Ovalbumin structure

The crystal structure of native hen ovalbumin shows an intact reactive center loop in the form of an exposed a-helix of three turns that protrudes from the main body of the molecule on two peptide stalks. The ovalbumin structure includes four crystallographically independent ovalbumin molecules and the position of the helical reactive center loop relative to the protein core differs by 2-3 A between molecules. Although this shift is probably due to the different environments of the helices in the crystal lattice, it suggested that the reactive center loop is flexible in solution. Structural studies of serpins in various conformations have shown how the exceptional mobility of the serpin reactive center loop and their unique flexibility is essential for function. In contrary to inhibitory serpins, ovalbumin does not show evidence for a large conformational change following cleavage at its putative reactive center and appears to have lost the extreme mobility which is characteristic for its inhibitory ancestors. 

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

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 resolution of these columns for protein mixtures, however, was comparably poor. The peak capacity for human serum albumin was near 3 during 20 min gradient elution. Improvement has been reached by covalent binding of PEI (M = 400-600) onto a 330 A silica of 5 pm particle size [38], The peak capacities of ovalbumin and 2a -arid glycoprotein were 30-40 (tgradienl = 20 min). Enhanced peak capacity and resolution probably were due to the more diffuse structure of PEI coupled to silane moieties than that of strictly adsorbed on silica and cross-linked (see Sect, 2.2). Other applications of covalently adsorbed PEI are discussed in Sect. 4.1. 

Squaraine dyes 10b, 39a, 39b, 41a, 41c, 41d, and 41e were used to measure different proteins such as BSA, HSA, ovalbumin, avidin from hen egg white, lysozyme, and trypsin (Fig. 12) [58]. It is difficult to predict correlations between the dyes structures and the affinity or sensitivity of the dyes for different proteins. All squaraine probes exhibit considerable fluorescence increases in the presence of BSA. Dicyanomethylene-squaraine 41c is the brightest fluorescent probe and demonstrates the most pronounced intensity increase (up to 190 times) in presence of BSA. At the same time, the fluorescent response of the dyes 10b, 39a, 39b, 41a, 41c, 41d, and 41e in presence of other albumins (HSA and ovalbumin) is, in general, significantly lower (intensity increases up to 24 times). Dicyanomethylene-squaraine 41a and amino-squaraines 39a and 39b are the most sensitive probes for ovalbumin. Dyes 41d, 10b, and 41e containing an A-carboxyalky I -group demonstrate sufficient enhancement (up to 16 times) in the presence of avidin. Nevertheless, the presence of hydrolases like lysozyme or trypsin has only minor effects on the fluorescence intensity of squaraine dyes. 

R. Montgomery and his colleagues108 fractionated, on Dowex 50, the aspartamidoglycan from ovalbumin. The five components obtained were each subjected to exhaustive hydrolysis with a-D-man-nosidase and 2-acetamido-2-deoxy-/3-D-glucosidase, used separately. The results are shown in Table X. One way in which to explain these results would be to change the position of the extra hexosamine residue in 1, to give the alternative structure 2. 

Further evidence for the presence of /8-D-linked D-mannose in the inner core of ovalbumin was provided by the results obtained on use of purified enzymes from hen oviduct.20 a-D-Mannosidase released four residues of D-mannose from the molecule of a glycopeptide having D-mannose hexosamine L-asparagine ratios of 5 2 1, whereas /3-D-mannosidase had no effect thereon. /3-D-Mannosidase did, however, remove the remaining D-mannose residue. It would appear that the resistant core has structure 3. 

R. Montgomery and coworkers108 and Yamashina and coworkers72 advocated the general structure 4 for the group of aspartamidogly-cans obtained after proteolysis of ovalbumin. 

A slightly larger fragment containing five D-mannose and two hexosamine residues per molecule seems to be common to all of the glycopeptides that have been isolated from ovalbumin. A useful step forward would be to ascertain whether this fragment has an invariant structure. 

The differences in the rates of hydrolysis of various linkage types by a particular glycosidase can be used to provide information about this aspect of structure. Jack-bean a-D-mannosidase cleaves a-(l- 2) and a-(l- 6) linkages much faster than a-(l -> 3). Oligosaccharides, obtained by endo-N-acetyl-/J-D-glucosaminidase hydrolysis of ovalbumin, were subjected to acetolysis, which selectively cleaved the a-(l - 6) bonds. A tetrasaccharide isolated after this treatment was then incubated with jack-bean o-d-... 

The elution pattern in IEC results from the charge distribution on the folded chain. Therefore, IEC was used for indication, whether the native structure of the protein had been affected by previous RPC or not. Ribonuclease was found to retain its native structure, whereas bovine serum albumin, horse radish peroxidase, and ovalbumin were much altered through RPC on a C 18 column with a gradient water/ (ethanol-butanol 80 20) containing 0.012 M HC1 in both eluent components 59>. 

Tedford, L.A., Smith, D., and Schaschke, C.J. 1999b. High pressure processing effects on the molecular structure of ovalbumin, lysozyme and P-lactoglobulin. Food Res. Int. 32 101-106. 

Fig. 5.—Structure of Hen-ovalbumin Glycopeptides GP-IV and GP-V (Ref. 96), GP-III-B (Ref. 97), and GP-VI (Ref. 98). [GP-V is identical to that of Taka-amylase A glycan (see Fig. 4) GP-IV and GP-V are identical to GP-5 and GP-6 glycopeptides from ovalbumin, as described by Conchie and Straehan.99]...

Occasionally, the branchings are incomplete and the formation of the N-acetyl-lactosamine residues is only started in outline, as in the glycans of ovotransferrin (see Fig. 15), ovalbumin (see Fig. 16), and ovomucoid (see Fig. 18). In other instances, glycan structures such as have just been described are enriched with supplementary monosaccharide residues for example, the occurrence of disialyl groups [a-NeuAc-(2—>8)-a-NeuAc], and of /3-Gal-(l- 3) residues linked to the terminal galactosyl residues, has been demonstrated in different tissues and cell membranes,114,115 and in calf-thymocyte membranes,118 respectively. 

Fig. 16.—Structure of Hen-ovalbumin Glycopeptide of Oligomannosyl-N-acetyl-lae-tosaminie Type.97,117 (GP-III-A is identical to GP-4-B as described by Conchie and Stra-chan.99)...

---