Hen Egg Albumin Research Articles

The effect of sodium dodecyl sulfate and Pluronic F127 on the electrophoretic separation of protein and polypeptide test mixtures at acid pH.

Using a test mixture consisting of standard proteins (cytochrome c, chymotrypsinogen A, hen egg albumin, bovine serum albumin, aldolase, catalase and ferritin) and synthetic polypeptides (polylysine, polyaspartic, polyglutamic acid and polyproline) it was revealed that using sodium dodecyl sulfate (SDS) as background electrolyte modifier at acid pH (2.5) allows selective separation of highly positively charged polypeptides (polylysine) provided that their relative molecular mass is sufficiently low (3300 Da). The altered elution sequence of standard proteins as compared to a separation done without SDS may help their identification. Addition of Pluronic F127 offers clear-cut separations of standard proteins up to a relative molecular mass of 5 x 10(4) Da and allows to reveal protein/polypeptide microheterogeneity where applicable. None of the systems tested is suitable for the separation of acidic polypeptides and polyproline.

Application of a modified version of Habeeb's trinitrophenylation method for the characterization of hapten–protein conjugates in a reversed micellar medium

We describe here a protocol for determining the epitope density of hapten–carrier conjugates at the nanomolar level. Conjugates of bovine serum albumin (BSA) and hen egg albumin (OVA) were prepared with two model haptens: 4-acetyl benzoic acid (ABA) as a chromophoric carboxylic hapten and cholesterol hemisuccinate (Chol HS) as a carboxylic derivative of a nonchromophoric hydroxylated hapten. Small-scale but valuable haptenization of carriers was achieved (≈4.3 nmol with an input molar ratio of hapten to carrier within the range from 50:1 to 100:1) that proved yet compatible with immunogenicity and antibody detection. We used a modified version of the amide bond-generating mixed anhydride method of Erlanger performed in a reversed micellar medium. Microsample aliquots of the coupling reaction (carriers plus activated haptens) or control experiment (carriers plus nonactivated haptens) mixtures were directly subjected to trinitrophenylation in the reversed micellar medium. The results for carrier substitution were strongly correlated ( r 2=0.94; n=12) with those obtained by other methods such as UV analysis (for ABA) and chromatographic determination (for Chol HS). This method was found directly applicable to other hapten–carrier conjugates coupled by the same procedure, provided that the haptens do not absorb at about 335 and 420 nm (absorption peaks of trinitrophenyl groups). With this simple, rapid, reproducible and low-cost analysis method, the separation of uncoupled hapten and conjugate is unnecessary. This facilitates the optimization of reaction conditions. Finally, using this procedure, kinetic studies of conjugate formation can be carried out in a very simple manner.

Isolation of Lysozyme from Hen Egg Albumen by Alcohol‐Insoluble Cross‐Linked Pea Pod Solid Ion‐Exchange Chromatography

ABSTRACT: Albumen from hen eggs was diluted 3‐fold with 0.05 M NaCl solution at pH 4.0 and was further treated with 30% ethanol for 8 h. The supernatant (77900 U/mg protein) thus obtained was further diluted (2.5‐fold) with distilled water and its pH value was adjusted to 8.0 before being subjected to alcohol‐insoluble cross‐linked pea pod solid (AICLPPS) ion‐exchange chromatography for lysozyme isolation. Results showed that AI‐CLPPS ion‐exchange chromatography increased the purification to 68‐fold with a 72% lysozyme recovery from the starting albumen.

Matrix proteins from insect pliable cuticles: are they flexible and easily deformed?

Proteins from pliable cuticle of locusts, Schistocerca gregaria, and silk moth larvae, Hyalophora cecropia, were studied in solution by means of a fluorescent probe, 8-anilinonaphthalene-1-sulphonic acid (ANS), which is much more fluorescent in non-polar media than in polar media. An intense ANS-fluorescence was observed in the presence of the cuticular proteins at pH-values close to their acidic isoelectric points, and the fluorescence decreased markedly when pH was increased to neutrality or when small amounts of denaturants were added. Aggregation and eventual precipitation of both H. cecropia and locust proteins were obtained by addition of neutral salts, and the aggregation was accompanied by an increased ANS-fluorescence intensity. A decreased ANS-fluorescence was observed at salt concentrations too low to cause visible aggregation of the H. cecropia proteins, probably due to weakened electrostatic interactions between chain segments, but such a decrease was not observed for the locust proteins. The changes in intensity of ANS-fluorescence induced by addition of small amounts of denaturants or salts to solutions of the proteins indicate that more hydrophobic residues are exposed to the solvent, when either hydrophobic interactions or electrostatic attractions between chain segments are weakened. The result is a less compact protein structure, where fewer and smaller hydrophobic clusters are available for protecting ANS-molecules from the quenching effects of water. The effects of denaturants on ANS-fluorescence in the presence of the cuticular proteins are different from those observed for globular proteins, such as hen egg albumen, and the differences can be explained by the suggestion that the cuticular proteins do not have a precisely folded and densely packed hydrophobic core comparable to that present in native globular proteins, and that accordingly they do not undergo a process of denaturation corresponding to that of globular proteins. The behaviour of the cuticular proteins resembles that decribed for unordered, randomly coiled, thermally agitated polymer chains, whose hydrodynamic volumes depend upon the composition of the medium. It is proposed that the major part of the peptide chains of the cuticular proteins are in an unordered, random structure both when the proteins are in solution and when present in the intact cuticle; probably only the chain regions involved in binding the proteins to chitin will have a well-defined spatial organisation.

Novel polyethylene glycol/levan aqueous two-phase system for protein partitioning

A novel aqueous two-phase system was made by mixing PEG (polyethylene glycol) and levan. The partitioning experiments were performed with six model proteins: horse heart cytochrome c, horse hemoglobin, horse heart myoglobin, hen egg albumin, bovine serum albumin and hen egg lysozyme. The addition of NaCI increased the partition coefficient of lysozyme but decreased that of albumin. The partition coefficients were sensitive to pH, increasing in the pH range of 6 to 8 for five of the proteins tested and decreasing slightly for lysozyme.

Effect of food restriction on life span and immune functions in long-lived Fischer-344 x Brown Norway F1 rats.

Life-long food restriction is known to slow aging and reduce the rate of occurrence of age-associated disease processes, but the mechanism by which this is accomplished is unknown. In this study we have examined the effect of food restriction on the proliferative response of spleen cells to mitogens and lymphokine production in 6-, 18-, and 30-month-old AL and FR Fischer-344 x Brown Norway (F-344 x BNF1) female rats whose average life span is 137 weeks on an ad libitum (AL) diet and 177 weeks on a food-restricted (FR) diet. In addition, the ability of food restriction to recall antigens was tested in 10-month-old rats by immunizing them with keyhole limpet and hen's egg albumin and measuring proliferative response of draining lymph node cells to these antigens. Our results indicated that the spleen-cell proliferative response to phytohemagglutinin and concanavalin A (Con A) was equal in 6- and 18-month-old rats but declined significantly in 30-month-old AL rats compared to FR rats. Although flow cytometric analyses did not reveal differences for CD4, CD8, and Ig+ cells with age, a significant rise in memory T cells (Ox-22low) in both CD4+ and CD8+ T-cell subset lineage was noted in AL-fed rats at 30 months of age. In FR rats, however, only a minimal shift of naive T cells (Ox-22high) to memory cells was observed. In FR rats, the observed changes in the naive and memory T-cell subsets correlate well with the observed higher levels of the antiinflammatory interleukin-2 (IL-2) and lower levels of the proinflammatory cytokines such as IL-6 and tumor necrosis factor-alpha. The ability of food-restricted animals to recall antigens was lower compared to their age-matched controls, though the proliferative response to T-cell mitogen Con A and superantigen staphylococcal enterotoxin B was higher. These findings indicate that food restriction may selectively act to maintain a lower number of antigen-induced memory T cells with age, thereby maintaining the organism's ability to produce higher levels of IL-2 with age. In summary, the increased cell-mediated immune function noted in aged FR rats appears to be due to the presence of a higher number of naive T cells, which are known to produce elevated levels of the antiinflammatory cytokines, which may in part be responsible for reducing the observed age-related rise in disease.

Toxicologic evaluation of a high-selenium hay diet in captive pronghorn antelope (Antilocapra americana).

Five captive-raised pronghorn antelope (Antilocapra americana) were fed an alfalfa-grass hay diet containing 15 ppm total dietary selenium (Se) for 164 days. Four additional captive-raised pronghorns fed a similar diet containing approximately 0.3 ppm total dietary Se served as controls. None of the pronghorns had clinical signs attributable to the high Se hay. Plasma Se increased more rapidly than blood Se concentrations, from baseline concentrations (< 0.15 g/ml) to > 0.40 g/ml within the first 50 days on the high selenium diet, but thereafter declined to approximately 0.30 microgram/ml. Mean primary antibody response to hen egg albumin was less in pronghorn on Se hay. No significant gross or histological lesions attributable to selenosis were found, nor was there any evidence of dystrophic hoof growth. The greatest Se tissue concentrations were found in liver and kidney (5.67 to 10.4 micrograms/g and 2.36 to 3.14 micrograms/g, respectively) from experimental animals; liver and kidney from the controls contained considerably less (< or = 0.52 microgram/g and < or = 0.61 microgram/g, respectively). Exposure of pronghorns for more than 5 mo to a diet containing 15 ppm Se caused significant increases in plasma, liver and kidney Se concentrations, in the absence of clinical disease or pathologic lesions due to selenosis. Based on these results, we propose that pronghorns are less susceptible to selenosis than previously reported and that diagnostic criteria for the disease should be modified.

Structure/function studies on QS-21, a unique immunological adjuvant from Quillaja saponaria.

QS-21 is an acylated triterpene glycoside isolated from the bark of the South American tree Quillaja saponaria Molina’. It is a potent immunological adjuvant. It has been shown to enhance antigen-specific antibody titers to a wide variety of T-dependent and T-independent antigens, including hen egg albumin (ovalbumin)2, recombinant Borrelia burgdorferi OspA and OspB3, recombinant human cytomegalovirus envelope protein gB4, and E. coli 018 polysaccharides’. Further, QS-21-adjuvanted subunit antigen vaccines stimulate a cytotoxic T lymphocyte (CTL) response6,7,8. This response is characterized by the induction of CD8+ T cells that kill target cells expressing specific antigen. Recently, QS-21 was shown to enhance antigen-specific IgM and IgG responses in a Phase I clinical study of a melanoma immunotherapeutic vaccine9.

Selective pre-enrichment of salmonella from eggs by siderophore supplements

Salmonella can overcome the iron limitation of iron-limited nutrient media and albumen by utilizing the siderophores ferrioxamine E and G und grow. 14 out of 18 food-poisoning Salmonella strains of different serotypes stored in hen's egg albumen (approx. 2 organisms/ml) could be detected after 6 h of pre-enrichment with buffered peptone water supplemented with ferrioxamine G (1 microgram/ml) on selective nutrient media. In contrast, only 4 of these Salmonella strains could be reisolated without supplementation. E. coli strains cannot be supplied with iron by ferrioxamines E and G. Therefore, Salmonella enteritidis phage type 4/6 could be isolated in great numbers from mixed cultures with E. coli kept in albumen. Buffered peptone water supplemented with ferrioxamine G also permitted a successful reisolation of Salmonella enteritidis from artificially contaminated eggs. Ferrioxamines E and G function as selective growth factors to enhance sensitivity in the isolation of Salmonella in other nutrient media, too.

A comparison of commercially available adjuvants for use in research

We evaluated an adjuvant, TiterMax, as an alternative to complete Freund's adjuvant (CFA) fo producing antisera in animals. TiterMax, consists of a microparticulate stabilized water-in-oil emulsion of a metabolizable oil, squalene, with the adjuvant block copolymer CRL89-41. This paper reports two evaluations of TiterMax versus CFA and other commercially available adjuvants. In the first study, mice were immunized with a hapten, trinitrophenol, conjugated to hen egg albumin (TNP-HEA) in one of several adjuvants: TiterMax, CFA, Adjuvax, Ribi adjuvant system (RAS), Alhydrogel or Lipovant. TiterMax induced higher longer lasting titers with fewer injections than any of the other adjuvants. The magnitude of the response to TNP varied with species and route of immunization. In the second study, CFA, TiterMax, Adjuvax and RAS were compared in rabbits, mice and goats. Animals were immunized with luteinizing hormone-releasing hormone (LHRH) conjugated to BSA in each adjuvant using comparable protocols. TiterMax induced titers against the peptide equivalent to CFA in all three species. The inflammatory responses induced by TiterMax were mild and transient compared with those induced by CFA. These data suggest that TiterMax is an effective alternative to CFA in many situations.

Thin Layer Chromatographic Analysis of Neutral Lipids in the Digestive Gland-Gonad Complex of Biomphalaria Glabrata Snails Maintained on Various Diets

Abstract Thin layer chromatography on channeled, high performance preadsorbent silica gel plates was used to study the neutral lipid composition of six diets/i.e., hen's egg albumen, hen's egg yolk, leaf lettuce, Tetramin fish food, lettuce/Tetramin (1:1), and Spirulina, and the digestive gland-gonad complex of Biomphalaria qlabrata snails maintained on these diets. Differences in the neutral lipid profiles between the diet and the DGG suggest that factors other than dietary uptake, presumably metabolic, play a role in determining the lipid composition of the snail.

Adjuvant activity of non-ionic block copolymers. V. Modulation of antibody isotype by lipopolysaccharides, lipid A and precursors

Non-ionic block copolymers and lipopolysaccharides are both effective immunological adjuvants which are thought to act via distinct mechanisms. We hypothesized that they might produce synergistic effects when used together. We prepared a series of lipopolysaccharide (LPS) preparations ranging from the smallest precursor, lipid X through complete LPS with O-polysaccharide chains. Three preparations with reduced toxicity, monophosphoryl lipid A, partially hydrolysed Ra-LPS and LPS of Rhodopseudomonas sphaeroides were also utilized. All LPS preparations except the smallest were effective adjuvants for inducing early antibody responses to trinitrophenyl-conjugated hen egg albumin (TNP-HEA) when injected in squalane-in-water emulsions with copolymer L141. Only the larger LPS preparations induced sustained antibody responses. By itself, emulsions of copolymer L141 induced a predominant IgG1 antibody isotype response with lesser amounts of IgG2a and IgG2b. Surprisingly, all of the LPS preparations tested increased the proportion of IgG2 isotypes even though some had little effect on overall titres. The detoxified Ra-LPS (Ra-detox) was the most effective preparation for both increasing antibody titres and inducing the desirable IgG2a and IgG2b isotypes. These results demonstrate that the combination of LPS and block polymer adjuvants can produce synergistic effects without unacceptable toxicities.

Permeability of the Isolated Atlantic Salmon (Salmo salar) Chorion to Ions as Estimated by Diffusion Potentials

The properties of the teleost chorion and perivitelline fluid may allow the embryo to develop in an ionic medium which differs from ambient. A knowledge of these properties facilitates assessment of the potential impact of environmental perturbations (e.g. low-pH episodes) on the developing embryo. Diffusion potentials are created when differing concentrations of a salt solution are imposed upon the opposite sides of isolated Atlantic salmon (Salmo salar) chorions. These diffusion potentials are related to the mobilities of the cations and anions. The magnitude of the diffusion potentials were measured with NaCl, CaC2, H2SO4, and HCl solutions. The results indicate that both anions and cations permeate the chorion. Chloride appears to have greater mobility through the chorion than does sodium, although the difference in mobility of such ion pairs moving through the chorion is not as great as the difference in their mobilities in aqueous solution. Diffusion potentials obtained with CaCl2solutions, on the other hand, indicate reduced calcium mobility through the chorion relative to chloride. The decreased mobility of Ca2+is probably due to adsorption to fixed negative charges on the chorion. The properties of the perivitelline potential of the intact egg could be simulated qualitatively by placing a solution of negatively charged colloid (hen's egg albumin) inside the isolated chorion.

Stimulation of prostaglandin formation by an antigen- and Ia-restricted T-cell-macrophage interaction.

Stimulation of macrophages by zymosan phagocytosis triggers the respiratory burst and induces an early release of lysosomal hydrolases and E-type prostaglandins (PGE). We have studied whether antigen presentation by macrophages to helper T-cells elicits a comparable sequence of events. Cloned T-helper cells specific for hen egg albumin (EA) were added to histocompatible or histoincompatible resident mouse peritoneal macrophages in the presence of EA or an unrelated antigen, and the changes in biochemical parameters were monitored. The interaction between macrophages. T-helper cells and EA induced the production of PGE, but no release of lysosomal hydrolases or activation of the respiratory burst. In addition T-cell proliferation was observed. By contrast, no proliferation and no biochemical changes were observed when histoincompatible macrophages or unrelated antigen were used. When the experiments were done in the presence of indomethacin to inhibit PGE release, T-cell proliferation was enhanced. These results suggest that the PGE released may exert a feed-back control of the T-cell response.

Isolation of phosphoproteins by immobilized metal (Fe 3+) affinity chromatography

Phosphoproteins and phosphoamino acids bind to ferric ions immobilized on iminodiacetate-agarose gel and can be eluted by increasing pH or by introducing phosphate ions to the eluant. Some other metals were found to resemble iron with regard to the interaction with protein-bound phosphate and phosphoamino acids. These observations were utilized to develop purification procedures for phosphoproteins. Hen egg albumin (ovalbumin) was fractionated into three components of varying phosphate contents. Porcine pepsin was purified in a similar manner.

Inhibition of immune precipitation by complement.

Normal human complement serum (NHS) inhibited precipitin reactions between tetanus toxoid and human or rabbit anti-tetanus toxoid IgG antibody, between bovine serum albumin (BSA) and rabbit anti-BSA IgG antibody, and between hen egg albumin and rabbit anti-egg albumin IgG antibody. Ethylene-diaminetetraacetic acid (EDTA) prevented this inhibition. Mg-ethyleneglycol-bis(aminoethyl)-tetra-acetic acid-(EGTA) also prevented the inhibition except with lower concentrations of antibody and antigen. Therefore, the inhibition of immune precipitation seemed to occur mainly through the classical pathway of complement activation. The alternative pathway was usually dispensable, but it augmented the inhibition. Guinea pig complement serum (NGS) was less effective than NHS in inhibiting immune precipitation. Guinea pig serum deficient in C4 (C4DGS) did not inhibit the immune precipitation. Mouse complement serum was effective for inhibiting precipitation, and C5-deficient serum was as effective as normal serum. Therefore, the inhibition of immune precipitation is considered to occur by activation of complement up to the step of C3. The size of the soluble immune complexes formed in the presence of NHS varied depending on the concentrations of antibody and antigen, even when the ratio of antigen to antibody was constant. On incubation at 37 degrees C immune precipitation was inhibited by 1/2 dilution of NHS for 2 to 3 hr and then gradually increased to the level in the absence of complement. When the immune complexes were formed in the presence of serum containing complement, fragments of C4 and C3 were incorporated into the soluble immune complexes. The C3 fragments incorporated into the soluble complexes were C3b, iC3b, C3c, and C3d, some of which were bound covalently with heavy chains of IgG antibody molecules. Some of the covalent linkages between C3 fragments and IgG seemed to be destroyed by alkali treatment, but not by hydroxylamine treatment. The formation of covalent bonds between IgG and C3 and probably C4 was essential for inhibition of immune precipitation, because inhibitors of their formation, such as putrescine, cadaverine, and salicylhydroxamic acid, effectively prevented the inhibition of precipitation. When antigen and antibody reacted in the presence of mixtures of various combinations of isolated complement components, C1, C4, C2, and C3 showed maximal inhibition of immune precipitation, whereas factors I and H had little effect.

The influence of incubation temperature and pH on the antimicrobial properties of hen egg albumen.

Gram positive bacteria, including lysozyme-resistant strains, and yeasts were killed in hen egg albumen with or without iron at 30 of 39.5 degrees C. The albumen was more toxic at 39.5 degrees C than at 30 degrees C for Gram negative bacteria. With the exceptions of Pseudomonas fluorescens, Acinetobacter sp. and Proteus vulgaris, iron caused the growth of Gram negative bacteria or protected them from being killed in hen albumen at 39.5 degrees C. At this temperature, however, maximal growth of and glucose utilization by Escherichia coli C20 only occurred in albumen supplemented with growth factors, trace metals, additional nitrogen and sufficient iron to quench ovotransferrin. The bactericidal properties of albumen could be negated by changing its pH from 9.0 or above to 7.5 or below. At 39.5 degrees C, enterochelin allowed growth of E. coli in albumen at pH 7.9, but not at 9.4, whereas iron allowed growth at both pH values.

Kinetics of oral tolerance: study of variables affecting tolerance induced by oral administration of antigen.

The induction of antigen-specific tolerance by oral administration of hen egg albumin (OVA) was shown to be time- and dose-dependent. The IgE response was the most effectively suppressed by antigen feeding prior to or after parenteral immunization. The effect on IgG response paralleled that observed on the IgE response, but the magnitude of suppression was less pronounced. The IgA response was enhanced rather than suppressed if the interval between feeding and parenteral immunization was short or if the animals were antigen-fed after the intraperitoneal priming.

Sulfated asparagine-linked sugar chains of hen egg albumin.

The fraction of hen egg albumin glycopeptides mixture, which passes through a Dowex 50-H+ column, contains two sulfate-containing glycopeptides. Based on the structural studies of oligosaccharides released from the glycopeptides by hydrazinolysis, their structures were elucidated as follows. (formula; see text)

Development in vitro of the metacercaria of Bucephaloides gracilescens (Trematoda: Bucephalidae)

The development of Bucephaloides gracilescens metacercariae was studied using a range of cultivation conditions. The most rapid development occurred at 18°C in a medium containing NCTC 135 supplemented with 25% chicken serum, 25% hen egg yolk and 25% hen egg albumen, with a gas phase of air. Under these conditions, shell-protein synthesis was triggered by day 3 in culture; secondary oocytes were apparent in the ovary by day 10; and egg production began by day 14. Survival of worms in media containing chicken serum was twice as long as that achieved with either whiting or angler fish serum. The ingestion of yolk (feeding) appeared to be a necessary prerequisite to development and egg production. The presence of yolk in the culture medium greatly increased the amount of 3H-thymidine incorporated by the reproductive system of freshly excysted metacercariae but had little effect on the uptake and incorporation of tyrosine. The eggs produced in vitro failed to embryonale and were abnormal in appearance, being non-operculate with irregularly thickened shells.