12 PHYSIOLOGICAL CHEMISTRY 



H. PROTEASES 



1. Preparation of Gastric Protease. 1 Treat the finely comminuted mucosa of 

 a pig's stomach with 0.4 per cent hydrochloric acid and extract at 38C. for 

 24-48 hours. The filtrate from this mixture constitutes a very satisfactory acid 

 extract of gastric protease (see page 143). 



2. Demonstration of Gastric Protease. Introduce some protein material 

 (fibrin, coagulated egg-white, or washed lean beef) into the acid extract of gastric 

 protease prepared as above described, 2 add an equal volume of 0.4 per cent 

 hydrochloric acid and place the mixture at 38C. for 2-3 days. Identify the 

 products of digestion according to directions given on page 144. 



Carmine-fibrin may also be used in this test. This is prepared 

 by running fibrin through a meat chopper washing carefully and placing 

 in a J^ per ( cent ammoniacal carmine solution (very little excess am- 

 monia should be present) until the maximum coloration of the fibrin 

 (a dark red) is obtained. The fibrin is then washed in water and water 

 acidified with acetic acid. It is preserved under glycerol. 



To 15 c.c. of the solution to be tested add a small amount of the 

 carmine fibrin and allow to digest at room temperature. Digestion will 

 be ^hown by the setting free of carmine with coloration of the solution. 

 This is a delicate test for pepsin. A control should be run using acid of 

 same strength as that of enzyme solution tested. 



3. Preparation of Pancreatic Protease. 3 A satisfactory extract of this 

 enzyme may be made from the pancreas of a pig or sheep according to the direc- 

 tions given on page 192. 



4. Demonstration of Pancreatic Protease. Into an alkaline extract of pan- 

 creatic protease, 4 prepared as directed on page 192, introduce some fibrin, coagu- 

 lated egg-white or lean beef and place the mixture at 38C. for 2-5 days. 6 At 

 the end of that period separate and identify the end-products of the action of pan- 

 creatic protease according to the directions given on page 192. 



Congo-red fibrin may be used in this test. This may be prepared 

 by placing fibrin in faintly alkaline congo-red solution and heating to 

 8oC. The fibrin is then washed. A small amount of this colored 

 fibrin is placed in the slightly alkaline solution of the enzyme. Diges- 

 tion is shown by a red coloration of the solution due to setting free of 

 congo red. 



1 Also called pepsin, pepsase, gastric protease and acid protease. For a discussion of this 

 enzyme see p. 140. 



2 If so desired, a solution of commercial pepsin powder in 0.2 per cent hydrochloric acid 

 may be substituted for the extract of mucosa. 



3 Also called trypsin, trypsase, pancreatic protease and alkali protease. For a discussion 

 of this enzyme see p. 189. 



4 A 0.25 per cent sodium carbonate solution of commercial trypsin or pancreatin maybe 

 substituted. 



6 A few c.c. of toluene or an alcoholic solution of thymol should be added to prevent 

 putrefaction. 



