STRUCTURE OF INSULIN 



enzyme may become attached to the less polar parts of the sub- 

 strate and catalyze a more general hydrolysis in that area. 



The action on fraction B of the protease obtained from 

 Bacillus subtilis, which catalyzes the specific conversion of 

 ovalbumin to plakalbumin, has been studied by Tuppy (25). 

 These results are also shown in Figure 2. Clearly in its action 

 on fraction B it appears to be very nonspecific in contrast to its 

 action on ovalbumin. 



Amino Acid Sequence in the Glycyl Chain 



Although it contains fewer residues, the elucidation of the 

 structure of fraction A proved more difficult than that of fraction 

 B. In fraction B there are several residues such as aspartic acid, 

 serine, and arginine that occur only once in the chain and assist 

 greatly in the interpretation of results. Fraction A, on the 

 other hand, contains very few such residues and has relatively 

 large amounts of cysteic acid, glutamic acid, and leucine; 

 this made it necessary to obtain a higher proportion of larger 

 peptides in order to work out the complete sequence. 



Particular difficulty was encountered with the large number 

 of cysteic acid peptides, especially those containing two such 

 residues, which are very water-soluble, and could not be well 

 separated by paper chromatography. They could, however, 

 be fractionated by paper ionophoresis in acetic acid (pH 2.75), 

 for under these conditions the — SO3H but not the — COOH 

 groups are charged. Thus peptides free of cysteic acid residues 

 are basic owing to the positively charged amino group, whereas 

 those containing one cysteic acid residue are neutral and may be 

 separated as a group free from all other peptides ; those with two 

 are acidic and may be fractionated according to their rate of 

 migration towards the anode. More recently, the separation 

 has been carried out in a pyridine acetate buff'er at /?H 3.3, 

 where some fractionation of the individual cysteic acid peptides 

 is obtained. Figure 3 is a diagram of a fractionation of a partial 

 hydrolyzate of fraction A obtained under these conditions. 



447 



