The Sequence of Amino Acid Residues in Bovine Pancreatic Ribonuclease: Revisions and Confirmations

Recent studies (l-3) have led to a revision of the published sequence of residues 11 to 18 in bovine pancreatic ribonuclease (4). In the experiments from this laboratory (1)) the use of improved conditions for the Edman degradation (Konigsberg and Hill (5)) provided an understanding of some of the limitations inherent in the procedure employed by Hirs, Moore, and Stein (4). It was noted that difficulties can be encountered particularly in the determination of sequences of peptides containing glutamine, asparagine, serine, and threonine residues. These observations have stimulated a new examination of the amino acid sequence of the whole molecule to determine whether such complications had affected any of the conclusions drawn in regard to other parts of the chain. Experience with the stepwise degradation has resulted in the following observations: (a) When the cyclization in the Edman degradation is performed in glacial acetic acid-anhydrous HCI, a newly liberated NH*-terminal glutamine residue cyclizes to a pyrrolidone carboxylic acid residue. Since such a peptide has no a-NH2 group, the degradation cannot proceed further. (b) When the process is followed by amino acid analysis of the residual peptide (the subtractive method), an incorrect conclusion can be drawn if any artifactual decomposition of amino acids occurs during hydrolysis, as has been observed for serine and threonine (1) and tyrosine (4). (c) These difficulties are avoided by the modified conditions of Konigsberg and Hill (5), in which the cyclization is performed at 25” in anhydrous trifluoroacetic acid and the residual peptide is purified by passage through a short column of Dowex 50-X2. (d) An internal asparagine or aspartic acid residue may form an imide in glacial acetic acid-HCI at 100” (1). Since the imide opens to give predominantly the P-aspartyl peptide, the stepwise degradation cannot proceed in good yield to the next residue in the sequence. This complication is avoided when the cyclization is performed at 25”. (e) The hydroxyl group of a serine or threonine residue is nearly quantitatively acetylated in acetic acid-HCl. When such an 0-acetylated residue becomes NHe