Enzymatic Digestion of C-Terminal 3 H-Labeled Peptides

Abstract

Selective tritiation of the C-terminal amino acids in polypeptide chains has recently been proposed by one of the present authors (1–5) as a new method for identifying the C-terminal amino acids in proteins.

Proteins are selectively tritiated at their C-terminal amino acids through racemization via oxazolone formation by the action of acetic anhydride and pyridine in a medium containing ³H₂O. The present paper describes the enzymatic digestion of C-terminal tritiated peptides which were readily obtained by the above method. This facilitates the detection of C-terminal fragments in enzymatic digests by radioactivity.

Our investigations were carried out with Scenedesmus ferredoxin and peptides derived from it during the course of structural studies of Scenedesmus ferredoxin, whose primary structure has recently been established [(6); also Sugeno and Matsubara, in preparation]. The results indicate the usefulness of tryptic and chrymotryptic digestion of the tritiated peptides in identifying C-termini.

Tryptic digestion of C-terminal tritiated peptide. As shown in Fig. 1, the peptide (C-V), one of the C-terminal fragments obtained by chymotryptic digestion of carboxymethylated Scenedesmus ferredoxin, had one lysine residue whose peptide linkage is susceptible to trypsin.

To protect the free amino group of lysine from acetylation which might occur during subsequent tritiation reaction, 0.337 μmole of peptide C-V was treated with F₃CCOSEt and 1 N NaOH at pH 9.8 as described by Goldberger and Anfinsen (7). This yielded the corresponding N-trifluoroacetyl peptide (TFA-C-V). Purification was carried out on a small Dowex 1 × 2 column equilibrated with a buffer containing 0.124 M pyridine and 0.003 M acetic acid by successive elutions with the same buffer and 30% acetic acid. Lyophilization of the fraction eluted by acetic acid gave a pure peptide, TFA-C-V (ninhydrin reaction: -, Pauly reaction: +), which was subjected to tritiation reaction by the method described by Matsuo et al. (2) as follows: Peptide TFA-C-V was dissolved in 0.1 ml (10 mCi) of ³H₂O and pyridine (0.2 ml), and acetic anhydride (0.05 ml) was added.