Ovalbumins

Ovalbumin. — Gly copeptides prepared from ovalbumin have been used as substrates for investigation of the specificity of /3-D-2-acetamido-2-deoxy-glucosidase from fig latex.  

The nature of the cross-linking of proteins by glutaraldehyde has been investigated using ovalbumin. Several quaternary pyridinium compounds were isolated from acid hydrolysates of glutaraldehyde cross-linked ovalbumin and their structures were confirmed by synthesis. 

Miscellaneous Glycoproteins. Bovine lutrophin, bovine thyrotrophin, and human chorionic gonadotrophin preparations with carbodi-imide-mediated covalently cross-linked sub-units have been isolated in high yield and characterized by c.d. spectroscopy, radioligand receptor assays, and hormone-specific bioassays. The c.d. spectra of the native and cross-linked hormones did not differ much, suggesting that there is little conformational change on cross-linking. 

The reaction of l-ethyl-3(3-dimethylaminopropyl)carbodi-imide with bovine lutrophin has been studied and the reaction conditions have been investigated. Spectrophotometric measurements, tryptic peptide maps, and sodium dodecyl sulphate-urea polyacrylamide gel electrophoresis banding patterns were obtained and compared for native and cross-linked derivatives of the glycoprotein hormone.  

Gonatas, C. Harper, T. Mizutani, and J. O. Gonatas, J. Histochem. Cytochem., 

Ovalbumin is the principal phosphoprotein in egg white (Chapter 12.4). It has a molecular weight of -45,000 and contains about 0.1% P in the form of phosphate groups bound to serine units. Three 

Thr-Val-GIn-Val-Thr-Ser-Thr-Ala-Val-OH FIGURE 10.24 Bovine K-casein—amino acid sequence. 

FIGURE 10.25 Bovine P-casein—amino acid seqnence. 

FIGURE 10.27 3-Lactoglobulin-amino acid sequence. May be replaced by other amino acids in genetic variants. Few, if any residues are phosphorylated. 

FIGURE 10.28 Ovalbumin—part amino acid sequence. 

Ovalbumin.—Structural studies of ovalbumin gjycopeptides I and II have been performed using ]8-D-2-acetamido-2-deoxyglucosidase.  

Using the DNP method it has been shown that ovalbumin has no A -terminal residue (Desnuelle and Casal, 1948 Porter, 1950a). Either the amino groups at the end of the chains are masked by the carbohydrate moiety or the protein contains one or more cyclopeptide units. 

This is the main albumen protein, crystallized by Hofmeister in 1890. It is a glycophospho-protein with 3.2% carbohydrates (Table 11.5) and 0-2 moles of serine-bound phosphoric acid per mole of protein (ovalbumin components A3, A2 and Ai, approx. 3, 12 and 85%, respectively). 

Ovalbumin consists of a peptide chain with 385 amino acid residues. It has a molecular weight Mr = 42,699 and contains four thiol and one disulfide group. The phosphoric acid groups are at Ser-68 and Ser-344. During the storage of eggs, the more heat-stable S-ovalbumin (coagulation 

Ovalbumin is relatively readily denatured, for example, by shaking or whipping its aqueous solution. This is an interphase denaturation which occurs through unfolding and aggregation of protein molecules. 

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Albumins. Soluble proteins both in water and in dilute aqueous salt solutions found in all living tissue. Typical albumins are ovalbumin from eggs and lactalbumin from milk. 

The elution volume, F/, and therefore the partition coefficient, is a function of the size of solute molecule, ie, hydrodynamic radius, and the porosity characteristics of the size-exclusion media. A protein of higher molecular weight is not necessarily larger than one of lower molecular weight. The hydrodynamic radii can be similar, as shown in Table 4 for ovalbumin and a-lactalbumin. The molecular weights of these proteins differ by 317% their radii differ by only 121% (53). 

The sulfur amino acid content of soy protein can be enhanced by preparing plasteins from soy protein hydrolysate and sources of methionine or cystine, such as ovalbumin hydrolysate (plastein AB), wool keratin hydrolysate (plastein AC), or L-methionine ethyl ester [3082-77-7] (alkaU saponified plastein) (153). Typical PER values for a 1 2 mixture of plastein AC and soybean, and a 1 3 mixture of alkah-saponified plastein and soybean protein, were 2.86 and 3.38, respectively, as compared with 1.28 for the soy protein hydrolysate and 2.40 for casein. 

The setpin fold comprises a compact body of three antiparallel p sheets, A, B and C, which ate partly coveted by a helices (Figure 6.22). In the structure of the uncleaved form of ovalbumin, which can be regarded as the canonical structure of the serpins, sheet A has five strands. The flexible loop starts at the end of strand number 5 of p sheet A (plS in Figure 6.22), then... 

Figure 6,22 Schematic diagram of the structure of ovalbumin which illustrates the serpin fold. The structure is built up of a compact body of three antiparallel p sheets,...

A, B, and C, surrounded by a helices. The polypeptide chain is colored in sections from the N-terminus to facilitate following the chain tracing in the order green, blue, yellow, red and pink. The red region corresponds to the active site loop in the serpins which in ovalbumin is protruding like a handle out of the main body of the structure. (Adapted from R.W. Carrell et al.. Structure 2 257-270, 1994.)... 

The flexible loop region in the active form of antithrombin (Figure 6.23a) is in the same general position as in ovalbumin but the first few residues form a short sixth p strand in p sheet A inserted between strands pS and pis. Furthermore there is no a helix in the loop which is extended outside the main body of the molecule, ready to be inserted into the active site of thrombin. 

Stein, P.E., et al. Crystal stmcture of ovalbumin as a model for the reactive centre of serpins. Nature 347 99-102, 1990. 

FIGURE 2.15 Influence of the pore size of Sephacryl HR on the separation of proteins of various molecular mass. The protein mixture is composed of ferritin, aldolase, ovalbumin, and chymotrypsinogen A. [Reproduced from Hagel et al. (1989), with permission.]... 

FIGURE 4.29 Relation between molecular weight of lipoproteins and elution volume for combination GFC columns. Column 7.5 mm i.d. X 60 cm. Sample Chylomicron, VLDL, LDL, HDLj, HDL3, albumin, and ovalbumin. Elution 0.1 hA Tris—HCI buffer (pH 7.4). Flow rate 1.0 ml/min. 

S mg), dimer (peak I) and monomer (peak 2), ovalbumin (S mg) (peak 3), and cytochrome c (3 mg) (peak 4) was loaded onto a Fractogel EMD BioSEC column (600 X 16 mm) with a bed height of 600 mm. PBS (pH 7.2) was used as the eluent at a flow rate of I ml/min the sample volume was O.S ml. (B) The same protein sample as in A was injected onto a column of identical dimensions packed with unmodified Fractogel HW 6S. Without the tentacle modification the base matrix displays only a poor resolution of the test mixture. 

FIGURE 7.4 Separation of a standard protein mixture on a Fractogel EMD BioSEC-column (600-16 mm) after incubation with 30% acetonitrile. The sample contained BSA ( ), ovalbumin ( ), and cytochrome c (A) (sample volume 500 ftl flow rate 1.0 ml/min). No significant shifts of the retention times and no loss of the resolution were observed even after 900 hr of exposure. 

FIGURE 7.6 Effect of column length on the separation efficiency. Two different Fractogel EMD BioSEC columns (A 600 X 16 mm, B 1000 X 16 mm) were tested using BSA, ovalbumin, and cytochrome c (S/S/3 mg/ml) as sample (20 m/VI sodium dihydrogen phosphate, 300 m/VI NaCI, pH 7.2 0.5 ml/min). Better resolution can be achieved using longer columns. 

The chromatogram of the protein mixture should show the partial separation of serum albumin and ovalbumin with a trough of at least 30% of height between their peak signals and baseline separation between ovalbumin and cytochrome c. If present in the sample, the dimeric form of serum albumin should also appear as an individual peak signal before elution of the monomeric form. 

FIGURE 7.10 Dependence of the resolution on the sample volume. A preparative Superformance column 1000-200 (bed volume 20 liters) packed with Fractogel END BioSEC (S) (bed height 63 cm) was loaded with 60 ml (top) and 300 ml of a mixture of bovine serum albumin (5 mg/ml), ovalbumin (5 mg/ml), and cytochrome c (3 mg/ml) (bottom) (20 m/VI sodium phosphate buffer, 0.3 M NaCI, pH 7.2 flow rate 100 ml/min corresponding to 19 cm/hr). When the sample volume is 300 ml the separation efficiency for BSA and ovalbumin is similar. Thus the column can be loaded with larger sample volumes, resulting in reasonable separations. 

FIGURE 7.17 Separation of a complex mixture on Fractogel EMD BioSEC (S) with a column dimension of 1000 X 50 mm (Superformance glass column). The sample contained ferritin (I), immunoglobulin G (2), transferrin (3), ovalbumin (4), myoglobin (5), aprotinin (6), and vitamin B, (7). Five milliliters of the mixture was injected onto the column at a flow rate of 3 ml/min (eluent 20 mAI sodium phosphate buffer, 0.1 M NaCI, pH 7.2). 

FIGURE 8.12 Effect of pore diameter on SEC of standards (nondenaturin > mobile phase). Nondenaturing" refers to the effect on the stationary phase. Most iarge proteins were in fact denatured by this mobile phase (which was optimized for use with peptides, not proteins). Accordingly, it was necessary to use polyacrylamide to demonstrate the approximate range and position of Vo under these conditions. The polyacryiamide standards both eiuted at V with the 300-A coiumn (not shown). Columns and flow rate Same as in Fig. 8.11. Mobile phase Same as in Fig. 8.1. Sample key (B) Ovalbumin (43,000 Da) 0) polyacrylamide (1,000,000 Da) (K) polyacrylamide (400,000 IDa) (L) low molecular weight impurity in the polyacrylamide standards. Other samples as in Fig. 8.11. 

Loading capacities in size exclusion chromatography are very low because all separation occurs within the liquid volume of the column. The small diffusion coefficients of macromolecules also contribute to bandspreading when loads are increased. The mass loading capacities for ovalbumin (MW 45,000) on various sizes of columns can be seen in Table 10.5. The maximum volume that can be injected in size exclusion chromatography before bandspreading occurs is about 2% of the liquid column volume. The maximum injection volumes for columns of different dimensions can also be seen in Table 10.5. 

Storage proteins Ovalbumin Casein Zein Phaseolin Eerritin... 

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