304 TITRIMETRIC METHODS 



Aminopoliipeptidase. Alanylglycylglycine or leucylglycylglycyl- 

 glycine; also glycylglycyl-D-alanine (Levy and Palmer, 1943). 

 Carboxypeptidase. Benzoylleucylglycine. 

 Prolinepeptidase. Glycyl-L-proline. 



Cathepsin. 4% edestin in 0.008 A'" hydrochloric acid, pH 4.4. 

 Pepsin. 4% edestin in 0.056 N hydrochloric acid, pH 2.1. 



PROCEDURE 



1. Pipette 7/xl. enzyme extraction medium into a 0.25 ml. reaction 

 tube. Place tissue section in the liquid, and introduce a mixing 

 "flea." 



2. After standing for 1-2 hr. at room temperature for enzyme ex- 

 traction, 7 ixl. substrate soln. is pipetted in. The liquid is then mixed. 



3. Cap the tube and suspend in thermostat for the chosen diges- 

 tion period. 



4. Set up a control experiment by carefully placing the substrate 

 soln. on the side of the tube as a separate drop not touching the 

 enzyme in the bottom. Since there is a tendency for the side drop 

 to run down into the bottom drop, hold the tube in a horizontal 

 position, cap, and suspend in the thermostat in a horizontal position. 



5. Stop the reaction, after removing from the thermostat, by 

 pipetting in 30 /xl 0.05 N alcoholic acid and mixing. The automatic 

 pipette shown in Figure 60 (page 175) is especially useful in this step. 



6. Add 150 111. acetone-naphthyl red soln. and titrate the yellow 

 liquid with 0.05 N alcoholic hydrochloric acid to an orange end point. 

 Place 200 [A. of a standard orange soln. in a reaction tube and mount 

 this beside the titration tube so that the end points may be brought 

 to the same color tone. 



Linderstr0m-Lang and Duspiva Alkalimelric Alcohol Method 

 for Proteolytic Enzymes 



SPECIAL REAGENTS 



Enzyme Extraction Medium. Glycerol-phosphate soln. prepared as 



for preceding method. 

 Absolute Alcohol Containing 0.05% Thymol Blue. 

 0.05 N 90% Alcoholic Tetramethylammonium Hydroxide. 



