Ovalbumin Molecules Research Articles

Protein-protein and protein-salt interactions in aqueous protein solutions containing concentrated electrolytes

Protein-protein and protein-salt interactions have been obtained for ovalbumin in solutions of ammonium sulfate and for lysozyme in solutions of ammonium sulfate, sodium chloride, potassium isothiocyanate, and potassium chloride. The two-body interactions between ovalbumin molecules in concentrated ammonium-sulfate solutions can be described by the DLVO potentials plus a potential that accounts for the decrease in free volume available to the protein due to the presence of the salt ions. The interaction between ovalbumin and ammonium sulfate is unfavorable, reflecting the kosmotropic nature of sulfate anions. Lysozyme-lysozyme interactions cannot be described by the above potentials because anion binding to lysozyme alters these interactions. Lysozyme-isothiocyanate complexes are strongly attractive due to electrostatic interactions resulting from bridging by the isothiocyanate ion. Lysozyme-lysozyme interactions in sulfate solutions are more repulsive than expected, possibly resulting from a larger excluded volume of a lysozyme-sulfate bound complex or perhaps, hydration forces between the lysozyme-sulfate complexes.

Impact of structural changes and aggregation on adsorption kinetics of ovalbumin at the water/air interface

Differential Scanning Calorimetry (DSC) was used to study the thermal stability of ovalbumin molecules at concentrations lying between 0.25% and 5% and at pH 3 and pH 7: critical temperatures such as temperature of the first increase in heat flow ( T i), peak temperature ( T p) and enthalpy change of heat reactions (Δ H app) were determined. The results indicated that T p did not vary significantly whatever the protein concentration, whereas the heat stability of protein conformational states was higher at pH 7 than at pH 3. These results were confirmed through the secondary structure changes upon heating, as monitored by circular dichroism measurements: at pH 3, we observed the formation of molecular species probably associated by intermolecular β-sheets at T⩽ T i. Electrophoretic patterns of preheated solutions indicated the formation of aggregates at T< T i (pH 3) and at T< T p (pH 7). The ability of the predenatured molecules to adsorb onto the air/water interface, as measured by the Wilhelmy balance, was shown to be closely related to their initial conformational states and aggregation properties before absorbing.

Induction of an antigen-specific, CD1-restricted cytotoxic T lymphocyte response In vivo.

The majority of T cell responses are restricted to peptide antigens bound by polymorphic major histocompatibility complex (MHC) molecules. However, peptide antigens can be presented to T cells by murine non-MHC-encoded CD1d (mCD1) molecules, and human T cell lines specific for nonpeptide antigens presented on CD1 isoforms have been identified. It is shown here that antigen-specific, mCD1-restricted lymphocytes can be generated in vivo by immunizing mice with a combination of plasmids encoding chicken ovalbumin, murine CD1d, and costimulatory molecules. Splenocytes from immunized mice have CD1d-restricted, MHC- unrestricted, ovalbumin-specific cytolytic activity that can be inhibited by anti-CD1 antibodies as well as a competing CD1-binding peptide. These results suggest a physiologic role for murine CD1d to present exogenous protein antigens.

Protein-protein and protein-salt interactions in aqueous protein solutions containing concentrated electrolytes.

Protein-protein and protein-salt interactions have been obtained for ovalbumin in solutions of ammonium sulfate and for lysozyme in solutions of ammonium sulfate, sodium chloride, potassium isothiocyanate, and potassium chloride. The two-body interactions between ovalbumin molecules in concentrated ammonium-sulfate solutions can be described by the DLVO potentials plus a potential that accounts for the decrease in free volume available to the protein due to the presence of the salt ions. The interaction between ovalbumin and ammonium sulfate is unfavorable, reflecting the kosmotropic nature of sulfate anions. Lysozyme-lysozyme interactions cannot be described by the above potentials because anion binding to lysozyme alters these interactions. Lysozyme-isothiocyanate complexes are strongly attractive due to electrostatic interactions resulting from bridging by the isothiocyanate ion. Lysozyme-lysozyme interactions in sulfate solutions are more repulsive than expected, possibly resulting from a larger excluded volume of a lysozyme-sulfate bound complex or perhaps, hydration forces between the lysozyme-sulfate complexes.

Highly ordered molten globule-like state of ovalbumin at acidic pH: native-like fragmentation by protease and selective modification of Cys367 with dithiodipyridine.

Structural characteristics of ovalbumin at acidic pH were investigated by a variety of analytical approaches. At pH 2.2, the protein appeared to assume a partially denatured, molten globule-like conformation as evaluated by the binding of a hydrophobic probe, anilino-1-naphthalene-8-sulfonate. The protein was, however, resistant to proteolysis with pepsin under conditions in which the urea-denatured form was extensively hydrolyzed. Furthermore, under more drastic proteolytic conditions, the acid ovalbumin was specifically proteolyzed at the N-terminal site of Ala351, which is located in close proximity to the canonical serpin cleavage site Ala352-Ser353 that is known to be the cleavage site at neutral pH with subtilisin and elastase in native ovalbumin. Among the four cysteine residues (Cys11, Cys30, Cys367, and Cys382), which are all known to be buried in the native ovalbumin molecule, only Cys367 was specifically modified with 2,2'-dithiodipyridine, generating a mixed-disulfide protein derivative. Upon incubation of the derivative with a high concentration of L-cysteine, the thiopyridine mixed disulfide did not undergo any bimolecular exchange reaction with the thiol in the absence of an added denaturant, indicating that the mixed disulfide group is inaccessible. The far-UV CD spectra indicated that the native secondary structure is retained in either the modified or non-modified protein; but as evaluated by the near-UV CD spectra, the asymmetric nature of aromatic side chains in the non-modified ovalbumin and of the mixed-disulfide group in the modified protein was almost lost at pH 2.2. These results are consistent with a highly ordered molten globule-like state for OVA at pH 2.2, in which side chains, but not the backbone chain, significantly fluctuate.

Refolding of urea-denatured ovalbumin that comprises non-native disulfide isomers.

Ovalbumin, which contains one cystine disulfide (Cys73-Cys120) and four cysteine sulfhydryls (Cys11, Cys30, Cys367, and Cys382) in the native state, undergoes intrachain sulfhydryl-disulfide exchanges at high concentrations of urea, generating many non-native disulfide isomers [E. Tatsumi, N. Takahashi, and M. Hirose (1994) J. Biol. Chem. 269, 28062-28067]. The refolding of ovalbumin from the urea-denatured state was investigated. When the denatured protein was diluted 20-fold with a refolding buffer (pH 8.2), an initial burst intermediate I(N) was produced within the 20 ms instrumental dead time; I(N) showed about 60% of the native CD ellipticity at 222 nm. The intrinsic tryptophan fluorescence of I(N) showed the same peak (338 nm), but with decreased intensity (57%), as compared to the native protein. After the rapid formation of I(N), most of the ovalbumin molecules correctly refolded into the native state with slow biphasic kinetics, as evaluated by far-UV CD, tryptophan fluorescence, and trypsin-resistance analyses. Furthermore, a peptide-mapping analysis revealed that sulfhydryl/disulfide exchange reactions occurred during the refolding, thereby increasing the formation of the native disulfide. The integrity of overall refolding was confirmed by a differential scanning calorimetry analysis. These data were consistent with the view that most, if not all, of the mispaired disulfide isomers in the urea-denatured ovalbumin can correctly refold into the native state via intrachain disulfide rearrangements.

Specific T cell lines for ovalbumin, ovomucoid, lysozyme and two OA synthetic epitopes, generated from egg allergic patients' PBMC

Proteins of hen egg whites are common ingredients in food and difficult to eliminate. Allergens of egg white induce allergic symptoms among relatively high numbers of patients suffering from food allergy. B cell epitopes to hen egg white major allergens have been reported. Considering that IgE antibody formation is mostly T cell dependent, the study of T cell epitopes is essential for both T cell dependent and independent IgE response. Little information on T cell epitopes recognizing food allergens has been reported. T cell responses to hen egg white allergens and two synthetic OA peptides located at amino acid residues No. 105-122 and 323-339 were investigated. Peripheral blood mononuclear cells from hen egg allergic patients were investigated. Various allergens of hen egg white were used for stimulation. Primary proliferation responses were detected followed by the generation of long-term cultures which were examined for their specificity, phenotype, cytokine profile and IgE production. The allergen specific T cell lines were mapped using a panel of 13 synthetic peptides of ovalbumin. Human T cells recognizing ovomucoid, lysozyme and ovalbumin epitope 105-122 are reported for the first time. The cell lines were enriched CD4+/CD8+ T cells (CD2+ > 95%). Ovomucoid and ovalbumin induced IgE synthesis by a small fraction of B cells (1%) present in the ovalbumin and ovomucoid specific T cell lines. Human T cells recognized several egg white allergens and epitopes within the ovalbumin molecule. Specific IgE was produced in cultures stimulated with ovalbumin and ovomucoid. OA peptides 105-122 and 323-339 have no affinity to the specific IgE of the two patients; an observation which could be of particular interest regarding the mechanisms of peptide-based immunotherapy.

Viscoelastic characterization of solid-like structure in aqueous colloids of globular proteins

The relationship between viscoelastic properties and the colloidal structure of aqueous colloids of globular proteins, such as ovalbumin (OA), bovine serum albumin (BSA), and rat serum albumin (RSA), has been studied. The OA colloidal systems show a remarkable yield stress σy and dynamic modulus G′, due to a solid-like structure, even in very dilute systems of 0.03 wt.%. The yield stress and rigidity increase sharply with gelation due to the heat denaturation of the OA molecule. They increase with increasing ionic concentration and then decrease, passing through a maximum. The serum albumin systems also have a certain solid-like structure and the values of σy and G′, which are approximately equal to 0.1 Pa for the BSA and 0.08 Pa for the RSA system, remain almost constant over a wide concentration range from about 0.01–10 wt.%. Although this rheological behavior is very characteristic, it seems to be a common property for the colloidal systems of the various globular proteins. The solid-like structure can be strongly related to the electrostatic interaction between the dispersing particles and the size of the particles.

Characteristics of Ovalubumin Gel Layer Formed on Ceramic Microfiltration Membranes.

Ovalbumin solution, which is known to form a gel layer on membrane surfaces, was filtrated in a cross-flow mode using ceramic microfiltration membranes. The influences of pH and ionic strength of the ovalbumin solution on the properties of the gel layer and membrane permeability were studied.At a pH near the isoelectric point of the ovalbumin solution, both the permeate flux and ovalbumin rejection showed the lowest value. The cohesion force acting among ovalbumin molecules became strongest because the electrostatic repulsion force among the molecules was weakened, and then the gel layer was tightly adsorbed onto the membrane surface. Therefore, the membrane permeate flux obtained at the pH of the isoelectric point was smaller than that obtained at the other pH. The ovalbumin rejection increased at the other pH, except for the isoelectric point, because of the electrostatic repulsion of the ovalbumin gel layer formed on the membrane surface. The influence of electric charge was also recognized in the case of the filtration of sodium L-glutamate solution using the ovalbumin gel layer.When the ovalbumin solution was filtered with a high ionic strength, the permeate flux and the ovalbumin rejection were almost constant, independent of the solution pH.The molecular weight cut-off values of the ovalbumin gel layer were about 200,000–300,000 as self-rejecting dynamic membranes.The specific resistance of the ovalbumin gel layer obtained in dead-end filtration increased in pH near the isoelectric point and with increasing ionic strength of the feed solution. This tendency of the specific resistances was in good agreement with the behavior of the permeate flux of the ovalbumin solution.

FACTORS AFFECTING THE VISCOELASTIC PROPERTIES OF ALBUMEN FOAMS1

ABSTRACTThe stability of foams prepared from egg albumen could be characterized by the changes in viscoelastic parameters obtained from measurements of stress relaxation and creep in compression. The initial relaxation modulus and instantaneous elasticity increased with time for about 40 min after the preparation of the foams because of the slow rate of adsorption of ovalbumin molecules to the air/water interface of the liquid film. These moduli then decreased with aging due to the thinning and rupture of the liquid films in the foam. The rate of decrease of the moduli was accelerated by reducing the concentration of sugar or by lowering pH in the aqueous phase of the liquid films. The presence of a certain amount of sugar seems to control the drainage rate of water from the liquid films, while the electrostatic repulsion between the inside surfaces of the liquid films may decrease in the acid region according to the reducing charged potential at the inside surfaces adsorbed ovalbumin molecules.

Behavior of the Idiotypic Network in Conventional Immune Responses: III. Detection and Enumeration of Cells Producing Idiotypic and Anti-Idiotypic Antibodies by a Spot ELISA Technique

The idiotypic (Ab1) and anti-idiotypic (Ab2) responses of spleen cells of mice immunized with DNP-Ficoll were assayed with the spot ELISA technique in wells coated with DNP-BSA or with affinity-purified rabbit anti-DNP antibodies, respectively. In agreement with results previously obtained with the hemolytic plaque technique, large numbers of Ab1 and Ab2 ELISA spots were found. The cytokinetics of the Ab1 and Ab2 responses were very similar and the peak responses occurred simultaneously. The isotypes of Abs1 and Abs2 were also similar. Both responses were specifically inhibited by soluble DNP-lysine. Similar results were obtained with spleen cells from mice immunized with the T-dependent antigen DNP-KLH and with the T-independent antigen fluorescein-Ficoll. In contrast, no Ab2 response was detected when spleen cells from mice immunized with ovalbumin were assayed, probably because the affinity-purified rabbit anti-ovalbumin antibodies used to coal the wells for the Ab2 assay contained molecules of several specificities, corresponding to the several epitopes of the ovalbumin molecule. The many similarities between the Ab1 and Ab2 responses to the haptens suggest that they reflected two different reactivities of the same antibody molecules.

Vaccination with immunodominant peptides encapsulated in Quil A-containing liposomes induces peptide-specific primary CD8+ cytotoxic T cells

Immunostimulating complexes (ISCOMs), containing lipids, the saponin Quil A, and proteinaceous antigens, have been proven to vaccinate effectively CD8+ cytolytic T cells in vivo. However, conventional ISCOM technology is restricted to hydrophobic proteins or fatty acid-derivatized proteins or peptides. We therefore analysed whether Quil A-containing liposomes are an effective vehicle to shuttle hydrophilic proteins or peptides into the MHC class I pathway of antigen presentation resulting in the in vivo induction of antigen-specific cytolytic T cells (CTL). Liposomes were formed by a lipid dry-down method followed by resuspension with an aqueous solution containing protein/peptide and Quil A and then an extrusion step. Quil A-containing liposomes are an effective means to elicit a CD8+ CTL response to peptide antigen in vivo. CTL could be raised in C57B1/6 mice against ovalbumin (OVA) peptide 257–264 and vesicular stomatitis virus nucleoprotein 52–59, as well as in Balb/c mice against listeriolysin peptide 91–99 and cytomegalovirus pp89 168–176, demonstrating the versatility of this approach. The elicited response was peptide-specific, peptide dose-dependent and Quil A was necessary. Vaccination with liposomes entrapping the whole ovalbumin molecule or an extended (OVA) peptide 254–276 also yielded a CTL responsive to the immunodominant OVA peptide 256–264, implying cellular internalization and correct processing. Thus Quil A-containing liposomes appear to be a versatile vehicle to vaccinate CD8+ T cells in vivo; in addition, they could rapidly enhance the understanding of subunit vaccines and rules of antigen processing and peptide-MHC class 1 binding.

Fusion proteins with heterologous T helper epitopes. Recombinant E. coli heat-stable enterotoxin proteins.

Fusion proteins containing specific B cell and T cell epitopes were used to examine how the intramolecular arrangement of T and B cell epitopes within a chimeric protein influences antigen-specific B cell antibody responses as well as specific T cell activation. Chimeric proteins, containing single or multiple copies of the Th epitope ovalbumin 323-339 (ova) linked at different positions to STa, the heat-stable enterotoxin of E. coli, were compared with respect to their ability to induce STa-specific antibody production and to induce ova-specific T cell activation. Chimeric proteins induced ova-dependent antibody production against STa at the amino terminal end, irrespective of the positioning of ova. Multiple tandem copies of ova in any position led to increased levels of antibody production against this epitope. In contrast, T cell help for antibody production against a second B cell epitope at the carboxy terminus of the fusion proteins was more effective after insertion of multiple copies of ova in a distal than in an adjacent position. A fusion protein, containing four copies of ova effectively elicited T cell help for antibody production against both examined B cell determinants, showing that activated Th cells recognizing a single epitope could simultaneously provide help for distinct sets of B cells specific for widely separated epitopes within a protein. T cell recognition of ova in all chimeric peptides, independently of its position, following the same pattern of genetic restriction (i.e. immunodominant in H-2d and nonimmunogenic in H-2k) as in the native ovalbumin molecule.(ABSTRACT TRUNCATED AT 250 WORDS)

Association State, Overall Structure, and Surface Roughness of Native Ovalbumin Molecules in Aqueous Solutions at Various Ionic Concentrations

The shape, size, and surface roughness of a globular molecule of ovalbumin (OA) were studied with small angle X-ray scattering over a wide concentration range from 0 to 300 m M of phosphate buffer solution. The Guinier approximation can be sufficiently applied over a relatively wide range of the small scattering angle. The radius of gyration of the native OA molecule can be obtained as ca. 27 Å and it is almost independent of the concentration of the buffer solution. The native OA molecule is spherical and the radius of the molecule is ca. 25 Å over the concentration range from 0 to 300 m M of buffer solution. The specific inner surface, O s, of the native OA molecule, which is obtained at low and high OA concentrations at various buffer concentrations, is approximately 1.8 times that of a sphere with a smooth surface. The value of O s is almost independent of the concentration of the buffer solution. The surface roughness of the OA molecule is also discussed in comparison with that of the micelles and vesicle.

Application of the Yukawa potential to the auto-controlled mechanism of the ovalbumin molecule in aqueous systems

The rigidity of ovalbumin (OA) aqueous colloids remains almost constant over a wide range of OA concentration c from ≈0.1 to ≈17 wt%; this is called the auto-controlled mechanism. The Yukawa potential was applied to explain the auto-controlled mechanism and revealed that the effective charge Z * of the OA molecule is relatively small. Further, The Z * value is almost constant at extremely low concentrations and is proportional to c − 1 4 in the concentration range where the rigidity remains approximately constant.

Auto‐controlled mechanism for rheological properties of native ovalbumin aqueous colloid (abstract)

Native ovalbumin (OA) molecules in aqueous colloids are more stable in an ordered arrangement state than in a random dispersion state. Rate of the stabilization from the random dispersion state to the ordered arrangement state is extremely accelerated by shear flow. Isotropic colloids of native OA molecules in buffer solutions show a remarkable yield stress σy and a rigidity G even in very dilute systems such as 0.027 wt. %. The values of σy and G remain almost constant over a wide concentration range from 0.1% to 17%. This suggests that the systems are spontaneously controlled (auto‐controlled) in order to suppress the increase in σy and G with increasing concentration of OA. This auto‐controlled mechanism disappears completely with gelation by heat denaturation.

Elastic Attributes of Heated Egg Protein Gels

ABSTRACTThe equilibrium stress of egg white (EW) and ovalbumin (OV) gels using the statistical theory of rubber elasticity suggested that 3.9 covalent disulfide cross‐links per OV molecule were formed on gelling. DTNB and PCMB reacted with approximately 4 sulfhydryls of OV in 2% SDS and/or at 82°C. Moreover, the equivalent of 3 secondary bonds, calculated from the initial stress and presumably a composite of numerous weak secondary bonds, contributed significantly to the protein gel stress.

Effect of pH on colloidal properties of native ovalbumin aqueous systems

The effect of pH on the molecular shape and dispersed state of native ovalbumin molecules in 20 mM phosphate and acetic acid buffer solutions has been studied using small-angle x-ray scattering (SAXS) and a rheological method The degree of association of the OA molecule at the 0.5% colloid system increases slightly with decreasing pH, i.e., 2.10 at pH 7.0, and 2.88 at pH 4.0, and the radius of the OA molecule decreases slightly with decreasing pH, i.e., 24.5 A at pH 7.0, and 22.0 A at pH 4.0. The OA colloid shows apparent yield stress and rigidity which are due to a certain ordered arrangement of the molecules. The yield stress and the rigidity increase abruptly at a pH value near to an isoelectric point (ca. pH 4.4). In the dilute system this increment is attributed to the change in the ordered arrangement or in the interparticle interaction, and not to the change in the association state of the OA molecules. The values of the yield stress and the rigidity remain almost constant over a wide concentration range and this feature (an auto-controlled mechanism) is kept over a certain range of pH.

Thermally Induced Epitope Regions of Ovalbumin Identified with Monoclonal Antibodies

ABSTRACTTo identify the structural regions of the ovalbumin molecule that change during heat denaturation, we used monoclonal antibodies that reacted specifically with the soluble heat‐denatured ovalbumin as probes. Five monoclonal antibodies recognizing different epitopes of the denatured ovalbumin were produced. Through the isolation and sequence analysis of the peptides derived from ovalbumin that reacted with the monoclonal antibodies, five parts of the ovalbumin molecule were identified as the regions that change structurally and provide new epitopes during heat denaturation. These findings could be useful information in understanding the structural properties of heat‐denatured ovalbumin.

Requirement of certain epitope specificities of glycosylation inhibiting factor for the suppression of in vivo IgE and IgG antibody responses.

The ovalbumin (OVA)-specific T cell hybridoma 71B1, which constitutively secretes glycosylation inhibiting factor (GIF) and is specific for the immunogenic epitope represented by amino acids 323-339 in the OVA molecules, failed to form GIF having affinity for nominal antigen upon stimulation with OVA-pulsed antigen-presenting cells (APC). However, the GIF produced by the antigen-stimulated 71B1 cells bound to the mAb 14-12, which is specific for the antigen-binding chain of effector type suppressor T cell factor (TseF), and to mAb specific for TCR. The GIF constitutively released from unstimulated 71B1 cells failed to bind to any of these antibodies. Gel filtration of GIF preparations showed that the 14-12+ GIF from the antigen-stimulated 71B1 cells are composed of 80-100 and 25-35 kDa species, while the GIF from unstimulated cells was 12-15 kDa. Reduction and alkylation treatment of the GIF from the antigen-stimulated cells resulted in the disappearance of the 80-100 and 25-35 kDa GIF, which was accompanied by the formation of the 12-15 kDa GIF. Thus, the GIF from the antigen-stimulated 71B1 cells was similar to the previously described OVA-binding GIF from the 231F1 cells with respect to their antigenic structures and molecular size, and both factors appear to be composed of the 14-12+ polypeptide chain and 12-15 kDa non-specific GIF. However, the GIF from the antigen-stimulated 71B1 cells lacked affinity for the native OVA or synthetic peptide 323-339, and failed to suppress the in vivo antibody response to dinitrophenyl (DNP)-OVA. In contrast, the OVA-binding GIF has affinity for native OVA and the peptide 307-317, to which the cell source of the factor is specific, and suppressed the in vivo anti-hapten antibody response to DNP-OVA. The results suggest that formation of antigen-specific TsF is confined to T cells with certain epitope specificities. It was also found that the OVA-binding GIF failed to suppress the in vivo anti-hapten antibody response to DNP-conjugates of urea-denatured OVA (UD-OVA), which does not bind OVA-binding GIF. However, APC pulsed with UD-OVA appear to express the epitope 307-317 for which the OVA-binding GIF has affinity. The results collectively suggest that the affinity of GIF for an immunizing antigen, rather than processed antigen, is required for immunosuppression.