STRUCTURE OF INSULIN 



(2), in which the free carboxyl groups are reduced to hydroxyl 

 groups and the resulting amino alcohols identified. When 

 applied to insulin the presence of alanine and asparagine C- 

 terminal residues was confirmed. 



Amino Acid Sequence in the Phenylalanyl Chain 



The results with the A^-terminal sequences showed for the 

 first time that the fractions A and B of the oxidized insulin were 

 essentially homogeneous preparations of single polypeptide 

 chains containing 20 and 30 residues, respectively. It thus 

 seemed that it would be worth while to investigate the peptides 

 produced on partial hydrolysis in order to determine amino 

 acid sequences within the chains (20). The method was 

 essentially that of Consden, Gordon, Martin, and Synge (3), 

 which made use of paper chromatography to separate the very 

 complex peptide mixture produced. 



Fraction B was subjected to partial hydrolysis with con- 

 centrated acid, and preliminary fractionation of the mixture 

 into less complex groups of peptides was carried out by iono- 

 phoresis, ion-exchange chromatography, and adsorption on 

 charcoal. The fractions thus produced contained up to about 

 20 different peptides, and could be further fractionated by two- 

 dimensional paper chromatography. The various peptide 

 spots were then cut out, eluted with water, and taken to dryness. 

 The residue was then hydrolyzed and the amino acids present 

 identified. To determine the order of amino acids in the 

 peptides it was necessary to identify the A^-terminal residues. 

 This was in general carried out by the DNP technique which 

 could be adapted to work on a small scale. In this way the 

 complete structure of dipeptides could be determined, and a 

 considerable amount of information about longer peptides 

 obtained. In certain cases the structure of higher peptides was 

 determined by degrading them further by partial hydrolysis to 

 dipeptides. With such methods 22 dipeptides, 13 tripeptides, 

 and 8 higher peptides were identified in the partial acid hydrol- 



441 



