Narrow-bore high-performance liquid chromatography of phenylthiocarbamyl amino acids and carboxypeptidase P digestion for protein C-terminal sequence analysis

Abstract

Carboxypeptidase P digestion followed by narrow-bore high-performance liquid chromatography of phenylthiocarbamyl amino acids is employed for polypeptide C-terminal end group and sequence determination. Carboxypeptidase P digestion of polypeptides provides specific cleavage of protein C-terminal amino acids. The digestion offers the advantage that it can be carried out in either 10 mM sodium acetate or water at pH 4.0 in the presence of an enzyme activator, Brij-35. The narrow-bore high-performance liquid chromatography of all 20 phenylthiocarbamyl-amino acids has provided quantitative analysis at low picomole levels. This efficient and sensitive procedure is particularly useful for examining in vivo excision of protein C-termini and for verifying the integrity of various protein products produced by recombinant DNA techniques.