Spontaneous Deamidation of a Protein Antibiotic, Neocarzinostatin, at Weakly Acidic pH

Abstract

The amide content of neocarzinostatin (NCS), an antitumor protein, has been determined by analysing asparagine and glutamine in the Pronase-artunopeptidase M digests of tetra-S-carboxymethyl-NCS and carboxyl-modified NCS (modified with a water-soluble carbodiimide and [¹⁴C]glycine methyl ester). Preneocarzinostatin (PRE) was separated and purified from a crude NCS preparation by CM-cellulose column chromatography. PRE was found to contain one mole less asparagine than NCS, and asparagine was deamidated to aspartic acid in PRE. A time-dependent conversion of NCS to PRE at pH 3.2 at 4° or in 0.1 M acetic acid at 26° was studied in two ways; first, by quantitative determination of NCS and PRE by CM-cellulose column chromatography and second, by following the release of free NH₃ during dialysis in an air-tight container. Within experimental error, PRE was indistinguishable from NCS in amino acid content after acid hydrolysis, as well as in apparent molecular weight as determined by SDS-disc gel electrophoresis (10% acrylamide), and N- and C-terminal amino acid residues. Both NCS and PRE shared a common antigenicity as determined by Ouchterlony's agar diffusion method. Only a slight difference between the two in electro-phoresis on a cellulose acetate membrane and on a peptide map of the tryptic digest was demonstrated. PRE, however, was completely devoid of biological activity. In addition to the chromatographic difference, a conformational difference was observed by CD spectro-scopy, namely, an apparently looser structure of PRE was indicated by the shallowness of the trough in the 240–265 nm region. This interpretation was supported by the finding that digestions by Pronase were more extensive with PRE than with NCS. These results indicate an important role of the single asparagine residue (Asn 83) of NCS in the biological activity, which is evidently governed by the conformation.