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Modification of an Arginyl Residue in Pepsin by 2,3-Butanedione

From the ¹ From the Laboratory of Protein Studies, Oklahoma Medical Research Foundation and the Department of Biochemistry and Molecular Biology, University of Oklahoma School of Medicine, Oklahoma City, Oklahoma 73190

2,3-Butanedione (biacetyl) was found to modify an arginyl residue in porcine pepsin at pH 6.0, 25°. In an inhibition study, half-inactivation occurred after 2 hours of reaction, and maximum inactivation of 80 to 85% was reached after 24 hours. Amino acid analysis of the modified pepsin revealed that 1 arginine residue remained, indicating that 1 arginine residue had been modified. The loss of activity and loss of arginine occurred concomitantly during the course of reaction. The presence of substrates retarded enzyme inactivation, as well as arginine loss, at corresponding rates. Porcine pepsinogen treated with biacetyl in a pH 7.0 solution did not lose activity significantly, as shown by its proteolytic activity after subsequent activation by acid. Biacetyl-modified pepsin continued to be susceptible to specific esterification reagents: diazoacetyl-dl-norleucine methyl ester, p-bromophenacyl bromide, and 1,2-epoxy-3-(p-nitrophenoxy)propane.

Peptides from a chymotryptic digest of pepsin and of biacetyl-treated pepsin were separated by high voltage paper electrophoresis at pH 6.5. Two peptides were isolated, containing one each of the only 2 arginine residues of pepsin. The sequences of these peptides were ascertained from amino acid compositions and amino-terminal determinations. One of the peptides (A₁) from biacetyl-pepsin contained almost no free arginine. An additional spot, apparently representing modified arginine, was observed for the digest of Peptide A₁ in high voltage electrophoresis. These results indicated that the arginyl residue in Peptide A₁, located 12 residues from the carboxyl terminus of pepsin, is the site of biacetyl modification. This arginyl residue apparently does not directly participate in the catalysis of the enzyme.

Biacetyl was also found to inactivate other gastric proteases in similar fashion, i.e. human gastricsin, human pepsin, and bovine rennin.

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