separation of ethyl acetate-extractable phenylthiohydantoin-amino acids by high-pressure liquid chromatography with organic solvent gradients have been modified to facilitate sequence determination of proteins labeled with several radioactive amino acids at a time.
Several laboratories
have recently determined the amino-terminal sequence of proteins which had been synthesized in vitro by translation of mRNA in a cell-free system (l-9). In most cases, translation was in the presence of 19 cold amino acids and a single radioactive amino acid. The radiolabeled protein was then subjected to consecutive Edman degradations. Discrete peaks of radioactivity were released from the radiolabeled protein after certain of the cycles of the Edman degradation. However, to determine amino acid residues for all of the positions in an amino-terminal sequence (e.g., of up to 50 residues in length) by this procedure may require as many as 20 separate syntheses, each of them with a different radioactive amino acid. An alternative approach has been taken (1) by translating mRNA in the presence of as many as 18 radioactive amino acids and by separating the phenylthiohydantoin (PTH) derivatives resulting from each Edman degradation by thin-layer chromatography. However, this approach was not wholly satisfactory, primarily because of difficulties in quantitating the data.
Recently high-pressure liquid chromatography (HPLC) has been introduced as an alternative to thin-layer or gas chromatography, commonly used for the analysis of PTHs. In this paper we describe our modifications of published procedures (10,ll) which resulted in an improved separation of PTHs.