218 C. H. W. HIRS, WILLIAM H. STEIN & STANFORD MOORE [12 

 equipment of the type described by Spackman, Stein, and Moore. ^^ The 

 numbers in parentheses adjacent to each peak are the molar ratios of 

 each component, and it will be noted that the hydrolysate contains nearly 

 equimolar quantities of cysteic acid, aspartic acid, alanine, and valine. 

 Lysine was not eluted in these analyses ; however, its presence at the carboxyl- 

 terminal position could be inferred from the specificity of trypsin, and was 

 confirmed by other means, as will be evident presently. After the first appli- 

 cation of the Edman degradation, carried through with 2-5 micromoles of 

 the peptide, the quantitative analysis shown on the second line of Fig. 4 

 was obtained. The molar ratios of the components are still unity, with the 

 exception of aspartic acid, for which the value has fallen from about 1 to 

 0-23, thus revealing that aspartic acid or asparagine must be the amino 

 terminal residue. After the second application of the degradation, the ana- 

 lysis on the third line of Fig. 4 was obtained, which showed that the molar 

 ratios of both aspartic acid and valine are now low. Valine was thus re- 

 vealed to be the second amino acid, while the third amino acid was shown 

 to be alanine as a result of a further degradation, as indicated by the last 

 curve of Fig. 4. The degradation, when extended to a fourth stage, indi- 

 cated a loss of cysteic acid, which established the sequence aspartyl- or of 

 asparaginylvalylalanylcysteicyllysine. 



The yield of lysine obtained by acid hydrolysis of peptides that have 

 undergone reaction with phenyl-wo-thiocyanate is not quantitative and falls 

 off unaccountably as the number of steps of the Edman degradation is 

 increased. For this reason, analyses for lysine have not been included in 

 Fig. 4. An independent demonstration of the presence of lysine at the 

 carboxyl-terminal position in the peptide was furnished by hydrazinolysis, 

 which gave lysine in a yield of 70 per cent as the only free amino acid. The 

 evidence was now sufficient to reveal completely the sequence of the resi- 

 dues in the peptide. 



The final question was whether an aspartyl or an asparaginyl residue is 

 present at the amino-terminal position. Analysis revealed, that on acid 

 hydrolysis, one equivalent of ammonia was released per molecule of pep- 

 tide, indicating that the amino-terminal residue is probably asparagine and 

 not aspartic acid. This was independently confirmed when it was found that 

 the action of the leucine aminopeptidase of Spackman, Smith, and Brown ^^ 

 on the peptide gave asparagine, identified by ion-exchange chromatography, 

 in yields of 27 and 91 per cent after 1 and 8 hours respectively of hydrolysis 

 with the enzyme. In addition, two further peaks were present on the effluent 

 curve; one at the valine position, and another some 25 effluent ml. behind it. 

 They presumably corresponded to the peptides derived from the starting 

 material by the successive removal of asparagine and valine. Similar results 

 have been obtained with some of the other peptides examined when hydro- 

 lysis with either leucine aminopeptidase or carboxypeptidase was studied. 

 In addition to providing conclusive evidence for the presence of the amides 



