On Determining the Chemical Structure of Proteins 285 



ACTH, for example, a similar bond was found by Bell 35 not to be 

 hydrolyzed by trypsin. 



Rechromatography of the next to last peak in Fig. 3 gave the two 

 peptides shown at the bottom of Fig. 4. One is a heptapeptide contain- 

 ing lysine found in good yield. The other, however, contains twenty- 

 four amino acid residues including two lysine and one argininc residue 

 and was obtained in low yield. Since all the lysine and argininc 

 residues in the molecule have already been accounted for, this peptide 

 must represent a combination of two other peptides. From its com- 

 position, it could be formulated as aspartylarginine (peptide 7) added 

 to peptide 9. Whether this peptide is an intermediate degradative 

 product or a rearrangement product cannot be determined for certain 

 at present, but, since the yield of aspartylarginine increases with time, 

 the former possibility seems more likely. 



The peptides characterized thus far account for 105 of the 126 

 amino acid residues in ribonuclease. Missing are four residues of 

 valine, three of proline and aspartic acid, two of alanine, isoleucine 

 and histidine, and one of glutamic acid, glycine, half-cystine, phenyl- 

 alanine, and tyrosine. Also missing is a peptide devoid of basic amino 

 acids that should arise from the carboxyl-terminal end of ribonuclease. 

 According to Anfinsen et al., 18 the last six residues in the molecule 

 are -met, tyr, ala, leu or ileu, phe, and val. It has not been possible 

 thus far to isolate such a carboxyl-terminal fragment from the Dowex 

 50-X2 columns. It may be that the "end piece" of ribonuclease is 

 particularly susceptible to the action of traces of chymotrypsin, which, 

 as has been noted above, may contaminate the trypsin. It is tempting 

 to assume that all the amino acids listed above as not accounted for 

 in the basic peptides should be assigned to the carboxyl-terminal end 

 piece, but only future work can decide the validity of this assumption.* 

 The balance sheet does prove, however, that isoleucine and not leucine 

 is present in the end fragment, because both leucine residues have been 

 found in peptides shown in Figs. 3 and 4. The existence of a residue 

 of methionine near the carboxyl end cannot be reconciled with the 

 analyses of the peptides reported above, because all four of the methio- 

 nines supposed to exist in the molecule have been accounted for. This 

 discrepancy will also have to be resolved by future work. 



Although the work on tryptic digests of ribonuclease is still far from 

 elucidating the complete structure of the molecule, it has demonstrated 



* A large peptide devoid of basic amino acids and believed to represent the 

 carboxyl-terminal segment has recently been isolated. Its composition is discussed 

 in ref. 39. 



