Pepsin inactivation

Iodination of PIR (147) showed 1 residue buried, Tyr 25, and all others iodinated at least to the monoiodotyrosyl form. Pepsin-inactivated RNase also has only one abnormal tyrosyl by titration which is thus assumed to be 25. Iodination of RNase-S is very similar to RNase-A in the early stages (lift). Extensive iodination leads to dissociation of the protein and peptide components. Direct iodination of S-protein indicated that all 6 tyrosyl residues were accessible, in this sense comparable to urea-denatured RNase-A. Substantial structural changes must be involved for both S-protein and PIR if Tyr 97, in particular, is to become susceptible to attack (see Section IV,B,3). 

Bigelow (1960) has found that all six of the phenolic groups of performic acid-oxidized ribonuclease behave normally, which is to be expected since the oxidized protein is believed to be highly unfolded. More interesting is his finding that a pepsin-inactivated preparation of ribonuclease can be prepared which contains five normal phenolic groups and one buried one. 

Ribonuclease Pepsin-inactivated RNase has 1 anomalous Tyr Oxidized RNase has none anomalous Bigelow (1960)... 

For analysis of pepsinogen and pepsin, inactivated serum is needed as a supplementary substrate. It is prepared by pooling remainders of sera of healthy subjects, or at least of subjects in whose electrophoretic pic-... 

Figure 15.8. Inactivation of Gly2Cys/Leul67Cys mutant pepsin. Inactivation of Gly2Cys/Leul67Cys mutant and the effect of the oxidizing reagent. At pH 7.5, the oxidization did not stabilize the wild-type ( without oxidization and with oxidization), while oxidization of Gly2Cys/Leul67Cys stabilized the enzyme (O without oxidization and with oxidization). Each data point represents the mean of three determinations.

For ribonuclease, removal by pepsin proteolysis of only a tetrapeptide sequence at the C-terminus (Asp-Ala-Ser-Val) lead to an inactive enzyme and unstable structure (Anfinsen 1956 Taniuchi, 1970). When this shortened enzyme, the so-called des(121-124) ribonuclease or pepsin inactivated ribonuclease (PIR), is reduced, it cannot reoxidize to 3deld the native pairing of disulfide bonds (Taniuchi, 1970). Removal of six C-terminal residues to form RNase 1-118 also yields a structureless and inactive enzyme (Lin, 1970 Andria and Taniuchi, 1978). The importance of the C-terminal end to the folding of these two proteins was also emphasized by the data reported in Chapter 9. The information contained in the C-terminal sequence of the two nucleases appears to be crucial for their refolding. It was proposed that the polypeptide chain of nuclease and ribonuclease cannot achieve the native structure, during biosynthesis, until the termination of the polypeptide chain. For RNase even the N-terminal end seems to be important for folding. After removal by proteolytic action of subtilisin of the first 20 amino adds (Richards, 1958), the RNase S protein is unstable and cannot refold correctly when disulfide bridges are reduced. 

Inactivation due to digestive proteases. Therapeutic proteins would represent potential targets for digestive proteases such as pepsin, trypsin and chymotrypsin. 

Magnesium carbonate is most water soluble and reacts with hydrochloric acid at a slow rate. Magnesium hydroxide has low water solubility. It reacts with hydrochloric acid promptly. Magnesium trisilicate has low solubility and has the power to adsorb and inactivate pepsin and to protect the ulcer base. 

Emmons (1970) experienced significant inactivation when commercial pepsin and pepsin-calf rennet mixtures were diluted with high-pH, hard water 10 min before adding them to the cheese vat. Mickelsen and Ernstrom (1972) reported that mixtures of porcine pepsin and calf rennet were stable between pH 5.0 and 6.0, but that pepsin activity was lost from the mixture aboire pH 6.0. This loss was shown to be entirely due to pepsin instability. Below pH 6.0 chymosin activity was destroyed by pepsin. 

Ribonuclease Ti is fairly resistant to proteases. The threonine residue at the carboxyl terminal of the enzyme can be removed by carboxy-peptidase A without loss of activity (67). Leucine aminopeptidase does not release amino acids from the amino terminal (68). Ribonuclease Ti is not inactivated by trypsin or chymotrypsin in the presence of 0.2 M phosphate (69), which probably binds the enzyme and protects it from inactivation (67). Treatment of the enzyme with trypsin in the absence of phosphate inactivates it (67). Ribonuclease Tj is hydrolyzed by pepsin with progressive loss of activity (69). 

The diazomethyl ketone functional group was first observed to be an affinity label by Buchanan and co-workers who showed that the antibiotic azaserine, an O-diazoacetyl derivative, 9 inhibited an enzyme in the biosynthesis of purine by alkylation of a cysteine residue. 10 The acid protease pepsin was then observed to be inhibited by peptidyl diazomethyl ketones in the presence of copper ions with the resulting esterification of an aspartate residue. 11 Two peptidyl diazomethyl ketones, Z-Phe-CHN2 and Z-Phe-Phe-CHN2, were found to irreversibly inactivate papain, a cysteine protease. 12 Since these reports, many peptidyl diazomethyl ketones have been prepared primarily as inhibitors of various cysteine proteases. 7 Peptidyl diazomethyl ketones are also synthetic intermediates and have been used to prepare chloromethyl ketones (Section 15.1.3), 13 bromomethyl ketones (Section 15.1.3), acyloxymethyl ketones, 14 and (i-peptides. 15 A few peptidyl diazoalkyl ketones have been reported. 16,17 ... 

Hypertensin is soluble in alcohol, glacial acetic acid, phenol, and water, and insoluble in ether (61). Because it is inactivated by tyrosinase it probably contains a catechol or phenol group, and by amine oxidase, an amine group on an a-carbon atom (Figure 2). Hypertensin is inactivated by certain phenolic, catecholic, and amine oxidases, by pepsin, trypsin, chymotrypsin, and carboxypeptidase, and by hypertensinase found in plasma. The nature of hypertensinase is unknown, but it is probably not an oxidative enzyme. Because it is heat-labile, hypertensinase can be removed from blood and renin preparations by heating hypertensin itself is heat-stable. Lack of pure preparations of hypertensin has delayed its further chemical identification. 

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. 

The stomach environment is acidic as a result of HC1 secretion by the parietal cells. The acidic pH serves to denature many proteins, thus making them susceptible to proteolysis. The chief cells of the stomach produce pepsinogen, which is activated to pepsin by the HC1 (see Table 20.3). The optimum pH of peptic activity is around 2, and pepsin is inactivated at neutrality. Another stomach enzyme is rennin or chymosin, which is present in infants but not in adults. It removes a glycopeptide from milk-K-casein, disrupting the casein micelle and promoting milk protein coagulation and digestion. 

Figure 3. Inactivation of ribonuclease (0) and ribonuclease-dextran conjugate on treatment with pepsin at pH 2.4 and 37°C.

The first experimental criteria to be met in a spectrophotometric titration are that the absorptivities be time-independent, and reversible. This is so fundamental that it should be obvious, but it still must be stressed. Proteins not infrequently possess marked alkali-sensitivity, as shown by the inactivation of pepsin at pH 7, the hydrolysis of one particular amide linkage in a-corticotropin at pH 9 (Shepherd et al, 1956), and the irreversible inactivation of bacitracin at pH > 7 (Weisiger et al, 1955). 

Acid proteases are inactivated by active-site specific reagents, diazoacetylnorleucine ethyl ester and other diazo compounds, and epoxy (p-nitrophenoxy)propane. Covalently labelled aspartic acid peptides have been isolated from pepsin, chymosin (= rennin), and penicillopepsin. The peptides labelled with the diazo compounds have similar sequences and differ from the epoxy (p-nitrophenoxy)pro-pane labelled peptides. These results indicate two aspartic acids at the active site and suggest homology between the enzymes. The latter is confirmed by a comparison of the sequence data. Studies of the action of porcine pepsin and penicillopepsin on some dipeptides with free N-terminal groups show transpeptidation involving a covalent acyl intermediate. It is proposed that there are differences in the mechanism of action of pepsin which are determined by the nature of the substrate. 

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