THE BIOCHEMISTRY OF PLANT VIRUSES 49 



2. Determination of the Nature of the Chain Ends 



a. N-Terminal Residues. The N-terminal amino acid (that having a free 

 — NHg group) may be identified by one of a number of methods. That most 

 suitable for general use is the reaction of the peptide with 1,2,4-fluoro- 

 dinitrobenzene, which may be effected in aqueous solution near neutrality 

 (Sanger, 1945). The N-terminal amino acid residue is converted to a dinitro- 

 phenyl amino acid residue, which may be identified after hydrolysis, particul- 

 arly as most dinitrophenyl amino acids are yellow in color and most are 

 soluble in ether. It is, of course, necessary to take account of the e-dinitro- 

 phenyl derivative of lysine, which reacts even when not terminal. The reagent 

 may also substitute some groupings such as — SH which do not contain 

 nitrogen. 



Other methods which may be used are the acetylation of the free — NHg 

 groups, for example, by ketene, or the deamination of the former by HNOg, 

 when they yield a-hydroxy acid residues. 



6. C-Terminal Residues. The C-terminal residue, or that bearing a free 

 a-carboxylic acid group, can also be identified in a number of ways. The 

 simplest is the hydrazinolysis method of Akabori et al. (1952), in which the 

 peptide is treated with anhydrous hydrazine. During the reaction all the 

 carboxyl groups involved in peptide linkages are converted to hydrazides, 

 while the C-terminal amino acid is released unchanged. 



3. Determination of Sequence 



The determination of the amino acid sequence is, of course, one of the 

 ultimate ends in the determination of the structure of a protein. Its accom- 

 plishment is a matter of extreme difficulty, increasing as a power of the 

 number of amino acid residues in the protein. While theoretically it is 

 possible to degrade a protein from one end, removing amino acid residues 

 successively, practical considerations rule this out, and the practical approach 

 is as follows: 



a. The protein must be hydrolyzed, preferably in more than one way, 

 to give a number of small polypeptides. This may be accomplished by 

 chemical hydrolysis, with acid or alkah, or by enzymes. In this hydrolysis 

 care must be taken to avoid the possibility of the synthesis of structures not 

 already present in the protein. The hydrolysis of polypeptide chains into 

 easily recognizable fragments is a matter of considerable difficulty. It can 

 be shown that any method which does not have absolute specificity, or at 

 least a very high degree of specificity for a particular set of bonds, is not 

 likely to be of much use for a large polypeptide. This more or less excludes the 

 use of acid- or base-catalyzed hydrolysis for most work, and one is forced to 

 use enzymatic hydrolysis, with the inevitable possibility of synthesis con- 

 comitant with degradation. Also there are few enzymes of the endopeptidase 



VOL. II — 4 



