Ovomucoid groups

While it is impossible to make any precise statement regarding the structure of this blood group A polysaccharide until our present investigations are more advanced, it does seem possible that the structure of the alkali-stable carbohydrate residue is of a ramified type, bearing some general relationship to that deduced for ovomucoid, which, however, is much less resistant to hydrolysis than is this blood group A polysaccharide. 

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. 

D-GIucosamine, N-acetyl-, in blood group substances, IV, 45, 46, 51 in ovomucoid, IV, 52 —, N-acetyl-, diethyl thioacetal, III, 384 —, pentaacetyl-, diethyl thioacetal, III, 384... 

The amino groups of ovomucoid, lysozyme, and ovotransferrin were alkylated extensively (40-100%) with various carbonyl reagents in the presence of sodium borohydride. Monosubstitution was observed with acetone, cyclopentanone, cyclohexanone, and benzaldehyde, while 20-50% disubstitution was observed with 1-butanal and nearly 100% disubstitution was observed with formaldehyde. The methylated and isopropylated derivatives of all three proteins were soluble and retained almost full biochemical activities. Recently amine boranes have been shown to be possible alternative reducing agents for reductive alkylation... 

Other conversions to unnatural residues occur when most proteins are exposed to high pH (80, 81,82). The high pH causes a -elimination of a cystine (see Figure 16) or O-substituted serine or threonine, with the formation of a dehydroalanine or a dehydro-a-aminobutyrate. Such products are subject to nucleophilic attack by the e-amino group of a lysine to form a cross-linkage, such as lysinoalanine, or attack by cysteine to form lanthionine. Walsh et al. (81) have taken advantage of the formation of these cross-links to produce avian ovomucoids that have nonreducible cross-links and have lost the antiprotease activity of one of their two inhibitory sites (see Figure 17). 

Figure 19. Semilogarithmic plot for the loss of amino groups and trypsin-inhibitory activity in turkey ovomucoid by modification with 2,4,6-tri-nitrobenzenesulfonic acid. The results suggest that one amino group is essential for activity (51).

B Elimination has been used to show differences among the disulfide bonds in various proteins including the ovomucoids (54). The e-elimination reaction has also been used to replace the hydroxyl group of the essential serine residue of subtilisin with a sulfhydryl group (55). The thiolsubtilisin had a small fraction of the activity of subtilisin but it has been quite useful in mechanistic studies of the serine and sulfhydryl proteases. 

Fraenkel-Conrat H, Bean RS, Lineweaver H. Essential groups for the interaction of ovomucoid (egg white trypsin inhibitor) and trypsin, and for tryptic activity. J. Biol. Chem. 1949 177 385-403. 

Since the same type of amino acid in a protein can have very different degrees of reactivity, these differences can sometimes be used to classify them into fast and slow, and sometimes even intermediate, degrees of reactivity. When this can be done, there may be only a few amino acids of a particular type in one of these classifications. If one of the amino acids in a classification is essential for the function of the protein, then it may be possible to do a kinetic analysis relating the loss of activity to the modification of residues in that particular class. It is then frequently a simple procedure to determine whether there are one, two, or more amino acid side chains required for activity. This was used to relate losses of activities with numbers of essential amino groups in ovomucoids. Turkey and penguin ovomucoids were easily shown to have one essential amino group (Table V) (27). 

Duck ovomucoid inhibits two trypsins simultaneously. It was not possible to separate the fast and intermediate amino groups. 

Chicken ovomucoid has an arginine at the reactive site and therefore no essential amino group. 

Immobilised protein columns. A further group of chiral stationary phases has been developed by immobilising naturally occurring asymmetrical peptides, e.g. bovine serum albumin, ai-acid glycoproteins, ovomucoid and... 

There are examples of inhibitors which are specific for only one protease within a group. The best known examples Include the Kunitz soybean (Glycine max) inhibitor (14) and isoinhibitors I and II of the Great Northern bean (Phaseolus vulgaris)(15). Even these two examples are not clear cut as there is some small non-stoichiometric combination and inhibition of a-chymotrypsin. Chicken (16) and Japanese quail (17) egg white ovomucoids only inhibit trypsin. 

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