CH. VIII.] TRYPSIN. 213 



opened, it may be necessary to add a little more toluol and to shake well before 

 returning it to store. 



Trypsin can also be obtained by extraction with dilute alcohol. The 

 material left over after the preparation of lipase (see p. 158) should be filtered. 

 The nitrate contains amylopsin and trypsin. The trypsin is more permanent 

 if i cc. of pure concentrated hydrochloric acid be added for every litre. If 

 amylopsin is required the acid should not be added. 



For the following experiments a i in 5 or even i in 10 

 dilution of the above extract can be used. 



257. Detection of Trypsin by the use of Calcified Milk. 



Measure 5 cc. of the prepared milk (see Ex. 252) into two test- 

 tubes labelled A and B, and place them in a water bath at 37 C. 

 To A add i cc. of the solution. Boil a little of the solution and add 

 i cc. of the cooled solution to B, using a clean pipette. Mix the 

 contents of the tubes separately, replace them in the bath and 

 observe at intervals. If trypsin is present the milk in A will be 

 precipitated or form a clot. The observation is valueless if the milk 

 in B also clots, as may happen with milk which has " turned sour," 

 or even with fresh milk if the fluid tested is very acid. 



NOTE. A positive result indicates the presence of trypsin, pepsin, rennin, 

 or other proteolytic enzyme. Further tests must be applied (Ex. 248, 256, 

 and 258). But it may be noted that trypsin will cause the clot to disappear 

 gradually, a phenomenon not obtained with the other enzymes. 



258. Detection of Trypsin by the use of casein solution. 



To 10 cc. of the casein solution (see below) add 2 cc. of the enzyme 

 solution, mix, label the tube C, and place it in the water bath at 37 C. 

 Boil a little of the enzyme solution, cool, and add i cc. to 5 cc. of the 

 casein. Label the tube D. At intervals of about 10 minutes 

 transfer about i cc. of C to a tube, and add i per cent, acetic acid, 

 drop by drop. If trypsin is present, it will be found that after a 

 certain interval, depending on the strength of the ferment, it is not 

 possible to produce a precipitate by the addition of acetic acid. 

 Should this stage be reached, confirm the result by obtaining a 

 precipitate of casein in D by careful acidification. 



NOTES. i. Casein solution. Weigh out i gram, of Hammersten's Casein 

 (which can be obtained from Casein, Ltd., Battersea, London, S.W.) into a 

 clean, dry beaker. Add about 20 cc. of distilled water and stir. Add 10 cc. 

 of o-i N. soda and stir well. Allow to stand for about 30 minutes, and make 

 the volume up to TOO cc. with distilled water. 



2. Casein is hydrolysed by trypsin in alkaline neutral or faintly acid 

 solution. The first product of hydrolysis is paracasein, which is precipitated 



