THE REMOVAL OF 2-OXOACYL RESIDUES FROM THE N-TERMINUS OF PEPTIDES AND CYSTATIN IN NONDENATURING CONDITIONS

The N-terminal residue of a protein or peptide may be converted into a 2-oxoacyl group by non-enzymic transamination. This group may then be removed, to obtain the peptide chain shortened by one residue, by treatment with phenylene-1,2-diamine. Hitherto this scission has required a pH of 4-5, but we find that the reaction will proceed well at pH 7 in the presence of concentrated phosphate buffer. We describe a method using reverse-phase HPLC for determining the extent of scission in model peptides; this method also allows products to be isolated and identified. The new scission conditions have been tested by removing the N-terminal residue from cystatin, an inhibitor of cysteine peptidases; electrospray mass spectrometry was used to assess how this protein reacted.