Ovalbumin Molecules Research Articles

Immunochemical Studies on the Denaturation of Ovalbumin Stored with Glucose

ABSTRACTThe ovalbumin denaturation induced by Maillard reaction with glucose was investigated by immunochemical methods. The storage for either 2 or 10 days at 50°C and 65% relative humidity decreased the maximum immunoprecipitation to 90 and 80% of that by native protein, respectively. Ten day‐stored ovalbumin was separated into two fractions with antigenicity (fr‐II) and without (fr‐I) by the immunoaffinity chromatography of antiovalbumin‐coupled Sepharose 4B. The circular dichroism (CD) analysis showed that fr‐II, containing amino groups in almost the same amount as fr‐I, retained a large amount of ordered structure. Ovalbumin molecules were presumably not denatured by only the modification of amino groups with glucose in an early stage of Maillard reaction.

Either high-mannose-type or hybrid-type oligosaccharide is linked to the same asparagine residue in ovalbumin

After pepsin digestion, all of the carbohydrates in ovalbumin were recovered in two glycopeptides, Glu- Glu -Lys-Tyr-Asn(CHO)-Leu-Thr-Ser-Val and Glu- Glu -Lys-Tyr-Asn(CHO)-Leu-Thr-Ser-Val. Almond glycopeptidase released quantitatively oligosaccharides from the glycopeptides. The products from both glycopeptides contained both the high-mannose-type oligosaccharides and the hybrid-type oligosaccharides in the same ratio. Thus, either the high-mannose-type or the hybrid-type oligosaccharide is attached to the unique asparagine residue in the ovalbumin molecule.

A method for the preparation of protein-protein conjugates of predetermined composition

A novel procedure for the synthesis of well-defined protein-protein conjugates is described using ovalbumin (OA) and IgG as test proteins. This procedure involves the highly selective and rapid reaction of alkyl halide and sulfhydryl groups, which have been grafted, respectively, onto the proteins to be conjugated. Accordingly, iodoacetylated IgG, (ICH 2CO) nIgG, was prepared by the reaction of the ϵ-amino groups of IgG with the N-hydroxysuccinimide ester of iodoacetic acid (NHIA), the degree of iodoacetylation (n) being proportional to the concentration of NHIA. OA was reacted with S-acetylmercaptosuccinic anhydride (SAMSA) under conditions yielding, on the average, a monosubstituted derivative. Following removal of the protective S-acetyl group, the resulting -SH derivative of OA was reacted with (ICH 2CO) nIgG. The OA x-IgG conjugates so produced were characterized by gel filtration, specific radioactivity (using tritiated OA) and immunodiffusion. It was found that the average number of OA molecules coupled per IgG molecule could be controlled by varying the degree of iodoacetylation of IgG.

Determination of the number of cross-links in a protein gel from its mechanical and swelling properties.

AbstractThe relation between the chemical structure of a protein and the physical properties of a heat‐set gel of that protein has been investigated. The physical properties of the gel are determined by means of mechanical experiments in which the viscoelastic properties of the gel are determined in terms of the storage shear modulus, the loss modulus and the stress‐strain curve. The storage shear modulus defined the solid (elastic) character of the gel. The chemical structure of the protein and the nature of the solvent determine the nature and number of cross‐links in the gel. The cross‐links in gels formed by heating concentrated solutions of ovalbumin in 6M urea solutions were found to be disulfide bridges and the mechanical properties of these ovalbumin/urea gels approximated those of an ideal rubber. The latter finding enables one to calculate the number of cross‐links per ovalbumin molecule from the value of the storage modulus, using the classical theory of rubber elasticity. This theory, together with the Flory‐Huggins lattice model, can also be used to calculate the number of cros‐links per ovalbumin molecule from the swelling behavior of ovalbumin/urea gels. The number of cross‐links per ovalbumin molecule calculated from these two types of experiments are in mutual agreement and correspond with the number of thiol groups in ovalbumin. We conclude, thereforee, that theories of polymer physics can be used to relate the chemical structure of a protein to the physical properties of its gel.

Biological process of carbohydrate attachment to ovalbumin.

Dithiothreitol or glutathione, essential for amino acid incorporation into polypeptides in cell-free systems, inhibited glucosamine incorporation into ovalbumin, a glycoprotein containing about three residues of N-acetylucosamine and about five residues of mannose. However, the thiol was necessary for mannose incorporation into ovalbumin in cell-free system prepared from hen's oviducts. The two monosaccharides were incorporated into soluble ovalbumin (intracellular ovalbumin released from the polysomes, corresponding to ovalbumin A3 which contains no phosphate and probably no carbohydrate) present in cell sap which was prepared from homogenates of minced oviducts by ultracentrifugation. It was difficult to study the incorporation of the carbohydrates into ovalbumin in connection with synthesis of the protein using the crude cell-free system. The incorporation of glucosamine and mannose into various forms of ovalbumin was therefore studied using minced oviducts. It was concluded from the incorporation of the two radioactive monsaccharides and of amino acids into the extracellular, soluble and nascent protein fractions that all the carbohydrate moieties were not incorporated into the growing polypeptides bound to polysomes, but were incorporated into ovalbumin molecules released from polysomes after synthesis had been completed.

Reaginic antibody formation in the mouse: VI. Suppression of IgE and IgG antibody responses to ovalbumin following the administration of high dose urea-denatured antigen

The major antigenic determinants in ovalbumin molecules (OA) were lost following denaturation in 8 M urea. The urea-denatured antigen (UD-OA) failed to combine with anti-OA antibody, but was capable of priming mouse T cells specific for OA. BDF 1 mice primed with alum-precipitated OA were given three intravenous injections of 100 μg UD-OA at 3, 5, and 7 days after the primary immunization. The treatment with UD-OA suppressed both IgE and IgG antibody responses to OA. The same treatment of OA-primed animals with intravenous injections of OA resulted in suppression of IgE antibody response but enhanced IgG antibody response. Intravenous injections of either OA or UD-OA suppressed both IgE and IgG anti-DNP antibody responses of DNP-OA-primed animals but failed to suppress anti-hapten antibody responses to DNP-keyhole limpet hemocyanin. The effect of the treatment on helper T cells and B memory cells in OA-primed animals was studied by adoptive transfer experiments. The results showed that the OA-treatment as well as UD-OA-treatment suppressed the development of both helper function of T cells and B memory cells in the spleen, but the UD-OA treatment was more effective than OA-treatment for the suppression of B cell development. Possible mechanisms for the suppression of the development of immunocompetent cells were discussed.

Steroid hormone regulation of specific messenger RNA and protein synthesis in eucaryotic cells.

Evidence is presented that the induction of specific proteins in the chick oviduct by the steroid hormones estrogen and progesterone, involves a primary effect at the level of gene transcription. The intracellular levels of mRNA's which code for the synthesis of the egg-white proteins, ovalbumin and avidin, have been quantitated in a heterologous protein synthesizing system. It is demonstrated that these levels are directly dependent upon the inducing steroid, estrogen or progesterone, respectively. Ovalbumin mRNA has been purified to apparent homogeneity. This ovalbumin mRNA was then used as a template for the synthesis of a complementary DNA copy catalyzed by the enzyme reverse transcriptase which was isolated from avian myeloblastosis virus. This radioactively labeled complementary DNA was used to demonstrate, by means of DNA excess hybridization, that the ovalbumin gene is represented only once in each haploid genome of the chick cell. Next the complementary DNA copy of the ovalbumin mRNA was used as a genetic probe to determine the precise number of sequences of ovalbumin mRNA present at any one time after the administration of estrogen. It was demonstrated that the unstimulated chick contained no sequences of ovalbumin mRNA. Within a very short period of time after estrogen is administered the ovalbumin sequences begin to appear and reach a steady state level of 140,000 molecules per tubular gland cell. It could also be calculated that each ovalbumin molecule is probably translated some 50,000 times during its life which explains why ovalbumin comprises some 60% of the total protein in the oviduct cell. Following withdrawal of the oviduct from estrogen treatment, ovalbumin mRNA sequences again drop to undetectable levels. However, following a single injection of estrogen to these withdrawn animals, new ovalbumin mRNA sequences could be detected within 30 minutes. These data suggest that estrogen controls the activity of the ovalbumin gene via a pure transcriptional control mechanism. It is also demonstrated that the efficiency of the complementary DNA as a means of quantitating specific mRNA sequences is some 1,000 times more sensitive than the best available in vitro translation system. Finally, the efficacy of four popular translation systems is compared. It is suggested that for initial studies involving hormonal control of mRNA levels, the translation system derived from wheat germ is the simplest and most sensitive.

Investigation of Protein Hydration by Proton Spin Relaxation Time Measurements

Summary Spin-lattice relaxation times T1 in aqueous solutions of dialysed egg white were measured for concentrations between 4.5 and 9.3 wt. % in the magnetic field range corresponding to proton Larmor frequencies from 0.01 to 160 MHz. A strong dependence of T1 on the magnetic field begins at approx. 0.5 MHz. It can be described by a log-Gauss distribution of rotational correlation times in the solvation layer with the maximum at 1.52·10−9 sec. The greater part of the distribution lies below the correlation time for the reorientation of the ovalbumin molecule itself. This means that the adsorbed water molecules have some freedom of motion with respect to the protein molecule. The width of the distribution is somewhat dependent on the concentration. This paper also contains the results of neutron activation, spectrochemical analysis and EPR measurements, which show that the amount of paramagnetic ions present in samples of protein solutions cannot be responsible for the decrease of T1 with respect to pure water.

Studies on the denaturation of biological macromolecules by chemical carcinogens. 3. Optical rotatory dispersion and light-scattering changes of ovalbumin during denaturation and aggregation by water-soluble carcinogens.

Abstract 1. 1. Optical rotatory dispersion and light-scattering measurements have been used for an investigation of the protein-denaturing ability of the water-soluble carcinogens, dimethylnitrosamine, diethylnitrosamine, ethylcarbamate, and dioxane. 2. 2. It is found that each of these carcinogens produces a helix-coil transition in ovalbumin, as revealed by the change in b 0 of the Moffitt-Yang equation. The reduced, non-carcinogenic derivative of dimethylnitrosamine, dimethylhydrazine, is unable to produce this unfolding in the ovalbumin molecule. The helix content of ovalbumin in dioxane measured as a function of time indicates an unfolding and partial refolding of the protein. 3. 3. Calculations of molecular weight from the light-scattering measurements show the formation of aggregates consisting of 2–9 ovalbumin molecules, for the range of denaturant concentrations used here. The magnitude of the time-dependent refolding of ovalbumin in dioxane appears to be limited by the aggregation process, since the helix content of ovalbumin in concentrated dioxane solution is not as high as is generally observed for other proteins and polypeptides in this solvent. 4. 4. Alteration of hydrophobic bonding alone in ovalbumin cannot account for the denaturing ability of these carcinogens, rather the results support the proposal of hydrogen bonding between the agent and functional groups of the protein.

Effect of ions on the dielectric dispersion of ovalbumin solution

AbstractThe dielectric dispersion of ovalbumin solution was studied in the presence of various ions. It was found that ions such as tetraethylammonium, ammonium, Na, and K which are not bound to the protein had no effect on the dielectric increment or on the relaxation time. On the contrary, the addition of ions such as Cu, Ca, Mg, and H ions had a considerable effect on the dielectric increment as well as on the binding of the ions to the protein. These results indicate that ions do not affect the dielectric dispersion parameters of this protein unless they have specific interaction with the polar sites of the ovalbumin molecule. The electric polarization of protein solutions is discussed and it is suggested that the ion fluctuation process including the Maxwell‐Wagner effect is not necessarily the essential mechanism of dielectric polarization of isoelectric protein solutions.

The Nature of the Chelate Structures Formed by Copper(II) and Ovalbumin in the Biuret Reaction

A spectrophotometric study was made of the interaction, in alkaline solution, of copper(II) with ovalbumin and several ligands of low molecular weight at several different atomic ratios of nitrogen/copper. The results indicate that the absorbance and chemical reactivity of the ovalbumin-copper(II) complex cannot be attributed to the presence of one particular chelate structure. It is suggested that the ovalbumin-copper(II) complex is a heterogeneous mixture of chelate structures in which the copper(II) is bonded to oxygen (in the enolate form) and nitrogen atoms in the several different combinations possible within the same polypeptide chain. It is also suggested that the actual arrangement of bonds in any one chelate structure is probably determined by both the nature of the amino acid residues and the configuration of the ovalbumin molecule in the immediate vicinity.

Hydrolysis of Irradiated Ovalbumin by Pepsin

Solid ovalbumin has been irradiated at three doses, 6.5, 25, and 40 megarads, under high vacuum. The native and irradiated samples have been hydrolyzed at pH 1.4 by pepsin, after centrifugation of the aggregates, if necessary. The number of bonds broken per ovalbumin molecule has been estimated by comparing the rate of protein destruction with the rate of formation of NH(2) groups. Both rates increase very much with the dose, but the number of bonds broken decreases. Sedimentation measurements show a strong shape modification of the soluble fraction in the case of the 25 and 40 megarad samples. The increase in asymmetry is bound to the increase in the rate of attack on gamma-irradiated ovalbumin by pepsin. Infrared spectra of the aggregates show small difference from those of the native samples.

The conversion of ovalbumin into plakalbumin, as followed in the pH-stat

The limited proteolysis accompanying the transformation of ovalbumin into plakalbumin has been measured using the pH-stat. The reaction consists of at least two steps, the first step being the opening of a single peptide bond in a closed chain in the ovalbumin molecule, leading to the formation of an open intermediary protein form which has lost the ability of crystallizing in needles. In the further reaction this form is converted into plakalbumin.

Stepwise degradation of the peptides liberated in the transformation of ovalbumin to plakalbumin.

IN the transformation of ovalbumin to plakalbumin by a proteinase from Bacillus subtilis, a small amount of material containing non-protein nitrogen is released, which has been separated into three fractions, designated1 A, B, and C. Amino-acid analysis of these fractions suggests that A and B are peptides composed chiefly of glycine, valine, aspartic acid and alanine, in the ratios 1 : 1 : 1 : 3 and 1 : 1 : 1 : 1, respectively, while C appears to be alanylalanine. As pointed out before1, it is reasonable to suppose that the three peptide fractions arise all from the same segment of the ovalbumin molecule, represented by A. Accordingly, B and C should be identical with parts of A. The present investigation furnishes some evidence for this scheme.

The Biological Action of Ions and the Concentration of Ions at Surfaces

ABSTRACT It is pointed out that at the surface of a cell the absolute concentration of ions, and the ratios of the concentrations of the individual ions, are usually different from the corresponding quantities in the environmental fluid. It is suggested that the necessity for a Na : Ca ratio of the order of 50:1 in a physiologically balanced fluid is found because such a ratio in the environmental fluid produces a Na : Ca ratio of the order of unity at the cell surface. The concentrations of calcium and sodium ions at the surface of ovalbumin molecules are calculated. It is shown that calcium is bound (a) by electrostatic forces, (b) by formation of apparently unionized complexes with carboxyl groups. The complex formation obeys the law of mass action. The ratio, total Na:total Ca at the surface of various cells in physiological media is calculated from the electrokinetic potentials of the cells. The calculated ratio is of the order of unity. In two cases calculation of the maximum possible total Ca at the cell surface by two other independent methods gives values of the same order of magnitude as the calculation from electrokinetic potentials. It is shown that, where the toxic action of ions is due to complex formation with anions present at a surface, the logarithms of the equitoxic concentrations of the metals should be a linear function of the standard electrode potentials of the metals. Calculations show that the concentration of colloidal Fe at the surface of organisms in sea-water should be far greater than the concentration in the environment: it is suggested that marine plants derive their iron from this layer of adsorbed colloidal iron. A mechanism is suggested which provides for intense adsorption of drugs at specific surfaces.

On the pH at the surface of ovalbumin molecules, and the protein error with indicators

On the <i>p</i>H at the surface of ovalbumin molecules, and the protein error with indicators