280 Essays in Biochemistry- 



step, only 15 mg. of material would remain after fifteen steps. Clearly, 

 more information can be derived from 1 gm. of protein by other means. 



The most appealing approach to the determination of the sequence 

 of amino acids in a peptide chain consists of specific hydrolysis of the 

 chain at selected linkages, separation of the relatively few fragments 

 formed, followed by further specific cleavage of the larger fragments 

 when necessary, and direct structural analysis of the smaller fragments. 

 Although specific chemical methods would be ideal, at the present 

 time proteolytic enzymes appear to offer the greatest promise as ana- 

 lytical reagents for the selective hydrolysis of peptide chains.* Some, 

 such as trypsin, have an extremely sharp specificity. All operate 

 under mild conditions of pH and temperature, so that after hydrolysis, 

 amide linkages are still intact and non-amino acid moieties such as 

 carbohydrates or porphyrins are likely to remain attached to that 

 portion of the peptide chain to which they were originally joined. 

 Sanger employed trypsin, pepsin, chymotrypsin, and papain in his 

 work with insulin. The enzymes were used as ancillary tools, how- 

 ever, to establish sequences of amino acids in the neighborhood of 

 peptide bonds that were particularly labile to acid hydrolysis. More 

 recently, Bell and his associates 35 used trypsin, pepsin, and chymo- 

 trypsin as primary hydrolytic agents in their studies with ACTH. 

 Trypsin has been employed by Tuppy and Bodo 36 in work with cyto- 

 chrome c, by Gorini, Felix, and Fromageot who studied lysozyme, 37 

 and by Monier and Jutisz 3S who isolated some basic peptides from 

 protamines. When enzymes are employed as primary hydrolytic 

 agents, it is, of course, essential to eliminate the possibility that the 

 products isolated might have been formed as a result of rearrange- 

 ments catalyzed by the enzymes. Unless this can be done with some 

 assurance relatively early in the investigation, an enormous amount 

 of labor could be wasted proving the structure of artifacts. 



In beginning partial degradation studies with ribonuclease, a tryptic 

 hydrolyzate of the oxidized protein was chosen for the first investi- 

 gations. 27 - 39 From the classic specificity studies of Bergmann, Fruton, 

 and Hofmann, taken in conjunction with the analytical results shown 

 in Table 1 and the data of Anfinsen et al. on the N- and C-terminal 

 amino acids, it would be anticipated that fourteen peptides should be 

 found in a tryptic hydrolyzate. Nine of them should terminate in 

 lysine, four in arginine, and one, corresponding to the C-terminal frag- 

 ment, should be devoid of basic amino acids. Free lysine, from the 



* The possibility of using proteolytic enzymes in this way was clearly envisaged 

 by Max Bergmann and stated in a Harvey Lecture delivered by him in 1935. 



