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PubMed1998

Specific interaction of food proteins with apical membranes of the human intestinal cell lines Caco-2 and T84.

A comparison between the intestinal epithelial cell lines Caco-2 and T84 was made to assess the influence of enterocytic differentiation on food protein binding capacities of the brush border membrane. Cell morphology and expression of brush border-associated enzymes were studied as differentiation markers. Food protein binding to isolated brush border membranes was measured with a dot blot chemiluminescence assay. Early at confluence, Caco-2 cells exhibited a more differentiated state compared to T84 cells. Brush border membranes of both cell lines bound gliadin peptides, beta-lactoglobulin and ovalbumin specifically. Binding capacities increased from gliadin peptides to ovalbumin to beta-lactoglobulin. There was correlation of membrane binding capacity with degree of cell differentiation. Due to their similarity to small intestinal epithelial cells, the colon carcinoma cell lines Caco-2 and T84 represent models for studying food protein-enterocytic brush border membrane interactions in relation to varying degrees of cell differentiation.

Caco-2 Cells↗
PubMed1997

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.

Caseins↗
PubMed1988

Tandem separation schemes for preparative high-performance liquid chromatography of proteins.

Preparative chromatography of protein mixtures was carried out by tandem separation schemes involving frontal chromatography followed by stepwise desorption or displacement. In this way, with columns and instruments generally employed in analytical high-performance liquid chromatography, proteins could be purified in quantities similar to those typically separated by a preparative-scale system. A mixture of beta-lactoglobulin A and B was loaded onto an anion-exchange column, and, in the process, a large fraction of the less-retained beta-lactoglobulin B was recovered in pure form. The column was then flushed with the carrier, and subsequent desorption of the substances bound on the stationary phase was carried out by single-step desorption, two-step desorption, or displacement. With this mixture, the last two methods yielded approximately the same results in terms of the amount of product obtained per unit column volume. Whereas stepwise desorption is a simpler technique than displacement, the latter is required for the separation of components having similar adsorption behavior. In another set of experiments, a protein mixture obtained by heat treatment of human growth hormone was fractionated on a reversed-phase column. After loading the column by frontal chromatography, which separated a large fraction of the main product from the other components retained by the column, four desorption steps were applied to recover the individual components. These separation schemes offer an approach to preparative chromatography of proteins that is superior to conventional linear elution in terms of column load capacity, low mobile phase consumption, simultaneous separation and concentration, as well as enrichment of trace components.

Chromatography, High Pressure Liquid↗
PubMed1975

Scanning isoelectric focusing in small density-gradient columns. IV. The use of deuterium oxide for preparing the density gradient and its effects on isoelectric points of proteins.

The use of deuterium oxide as a substitute for sucrose in preparing density gradients for small isoelectrofocusing columns has been investigated. A density gradient was created directly in a 1.5-ml column by free inter-diffusion of three deuterium oxide solutions for 3 min. The resulting deuterium oxide concentration course (as shown by measurement of the refractive-index gradient) had a very high degree of linearity. Test runs with beta-lactoglobulin and sperm-whale myoglobin showed that the strength and stability of the deuterium oxide density gradient should normally be sufficient for stabilization of protein zones against convection during isoelectric focusing. The isoelectric points of beta-lactoglobulins A and B were found to increase as the concentration ratio of deuterium oxide to water at focusing level increased. Within the limits of experimental error, the pI shift corresponding to a given change in solvent composition was equal for both components. These findings are accounted for in terms of the deuterium isotope effect on the dissociation constants of protolytic groups and the shift in the asymmetry potential of the glass electrode in deuterium oxide as compared with water. The spontaneous reduction of sperm-whale metmyoglobin to ferrous myoglobin, which occurs on prolonged isoelectric focusing, is discussed, as are the benefits and drawbacks of deuterium oxide as a density-gradient solute.

Animals↗
PubMed1998

Preparative ion-exchange chromatography of proteins from dairy whey.

A preparative-scale ion-exchange chromatographic process is described for the separation of the four major proteins and lactose from sweet dairy whey. Experiments using a commercial anion-exchange resin were carried out to determine the optimum conditions for initially separating the proteins alpha-lactalbumin, beta-lactoglobulin, bovine serum albumin, immunoglobulin G and lactose from a sweet dairy whey mixture. The separation was accomplished with simultaneous step elution changes in salt concentration and pH. It was found that the anion-exchange step was most effective in separating beta-lactoglobulin from the feed mixture. Following the anion-exchange separation, its breakthrough curve was processed using a commercial cation-exchange resin to further recover the valuable immunoglobulin G. The whey output from an east Tennessee cheese manufacturer was used as a feedstream for the preparative scale experiments and as a reference in scaling to an economically optimized production level operation.

Animals↗
PubMed1999

Detection of bovine whey proteins by on-column derivatization capillary electrophoresis with laser-induced fluorescence monitoring.

1-Anilinonaphthalene-8-sulfonic acid (1,8-ANS), 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid (bis-ANS) and 2-(p-toluidino)naphthalene-6-sulfonic acid (2,6-TNS) were evaluated as additives in different buffers for the detection of bovine whey proteins using laser-induced fluorescence (LIF) monitoring in capillary electrophoresis (CE). These N-arylaminonaphthalene sulfonates furnish a large fluorescence emission when associated to some proteins whereas their emission in aqueous buffers, such as those used in CE separations, is very small. To select the best detection conditions, the fluorescence of these probes was first compared using experiments carried out in a fluorescence spectrophotometer. Using bovine serum albumin (BSA) as a model protein, it was demonstrated that 2-(N-cyclohexylamino)ethanesulfonic acid (CHES) buffer (pH 8 and pH 10.2) and the fluorescent probe 2,6-TNS gave rise to the highest increase in fluorescence for BSA. When the composition of these separation buffers was optimized for the electrophoretic separations, CHES buffer, pH 10.2 was chosen as the most suitable buffer to detect bovine whey proteins. The limit of detection obtained for some whey proteins in CE separations was about 6.10(-8) M for BSA, 3.10(-7) M for beta-lactoglobulin A (beta-LGA), 3.10(-7) M for beta-lactoglobulin B (beta-LGB), and 3.10(-6) M for alpha-lactalbumin (alpha-LA). These detection limits were compared to those achieved using UV detection under the same separation conditions. The results showed that the detection limits of BSA, beta-LGA and beta-LGB were twice as good using LIF than with UV detection. However, the limit of detection for alpha-LA was better when UV was used. The applicability of LIF detection to CE separation of whey proteins in bovine milk samples was also demonstrated.

Anilino Naphthalenesulfonates↗
PubMed1998

Aging, calorie restriction and ubiquitin-dependent proteolysis in the livers of Emory mice.

Calorie restriction (R), the only known method to delay the aging process and extend mean and maximal lifespan, has been shown to delay the age-related decline in protein degradation. There are several proteolytic pathways. The ubiquitin- and ATP-dependent proteolytic pathway (UPP) is frequently associated with degradation of damaged abnormal and/or regulatory proteins. We examined the effect of aging and R on supernatants of livers taken from young (4.5 months) and old (23 months) Emory mice. Aging was associated with increased levels of endogenous ubiquitin conjugates, enhanced ability to form high molecular weight conjugates and ubiquitin activating (E1) and ubiquitin conjugating (E2) activity in the control (C) liver supernatants. The age-related increase in levels of endogenous ubiquitin conjugates in liver appears to be primarily due to increased E1 and E2 activities. R prevented the age-related increase in E1 and E2 activity, and thus prevented the age-related increase in levels of ubiquitin conjugates. In spite of the age-related increase in ubiquitin conjugates, no age-related changes in ubiquitin-dependent proteolytic pathway were observed in the C animals. R was associated with an enhanced ability (130%) to degrade beta-lactoglobulin by the ubiquitin-dependent proteolytic pathway in livers from 4.5-month-old animals relative to age-matched C livers. However, rates of the ubiquitin-dependent degradation of beta-lactoglobulin in the 23-month-old C and R animals were indistinguishable. There were no age- or diet-related differences in the ability to degrade another substrate, oxidized ribonuclease (RNase).

Adenosine Triphosphate↗
PubMed1998

Calorie restriction, stress and the ubiquitin-dependent pathway in mouse livers.

Calorie restriction (R) is the only known method to delay the aging process and extend mean and maximal lifespan in rodents. R has been shown to delay the age-related accumulation of damaged proteins and to protect organisms from various stresses which can produce damaged proteins. Such stresses include irradiation, heat shock, and oxidative stress. The ubiquitin- and ATP-dependent proteolytic pathway (UPP) has been associated with the degradation of abnormal and/or damaged proteins. We examined the effect of diet and oxidative stress on activities of the UPP in supernatants from livers taken from 23-month-old Emory mice which had been exposed to an in-vivo injection of paraquat. Paraquat induces oxidative stress by generating superoxide radicals. In livers from non-stressed animals, steady-state levels of endogenous ubiquitin conjugates, de novo conjugate formation, and E1 and E2 activities were significantly lower in R animals than in control (C) animals. However, after exposure to paraquat, levels of endogenous ubiquitin conjugates were significantly higher in R versus C animals, and de novo conjugate formation and E1 and E2 activities in R animals rose to levels which were indistinguishable from levels of these activities noted in C animals. R was associated with an increased ability to degrade beta-lactoglobulin by the UPP after an oxidative stress was imposed. Ability to degrade beta-lactoglobulin by the C or R livers in non-stressed animals was not significantly different. Taken together, these data indicate that oxidative stress in R animals is associated with enhanced levels of ubiquitin conjugates and that this enhancement may be due to an increase in UPP activity. These data also indicate that the ability to form ubiquitin conjugates and the UPP system does not change with oxidative stress in C animals. The latter is consistent with prior reports that suggests that older C animals may already be in a state of enhanced oxidative stress and that activities of the UPP provide a sensitive indicator of levels of cellular redox status.

Adenosine Triphosphate↗
PubMed1986

Cow's milk allergy in breast-fed infants: the role of allergen and maternal secretory IgA antibody.

The protective effect of breast-feeding in allergy prophylaxis remains controversial despite many epidemiologic studies. One reason for confusion could be heterogeneity of breast milk with regard to its protective capacity. To clarify this issue, we studied 57 mother-infant pairs where breast-feeding was the sole source of infant nutrition. Family history and infant symptom scores suggestive of allergic disease were noted. The presence and quantity in breast milk of a prominent food allergen (beta-lactoglobulin), total IgA, and IgA antibodies to whole cow's milk and casein were measured serially. Eleven infants had symptom scores highly suggestive of allergic disease. The breast milk from mothers of these 11 infants was found to have lower total IgA (p less than 0.01) and IgA antibodies to both whole cow's milk and casein (p less than 0.001 and p less than 0.005, respectively) than milk from mothers whose infants had few or no symptoms. Beta-lactoglobulin was detectable in 45% of breast-milk specimens at concentrations up to 6.4 ng/ml and persisted up to 3 days after maternal dietary milk exclusion but was unrelated to antibody levels or symptom scores. Inadequate quantities of maternal IgA antibodies to food allergens appear to play a permissive role in development of infantile allergic disease in breast-fed infants and are more closely associated with symptoms than parental atopic history.

Allergens↗
PubMed1999

Transforming growth factor-beta1 in mothers' colostrum and immune responses to cows' milk proteins in infants with cows' milk allergy.

BACKGROUND: Breast milk contains immune factors that compensate for the underdeveloped defenses of the gut of the newborn infant. OBJECTIVE: We sought to study the importance of these factors in the immune responses of infants with cows' milk allergy (CMA) to the proteins in cows' milk (CM). METHODS: We prospectively followed the development of CMA in 6209 healthy infants and collected samples of colostrum from mothers. Samples from mothers of infants with CMA and from control subjects were analyzed for immunoglobulins, CM-specific antibodies, and cytokines. In infants with CMA, correlations between the concentration of transforming growth factor (TGF)-beta1 in colostrum and the extent of the immune response to CM proteins were studied. RESULTS: The concentration of TGF-beta1 in colostrum samples from mothers of infants with IgE-mediated CMA (n = 65) was lower (mean, 589 pg/mL; 95% confidence interval [CI], 413-840) than from mothers of infants with non-IgE-mediated CMA (n = 37; mean, 1162 pg/mL; 95% CI, 881-1531; t = 2.57, P =.012). In 126 control subjects the mean concentration was 807 pg/mL (95% CI, 677-963). In the infants with CMA (n = 96-100), the concentration of TGF-beta1 in colostrum was positively correlated with IgA antibodies to beta-lactoglobulin and IgG antibodies to alpha-casein and whole formula and negatively with the diameter of a skin prick test response to CM and lymphocyte stimulation indices to alpha-casein and beta-lactoglobulin. CONCLUSIONS: In an infant prone to having CMA, the TGF-beta1 content of mother's colostrum may promote IgG-IgA antibody production and inhibit IgE- and cell-mediated reactions to CM.

Animals↗
PubMed2001

Susceptibility to trypsinolysis of esterified milk proteins.

Methyl-, ethyl- and propyl-esters of beta-lactoglobulin, alpha-lactalbumin and beta-casein were prepared and then hydrolyzed with trypsin in various conditions. Resulting hydrolysates were analysed by SDS electrophoresis and RP-HPLC. The degree of hydrolysis of esterified samples was generally lower than those of the non-modified proteins. The highest degrees of hydrolysis were obtained at pH 7--8 with native and esterified protein samples. beta-Lactoglobulin propyl ester and beta-casein methyl ester yielded the lowest degrees of hydrolysis. Ethyl- and propyl-esters of beta-casein showed high resistance towards tryptic attack, even after 20 h of hydrolysis. SDS electrophoretic patterns of tryptic hydrolysates of native proteins showed bands corresponding to low molecular weights. Tryptic hydrolysates of esterified proteins showed bands with higher sizes. RP-HPLC profiles of tryptic hydrolysates of esterified samples showed peaks with longer elution times than those obtained with native proteins, indicating the presence of more hydrophobic peptide populations. A peptic pre-treatment improved tryptic action on esterified proteins. It resulted in a better resolution of RP-HPLC profiles and in a complete disappearance of the protein after 20 h tryptic hydrolysis.

1-Propanol↗
PubMed1998

Capillary electrophoretic behavior of milk proteins in the presence of non-ionic surfactants.

The electrophoretic behavior of alpha-lactalbumin and beta-lactoglobulins (A and B) in the presence of non-ionic surfactants was studied by capillary electrophoresis (CE), using a poly(ethylene glycol) coated capillary column. The surfactants (Tween 20, Brij 35 and 78) were used as buffer additives. The separation is based on the difference in the strength of protein-surfactant association complexes, which results in a change of the effective electrophoretic mobility. The modification of the electrophoretic mobilities of proteins was observed and this variation permitted the estimation of the interaction between protein and surfactant. The effect of surfactant type and concentration on the migration behavior of protein in CE is discussed. It is found that the retention behavior of the milk proteins (the alpha-lactalbumin and the beta-lactoglobulins) in CE is very different. The pH of the buffer and the surfactant type influence significantly the protein-surfactant interactions.

Animals↗
PubMed2001

Comparative study of protein molecular weights by size-exclusion chromatography and laser-light scattering.

High-performance size-exclusion chromatography (SEC) based on UV-Vis detection is a relative technique for molecular weight determination whereas procedure based on multi-angle laser light scattering (MALLS) is both rapid and absolute. The two methods using recombinant human growth hormone (rHGH) and beta-lactoglobulin samples were compared. A calibration curve for the chromatographic system was generated based on standard proteins and the data were fitted by least squares to a third order polynomial model. The molecular weight from the conventional SEC method for both proteins was higher than the reported values. The molecular weight of rHGH from MALLS was 23.1+/-0.57 and 21.2+/-0.80 kDa using differential refractive index (SEC-MALLS/RI) and UV (SEC-MALLS/UV-Vis) detectors as mass detectors. Both values agree, within experimental error with the molecular weight sequence of rHGH, 22.1 kDa. In contrast, the molecular weight from LS for beta-lactoglobulin was 22.5+/-0.55 kDa by SEC-MALLS/RI and 23.0+/-1.22 kDa by SEC-MALLS/UV-Vis, respectively, values always higher than those supplied by the manufacturer, 18.4 kDa. The reproducibility of the SEC-MALLS/UV-Vis method versus the SEC-MALLS/RI method was performed using the concordance correlation coefficient. The method's reproducibility was accepted by assuming a precision of 98% and a 1% loss in precision.

Chromatography, Gel↗
PubMed1997

Formation of long-lived protein radicals in the reaction between H2O2-activated metmyoglobin and other proteins.

Free radicals formed during the reaction of H2O2 and metmyoglobin in the presence of bovine serum albumin (BSA) were investigated using freeze quench and spin-trap ESR spectroscopy. Increasing concentrations of BSA (0-300 microM) resulted in drastic changes in the characteristic freeze quench ESR signal of H2O2-activated metmyoglobin (perferryl protein radical) under physiological conditions (pH = 7.4; I = 0.16). The radical species formed during reaction of 100 microM H2O2, 100 microM metmyoglobin, and 200 microM BSA have half-lives of approximately 13 min at 25 degrees C, in contrast to the perferryl protein radical that has a half-life of approximately 28 s at 25 degrees C. The radical species formed in the presence of BSA were reactive towards ascorbate, glutathione, cysteine, and tyrosine. Substitution of BSA with defatted BSA, gamma-globulin or beta-lactoglobulin also resulted in formation of long-lived free radical species (half-lives: 13-18 min); however, the ability to form these was dependent of the specific protein and decreased in the following order: BSA > defatted BSA > gamma-globulin > beta-lactoglobulin. The spin-trap alpha-phenyl-tert-butylnitrone (PBN) showed the presence of transient protein radical species formed in the reaction between MMb, H2O2, and BSA. Transient radical species that could be proposed as intermediates in the formation of the long-lived protein radicals detected by freeze-quench ESR. Dityrosine was formed in the reaction between MMb, H2O2, and BSA, showing the involvement of tyrosine residues in the present reaction. The described chemical interaction between H2O2-activated myoglobin and other proteins have major consequences on future interpretations of the significance of the perferryl protein radical in biological systems where proteins are abundant.

Animals↗
PubMed2003

Allergenicity of a hydrolyzed rice infant formula in a guinea pig model.

BACKGROUND: Because sensitization to cow's milk is a common finding in children, the identification of safe alternative protein sources is important in the management of childhood allergy. OBJECTIVE: To evaluate, in an animal model, the allergenicity of a novel formula based on hydrolyzed rice proteins. METHODS: We conducted an experiment involving 130 guinea pigs, from 7 to 12 days old at the onset of the study. The animals were divided into 13 groups and were given, ad libitum, one of the following liquids to drink: (1) rice hydrolysate formula (RF), (2) a conventional cow's milk formula (CMF), or (3) water. After a 37-day sensitization period, a challenge was given, consisting of an intravenous injection of either isolated proteins or ultracentrifuged formulas (uCMF and uRF). Specific IgG antibodies to beta-lactoglobulin, casein, and whole rice protein were measured by enzyme-linked immunosorbent assay. RESULTS: When animals fed CMF were challenged with beta-lactoglobulin, casein, or whole uCMF, they showed significantly more reactions than did those fed RF when challenged with the same proteins (P < 0.001). In the groups fed RF, no reaction was observed after challenge with uRF, and only 2 mild reactions occurred after challenge with rice protein. Very low levels of specific IgG antibodies to rice protein were noted in all the groups, including the RF-fed animals, and no significant differences were evident between groups. CONCLUSIONS: The findings suggest that this new formula based on hydrolyzed rice proteins has a very low sensitizing capability.

Anaphylaxis↗
PubMed2002

Chelator, metal ion and buffer studies for protein C separation.

Protein C (PC) is the pivotal anticoagulant and antithrombotic in the human coagulation cascade. PC deficiency can result in major medical problems such as deep vein thrombosis (DVT), leading to tissue oxygen deprivation. PC treatment has no bleeding or skin necrosis problems because it circulates in the blood as a zymogen and is only activated when and where it is needed. One source of PC is transgenic animal milk. The major components in the milk, such as alpha-casein, beta-casein, kappa-casein, alpha-lactalbumin and beta-lactoglobulin, are proteins that must be separated from PC. Immobilized metal affinity chromatography (IMAC) is an inexpensive separation technology with relatively high specificity, and it has great potential for difficult protein separations. After systematic studies of different chelator, metal ion and buffers, the combination of iminodiacetic acid (IDA) and Fe was found to be effective to separate PC from major milk components. alpha-Lactalbumin and beta-lactoglobulin fell through the column in the starting buffer. PC was eluted. alpha-Casein, beta-casein, kappa-casein remained bound in the column after PC elution. This technology might be applied for PC separation from transgenic animal milk. It is very important for PC production in large quantities and at low cost to treat PC-deficient patients.

Animals↗
PubMed2002

A functional comparison of ovine and porcine trypsins.

Trypsin was isolated from ovine and porcine pancreas using affinity chromatography on immobilized p-aminobenzamidine. Molecular masses of the two proteins were 23900 and 23435 Da, determined by matrix-assisted laser desorption/ionisation time of flight (MALDI-TOF) mass spectrometry. The purified trypsins were compared using the kinetic properties K(m) and k(cat) which were determined at pH 8.0 and between 25 and 55 degrees C. Comparison of the Michaelis constants for ovine and porcine trypsins toward N-alpha-benzoyl-arginine-p-nitroanilide (BapNA) indicated that ovine trypsin had higher affinity for this substrate than the porcine enzyme. The rates of the reactions catalysed by the two enzymes correlated strongly over the range of temperatures and substrate concentrations tested, as did the k(cat) values. The specific activity of ovine trypsin for BapNA was, on average, approximately 10% higher than that of the porcine enzyme over the range of conditions tested. Porcine trypsin was less susceptible to denaturation at low pH or high temperature than was ovine trypsin. Porcine and ovine trypsin produced seven identically sized fragments from auto-catalytic hydrolysis. Proposed regions of identity between ovine and porcine trypsins were I(54)-K(77), L(98)-R(107), S(134)-K(178) and N(209)-K(116). Hydrolysis of beta-lactoglobulin, egg white lysozyme or casein by ovine or porcine trypsin yielded virtually identical patterns of fragments although the rate at which fragments were produced, in the case of beta-lactoglobulin, differed between the two enzymes. On balance the two enzymes appear to be functionally identical in their action.

Animals↗
PubMed2002

Multiplexed on-column protein digestion and capillary electrophoresis for high-throughput comprehensive peptide mapping.

A novel scheme based on multiplexed capillary electrophoresis (CE) has been developed for high-throughput, low-cost and comprehensive peptide mapping. Orthogonal peptide maps of the protein of interest were obtained by using multiple reaction conditions with three different enzymes (trypsin, pepsin, and chymotrypsin), and multiple separation conditions with six zone electrophoresis buffers and two micellar electrokinetic chromatography (MEKC) buffers. Fifteen nanoliters of two protein samples (beta-lactoglobulin A and beta-lactoglobulin B) were separately mixed on-column and digested independently at 37 degrees C for 10 min to produce peptides in a 20-capillary system. The resulting peptides were detected simultaneously at 214 nm by a photodiode array detector. The overall analysis time from reaction to detection was about 40 min.

Buffers↗