The Determination of the Order of Lysine‐containing

Abstract

A new diagonal electrophoretic technique for determining the order of the lysine‐containing tryptic peptides of a protein is described. The protein is converted into its trifluoracetyl derivative, digested enzymatically (or chemically), and the resulting peptides separated by paper electrophoresis. The paper is then treated with ammonia vapour, which re‐exposes the ɛ‐amino groups of the lysine residues, and submitted to a second electrophoresis at right angles to the first direction. Peptides containing lysine residues, together with the N‐terminal peptide of the protein, are found to lie off a diagonal formed by all other peptides, whence they may be readily purified. A study of these peptides enables the order of the lysine‐containing tryptic peptides in the protein to be deduced. The technique has been successfully tested with insulin. When the method was applied to porcine pepsin, the four tryptic peptides isolated were easily ordered and the carboxyl‐terminal sequence of the protein shown to be Arg‐Gln‐Tyr‐Tyr‐Thr‐Val‐Phe‐Asp‐Arg‐Ala‐Asn‐Asn‐Lys‐Val‐Gly‐Leu‐Ala‐Pro‐Val‐Ala. The three basic amino acid residues in the molecule are thus found clustering towards the C‐terminus of the polypeptide chain. A common ancestral gene for porcine pepsin and bovine (calf) rennin is suggested by the close homology between the C‐terminal sequences of the two proteins.