Ribonuclease pepsin inactivated

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

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. 

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. 

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). 

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

Several studies with ribonuclease suggest that ( ertain peptide bonds of a given kind are more available to proteolysis than others (reviewed by Scheraga and Rupley, 1962). Thus, pepsin hydrolyzes one bond between residues 120 and 121 which leads to inactivation of the molecule (Anfinsen, 19.56). Subtilisin (Richards and Vithayathil, 1959) rapidly splits a single bond between alanine and serine at residues 20 and 21. Carboxypeptidase... 

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