The Interaction between Ovomucin and Egg White Proteins

The interaction between ovomucin and egg white proteins has been investigated. Ovomucin interacted not only with lysozyme but also with such proteins in egg white as ovalbumin and conalbumin. The interaction increased correspondingly as the lysine content of proteins become higher. The maximum turbidity of ovomucin-lysozyme aggregates was proportionally dependent on the rate of acetylation of lysozyme, but that of ovomucin-ovalbumin aggregates was slightly dependent on the rate of acetylation of ovalbumin. The ovomucin-protein interaction decreased remarkably by the removal of sialic acid in ovomucin, but increased by the removal of polyhydroxyl group in sialic acid. From these results, the binding site of interaction was considered in detail.     

Studies on Changes in Stored Shell Eggs

The intrinsic viscosity of ovomucin(B) from the thick white was higher than that of the thin white. The relative area of the fast moving component in the electrophoretic pattern of ovomucin(B) from the thick white was about twice that of the thin white. The hexose, hexosamine and sialic acid contents of ovomucin(B) from the thick white were higher than those of the thin white. The intrinsic viscosity and carbohydrate content of ovomucin from the egg white previously freed from lysozyme were low in comparison with those of ovomucin(B). The carbohydrate contents of crude lysozyme from the thick white were higher than those of the thin white, and crude lysozyme obtained from the thick white contained larger amounts of ovomucin-iike material than the thin white. From these results, discussion were made about the relation between the properties of ovomucin(B) and ovomucin-lysozyme interaction.     

Studies on Changes in Stored Shell Eggs

Being solubilized by treatment with 0.01 m mercaptoethanol, the ovomucin gel(B) was found in free boundary electrophoresis to contain subunits which were consisted of two components. Changes in the physicochemical properties of all the insoluble ovomucin gel(B) and sol(B) obtained from stored egg white were studied after this treatment.

The fast moving component of the ovomucin gel(B) in free boundary electrophoresis decreased during storage and disappeared completely after 30 days. On the other hand, the fast moving component of the ovomucin sol(B) increased during storage.

The acid mucoprotein concentration of the ovomucin gel(B) in acrylamide gel electrophoresis decreased and that of the ovomucin sol(B) increased during storage, although the protein pattern did not show significant changes.

The interaction of the ovomucin gel(B) with lysozyme decreased whereas that of the ovomucin sol(B) increased during storage.

By summarizing these results, a model of ovomucin gel structure and a mechanism of egg white thinning were proposed.     

N-Terminal sequencing by mass spectrometry through specific fluorescamine labeling of α-amino groups before tryptic digestion

We present a single-step procedure for the specific mass labeling of unblocked protein N termini. We show that the dye fluorescamine, which is commonly assumed to require mildly alkaline conditions for undergoing a nonspecific reaction with α- and ε-amino groups associated with amino acids, in fact shows a specific reaction only with α-amino groups present at protein N termini when mildly acidic conditions are used. We use this finding to label, identify, and sequence the trypsinolysis-derived N-terminal peptide of lysozyme, using only mass spectrometry, to illustrate how this method could be used with other proteins.     

Requirements for Nutrients and Minerals by Candida brumptii IFO 0731 Producing Succinic Acid from n-Paraffin

The effect of several materials on viscometric behaviour of the soluble ovomucin was determined with a cone plate viscometer.

The soluble ovomucin showed a rapid increase in viscosity above 1.5 mg/ml, and a high concentration of the soluble ovomucin led to gel whose viscosity was comparable to that of the insoluble ovomucin. The apparent viscosity of the soluble ovomucin decreased with an increase in NaCl concentration and upon addition of lysozyme as well as of CaCl₂. The soluble ovomucin in the presence of the sonicated β-ovomucin showed an increase in viscosity on addition of a small amount of CaCl₂.

It is assumed that a great increase in viscosity of egg white may result from removal of sodium ion and lysozyme.     

Reductive Methylation of the Lysyl Residues in the fd Gene 5 DNA-Binding Protein: CD and 13C NMR Results on the Modified Protein

Abstract

The ∈-amino groups of the six lysyl residues of the fd gene 5 DNA-binding protein have been modified by reductive methylation to form N^∈, N^∈-dimethyl lysyl derivatives containing ¹³C-labeled methyl groups. The α-amino terminus of the protein was not accessible to methylation. Circular dichroism studies show that the modified protein binds to fd DNA, but with a slightly reduced affinity compared with that of unmodified gene 5 protein. We also find that both the modified and unmodified proteins bind to an oligodeoxynucleotide, d(A)₇, but in neither case does binding cause a decrease in the 228 nm CD band of the protein as occurs when the protein binds to long DNA polymers. ¹³C NMR spectra at 50.1 MHz of [¹³C]methylated gene 5 protein show five distinct resonances between 43.30 and 42.76 ppm originating from the six N^∈, N^∈-dimethyl lysyl residues. We attribute one of the resonances to two solvated lysyl residues and the other four to individual lysyl residues in different microenvironments. All four of these latter resonances are affected by the binding of d(A)₇. However, since two of these resonances are similarly affected by the presence of salt in the absence of DNA, only two are uniquely affected by DNA binding.     

Mode of interaction between lysozyme and the other proteins of the hen's egg vitelline membrane

Salt solutions and charged detergents are efficient solubilizing agents for ovovitelline membrane lysozyme. Reassociation experiments with chemically modified lysozymes indicate that positively charged amino acid residues of lysozyme (the epsilon-amino group of lysine and the guanidino group of arginine) are involved in the interaction with other proteins of the vitelline membrane. Exogenous proteins are adsorbed to lysozyme-free vitelline membranes, only if they have a high pI, comparable to that of lysozyme. It is concluded that the lysozyme-ovovitelline membrane interaction is predominantly ionic. An ovomucin-lysozyme complex is postulated as the major component of the outer layer of the membrane.     

The Release of Carbohydrate Rich Component from Ovomucin Gel during Storage

The fractionation pattern of OMG₀, ovomucin gel(B) in fresh egg white, by density gradient column electrophoresis showed two peaks. Each peak was shown to migrate as a single component, with a mobility of either the fast or slow moving component of ovomucin gel(B). Each peak was named as F-component and S-component.

Carbohydrate and sulfate contents of F-component were much higher than these of S-component. The carbohydrate content of F-component and S-component was found to be about 50 and 15 percents of dry matter, respectively. Serine and threonine contents in F-component were much higher than those in S-component.

The fractionation pattern of OMG₂₀, ovomucin gel(B) in egg white stored for 20 days at 30°C, by density gradient electrophoresis showed only one peak which corresponded to S-component, and that of OMS₂₀, ovomucin sol (B) in egg white stored for 20 days at 30°C, showed two peaks which corresponded to F- and S-component.

Ability of F-component to interact with lysozyme was greater than that of S-component.     

Role of magnesium chloride on the purity and activity of ovomucin during the isolation process

Purification of ovomucin is still an empirical technique and sometimes insufficient quantities of ovomucin are purified to allow characterization. Here we aimed to investigate the effect of MgCl(2) on the purity and bioactivity of ovomucin during isoelectric precipitation process and to develop an effective protocol to prepare pure ovomucin with high bioactivity. It was found that addition of MgCl(2) is an alternative approach to remove lysozyme from ovomucin, and that the hemagglutination inhibition (HI) activity of ovomucin with MgCl(2) against New Disease Virus (NDV) was about two times higher than the protein without salts. Thus, an improved procedure comprises a precipitation with 0.05 mol/L CaCl(2) followed by precipitation with 0.05 mol/L MgCl(2) was developed for the isolation of ovomucin. Better adhesion property of ovomucin was observed when low concentration of MgCl(2) was added in the designed ELASA test, whereas the adhesion property of the pure ovomucin without salts to NDV was lower. Thus, magnesium (II) plays an important role in the activity of ovomucin, and the alternative method developed in this study may significantly facilitate the further research on the mechanism of ovomucin activity.     

Effect of operating conditions on the extraction of ovomucin

Ovomucin, mainly responsible for the gelatinous property of egg white, has potential applications as a functional food and nutraceutical ingredient. A 2-step method for ovomucin preparation was recently developed. The purpose of this study was to determine the effects of various operating conditions, such as pH, NaCl concentrations and extraction volume at the second extraction, temperature, and centrifugation force, on the purity and yield of ovomucin. Our results showed that pH has a significant effect on the purity and yield of ovomucin extracts. Increasing the extraction pH from 4.0 to 5.0 could significantly (p < 0.05) increase the purity and yield of ovomucin; at pHs higher than 5.0, the purity was not affected but the yield was significantly decreased. The highest yield of ovomucin extract (308 mg/100 g of egg white) was achieved at pH 5.0 while the highest purity was achieved at pH 7.0. There is a trend that the purity of ovomucin increased (p < 0.05) but the yield of ovomucin decreased (p > 0.05) at increasing salt concentrations. Reducing extraction volume did not affect the yield of ovomucin whereas its purity was significantly decreased. The yield of ovomucin however was significantly increased at increasing settling time or centrifugation force, but the purity was less affected. Extraction of ovomucin at room temperature could significantly reduce the extraction yield compared to that at lower temperature (4 °C) but the purity was not affected.     

Solubilization and Characterization of Ovomucin without Chemical Modification

1. The egg white, thick and thin fractions, was solubilized in 1.0% SDS solution by vigorous mixing and subjected to gel filtration on a Sepharose 4B column, eluted with 1.0% SDS. The isolated thick and thin ovomucins were found by analytical disc electrophoresis to be free from contamination with lysozyme.

2. In the velocity sedimentation the two ovomucin fractions behave similarly, both comprising at least two components with sedimentation coefficients 35 S and 30 S.

3. The chemical compositions of the two ovomucin fractions showed only notable difference in that the carbohydrate content of the thick white ovomucin was somewhat higher than that of the thin white ovomucin. The amino acid profiles of the two fractions were similar.

     

Interactions involved in ovomucin gel-forming properties: a rheological-biochemical approach

Different kinds of interactions involved in the properties of ovomucin gel formation from hen egg white were studied by combining physical and biochemical methods. A decrease in viscosity of the ovomucin gel was observed when it was subjected to chymotrypsin or sonication treatment. The viscosity decrease correlated with a change from non-Newtonian to Newtonian properties of the ovomucin gel. By treatment of the gel with either 5 M guanidinium HCl, 6 M urea, or 5% sodium dodecyl sulfate a change from non-Newtonian to Newtonian properties was also obtained. Although high ionic strength or sialic acid liberation from the ovomucin gel by neuraminidase treatment provoked a decrease in viscosity, it was not followed by a change in non-Newtonian properties. The results obtained suggest that different noncovalent interactions might be involved in gel formation. Electrostatic interactions (partially destroyed by sialic acid removal or 2 M NaCl) and hydrophobic interactions might be responsible for protein-mucin and mucin-mucin interactions. Other bonds susceptible to chymotrypsin treatment and sonication would be involved in the interaction between mucin subunits.     

Studies on the Dissociation of the Soluble Ovomucin by Sonication

The soluble ovomucin obtained from the liquid part of thick white by gel filtration on a Sepharose 4B was an aggregated and polymerized molecule (intrinsic viscosity was 365 ml/g and molecular weight was 8.3 × 10⁶) and it was unable to dissociate the soluble ovomucin into two components without modifications.

Molecular weight and reduced viscosity of the soluble ovomucin decreased markedly with time of sonication. By the sonication for 10 min, it was successful to fractionate it into carbohydrate rich and poor component by density gradient electrophoresis, cellulose acetate electrophoresis and DEAE-cellulose column chromatography.

Concerning carbohydrate and amino acid compositions of two components obtained from the sonicated soluble ovomucin, it was found that the carbohydrate poor component corresponded to the reduced S-component or the reduced α-ovomucin, and the carbohydrate rich component to the reduced F-component or the reduced β-ovomucin.

It was considered that the sonicated soluble ovomucin was an intermediate of the aggregated, polymerized ovomucin (the soluble ovomucin) and the monomeric ovomucin (the sonicated and reduced soluble ovomucin).     

Fromation and stoichiometry of a lysozyme-phospholipid-mitochondrial protein ternary complex.

Mitochondrial membranes reconstituted from lipid-depleted mitochondria and aqueous phospholipid dispersions still have the phospholipid negative charges available for ionic interaction with the basic protein, lysozyme. The stoichiometry of the binding is of about 6 nmoles of lysozyme per 100 nmoles of phospholipid in membranes reconstituted with Asolectin, and of 10 nmoles of phospholipid phosphorus in membranes reconstituted with cardiolipin. Unextracted submitochondrial particles ETP also bind lysozyme (about 3 nmoles per 100 nmoles of phospholipid). These observations indicate that the phospholipid anionic groups are not completely shielded by the mitochondrial proteins, which might occupy areas between the nonpolar groups of the lipid molecules.     

Inactivation of the ribosomal protein S1 in polyuridylate binding by reductive methylation of the lysyl-ammonium groups.

The ribosomal protein S1 was modified by reductive methylation of some of its lysyl ammonium groups (S1). With 6 out of 30 groups methylated the protein lost its capacity to form stable complexes with polyuridylate. Addition of excess polyuridylate inhibited the methylation of the lysyl groups. In equilibrium dialysis experiments it was shown that the binding constant between S1 and U15 was lowered 10-fold as compared to the native protein. The pH-dependence of the complex formation between S1 and U15 confirms a participation of the lysyl residues. When S1 depleted 30-S ribosomes were reconstituted with methylated S1 these ribosomes were inactive in the poly(U) stimulated Phe-tRNA binding. The data are discussed with respect to a grid-like interaction between the lysyl groups of the protein and the phosphodiester bonds of the polynucleotide as a molecular basis of protein nucleic acid interaction.     

Chemosynthesis of poly(ε-lysine)-analogous polymers by microwave-assisted click polymerization

Poly(ε-lysine) (ε-PL)-analogous click polypeptides with not only similar α-amino side groups but also similar main chain to ε-PL were chemically synthesized for the first time through click polymerization from aspartic (or glutamic)-acid-based dialkyne and diazide monomers. With microwave-assisting, the reaction time of click polymerization was compressed into 30 min. The polymers were fully characterized by NMR, ATR-FTIR, and SEC-MALLS analysis. The deprotected click polypeptides had similar pK(a) value (7.5) and relatively low cytotoxicity as ε-PL and could be used as substitutes of ε-PL in biomedical applications, especially in endotoxin selective removal. Poly(ethylene glycol) (PEG)-containing alternating copolymers with α-amino groups were also synthesized and characterized. After deprotection, the polymers could be used as functional gene vector with PEG shadowing system and NCA initiator to get amphiphilic graft polymers.     

Effects of chemical modification of lysine residues on the sweetness of lysozyme

Lysozyme is a sweet-tasting protein with a sweetness threshold value of around 7 microM. To clarify the effect of basicity at the side chain of lysine residues on the threshold values of sweetness, charge-specific chemical modifications such as guanidination, acetylation and phosphopyridoxylation of lysine residues were performed. Sensory analysis showed that the sweetness threshold value of lysozyme was not changed by guanidination, whereas it was increased markedly by acetylation and phosphopyridoxylation. To confirm the importance of the basicity in the lysine residues in detail, purification of acetylated (Ac-) and phosphopyridoxylated (PLP-) lysozymes using SP-ion exchange column chromatography was performed. The threshold values were not changed by modification with fewer than two residues (approximately 7 microM), whereas the threshold values significantly increased to 15 and 34 microM when tetra-Ac and tri-PLP, respectively. Furthermore, sweetness was not detected at 30 microM (hexa-, penta-Ac and tetra-PLP). It should be noted that removal of the negative charges of the phosphate groups in the tri-PLP lysozyme by acid phosphatase resulted in the recovery of sweetness (6.4 microM), indicating that basicity at the position of the lysine residues is responsible for lysozyme sweetness and that strict charge complementarities might be required for interaction to its putative receptor.     

Evaluation of colorimetric assays for analyzing reductively methylated proteins: Biases and mechanistic insights

Colorimetric protein assays, such as the Coomassie blue G-250 dye-binding (Bradford) and bicinchoninic acid (BCA) assays, are commonly used to quantify protein concentration. The accuracy of these assays depends on the amino acid composition. Because of the extensive use of reductive methylation in the study of proteins and the importance of biological methylation, it is necessary to evaluate the impact of lysyl methylation on the Bradford and BCA assays. Unmodified and reductively methylated proteins were analyzed using the absorbance at 280 nm to standardize the concentrations. Using model compounds, we demonstrate that the dimethylation of lysyl ε-amines does not affect the proteins' molar extinction coefficients at 280 nm. For the Bradford assay, the responses (absorbance per unit concentration) of the unmodified and reductively methylated proteins were similar, with a slight decrease in the response upon methylation. For the BCA assay, the responses of the reductively methylated proteins were consistently higher, overestimating the concentrations of the methylated proteins. The enhanced color formation in the BCA assay may be due to the lower acid dissociation constants of the lysyl ε-dimethylamines compared with the unmodified ε-amine, favoring Cu(II) binding in biuret-like complexes. The implications for the analysis of biologically methylated samples are discussed.     

The involvement of ionic interactions during asbestos-induced enzyme release from polymorphonuclear leukocytes

Chrysotile asbestos permeabilizes the plasma membrane of rabbit polymorphonuclear leukocytes (PMNs) which is evident from the release of the cytoplasmic enzyme lactate dehydrogenase (LDH) from the cell. When Ca2+ is present in the medium exocytosis is observed, evident from the release of the granule associated enzyme lysozyme which is not liberated in the absence of Ca2+. Asbestos-induced enzyme release is inhibited by polyanions or by removal of positive charges on asbestos, and resembles enzyme release induced by synthetic polycations. Pretreatment of PMNs with neuraminidase does not affect the ability of asbestos to induce enzyme release from these cells. Asbestos induces release of glucose from glucose-loaded liposomes, and this effect can be inhibited by the polyanion poly-D-glutamic acid. The results are compatible with the view that positive charges play a decisive role in the interaction between PMNs and asbestos, and that the primary target of asbestos could be the lipid bilayer of the membrane. The interaction results in a permeabilized plasma membrane. When Ca2+ is present in the medium it moves into the cell and causes exocytosis of the granule enzyme lysozyme. Inhibition of cytotoxicity by polyanion may cause a diminished Ca2+-influx and hence inhibition of lysozyme release.