Proteomic analysis of egg white proteins during storage

Egg storage causes egg white to lose its viscous nature to form a thin liquid, commonly referred to as egg white thinning. To understand the mechanisms underlying egg white thinning, white-shell eggs were used in the present study to determine the proteome-level changes of egg white proteins occurred during storage. Egg white thinning was observed visually after 20 days of storage at ambient temperature (22 ± 2 °C) when the maximum number of proteome-level changes occurred. The proteins that showed significant changes in abundance during storage included ovalbumin, clusterin, ovoinhibitor, ovotransferrin, and prostaglandin D2 synthase. Among these, only the abundance of clusterin was observed to change continuously during the storage period. Hence, it is expected that the increase in the concentrations of clusterin and ovoinhibitor along with the change of ovalbumin content during storage might contribute to egg white thinning. Degradation of ovalbumin/clusterin during egg storage may be due to the combined effect of proteolysis and increase in pH; this may also be partly responsible for egg white thinning phenomenon.     

Co-extraction of egg white proteins using ion-exchange chromatography from ovomucin-removed egg whites

Efficient isolation of egg white components is desired due to its potential uses. Existing methods mainly targeted on one specific protein; an attempt has been made in the study to co-extract all the valuable egg white components in a continuous process. Ovomucin was first isolated by our newly developed two-step method; the resultant supernatant obtained after ovomucin isolation was used as the starting material for ion-exchange chromatography. Anion-exchange chromatography of 100 mM supernatant yielded a flow-through fraction and three other fractions representing ovotransferrin, ovalbumin and flavoproteins. The flow-through fraction was further separated into ovoinhibitor, lysozyme, ovotransferrin and an unidentified fraction which represents 4% of total egg white proteins. Chromatographic separation of 500 mM supernatant resulted in fractions representing lysozyme, ovotransferrin and ovalbumin. This co-extraction protocol represents a global recovery of 71.0% proteins.     

Raman spectroscopic study of the proteins of egg white

The Raman spectra of ovalbumin, ovomucoid, and conalbumin are reported. Spectral shifts in the conformationally sensitive amide I and amide III lines as a result of thermal denaturation indicate the formation of intermolecular β- sheets. A medium intensity line at 1260 cm^?1 in the spectra of ovomucoid and ribonuclease is demonstrated to contain a substantial contribution from tyrosine residues.     

Vasodilator effects of peptides derived from egg white proteins

The aim of this work was to investigate the effect of several peptides, identified before and after simulated gastrointestinal digestion of an egg white hydrolysate, on the vascular function, in rat aorta. The sequences IVF, RADHPFL and YAEERYPIL (0.1 mM) induced vasodilatation in intact aortic rings, with the maximum percentage of dilation corresponding to RADHPFL (40.5 ± 7.0%). Two of the end products of the gastrointestinal digestion, RADHP and YPI, also showed vasodilator activity with degrees of relaxation higher than 50%. However, all these peptides failed to induce relaxation in endothelium-denuded aortic rings. The relaxation induced by RADHP was concentration-dependent and it was partially blocked by the nitric oxide synthase inhibitor l-NAME (100 μM) and by the B₁ bradykinin receptor antagonist Des-HOE 140 (30 nM), thus showing that it was mediated by NO production through the activation of B₁ bradykinin receptors. These findings suggest that these peptides could reduce the vascular resistance and could be used as functional food ingredients in the prevention and treatment of hypertension.     

One-step purification of proteins from chicken egg white using counter-current chromatography

Proteins present in chicken egg white are separated by counter-current chromatography (CCC) in one step using a cross-axis coil planet centrifuge (X-axis CPC). The separation was performed with an aqueous polymer two-phase system composed of 16% (w/w) poly(ethylene glycol) 1000 and 12.5% (w/w) dibasic potassium phosphate by eluting the lower phase at a flow-rate of 1.0 ml/min. From about 20 g of the crude egg white solution, lysozyme, ovalbumin, and ovotransferrin were resolved within 5.5 h. Each component was identified by 12% SDS gel electrophoresis with Coomassie brilliant blue staining.     

Precipitation of egg white proteins below their isoelectric points by sodium dodecyl sulphate and temperature.

The precipitating of effect of sodium dodecyl sulphate (SDS) on the egg white proteins ovalbumin, conalbumin and lysozyme was studied at 25 degrees C and at different pH values. The proteins precipitated below their respective isolectric points, provided the detergent to protein ratio was appropriate. The pH profile of precipitation was different for the three proteins reflecting net charge differences. The binding of SDS to the proteins was studied with [35S]-labelled SDS and for ovalbumin a ratio of 21--28 SDS molecules/protein molecule, dependent on the concentration of SDS initially used, seem to be required for precipitation at pH 4.5. This number, however, is dependent on pH and increases with an increased positive net charge of the protein. The precipitating effect of SDS was identical for ovalbumin, conalbumin and lysozyme when compared on a gram to gram basis (0.1--0.15 g SDS/g precipitated protein). The precipitated protein was denatured as measured by differential scanning calorimetry, but was also completely redissolved if pH was increased to above the isoelectric point. The precipitating effecto f SDS was also examined at elevated temperatures. The two-phase systems of the proteins induced by SDS at 25 degrees C were heated from 25 degrees C to 90 degrees C at a rate of 1.25 degrees C/min. The precipitation behaviour was similar for the three proteins upon heating. When the SDS concentration was increased the precipitation curves were transferred towards lower temperatures and the courses of precipitation became less sharp. The synergistic effect of SDS and heat on protein precipitation was differentiated by denaturation measurements and radioactive labelling. The ratio SDS to precipitated protein gradually diminished towards higher temperatures but no purely thermal precipitation was found.     

Egg white proteins.

1. Egg white proteins are the principal solutes present in egg white, making up approximately 10% of its weight. 2. They are globular proteins and most have acidic isoelectric points. 3. Many are glycoproteins with carbohydrate contents ranging from 2 to 58%. 4. Of the major egg white proteins, lysozyme is the only one having catalytic activity, but many have specific binding sites, e.g. for vitamins such as biotin, riboflavin and thiamin, or for metal ions such as FeIII. 5. A major group are those showing proteinase inhibitory activity, and they include ovomucoid, ovoinhibitor, cystatin and ovostatin. 6. The synthesis of egg white protein occurs in the oviduct, and is hormonally controlled either by oestrogens or progesterone. 7. Extensive studies have been carried out in the genes coding for egg white proteins.     

Activity of crystalline turkey egg white lysozyme.

Hexagonal crystals of turkey egg white lysozyme have been examined for activity in order to evaluate their potential for use in time-resolved X-ray crystallographic experiments. Substrates used in this study were hexa-N-acetylglucosamine (hexa-GlcNAc) and a modified analogue of hexa-GlcNAc where the terminal sugar ring was opened by reduction with tritiated sodium borohydride. This gave a labeled beta-N-acetylglucosaminitol unit at the sixth position of the sugar chain and allowed easy quantitation of enzymatic cleavage on TLC plates. Using these substrates, it has been shown that turkey egg white lysozyme is enzymatically active in the crystal. Enzyme dispersed in the buffer surrounding the crystal does not show detectable activity under conditions relevant to an X-ray experiment. Unmodified hexa-GlcNAc is hydrolyzed into di-, tri-, and tetrasaccharides in the crystal. This cleavage pattern is different from that obtained with hen egg white lysozyme in solution and likely causes of the differences are discussed. The reduced radiolabeled oligosaccharide has a unique cleavage pattern with trisaccharides as the products. The specific activity of the enzyme with the radiolabelled analogue was 9.8 (+/- 1.0) x 10(-7) mmol/min/mg protein at 22 degrees C in the crystal.     

Proteomic analysis of egg white proteins during the early phase of embryonic development

Avian egg albumen participates in embryonic development by providing essential nutrients as well as antimicrobial protection. Although various biological functions of egg white proteins were suggested during embryogenesis, global changes of these proteins under incubation conditions remained uninvestigated. This study presents a proteomic analysis on the change of egg white proteins during the first week of embryonic development. By using 2-DE, together with MALDI-TOF MS/MS, thirty protein spots representing eight proteins were identified showing significant changes in abundance during incubation. An accelerating degradation of ovalbumin was observed in a wide range of molecular weight. In addition, four protein complexes were predicted according to the detected molecular weight increase. Among these speculated protein complexes, an ovalbumin spot coupled with RNA-binding protein was detected. The absence of these protein complexes before incubation, followed by the constant increase in abundance during incubation indicates conceivable pivotal roles in embryonic development. To better understand the function of the proteins identified in this study, discrepancies of egg white protein changes between fertilized and unfertilized chicken eggs were additionally demonstrated. These findings will provide insight into the embryogenesis process to improve our knowledge of egg white proteins in regulating and supporting early embryonic development.     

Structural and physicochemical characteristics of novel basic proteins isolated from duck egg white

Novel basic proteins, duck basic protein small 1 (dBPS(1)) and 2 (dBPS(2)), were isolated from duck egg white by cation-exchange and gel filtration chromatography. Protein sequence analyses indicated that they possessed 39 amino acid residues with three disulfide bonds. The amino acid sequence of dBPS(1) showed 45% identity with dBPS(2). The amino acid sequence of dBPS(2) was the same as cygnin, a small protein from black swan, and strongly homologous with meleagrin from turkey and chicken. Phylogenic relationships implied that dBPS(1) and dBPS(2) share a common ancestry with cygnin and meleagrin. Based on MALDI-TOF mass spectra, the molecular masses of dBPS(1) and dBPS(2) were 4,373, and the 4,486 Da. pI of dBPS(1) and dBPS(2) elucidated by isoelectric focusing were 9.35 and 9.44. FT-IR spectra classified these proteins as (beta) proteins. Both dBPS(1) and dBPS(2), possessed high heat stability, Td 101.2 and 98.3 degrees C. Indirect ELISA results showed that the dBPS(1)/dBPS(2)-related proteins were distributed in the oviduct and gallbladder.     

Sulfhydryl oxidase from egg white. A facile catalyst for disulfide bond formation in proteins and peptides.

Both metalloprotein and flavin-linked sulfhydryl oxidases catalyze the oxidation of thiols to disulfides with the reduction of oxygen to hydrogen peroxide. Despite earlier suggestions for a role in protein disulfide bond formation, these enzymes have received comparatively little general attention. Chicken egg white sulfhydryl oxidase utilizes an internal redox-active cystine bridge and a FAD moiety in the oxidation of a range of small molecular weight thiols such as glutathione, cysteine, and dithiothreitol. The oxidase is shown here to exhibit a high catalytic activity toward a range of reduced peptides and proteins including insulin A and B chains, lysozyme, ovalbumin, riboflavin-binding protein, and RNase. Catalytic efficiencies are up to 100-fold higher than for reduced glutathione, with typical K(m) values of about 110-330 microM/protein thiol, compared with 20 mM for glutathione. RNase activity is not significantly recovered when the cysteine residues are rapidly oxidized by sulfhydryl oxidase, but activity is efficiently restored when protein disulfide isomerase is also present. Sulfhydryl oxidase can also oxidize reduced protein disulfide isomerase directly. These data show that sulfhydryl oxidase and protein disulfide isomerase can cooperate in vitro in the generation and rearrangement of native disulfide pairings. A possible role for the oxidase in the protein secretory pathway in vivo is discussed.