Hydrolysis by pepsin

Fig. 3.10. Mechanism of peptide bond hydrolysis by pepsin, an aspartic endopeptidase [2]...

The various procedures used to differentiate the albumoses and the peptones are all based on the method of fractional precipitation. Before considering how the course of hydrolysis by pepsin is effected, and in order to be able to follow all the changes that occur, it is necessary first to consider various reagents utilized for this study. We will first examine the effect of salts. 

For hydrolytic action on proteins pepsin works optimally at pH 1.8 to 2. Earlier experiments indicated that pepsin displayed its optimum peptidase activity at pH 4, but recently Baker has shown that, for many peptides, hydrolysis by pepsin takes place most rapidly at pH 2. 

Since FPIAs are conducted as homogeneous immunoassays, they are susceptible to effects from endogenous fluorophores and from intersample variations. Such problems and others due to the sample matrix are largely avoided by sample dilutions of several hundredfold. Low-affinity, nonspecific binding of tracers to sample proteins, when present in sufficiently high concentrations, can result in a falsely elevated polarization signal. Interference from sample proteins can be eliminated when warranted, by proteolytic hydrolysis with pepsin.(46)... 

The question at once arose whether this a-pyrrolidine carboxylic acid, or a-proline as Fischer termed it in 1904, was a primary product or a secondary product formed by the action of mineral acids upon other products, but its formation by hydrolysis by alkali and by the action of pepsin followed by trypsin decided that it was a primary product and therefore one of the units of the protein molecule. Sorensen, in 1905, suggested that it might arise from an a-amino-S-oxyvalerianic acid which he synthesised, but the fact that this amino acid has not yet been obtained by hydrolysis of protein and the above facts seem to exclude this possibility. 

Figure 1. Schematic representation of the relationships between proposed catalytic and inhibitory mechanisms. A. Postulated general acid-general base catalyzed mechanism for substrate hydrolysis by an aspartyl protease. The water molecule indicated is extensively hydrogen bonded to both aspartic acid residues plus other sites in the active site (see Reference 16 for details). Hydrogen bonds to water are omitted here. B. Kinetic events associated with the inhibition of pepsin by pepstatin. The pro-S hydroxyl group of statine displaces the enzyme immobilized water molecule shown in Figure lA. Variable aspartyl sequence numbers refer to penicillopepsin (pepsin, Rhizopus pepsin), respectively.

Similarly, lysine has been incorporated into gluten hydrolyzate and lysine, threonine and tryptophan have been individually incorporated into zein hydrolyzates. Lysine, methionine, and tryptophan were incorporated simultaneously into hydrolyzates of protein from photosynthetic origin. A very interesting application of this procedure involved the preparation of low-phenylalanine plasteins from a combination of fish protein concentrate and soy protein isolate by a partial hydrolysis with pepsin then pronase to liberate mainly phenylalamine, tyrosine, and tryptophan, which were then removed on sephadex G-15. Desired amounts of tyrosine and tryptophan were added back in the form of ethyl esters and a plastein suitable for feeding to infants afflicted with phenylketonuria was produced. 

Partial hydrolysis of the LF molecule by heat as well as by pepsin results in the formation of an antibacterial peptide referred to as lactoferricin, which exerts a much stronger antimicrobial effect than the intact molecule (Section III.A.5.). 

Such isopeptides also have been found naturally present in keratin (77,78,85) and in polymerized fibrin (79). Their quantitative determination requires an enzymic hydrolysis using pepsin, pronase, amino-peptidase, and prolidase as described by Cole et al. (135) followed by a chromatographic separation using an amino acid analyzer under very specific conditions (80). 

The rate of hydrolysis of egg albumin by pepsin exceeded that of soy proteins and decitraconylated yeast proteins (Figure 6). More than 90% egg albumin was hydrolyzed within 3 h of incubation whereas only 60% of the soy proteins and decitraconylated yeast proteins were hydrolyzed under the same conditions. Hydrolysis of egg albumin was not impaired by the presence of added citraconic acid (0.01M and 0.05M). So the hydrolysis of the citraconylated yeast proteins by pepsin was measured (see Figure 6). No differences occurred either in the rate or in the extent of hydrolysis compared with decitraconylated yeast proteins. More thorough nutritional studies and a safety evaluation of the citraconic acid must be conducted before either citraconylated or decitraconylated yeast proteins are used for human consumption. 

Figure 6. Rate of hydrolysis of citraconylated and decitraconylated yeast proteins by pepsin egg albumin (( ) soy proteins (O) decitraconylated yeast proteins ( ) citraconylated yeast proteins (A).

The present study indicates that the extracellular enzyme, pepsin, exhibits striking differences from its mammalian homologue with respect to optimum pH, Ea for catalysis, thermal stability, and substrate affinity. These data are interesting from the viewpoint of biological adaption at low temperatures, but they also provide some substance to our contention that enzymes from fish plant wastes can have sufficiently unique properties to justify their use over conventional sources of enzymes used as food-processing aids. The relatively low Eas for protein hydrolysis by fish pepsins indicate they may be especially useful for protein modifications at low temperatures. Alternatively, the poor thermal stability of the fish pepsins studied indicate that the enzymes can be inactivated by relatively mild blanching temperatures. The reality of this concept will have to await studies where the pepsins are used as food-processing aids. Such studies are currently underway in our laboratory. 

Tam, J.T., Whitaker, J.R. 1972. Rates and extents of hydrolysis of several caseins by pepsin, rennin, Endothia parasitica and Mucor pusillus proteinase. J. Dairy Sci. 55, 1523-1531. 

The improvement of the whipping properties of enzymatically modified soy proteins and casein has already been of use in the baking industry. For example, Gunther (25) has patented a method for producing these products by pepsin hydrolysis. 

An additional factor that could limit peptic hydrolysis is the inhibitory effect of a free a-amino group adjacent to a potentially susceptible bond. It has been found that aeylated dipeptides are split more readily by pepsin than corresponding unprotected dipeptides. Thus, amino-terminal aromatic residues or leucine would not be released readily by pepsin. The lack of significant hydrolysis of the amino-terminal leucyl bond in the peptide shown in Fig. 3 might be the result of the inhibitory effect of the a-amino group. 

Fiq. 4. Hydrolysis of casein by several proteinases. I. Hydrolysis of casein by trypsin (A), chymotrypsin (Q), or subtilisin ( ) followed by S. griseus protease (O)- Curves B, C, and D indicate the extent of hydrolysis by individual enzymes without addition of S. griseus protease. Curve A indicates the extent of hydrolysis when S. griseus protease is added to the hydrolyzate of one of the other proteinases after approximately 47 hr. II. Hydrolysis of casein by S. griseus protease followed by hydrolysis with trypsin, chymotrypsin, subtilisin, or pepsin. The latter enzymes were added to the protease hydrolyzate after approximately 47 hr. From Nomoto et al. (1960a,b). 

Proteins are hydrolyzed very slowly with storage in water at neutral pH. However, addition of proteases can increase the rate of hydrolysis about 10 billion times over the spontaneous rate. The chymotrypsin mechanism depicted in Figure 2.53 is shared by trypsin and elastase. These three proteases are members of a family called the serine proteases (named after Ser 195), Carboxypeptidase Aand pepsin catalyze peptide hydrolysis by different mechanisms and are not part of this family. 

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