Subtilisin BPN'

In attempts to complete the amino acid sequence of subtilisin BPN', the diisopropylphosphoryl derivative was cleaved with cyanogen bromide, and the peptides were separated by gel filtration on Sephadex G-75. Four of the expected six fragments were isolated in pure form; the other two were purified together but could not be separated from each other. Tryptic digestion of the mixture of the two cyanogen bromide fragments produced six of the expected seven peptides. The remaining peptide was obtained by tryptic digestion of the diisopropylphosphoryl derivative of subtilisin BPN' followed by BrCN cleavage of the tryptic peptides. This procedure permitted the isolation of the peptide missing from the former procedure as well as the other expected peptides. This information combined with that previously obtained from the tryptic, chymotryptic, and peptic digests has provided the evidence to construct a unique amino acid sequence for the 275 residues in subtilisin BPN'. Proof of this structure is provided along with a discussion of some of the features of the sequence.