STRUCTURE OF INSULIN 



Thus for instance an unbroken line beneath PheVal indicates 

 that the peptide Phe Val had been detected. These sequences 

 account for all the residues of fraction B except for one leucine 

 and tyrosine and two phenylalanine residues. 



The chief obstacle to working out the complete amino acid 

 sequence from these small peptides was the fact that in frac- 

 tion B the less polar residues (phenylalanine, leucine, and 

 tyrosine) are grouped closely together and give rise on hydrol- 

 ysis to a large number of similar nonpolar peptides which are 

 particularly difficult to fractionate one from another. A 

 further obstacle with both fractions A and B was the great 

 lability of the bonds involving the amino groups of serine and 

 threonine residues, since these bonds were never found intact in 

 any peptide from a partial acid or alkaline hydrolyzate. 



In order to determine the complete sequence it was neces- 

 sary to study hydrolyzates obtained by the action of proteolytic 

 enzymes (21), which show an entirely different and much more 

 exacting specificity than does acid and give rise to a relatively 

 simple mixture of longer peptides containing up to about 15 

 residues. Such peptides could be separated by paper chromatog- 

 raphy although less satisfactorily than the smaller peptides, 

 and were studied by similar methods. Recently paper iono- 

 phoresis (13) has proved most useful for the fractionation of 

 larger peptides. In Figure 1 is shown a tracing of an iono- 

 phoretic fractionation of a peptic hydrolyzate of fraction B, 

 where the different peptides show a good separation and form 

 well-defined bands; this is frequently not the case with paper 

 chromatography. 



As an example we can consider the peptide Bp3 and see 

 what further information may be deduced from its composition 

 concerning the sequence of fraction B. It contained a phenyl- 

 alanine A^-terminal residue and the amino acids [CySOsH, 

 Asp, Glu, Ser, Gly, Val, Leu, His], of which the most significant 

 are aspartic acid and serine. Fraction B contains only one 

 aspartic acid residue which is present in the A^-terminal sequence 

 (sequence 1, Table III). This sequence must be intact in 



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