104 



UNITY AND DIVERSITY IN BIOCHEMISTRY 



(d) Determination of the Amino Acid Sequence 



A number of methods allow the removal of an amino acid residue from 

 the N-terminal end of a polypeptide terminal residue : 



+ I 

 NH,-CH-CO-- 



R 



Such a method is that of Edman (1950) in which phenylisothiocyanate 

 in pyridine at pH 9-0 is used. The principle is as follows : 



NCS NH,CHCO— pept. 

 A R 



V 



+ 



Pyridine 



NHCSNHCHCONH— pept. 

 /\ R 



V 



Phenylisothiocyanate 



Phenylthiocarbamylpeptide (PTC-pept.) 



Anhydrous HCl 



V 



N-CS 



CO NH 



\ / 



CH 



I 



R 



+NH2 — pept. 



R 



Alkali 



- NH,-CH-COOH 



N-terminal amino acid 



The phenylthiocarbamyl derivative of the peptide when treated with 

 anhydrous HCl gives the phenylthiohydantoin of the N-terminal amino 

 acid which, when treated with Ba(0H)2 in alkaline solution, liberates this 

 terminal amino acid. 



This method is applied to the polypeptides obtained from proteins by 

 various means. It is possible to remove the N-terminal amino acids one 

 after the other and identify them while still keeping the rest of the chain 

 intact for a further shortening and further identification of the amino acids 

 obtained. There are also methods which allow amino acid residues to be 

 chopped off from the c-terminal end of the chain. 



To obtain these polypeptides from the protein, it may be hydrolysed 

 carefully with a 10 n mixture of hydrochloric and acetic acids for several 

 days at 37°, or hydrolysed with alkali or by the action of enzymes. 



A witness to the success of these methods is the determination, by 

 Sanger, of the amino acid sequence of a part (fraction B, one of the two 



