5] STUDIES ON PROTEIN MICROSTRUCTURE 85 



Strongly acidic portion was roughly fractionated on Dowex 2 and the indi- 

 vidual peptides were isolated by repeated paper chromatography. The in- 

 dividual cysteic acid peptides obtained from both proteins are listed in 

 Table 6.' It is evident at first sight that our partial hydrolysates of trypsin 

 yielded a far smaller number of peptides than the partial hydrolysates of 

 chymotrypsinogen; this may be due to differences in the extent of hydro- 

 lysis in the two cases. 



It is further evident from Table 6 that, taking in conjunction those pep- 

 tides which are certainly or probably connected, it is possible to make out 

 nine different sequences for the immediate environment of the cysteic acid 

 residues in chymotrypsinogen; this corresponds exactly to the number of 

 cysteine (half-cystine) residues thought to be present in the chymotrypsino- 

 gen molecule. It may also be seen that, although the number of cysteic acid 

 peptides isolated from the partial hydrolysate of trypsin is much smaller, 

 their structures conform very closely to four of the peptide sequences found 

 in the case of chymotrypsinogen. 



For the isolation of the peptides of arginine, histidine and lysine from the 

 partial acid hydrolysates of chymotrypsinogen and trypsin we used the basic 

 peptide fractions obtained in a five-compartment electrophoretic apparatus. 

 These were further subjected to electrophoresis in a multi-compartment 

 apparatus under conditions permitting the direct separation of a weakly 

 basic fraction composed predominantly of histidine-containing peptides, and 

 at the same time a rough fractionation of the strongly basic peptides of 

 arginine and lysine. The final isolation of individual peptides was achieved 

 by repeated paper chromatography in several solvent systems. 



The compositions or structures of the individual peptides isolated in this 

 manner are summarized in Table 7 f- ^^ the results show that we have suc- 

 ceeded in establishing the amino acid sequence in the immediate vicinity 

 of two of the four arginine residues present in chymotrypsinogen, and con- 

 firm a third sequence (leu.ser.arg.ileu.)* which had previously been estab- 

 lished from activation studies. The amino-acid sequence about the fourth 

 residue is probably val.arg.asp or val.arg.glu, since we found bound arginine 

 to occur in the neutral peptide fractions of the hydrolysate. Of the dipeptides 

 isolated from partial hydrolysates of trypsin, two correspond in structure 

 with sequences identified in chymotrypsinogen. 



Table 7 also shows the structures of the histidine peptides isolated in this 

 work.^ Here again we were able, to a fair degree of certainty, to establish 

 the immediate environment of both the histidine residues present in chymo- 

 trypsinogen. The peptides isolated from trypsin show agreement with 

 chymotrypsinogen only for one of the three histidine residues; it further 

 appears that the sequence phe.his in chymotrypsinogen may be replaced by 

 tyr.his in trypsinogen. 



* This sequence has been confirmed by enzymic hydrolysis just before this paper was 

 given. 



