306 TITRIMETRIC METHODS 



Weil Forniol Method for Tryptic Activity 



SPECIAL REAGENTS 



Enterokinase Solution. Prepared according to Waldschmidt-Leitz, 

 (1924). Dehydrate and defat scrapings of hog duodenum mucosa 

 with acetone, acetone-ether, and ether in this order. Grind and 

 sieve the dried material, and extract it at 30° for 2 hr. with 50 ml. 

 0.04 iV ammonium hydroxide per gram solid. Centrifuge, and con- 

 centrate the supernatant by evai)oration at about 35°. 



Veronal Buffer (pH 8.4). Add 8.23 ml. M sodium diethylbarbi- 

 turate to 1.77 ml. 0.1 A'" hydrochloric acid. 



Substrate Solution. Bring 4% casein (Hammarsten) to pH 8.4 with 

 1 N sodium hydroxide. 



Formol Solution. Neutralize 4 ml. 40% formaldehyde, to which 2 

 ml. 0.1% alcoholic phenolphthalein is added, with 0.1 N sodium 

 hydroxide to the first pink color, and make up to 25 ml. with dis- 

 tilled water. Prepare fresh before use. 



0.05 N^ Tetramethylammonium Hydroxide. 



PROCEDURE 



1. Place tissue section or enzyme preparation in 7 fA. entero- 

 kinase soln. in a reaction tube and let stand for 1 hr. at room 

 temperature. 



2. Add 7 ;ul. Veronal buffer and 7 /xl. substrate soln. 



3. Close the vessel with a soda lime tube (Fig. 47, page 171) and 

 place in thermostat for the required digestion period. 



4. Set up a control in which the buffer and substrate are placed 

 on the side of the tube as a drop not touching the enzyme drop in 

 the bottom of the tube. Place horizontally in thermostat to prevent 

 the drops from touching. 



5. Add 1000 fA. formol soln. and titrate with 0.05 N tetramethyl- 

 ammonium hydroxide to a marked red color matching against a 

 standard. Protect from carbon dioxide in the air during the titration 

 by one of the arrangements shown in Figs. 85 and 86, (pp. 257, 258). 



ARGINASE 



Two methods for the micro estimation of arginase were reported 

 by Linderstr0m-Lang, Weil, and Holter ( 1935) . The hydrolysis of 

 the very strong base arginine yields the fairly strong base ornithine 



