table 



403 

 The results of the preceding experiments are expressed in the following 



Precipitates 



Proteolytic activity 



Trypsin. 



of precipitate. 



Yes 



Pos. 



Yes 



Pos. 



Yes 



Pos. 



Yes 



Pos. 



No 



Neg. 



Azine Base. 

 Dimethyl-diamino-tolu-phenazine hydrochloride 

 Dimethyl-diamido-phenazine hydrochloride 

 SafTranine, diamido-phenyl-toluazonium chloride 

 Dimethyl-diamino-phenyl-phenazoniiim chloride 

 Leuko saffranine 



Discussion. 



The precipitation of the proteolytic enzyme trypsin completely from solu- 

 tion by the azine bases suggests that a compound of the enzyme and the base 

 is formed. In the case of the azonium base, saffranine, the linkage would be 

 represented thus : — 



N 



h,nW\/nh 

 [/Ml 



The enzyme compound would alter the constitution of the latter compound 

 from the ortho- to the para-quinoid structure. This would institute a tautomeric 

 change and would be accompanied by a change in colour. The compound pro- 

 duced from saffranine (which is itself red in colour) is reddish-violet when 

 redissolved. This is possibly due to the occurrence of such a tautomeric change. 

 The analogous non-acid salt of saffranine, violet in colour, occurs in solutions 

 of moderately high hydrogen-ion concentration. 



Conclusions. 



It has been shown that the proteolytic enzyme trypsin is precipitated wholh 

 from solution by the azine bases. 



The nature of the groupings involved has been studied. It has been 

 demonstrated that the heterocyclic azine ring is functional in this precipitation. 

 The work is being extended. 



I wish to acknowledge the kindly interest and helpful suggestions of 

 Professor T. B. Robertson, and also the kindness of Professor H. M. Evans, 

 of the University of California, in supplying two of the azonium bases employed 

 in these experiments. 



Darling Laboratories, University of Adelaide, 

 May 10, 1923. 



