was compared with the activity of mucin, a milk glycoprotein known to affect rotavirus infection. Results obtained demonstrated that beta-lactoglobulin, apo- and Fe(3+)-lactoferrin are able to inhibit the replication of rotavirus in a dose-dependent manner, apo-lactoferrin being the most active. It was shown that apo-lactoferrin hinders virus attachment to cell receptors since it is able to bind the viral particles and to prevent both rotavirus haemagglutination and viral binding to susceptible cells. Moreover, this protein markedly inhibited rotavirus antigen synthesis and yield in HT-29 cells when added during the viral adsorption step or when it was present in the first hours of infection, suggesting that this protein interferes with the early phases of rotavirus infection.

Animals↗
PubMed2006

Improvement of interfacial protein stability by CHAPS.

Emulsification of aqueous protein solutions in methylene chloride triggered the formation of water-insoluble aggregates at a water/methylene chloride interface. As a result, the amounts of beta-lactoglobulin and ovalbumin recovered in water were 36 and 44%, respectively. Addition of 5 mM: CHAPS in the aqueous phase raised the degree of beta-lactoglobulin recovery to 96%. Sodium taurocholate, however, failed to improve protein recovery. The stabilizing effect of CHAPS was also protein-specific and concentration-dependent: at >or=5 mM: , the surfactant caused unfolding of ovalbumin to make a water-soluble oligomer. CHAPS thus stabilizes proteins at an interface.

Cholic Acids↗
PubMed1988

The measurement of insoluble proteins using a modified Bradford assay.

A technique for determining the amount of thermally denatured, insoluble protein is described. The assay has been validated using four globular proteins, bovine serum albumin, beta-lactoglobulin, lysozyme, and ovalbumin. It consists of a resolubilization protocol, using 8 M urea and 5% 2-mercaptoethanol, linked to the Bradford dye binding assay. The resolubilization protocol was carried out at 100 degrees C to enable complete recovery of all insoluble proteins. Beta-Lactoglobulin resolubilization was completed after heating for 1 min, whereas samples of bovine serum albumin, lysozyme, and ovalbumin required heating for 1.5 min. The assay can measure protein concentrations as small as 10 micrograms, typically with standard deviations of 3%, thus comparing favorably with the standard Bradford assay. Other types of denaturation, such as chemical denaturation causing subsequent insolubility, may be studied with this technique providing that there is no interference with the Bradford assay.

Hot Temperature↗
PubMed1996

Cadmium uptake by Caco-2 cells. Effect of some milk components.

The effect of some milk components on the cellular uptake of cadmium has been studied using a human intestinal cell line (Caco-2). Cadmium uptake by Caco-2 cells increased with the concentration of this metal in the culture medium, in a saturable way. These cells were exposed to different concentrations of cadmium and the synthesis of metallothionein was studied by a cadmium-saturation method. The levels of metallothionein increased with the cadmium concentration in the medium up to 20 microM of metal. Supplementation of the culture medium with 10% bovine milk caused a 25% decrease in the uptake of cadmium with respect to that internalized by the cells maintained in the culture medium alone. However, the uptake of cadmium from the medium supplemented with 10% human milk was similar to that with serum-free medium. beta-Lactoglobulin interacted with cadmium when studied by equilibrium dialysis, showing a stoichiometric binding constant of 5 x 10(4) l/mol. Interaction of lactoferrin with cadmium, however, was negligible. When Caco-2 cells were incubated in culture medium containing lactoferrin, cadmium uptake decreased with respect to that observed incubating the cells in a medium containing beta-lactoglobulin or in the free-protein medium. The inhibitory effect of lactoferrin on the uptake of cadmium might be due to a reduction of the cell surface charge, through its binding to the membrane.

Animals↗