401 



Preparation of the Saffranine Precipitate from Crude Trypsin. 



Fifty grains of dried pancreas tissue was extracted with 500 cc. of water, 

 the reaction being adjusted to pH 78. The insoluble portion was then separated 

 by filtration. To the amber-coloured filtrate 500 cc. of '5 per cent, saffranine 

 was added, and the resulting precipitate allowed to flocculate for half an hour. 

 It was then separated by centrifugalization and the sedimented precipitate 

 washed twice with 5 per cent, saffranine. The excess of saffranine was then 

 removed by washing five times with dry acetone. The precipitate was then 

 dried over sulphuric acid at 40 deg. Cent. 



The precipitate produced in this manner is a reddish-violet finely-grained 

 powder. It emulsifies in water and goes partially into solution on adjusting 

 the reaction to neutrality. The solution is reddish-violet in colour and is 

 remarkably active in hydrolising protein substrates. 



The yield of precipitate was about 2 per cent, of the original pancreas 

 tissue employed. 



The precipitate is hydrolysed by dilute acids. Hydrochloric acid, in half 

 normal concentration, will decompose the precipitate and liberate the saffranine 

 as the dye hydrochloride, which may be extracted by butyl alcohol. 



On the examination of the precipitating base, saffranine, it will be observed 

 that there are two possibilities for the union of acidic groups. Saffrine has the 

 constitution diamino-phenyl-toluazonium chloride. 



N 



It has two amino groups free to exert their activity towards electro-positive 

 substances. The heterocyclic azine nucleus is also a decidedly reactive group. 

 So there exist two possible points of union for a substance that is predominantly 

 electo-positive. That trypsin is acidic in nature was shown by Bayliss,^®) who 

 demonstrated the migration of trypsin to the anode when subjected to the 

 influence of an electric current. 



With a view to ascertaining the nature of the linkage between the enzyme 

 and the saffranine molecule the activity towards trypsin of a series of similar 



(6) Bayliss, Wm., Jour. Biol. Chem., vol. i., p. 225, 1906. 



