UREA AND UREASE 287 



cell (Fig. 43, page 168). Add 1 drop urease soln., stir with a "flea" 

 or otherwise, and allow to react for 20 min. at room temperature. 



2. Place a few drops of mineral oil in the well, and pipette 5 

 fx\. glycerol-boric acid into the center of the covering vial. 



3. When the digestion is completed, add 1 drop metaborate soln. 

 to the reaction mixture, and immediately place the covering vial over 

 the central container. Stir the digestion mixture and allow 60 min. 

 at 30° for the diffusion. 



4. Remove the covering vial, blot off the adhering mineral oil, 

 add 0.5 ml. diluted indicator, and titrate with 0.002 A'" hydrochloric 

 acid to the color of a control containing no urea. (The latter should 

 be yellow; a greenish appearance indicates some ammonia has 

 been introduced, probably from the sodium chloride used in the 

 urease soln. or from the potassium chloride-metaborate soln.) Allow 

 about 1 min. between the addition of the final increment of acid 

 and the matching of the end point. 



note: The colorimetric procedure of Russell (1944) (page 238) may be 

 substituted for the titration: Wash out the glycerol drop from the vial 

 with three 0.5 ml. portions of water into colorimeter tubes. Add 0.05 ml. 

 0.003 M manganese sulfate or chloride to each tube. Chill the tubes and 

 add 1.0 ml. alkaUne phenol reagent (25% phenol in 2.7 N sodium 

 hj^droxide) and 0.5 ml. hypochlorite soln. Place the tubes in a boiling 

 water bath for 5 min., cool, dilute to a convenient vol. and read in a 

 colorimeter. 



UREASE 



Procedures for the determination of urea can, of course, be adapted 

 to the measurement of urease. This was done by Linderstr0m-Lang 

 and Holter (1940, page 1144), who based the method on their 

 ammonia determination (page 230). 7 fA. enzyme soln. and 7 fA. 

 substrate (1.2 g. urea, 50 ml. 0.15 N sodium hydroxide, and 50 ml. 

 0.3 M primary potassium phosphate, pH 6.8) were employed. The 

 ammonia was liberated from the digested mixture by drawing into 

 it 7 /xl. 2 A'" sodium hydroxide, which had previously been placed on 

 the side of the reaction tube. The distillation of the ammonia and 

 the titration were then carried out (page 285). 



The determination can also be carried out by measuring the am- 

 monia in an acetone titration according to the procedure used by 

 Linderstr0m-Lang and S0eberg-Ohlsen (1936). To the reaction 



