10] ACTIVATION OF TRYPSINOGEN 171 



a peptide bond involving a more basic amino group, such as the e-amino 

 group of lysine, could not be detected by these measurements. 



Trypsinogen contains 16 peptide bonds which, on the basis of the speci- 

 ficity of trypsin, are potentially susceptible to hydrolysis (14 lysyl and 

 2 arginyl bonds). In order to exclude the possible opening, during activa- 

 tion, of a peptide bond involving the carboxyl group of arginine, acetylated 

 trypsinogen was prepared, by the use of acetic anhydride, and subjected to 

 ion exchange chromatography. There appeared to be an inverse relation 

 between the degree of acetylation and the degree of activation of tryp- 

 sinogen, suggesting that fully acetylated trypsinogen cannot be activated 

 at all. 



Whereas during activation the A^-terminal valine of trypsinogen is re- 

 placed by an isoleucine group,'^ the C-terminal sequence appears to be 

 unaffected during this process. This conclusion is based primarily on the 

 negative evidence that the C-terminal group of trypsinogen appears to re- 

 main unreactive, before and after activation, toward carboxypeptidase, 

 'basic carboxypeptidase' or toward hydrazinolysis. The lack of reactivity of 

 the C-terminal group in trypsinogen and activated trypsinogen is a structural 

 feature which is shared by chymotrypsinogen as well and which permits 

 various interpretations, as shown in Fig. 2. Structure 1 contains a peptide 



0=0 



I 



NH- 



HOOC-CH- 

 I 



NH 

 I 

 00- 



Hooc-r 



8 



CONHg 



Fig. 2. C-terminal structures which would yield negative results by enzymatic (carboxy- 

 peptidases) assay or by the hydrazinolysis method. 



bond between the C-terminal carboxyl group and an e-amino group else- 

 where on a side chain. Structure 2 involves a peptide bond between the 

 €-amino group of a C-terminal lysine and a side chain carboxyl group else- 

 where along the polypeptide chain. Structure 3 has a C-terminal cystine; 

 and structure 4, a C-terminal a-amide. All of these structures may be 

 expected to yield negative results by the enzymatic or chemical end-group 

 analyses previously mentioned. The lack of reactivity of activated trypsinogen 

 toward basic carboxypeptidase is to be contrasted with recent work of 

 Gladner, Folk, and Laki® who have reported that crystalline trypsin yields 

 approximately three equivalents of lysine when incubated with this enzyme. 

 Since, in the present experiments, end-group analysis was carried out on 

 freshly-prepared activation mixtures as soon as maximum tryptic activity was 

 reached, it seems likely that during the process of isolation of crystaUine 



