286 Essays in Biochemistry 



several things in quite a clear-cut fashion. In the first place, it seems 

 justifiable to conclude that under the proper circumstances, proteolytic 

 enzymes will prove to be remarkably general and useful tools for 

 determining the structure of proteins. The principal theoretical draw- 

 back to their use, namely, that they catalyze rearrangements, seems 

 not to be operative, at least in the case of trypsin.* It is difficult to 

 conceive that the group of peptides shown in Figs. 3 and 4 could be 

 artifacts. Practically all of them were obtained in yields of from 

 50 to 90% of theory after only 3 hours of tryptic digestion. It hardly 

 seems credible that rearrangements could proceed so rapidly and so 

 completely in homogeneous solution. The absence in most of the 

 peptides of more than one residue of a basic amino acid also argues 

 against transpeptidation. Of course it cannot be assumed that, because 

 transpeptidation does not occur to a significant extent when trypsin 

 acts on ribonuclease, it will never occur. By the use of quantitative 

 procedures, however, it should be possible to insure against undetected 

 rearrangements. When the peptides can be separated quantitatively 

 and their amino acid composition determined quantitatively, there is 

 far less likelihood of being misled. In the final analysis, proof for the 

 validity of the results obtained with trypsin can only emerge from an 

 examination of the products formed by the action of other enzymes. 

 Such studies with ribonuclease are now under way. It can be said 

 already that chymotrypsin splits oxidized ribonuclease into twenty 

 to thirty fragments that can be separated on the Dowex 50-X2 columns. 

 About fifteen of these fragments are found in major quantities. Pepsin 

 also attacks oxidized ribonuclease but with the formation of more 

 peptides in poorer yield. f 



The work with tryptic digests has also demonstrated quite clearly 

 that columns of Dowex 50-X2 provide extremely effective means for 

 separating mixtures of peptides. The resolving power is great, and 

 even peptides containing twenty or more amino acid residues can be 

 handled without difficulty. Moreover, such columns possess the great 

 advantage that they can be scaled up readily to a size sufficient to 

 permit the isolation of enough of a peptide for subsequent structural 

 studies. In the present work, the columns have been operated in the 

 sodium form. Doubtless quite similar separations could be achieved 

 with the ammonium form of the resin and NH 4 OAc or formate as 



* The subject of the synthesis of peptide bonds by proteolytic enzymes has 

 been elegantly summarized by J. S. Fruton in a Harvey Lecture given November 

 17, 1955. 



t J. L. Bailey. 



