Chymotrypsin oxidation

The cysteinyl residue in the core was oxidized with performic acid according to the method of Hirs ( ) and the oxidized core was digested with chymotrypsin for 8 hours at room temperature in a pH stat at pH 8.9. Enzyme equal to 0.5% of the substrate by weight was added at 0 and at 2 hr. The chymotryptic peptides were also separated by Dowex 50-X4 chromatography. 

Increased understanding of reaction mechanisms in the 1940s and 1950s pinpointed general acid or base catalysis as likely to be of importance in many hydrolytic reactions. The imidazole nucleus in histidine was the obvious center in proteins to donate or accept protons at physiological pH. The involvement of histidine was shown by photochemical oxidation in the presence of methylene blue (Weil and Buchert, 1951) which destroyed histidine and tryptophan and inactivated chymotrypsin and trypsin. 

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 function, if any, of methionine sulfoxide residues in peptides or proteins is a matter of conjecture. There is no evidence that they are of any structural significance. Indeed, since various enzymes such as ribonuclease and chymotrypsin are either partially or completely inactivated by oxidation of the methionine residues (15, 16), one hesitates to suggest any functional role for the sulfoxide. However, a role in the maintenance of oxidation-reduction potential of a biological system, as suggested by Dent (3), is conceivable. 

Figure 16. Hydrolysis rate of bovine ribonuclease relative to that of performic acid-oxidized ribonuclease as a function of temperature (155). The follotving enzymes were used O, aminopeptidase , carboxy-peptidase A A, trypsin and , chymotrypsin.

Fig. 8. Purification of chain C by chromatography on Sephadex G-50 (79). Sepha-dex G-50 column (1.8 X 35 cm) equilibrated with 0.05 M HCl. Lyophilized aqueous extract of 30 mg of DFP-inhibited, performic acid-oxidized a-chymotrypsin (A4) is dissolved in 1 ml 0.05 M HCl, put into the column, and eluted (5 ml/hr) by 0.05 M HCl. Solid line, absorption of the fractions at 280 mu. Dotted line, absorption at 230 mil. Ordinates, optical density. Abscissas, volume of eluatein milliliters. A, chain A C, chain C.

Better results are obtained for chain C, which corresponds to the chymotrypsinogen COOH-terminal sequence, when aqueous extracts of oxidized a-chymotrypsin (A4) are chromatographed on Sephadex G-50 equilibrated with 0.05 M HCl (79). Figure 8 shows that three well-separated peaks emerge. The first one is an ill-defined mixture containing chains B and C. The second is chain C in an apparently pure form. The third is the small chain A which does not absorb at 280 m/t. Chromatographically purified chain C can be obtained in an over-all yield of 30%. It contains alanine as the single NH2-terminal residue and all amino acids except histidine and phenylalanine. 

Fig. 27. Change in activity of chymotrypsin as a function of tryptophan oxidation. (A) Treatment with NBS (B) treatment with NBA. From Viswanatha and Lawson (1961).

N-bromoacetamide, which is less reactive than NBS at pH 4.0 and consequently requires more time to oxidize tryptophan residues, similar results were obtained (Fig. 27, curve B). Extrapolation of the initial steep portion of the inactivation curve obtained with NBA (Fig. 26) gives a value for tryptophan destruction of about 1 mole per mole of protein. This might suggest the association of one of the seven tryptophan residues present in the chymotrypsin molecule with the maintenance of catalytic activity. A similar involvement of one tryptophan residue in the reaction of diisopro-pylphosphorofluoridate with a-chymotrypsin is suggested by comparison of... 

Reaction with DFP. Chymotrypsin preparations oxidized with NBS to produce varying degrees of inactivation were treated with DFP and their phosphorus content then determined. An enzyme sample which had lost more than 60 % of its original activity was still able to incorporate 1 mole of phosphorus (DIP-group) per mole of protein. Samples with enzymatic activity equal to 15 % of the initial value or less incorporated 0.6 mole of phosphorus per mole of protein. The data on the extent of phosphorus... 

Phosphorus Content of Oxidized Chymotrypsin (CRT) Samples Reacted with DFP ... 

Reaction of NPA. Chymotrypsin samples oxidized with NBS to different degrees of inactivation were treated with NPA or C -labeled NPA at pH 5.0 or at pH 6.0. The acetylation of the enzyme by NPA is retarded by the NBS oxidation of the protein. The rates of acetylation of various oxidized chymotrypsin samples at pH 5.0 paralleled their relative activities toward V-acetyl-L-tyrosine-Ethyl ester (ATE). However, the net release of p-nitrophenol, measured after the rate of its liberation had approached that of spontaneous hydrolysis, corresponded to a considerable acetylation... 

Data Summarizing the Reaction of Oxidized Chymotrypsin Samples with NPA at pH 5.0 ... 

Data Showing the Incorporation of C -Acetate by Oxidized Chymotrypsin Samples during the Reaction with C -Labeled NPA"- ... 

Similar results were obtained when oxidized chymotrypsin samples were treated with C Mabeled NPA at pH 5.0 for 1 hr and the C -content of the isolated acetyl enzymes were measured. The data obtained are given in Table XXVIII. 

The C Mabeled acetyl derivatives of various oxidized chymotrypsin samples were allowed to deacetylate at pH 8.0. It was found that the oxidized enzymes lost the acetate much slower than the unoxidized enzyme. 

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