Ovomucoids, protease

Encapsulation within an enteric coat (resistant to low pH values) protects the product during stomach transit. Microcapsules/spheres utilized have been made from various polymeric substances, including cellulose, polyvinyl alcohol, polymethylacrylates and polystyrene. Delivery systems based upon the use of liposomes and cyclodextrin-protective coats have also been developed. Included in some such systems also are protease inhibitors, such as aprotinin and ovomucoids. Permeation enhancers employed are usually detergent-based substances, which can enhance absorption through the gastrointestinal lining. 

About 20 families of protein inhibitors of proteases have been described.488 The egg white ovomucoids comprise one family. Turkey ovomucoid is a three-domain protein whose 56-residue third domain is a potent inhibitor of most serine proteases.455 489 The 58-residue pancreatic trypsin inhibitor490 is a member of another family of small proteins. A 36-residue insect (locust) protease inhibitor is even smaller.491... 

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. 

The concept of a proteolytic mechanism in the production of inflammatory manifestations is supported by the demonstration of anti-inflammatory activity in various protease inhibitors, e.g. the trypsin inhibitors of the pancreas, soya bean, ovomucoid and potato . It is interesting to note that the inhibitor from potatoes exerts its effect even after the inflammation is well established , suggesting that protease action is a continuing feature of inflammation and not simply an initiating process. Various esterase inhibitors (dyflos, quinine, quinidine and chloroquine) also reduce capillary permeability induced by heat and the permeability globulins . 

The histogram of the interface areas observed in a sample of 75 protein-protein complexes analyzed by Lo Conte etal. (1999) is shown in Fig. 8. In this sample, the majority of the complexes (70%) have interfaces in the range 1200-2000 A. This range includes 19 of 24 protease-inhibitor complexes, 18 of 19 antigen-antibody complexes, and 15 of 32 other types of protein-protein complexes in the sample analyzed by Lo Conte etal. (1999), who define a standard size interface as having = 1600 400 A. General characteristics of such interfaces are listed in Table IV two typical examples are shown in Fig. 6 the chymotrypsin-ovomucoid inhibitor and the FvD1.3-Fv E5.2 interfaces. A standard size interface... 

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. 

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. 

They are proteins of 28,000 daltons and contain 20% carbohydrate. They are quite similar in other properties as well. However, they can be quite different in their inhibitory properties against proteases. Chicken (16) and Japanese quail (17) ovomucoids inhibit only trypsin. Tinamou ovomucoid inhibits chymotryp-sin and subtilisin (66) and turkey ( 67) and penguin ( 68) ovomucoids inhibit trypsin, chymotrypsin and subtilisin. The reason for this difference among the ovomucoids is due to more than one binding site for proteases in the ovomucoid molecule. As shown by... 

Figure 4. Schematic representation of multiple binding sites for proteases, and Eg, on an inhibitor, I. In the case of duck ovomucoid, is trypsin and E ...

Molecular Heterogeneity. The last type of heterogeneity we shall discuss is that of molecular heterogeneity. This type of heterogeneity was mentioned under the section on inhibitors from different varieties of a species. It is well known that many of the inhibitors have more than one binding site for proteases, either for the same protease or for different proteases. Examples of this molecular heterogeneity are shown in Table V. In the case of the bird egg white ovomucoids, Laskowski et al. (69) have shown that... 

The distinct nature of these multiple binding sites are shown by (a) the simultaneous binding of more than one mol/mol of a given protease or of two or more different proteases (b) amino acid sequence work (69) and (c) by fragmentation of the inhibitor molecule into separate, active parts Cyanogen bromide has been used effectively to cleave the inhibitor at methionine residues to give active fragments. Examples include the Bowman-Birk soybean inhibitor (73), turkey ovomucoid (86), the three isoinhibitors of Brazilian pink beans (77), potato inhibitor Ila (60), and potato inhibitor Ilb (62) ... 

Pspsin (EC 3.4.23.1) a protease in the stomach of all vertebrates with the exception of stomachless fish (e.g. carp). Purified P. shows maximal activity at pH 1-2, but in the stomach the optimal pH is 2-4. Above pH 6, P. is inactivated by denaturation. It preferentially catalyses hydrolysis of peptide bonds between two hydrophobic amino acids (Phe-Leu, Phe-Phe, Phe-TyrT With the exception of protamines, keratin, mucin, ovomucoid and other carbohydrate-rich proteins, most proteins are attacked by P. The products of P. action are peptone, i. e. mixtures of peptides in the M range 300-3,000. P. is a highly acidic (pi 1), single chain phosphoprotein (327 amino acid residues of known primary sequence, M, 34,500), which is released from its zymogen (pepsinogen, 42,500) by autocatalysis in the presence of hydrochloric acid. 

Native ovoflavoprotein (49 kDa, pf=5.1) has, as does ovomucoid, certain antinutritional effects, as it inhibits serine proteases (trypsin, chymotrypsin and also microbial proteases) and has antiviral activity. Ovomacroglobulin (ovostatin) is an inhibitor of serine, cysteine, thiol and metalloproteases and shows antimicrobial activity. Some antinutritional effects are also seen in the basic glycoprotein avidin in raw egg white (relative molecular weight of the monomer is 15.6 kDa). It contains four identical subunits (pf = 9.5), each of which binds one molecule of biotin to give an unavailable complex. However, the denatured avidin, for example in hard-boiled eggs, does not interact with biotin. The interaction of riboflavin with flavoprotein (32 kDa, pf = 4.0) has, on the contrary, a positive influence on vitamin stability. Cystatin acts as cysteine protease inhibitor, and shows antimicrobial, antitumor and immunomodulating activities. 

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