316 BIOCHEMICAL GENETICS 



DNP confers a yellow color upon the DNP-amino acid complex, which is useful in 

 following the fractionation and subsequently in identifying the N-terminal amino 

 acid by methods of paper or column chromatography. 111 - 777 In addition to the 

 terminal a-amino group, the <5- and e-amino groups of arginine and lysine, which 

 are not involved in the peptide linkage, will also react to form DNP complexes. This 

 must be considered in determining which labeled amino acid is actually in the terminal 

 position. Keil 698 has described a modification of Sanger's procedure for analysis of 

 microgram quantities. The dinitrophenylation, extraction, and hydrolysis can be 

 carried out in a single vessel, eliminating the possibility of loss of the peptide during 

 extraction of the DNP complex from reagents and degradation products formed during 

 the procedure. 



The free a-amino acid will also react with thioisocyanates (for example, phenyl- 

 thioisocyanate) to form a phenylcarbamyl-peptide, a principle applied by Edman 318 

 for the N-terminal analysis of proteins. In this complex, the terminal peptide bond 

 adjacent to that upon which the phenylcarbamyl substitution takes place is more 

 susceptible to acid cleavage than the other peptide bonds of the protein. Controlled 

 acid hydrolysis splits off the N-terminal amino acid in a phenylthiohydantoin form 

 soluble in 5 per cent NaHCOg, while the other peptide bonds of the insoluble peptide 

 are left untouched. The amino acid is freed from the phenylthiohydantoin complex 

 by alkaline hydrolysis and can be identified by paper or column chromatography. 



An important adaptation of N-terminal analysis has been its use in analyzing 

 the sequence of amino acids in polypeptide chains. The method of Edman is suited 

 for analysis of amino acid sequence through stepwise degradation of the polypeptide. 

 Ingram 638 has adapted the DNP-labelling method for stepwise degradation through 

 catalytic reduction of the DNP peptide. The sequence of the amino acids in the pep- 

 tides is established through identification of the amino acid released after each stepwise 

 degradation procedure. Hunt and Ingram 618 and Hill and Schwartz 579 have analyzed 

 the use of the two methods for studying the sequence of amino acids in peptide number 4 

 of human hemoglobins. 



With the use of the methods described above, the N-terminal amino acids and, 

 in some cases, the sequence of adjacent amino acids have been established for hemo- 

 globins of man, 581 ' 616, 667, 1053, 1054, 1168 ' 1196 horse, dog, cow, pig, goat, sheep, 

 rabbit, and guinea pig. 785 ' 987 ' 988 - 1156 - 1157 



Enzymatic methods for determining N-terminal residues with the use of leucine 

 aminopeptidase are under development. 580 Because of the rapid rate of the reaction, 

 it is difficult to obtain information on the sequence of the amino acids released. Further- 

 more, leucine aminopeptidase may not react on all native or even denatured proteins ; 

 for example, performic acid oxidation was required before the N-terminal residue of 

 albumin could be hydrolyzed by the enzyme. 



C-terminal analysis. — Procedures for the chemical analysis of C-terminal amino 

 acids have developed more slowly than those for N-terminal analyses. The method 

 developed by Akabori 3 and Ohno 960, 961 is performed by hydrazinolysis of the protein 



