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PubMed1997

Density gradient isoelectric focusing of proteins in artificial pH gradients made up of binary mixtures of amphoteric buffers.

A density gradient electrophoresis apparatus made of Perspex (7 cm, O 2.2 cm) with a circular platinum anode and a palladium cathode was used for the separation of proteins in free liquid. Following a concept developed by M. Bier et al. (Electrophoresis 1993, 14, 1011-1018), mixtures of two suitable amphoteric buffers I and II provide for media with a fixed and electrophoretically stable pH or were used for the generation of preformed (electrophoretically stable) pH gradients covering about 1 pH unit. Amphoters I and II are considered suitable if there is overlap between (pK(1,1)-1-2) and the pK(2,II)+1+2) region. 3-(N-Morpholino)propanesulfonic acid (MOPS) and gamma-amino-n-butyric acid (GABA) were used as an example. Two approaches were followed: (i) rate-zonal separation of test proteins in a pH window, formed by a fixed ratio of MOPS/GABA. (ii) Isoelectric focusing in a shallow preformed pH gradient, made up of inverse reciprocal linear gradients of MOPS and GABA. At isopH, test proteins (bovine serum albumin, cytochrome c, ferritin, hemoglobin, lactoglobulin, myoglobin, and transferrin) were rate-zonally separated within a short time. Even the separation of the A and B forms of lactoglobulin was feasible at isopH. The glycoforms of transferrin were separated and enriched on a pH 5.2-6.1 pH gradient, indicating that pH differences of about 0.01 still permit resolution. Contrary to the ill-defined Ampholines, the cost of these well-defined amphoters is low.

Buffers↗
PubMed1991

[The determination of protein hydrophobicity. 1. Determination of the hydrophobicity of selected cereal and milk proteins using their sodium dodecyl sulfate binding capacities].

The sodium dodecylsulphate (SDS) binding capacities of secalin, gliadin and gluten in the presence of a very low SDS concentration were determined and compared to the SDS binding capacities of bovine serum albumin (BSA), beta-lactoglobulin, ovalbumin und beta-casein. The SDS binding capacities of endosperm proteins determined in phosphate buffer (pH 6.0) are very low. Only 0.6 microgram .. 0.8 microgram SDS were bound to 500 micrograms of the proteins. This low SDS binding capacities do not correlate with the expected hydrophobicity of these proteins. In comparison, 500 micrograms of ovalbumin, beta-lactoglobulin and BSA each bind 0.5, 5.9 and 13.5 micrograms SDS, respectively. According to literature the SDS binding capacities of these proteins are in correlation with the surface hydrophobicity determined with cis-parinaric acid using the fluorescence probe method. The SDS binding capacities of endosperm proteins increased in the presence of 0.1 N acetic acid and consequently 6.2 micrograms .. 6.9 micrograms SDS were bound to 500 micrograms of the corresponding proteins. beta-casein described as a highly hydrophobic protein binds only 0.9 micrograms SDS to 500 micrograms of it in phosphate puffer (pH 6.0) and 1.2 micrograms SDS in 0.1 N acetic acid, respectively.

Chemical Phenomena↗
PubMed2004

Differential protein composition of bovine whey: a comparison of whey from healthy animals and from those with clinical mastitis.

During clinical mastitis in dairy cows, the quantity of milk produced decreases and the composition of the milk is altered. As the severity of inflammation associated with the disease increases, the chemical composition of milk approaches that of blood as a consequence of increased permeability of the blood mammary barrier, or de novo intramammary synthesis, as has been suggested for mammary associated serum amyloid A3. A better understanding of these events may provide new approaches for the diagnosis and treatment of mastitis. The objective of this study was to document the changes in the protein composition of milk during clinical mastitis using a proteomic approach, with the objective of identifying new diagnostic markers of mastitis. Whey from dairy cows with clinical mastitis was compared to whey from healthy animals by two-dimensional gel electrophoresis (2-DE) with colloidal Coomassie staining and matrix-assisted desorption/ionization mass spectrometry. Increases in the concentrations of proteins of blood serum origin, including serotransferrin and albumin, were identified in mastitic whey compared to normal whey, while concentrations of the major whey proteins alpha-lactalbumin and beta-lactoglobulin were reduced in mastitic whey. Mass spectrometry subsequently confirmed the location of albumin, alpha-lactalbumin and beta-lactoglobulin on the 2-DE gels at M(r)/pI of 69 294/5.8, 14 200/4.5 and 19 883/4.9 respectively.

Animals↗
PubMed1999

Quantification of glutamine in proteins and peptides using enzymatic hydrolysis and reverse-phase high-performance liquid chromatography.

An enzymatic method for hydrolyzing bovine milk proteins was developed. Purified milk proteins (alpha-lactalbumin, beta-lactoglobulin, and beta-casein) were hydrolyzed in 0.1 M Hepes buffer (pH 7.5) containing pronase E, aminopeptidase M, and prolidase at 37 degrees C for 20 h. Free glutamine and other amino acids were derivatized with phenylisothiocyanate and separated using a C18 Pico-Tag column. Amino acids were eluted from the column with an aqueous sodium acetate-acetonitrile gradient with detection at 254 nm. Glutamine recoveries from hydrolyzed alpha-lactalbumin, beta-lactoglobulin, and beta-casein were 78 +/- 4, 98 +/- 3, and 101 +/- 3% of the theoretical values, respectively. The recoveries of most amino acids were comparable with those obtained using acid hydrolysis, except for the recoveries of proline and acidic amino acids. These peptide bonds appeared to be resistant to enzymatic hydrolysis and also to inhibit the hydrolysis of adjacent amino acids. Free glutamine was found to be very stable (97% recovery) under the enzymatic hydrolysis conditions.

Amino Acids↗
PubMed1999

Orogenic Displacement of Protein from the Air/Water Interface by Competitive Adsorption.

The displacement of proteins from an air/water interface by surfactant has been visualized by atomic force microscopy (AFM) through the imaging of Langmuir-Blodgett films formed on mica. Three different proteins were studied: beta-casein, a largely random coil protein, and two globular proteins, beta-lactoglobulin and alpha-lactalbumin. The proteins were displaced from both spread and coadsorbed films using the nonionic surfactant Tween 20. The combined use of AFM with studies of surface tension and surface rheology have revealed the mechanism of protein desorption from the air/water interface. The surfactant is found to adsorb at defects in the protein network and these nucleated sites then grow, compressing the protein network. At sufficiently high surface pressures the network fails, releasing proteins that then desorb from the interface. We have called this mechanism orogenic displacement. Stress propagation through beta-casein films is homogeneous resulting in the growth of circular surfactant domains. beta-Lactoglobulin and alpha-lactalbumin form stronger networks and stress propagation is restricted resulting in the growth of irregular (fractal) surfactant domains. The AFM images also provide direct evidence for the formation of elastic (gel-like) protein networks at the air/water interface. Copyright 1999 Academic Press.

Journal Article↗
PubMed2002

Network forming properties of various proteins adsorbed at the air/water interface in relation to foam stability.

A series of proteins was studied with respect to their ability to form a network at the air/water interface and their suitability as foaming agents and foam stabilizers. Proteins were chosen with a range of structures from flexible to rigid/globular: beta-casein, beta-lactoglobulin, ovalbumin, and (soy) glycinin. Experiments were performed at neutral pH except for glycinin, which was studied at both pH 3 and pH 6.7. The adsorption process was followed with an automated drop tensiometer (ADT). Network forming properties were assessed in terms of surface dilational modulus (determined with the ADT), the critical falling film length (L(still)) and flow rate (Q(still)) below which a stagnant film exists (as measured with the overflowing cylinder technique), and the fracture stress and fracture strain measured in surface shear. It was found that glycinin (pH 3) can form an interfacial gel in a very short time, whereas beta-casein has very poor network-forming properties. Hardly any foam could be produced at the chosen conditions with glycinin (pH 6.7) and with ovalbumin, whereas beta-casein, beta-lactoglobulin, and glycinin (pH 3) were good foaming agents. It seems that adsorption and unfolding rate are most important for foam formation. Once the foam is formed, a rigid network might favor stabilizing the foam.

Adsorption↗
PubMed1991

The human placental protein 14 (PP14) gene is localized on chromosome 9q34.

PP14 protein (placental protein 14) is abundantly secreted by the human endometrium under the influence of progesterone. Human PP14 is homologous to beta-lactoglobulin, the main component of equine, bovine, and ovine milk whey. A genomic PP14 probe (PP14G1) was used for the chromosome assignment of the PP14 gene. Somatic hybrid cells enabled PP14G1 to be assigned to chromosome 9. In situ hybridization further refined this assignment to 9q34. The localization of the PP14 gene in the region of the ABO locus is consistent with the linkage described in bovines between beta-lactoglobulin and the J blood group (homologous to the human ABO group).

Animals↗
PubMed1991

Stimulation of proliferation and immunoglobulin production of human-human hybridoma by various types of caseins and their protease digests.

We have studied the effect of such milk proteins as caseins, lactalbumin, and lactoglobulin, on proliferation and immunoglobulin production of human-human hybridoma HB4C5 cells. It was found that alpha-, beta-, and kappa-caseins stimulated both proliferation and IgM product ion of human-human hybridoma HB4C5 cells, while the activities of alpha-lactalbumin and beta-lactoglobulin were negligible. To localize the active sites of these caseins, effect of protease treatments on the activities were examined. When caseins were digested with trypsin, casein digests stimulated proliferation of the hybridoma, but not their IgM production. When kappa-casein was digested with chymosin and fractionated to p-kappa-casein and glycomacropeptide, both fragments stimulated proliferation of the cells, but only p-kappa-casein fragment stimulated IgM production. These results indicate that kappa-casein has at least two proliferation stimulating sites and an IgM production stimulating site in the p-kappa-casein region.

Antibodies, Monoclonal↗
PubMed1986

Secretory antibodies in IgA-deficient and immunosuppressed individuals.

Total levels of IgM and secretory IgM as well as specific antibodies to poliovirus type I antigen, Escherichia coli O antigens, and beta-lactoglobulin were measured in unstimulated and stimulated saliva as well as nasal secretion using an enzyme-linked immunosorbent assay (ELISA). The levels of these antibodies in IgA-deficient adults with and without frequent respiratory infections and children under immunosuppressive therapy for malignant disease were compared to those in normal adults and infants 1-7 months of age. The IgA-deficient adults had significantly higher IgM levels (P less than 0.002) than the normal adults as well as higher levels of IgM antibodies to poliovirus type I (P less than 0.05) and E. coli O antigen (P less than 0.002). There was a less pronounced IgM anti-beta-lactoglobulin compensation. Secretory component (SC)-carrying antibodies against all three antigens were lower than in normal adults. The infants studied had levels of IgM in secretions close to those of the normal adults and significantly lower than those of the IgA-deficient adults (P less than 0.001) but with a higher proportion of SC-carrying IgM. The increase in total IgM and specific bacterial and viral IgM antibodies in saliva above that of the normal adults was significant (P less than 0.001-0.005) for those IgA-deficient individuals without, but not for those with, frequent infections. There was, however, no significant difference between the levels in the two groups of IgA-deficient adults.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗
PubMed1991

Production of pharmaceutical proteins in milk.

There is every reason to expect that it will be possible within the next few years to begin to use farm animals to produce large quantities of some of the human proteins that are needed for the treatment of disease. Revolutionary new opportunities for the production of novel proteins in milk have been created by the development of methods for gene transfer. Exploitation of these opportunities depends upon selection and cloning of milk protein genes and identification of the sequences that govern tissue specific hormonally induced expression in the mammary gland. Studies with three genes, ovine beta-lactoglobulin, rat beta-casein and whey acidic protein of rat and mouse, suggest that they may all meet this requirement. Fragments of the ovine beta-lactoglobulin, murine whey acidic protein and rabbit beta-casein genes have directed production of novel proteins in the milk of transgenic mice, sheep, rabbits and pigs. The proteins were biologically active and usually co-migrated with authentic proteins. In early experiments, protein concentration was low, but our recent observations suggest that fusion genes containing genomic clones direct production of concentrations of protein that are suitable for commercial exploitation. In the longer term, two approaches may offer the potential of more reliable expression. Control elements capable of directing expression that is independent of site of insertion of the gene, but dependent on the number of copies of the gene, have been identified for a small number of genes. The availability of such elements for the milk protein genes would increase the reliability of gene expression considerably. Alternatively, targeted mutation of genes may allow the insertion of coding sequences within an existing gene so avoiding position effects.

Animals↗
PubMed1995

Characterization of antigens and allergens in hypo-allergenic infant formulae.

The antigenicity and allergenicity of so-called hypo-allergenic infant formulae is mainly determined by the degree of hydrolysis and ultrafiltration. Five different formulae were investigated by means of immunoblotting and RAST in order to characterize the antigens and allergens regarding their molecular weights, molecular origin and their ability to bind human IgG and IgE antibodies: A non hydrolysed infant formula (I-F), a mixture of the major cow's milk proteins (PM), a whey-based infant formula (W-H), a whey-based and ultra-filtrated infant formula (U-H), a casein/whey-based infant formula (CW-H). By immunoblotting we demonstrated that all tested formulae still contain antigens with molecular weights from 3 to 67 kD. But when compared with I-F and PM the antigen content of the hydrolysed formulae was considerably lower. The lowest antigen content could be demonstrated in U-H, which contains casein fragments (3-6 kD) and beta-lactoglobulin and its fragments (6-18 kD). W-H and CW-H contain bovine serum albumin, beta-lactoglobulin, casein and their fragments (3-67 kD). All hydrolysed formulae tested showed a reduced IgE-binding capacity. Three out of 12 cow's milk allergic children possessed IgE binding to U-H or W-H, and 5 of them IgE against CW-H. Conclusion. The enzymatic hydrolysis plus ultra-filtration seems to be the most efficient method to reduce the antigen content of so-called hypo-allergenic infant formulae.

Allergens↗
PubMed1995

Expression of genomic and cDNA transgenes after co-integration in transgenic mice.

In general, genomic transgenes are expressed efficiently in mice, while their cDNA-based transgenes are frequently silent. Clark et al. (1992) have shown that silent cDNA transgenes under the control of the sheep beta-lactoglobulin promoter can be activated after co-injecting them with a genomic sheep beta-lactoglobulin transgene. We have tested the general utility of this concept using mouse whey acidic protein (WAP) transgenes. Here we show that WAP cDNA transgenes are virtually silent in transgenic mice. In contrast, WAP transgenes containing all introns are expressed in approximately 50% of the lines at levels ranging from 1% to more than 100% of the endogenous RNA (McKnight et al., 1992). When a WAP-genomic transgene was co-injected with a WAP-cDNA, basal activation of the cDNA was possible. However, the activity of the WAP-cDNA transgene did not exceed 1% of the WAP-genomic transgene. This suggests that a permissive integration site capable of supporting basal level transcription can be established, but further events are required for full activation of the transgene.

Animals↗
PubMed1999

Inhibition of poliovirus type 1 infection by iron-, manganese- and zinc-saturated lactoferrin.

In this study we investigated the inhibitory activity of different milk proteins on poliovirus infection in Vero cells. Proteins analyzed were mucin, alpha-lactalbumin, beta-lactoglobulin, and bovine and human lactoferrin. Viral cytopathic effect was not prevented by mucin, alpha-lactalbumin or beta-lactoglobulin, whereas the lactoferrins tested were able to inhibit the replication of poliovirus in a dose-dependent manner. Further experiments were carried out in which apo- and native lactoferrin or lactoferrin fully saturated with ferric, manganese or zinc ions were added to the cells during different phases of viral infection. Results obtained demonstrated that all lactoferrins were able to prevent viral replication when present during the entire cycle of poliovirus infection or during the viral adsorption step. Only zinc lactoferrin strongly inhibited viral infection when incubated with the cells after the viral attachment, being the inhibition directly correlated with the degree of zinc saturation. Our results demonstrated that all lactoferrins interfered with an early phase of poliovirus infection; zinc lactoferrin was the sole compound capable of inhibiting a phase of infection subsequent to virus internalization into the host cells.

Animals↗
PubMed1991

Functionalized membrane supports for covalent protein microsequence analysis.

Methods were developed for high yield covalent attachment of peptides and proteins to isothiocyanate and arylamine-derivatized poly(vinylidene difluoride) membranes for solid-phase sequence analysis. Solutions of protein or peptide were dried onto 8-mm membrane disks such that the functional groups on the surface and the polypeptide were brought into close proximity. In the case of the isothiocyanate membrane, reaction between polypeptide amino groups and the surface isothiocyanate moieties was promoted by application of aqueous N-methylmorpholine. Attachment of proteins and peptides to the arylamine surface was achieved by application of water-soluble carbodiimide in a pH 5.0 buffer. Edman degradation of covalently bound polypeptides was accomplished with initial and repetitive sequence yields ranging from 33 to 75% and 88.5 to 98.5%, respectively. The yields were independent of the sample load (20 pmol to greater than 1 nmol) for either surface. Significant loss of material was not observed when attachment residues were encountered during sequence runs. Application of bovine beta-lactoglobulin A chain, staphylococcus protein A, or the peptide melittin to the isothiocyanate membrane allowed for extended N-terminal sequence identification (35 residues from 20 pmol of beta-lactoglobulin). A number of synthetic and naturally occurring peptides were sequenced to the C-terminal residue following attachment to the arylamine surface. In one example, 10 micrograms of bovine alpha-casein was digested with staphylococcal protease V8 and the peptides were separated by reverse-phase chromatography. Peptide fractions were then directly applied to arylamine membrane disks for covalent sequence analysis. From as little as 2 pmol of initial signal it was possible to determine substantial sequence information (greater than 10 residues).

Amines↗
PubMed1984

Inhibition of pancreatic and microbial lipases by proteins.

We have compared the effect of several proteins, including melittin, beta-lactoglobulin A, serum albumin, ovalbumin and myoglobin, on the hydrolysis of tributyrin and triolein by lipases from various origins. All proteins tested inactivate pancreatic lipase in absence of colipase and bile salt. Inhibition is not significantly reversed by colipase in absence of bile salt except in systems containing tributyrin and melittin or triolein and beta-lactoglobulin A. In all other cases, activation of pancreatic lipase by colipase in presence of inhibitory protein requires the presence of bile salt. Lipase from Rhizopus delemar is also inhibited by the proteins that inactivate pancreatic lipase. In contrast, the activity of lipase from Rhizopus arrhizus is not affected by the proteins in the same concentration range. Inhibition of lipase activity by amphiphiles such as proteins or detergents appears to be a general phenomenon not directly related to a decrease in tension at the triacylglycerol-water interface. Inhibition could be the result of desorption of lipase from its substrate due to a change in interfacial quality.

Animals↗
PubMed1987

Serum antibodies to cow's milk proteins in ulcerative colitis and Crohn's disease.

Serum antibodies of immunoglobulin G, immunoglobulin M, and immunoglobulin A isotypes to five major proteins of cow's milk, casein, bovine serum albumin, alpha-lactalbumin, beta-lactoglobulin A, and beta-lactoglobulin B, were measured using enzyme-linked immunosorbent assay in 51 patients with ulcerative colitis, 49 with Crohn's disease, and 20 age-matched controls. Immunoglobulin G and immunoglobulin M antibodies to cow's milk proteins were significantly elevated in patients with inflammatory bowel disease as compared to controls. In contrast, no significant increase in immunoglobulin A antibodies to 3 of 5 proteins was noted. The increased titers of antibodies to milk proteins seem to be specific and not due to a polyclonal immunoglobulin activation, as naturally occurring blood group antibodies were not elevated in patients with ulcerative colitis and Crohn's disease. A good correlation of disease activity, as measured by serum alpha 1-acid glycoprotein concentrations, and immunoglobulin G and immunoglobulin A antibody titers against certain cow's milk proteins could be demonstrated in Crohn's disease, but not ulcerative colitis. These findings suggest that production of antibodies to cow's milk proteins reflects specific immunization with these antigens. The study of antibody isotypes and correlation with disease activity may provide better insight into the immune response to dietary antigens and its possible role in the pathogenesis of inflammatory bowel diseases.

Adult↗
PubMed1996

Perfusion liquid chromatography of whey proteins.

A perfusion reversed-phase (RP) HPLC method was developed for the rapid separation of the main bovine whey proteins: alpha-lactalbumin (alpha-LA), serum albumin (BSA) and the genetic variants of beta-lactoglobulin (A and B) (beta-LG A and beta-LG B). For the method development, the influence of factors favouring structural changes of proteins (temperature and organic acid concentration in the mobile phase), gradient and other chromatographic conditions and the mass of protein injected was examined. The optimized method allowed the separation of proteins in about 1.5 min (cycle time 3.5 min) with resolution around 1.0 for the beta-lactoglobulins. The method was applied to the determination of proteins in a whey from raw bovine milk. The precision of the determinations was < or = 3.75 mg per 100 ml (S.D.). With respect to the accuracy, errors < or = 7.0% in the determination of alpha-LA, beta-LG A and beta-LG B were obtained, compared with an RP-HPLC reference method. However, higher errors in the quantification of BSA were found owing to the lack of purity of the peak assigned. In addition, the proposed method has proved to be very useful in the detection of homologous whey proteins from different species (cow, sheep and goat) in milk mixtures.

Animals↗
PubMed1992

IgG subclass antibodies to dietary antigens in IgA deficiency quantification and correlation with serum IgG subclass levels.

IgG subclasses of antibodies to the dietary antigens ovalbumin, beta-lactoglobulin, casein, bovine IgM, and glycgli (a gluten component) were quantified in 20 adults and 10 children with IgA deficiency and healthy controls (21 adults and 7 children). In the IgA-deficient subjects the levels of IgG subclasses in serum were determined. Detectable antibody levels were observed in the majority of the subjects in IgG1 and IgG4 for anti-ovalbumin and beta-lactoglobulin, and in IgG1, followed by IgG2, IgG3, and IgG4 for antibodies to casein, bovine IgM, and glycgli. Levels of IgG1 and IgG2 antibodies to bovine IgM were higher in the IgA-deficient adults than in controls (P less than 0.00005, P = 0.0007, respectively), whereas the other antibody levels did not differ significantly between the two groups. An analysis of correlation between the IgG subclass antibody levels did not provide evidence for a particular IgG subclass antibody response pattern against different protein antigens within the single individual. Serum IgG4 levels correlated positively with the summed IgG4 antibody levels (Spearman's p = 0.673, P = 0.0051). The IgA-deficient subjects, when compared with healthy controls, did not show a particular IgG subclass pattern or restriction of antibodies to dietary antigens.

Adolescent↗