Ovomucoid isolation

One popular strategy to isolate and identify the binding domain of a protein type CSP is to compare the retention and enantioselectivity behaviour of CSPs prepared with whole proteins and with isolated protein domains. Such a study has been performed by Pinkerton et al. [204) with turkey ovomucoid. Columns made from whole-turkey ovomucoid displayed chiral activity toward many racemates, whereas the fused first and second domain resolved only a selected number of aromatic weak bases. The first and second domains independently expressed no appreciable chiral recognition activity. The third domain, however, exhibited enantioselective protein binding for fused-ring aromatic weak acids, and glycosylation of this domain did not affect chiral recognition. 

Isolation from Different Strains (Varieties) of a Species. Very similar protease inhibitors have been Isolated from several different strains (varieties) of the same species or from similar species. In some cases, the inhibitors are very similar, in others they are quite different. Three examples will Illustrate this. Ovomucoids are probably found in all species of birds. 

Laskowski, M., Kato, 1., Ardelt, W. et al (1987). Ovomucoid third domain from 100 avian species isolation, sequences, and hypervariability of enzyme inhibitor contact residues. Biochemistry, 26, 202-21. 

C18H33NO15 503.456 Constit. of glycolipid from the spermatozoa of bivalve Hyriopsis schlegelii. Isol. from the acetolysis product of a sialogly-copeptide p obtd. by enzymatic hydrolysis of avian ovomucoid. 

LBI (Fraenkel-Conrat et al., 1952), proteinase inhibitor II from potato (Bryant et al., 1976 Huang et al., 1981) and chicken ovomucoid (see below). Substantial differences in stability have been reported for STI (Ellenrieder et al., 1980), trypsin-chymotrypsin inhibitor from chick pea (Belew et al., 1975), inhibitors from some other beans (Sgarbieri and Whitaker, 1982), peanut trypsin inhibitor (Tur-Sinai et al., 1972 Perkins and Toledo, 1982), proteinase inhibitor I from potato (Ryan, 1966 Huang et al., 1981), and chicken ovoinhibitor (see below). Although stabilities of the crude and purified inhibitors may differ, characterization of intrinsic stability requires isolation of the inhibitor protein. Effects of other factors on stability of the crude inhibitor may be difficult to define. 

Matsuda, T., Watanabe, K., and Nakamura, R. (1981T. Ovomucoid and ovoinhibitor Isolated from chicken egg white are limunologlcally cross-reactive Blochem. Blophys. Res. Commun. 110, 75-81. Matsuda, T., Watanabe, k., and Sato, V. (l981a). Independent thermal unfolding of ovomucoid domains. Blochim. Blophys. Acta 669, 109-112. 

Ovomucoid was isolated originally by Momer (1) from egg white by precipitation with weakly acidic alcohol after heat coagulation of other proteins. More recent isolation methods are based on fractionation with... 

The first fraction contains globulin, then follows crystallized albumin and finally amorphous albumins mixed with ovomucoids. Ovomucoid has the peculiar property of not coagulating when the slightly acid e solution is boiled, and can therefore be isolated. Globulins are insoluble in pure water but require a certain amount of salt before they will go into solution. The gold number of globulin lies between 0.02 and 0.05 while that of the ovomucoids lies between 0.04 and 0.08. 

The separation of proteids is made easier by the fact that specific liquids of the body contain only certain of the proteids. Thus egg albumin contains globulin, albumin and ovomucoid blood plasma contains fibrinogen, globulin and albumin. As an example of how the separations are carried out the isolation of certain members from the blood plasma may be cited. 

Beeley J.G. The isolation of ovomucoid variants differing in carbohydrate composition. Biochem. J. 1971,123, 399-405. 

Krysteva et aL (1973) have studied the environment of the most exposed tyrosine of chicken ovomucoid. The glycoprotein was treated with tetranitro me thane, and the nitrotyrosine peptide was isolated from the degraded fragments. The peptide which contained neither neutral sugar nor glucosamine was positive for sialic acid. The authors proposed that the sialic acid residue was linked to the hydroxyl group of tyrosine as an 0-glycoside. 

---