50 R. MARKHAM 



type which show a high degree of specificity or, at least, a specificity which 

 is fully understood. Outstanding, of course, is trypsin which breaks only 

 arginine and lysine peptide bonds. Thus, a polypeptide which had, say, 

 10 arginine and 3 lysine residues would be broken into 14 fragments, 10 

 containing a terminal arginine residue and 3 having a terminal lysine residue, 

 the basic amino acids being at the — COOH termination of the chains. 

 The last fragment will contain neither argmine nor lysine. Fragments 

 containing arginine can, of course, easily be detected by the specific Sakaguchi 

 reaction. Furthermore, the enzyme may be induced to split only the arginine- 

 containing linkages by the simple procedure of blocking the e-amino groups 

 of the lysine residues by carbobenzoxy groups which may later be removed 

 by means of HBr (Anfinsen et al., 1956). This simple trick has not been 

 exploited to the full, but of course will give some overlapping regions. Also, 

 it should be possible to regenerate the lysine residues and then apply trypsin 

 a second time. 



a-Chymotrypsin, which is also much used, has a specificity which is as 

 yet not fully defined, but it does give rise to a number of recognizable 

 fragments. Pepsin, which has also been used, is less promising in general. 

 Its splitting activity tends to be toward the aromatic amino acid linkages, 

 but is by no means specific for these only. It goes without saying that the 

 enzymes used for this type of work have to have an extremely high degree of 

 purity. This is often difiicult to obtain. 



b. Having been formed, the peptides must then be separated in a relatively 

 pure form from each other. This is the step which involves most of the labor. 

 Techniques used may involve electrophoresis, paper clu-omatography, ion 

 exchange chromatography or even countercurrent distribution, although 

 the latter has relatively low resolution and is extremely time-consuming. 



c. The individual small polypeptides must then be analyzed. For di- and 

 tripeptides the procedure is, of course, relatively simple, but for the larger 

 ones recourse must be had to more specialized techniques. One of the most 

 promising is the Edman (1950) degradation, which involves the successive 

 treatment of the polypeptide with phenyl isothiocyanate which reacts with 

 the free a-amino group to give a phenyl isothiocarbamyl amino group. On 

 treatment with anhydrous HCl the terminal amino acid residue is split off 

 as a phenyl thiohydantoin which absorbs ultraviolet light and which may 



^CS NH 



CO CH— R 



(I) 

 be identified, leaving the remainder of the polypeptide intact. Thus this 



