308 TITRIMETRIC METHODS 



PROCEDURE 



1. Pipette 7 /xl. enzyme soln. and 7 (A. substrate into the bottom 

 of the reaction tube, add a "flea," and mix. Close the tube with a 

 rubber stopper and allow the reaction to proceed for a suitable time 

 at an appropriate temperature. (Note that the type of tube origi- 

 nally used. Fig. 40, page 167, and the accompanying technique have 

 been replaced by the method of Briiel et al., 1946.) 



2. Place the vessel in boiling water for about 15 min., cool, add 

 20 ^1. urease-buffer soln., and stir with the "flea." 



3. After allowing the vessel to stand 1 hr. at 20°, measure the 

 ammonia formed using the method of Briiel et al. ( 1946) (page 283) . 



B. Acetone-Alcohol Titration Method 



SPECIAL REAGENTS 



Substrate Solution. Same as for urease method. 



Acetone- Alcohol-Indicator Mixture. Prepare 100 ml. by adding a 



mixture of equal vol. acetone and absolute alcohol to 5 ml, of 0.1% 



alcoholic thymol blue. 

 0.05 N Tetramethylammonium Hydroxide in 90% alcohol. 

 End Point Color Standard. See page 292, step 8. 



PROCEDURE 



1. Pipette 7 /xl. enzyme soln. and 7 /xl. substrate into the bottom 

 of a simple 250 [xl. reaction tube (Fig. 35, page 167). Add a "flea," 

 stir, stopper with a soda lime tube (Fig. 47, page 171), and allow 

 the reaction to proceed at an appropriate temperature for a suitable 

 period. 



2. Stop the reaction by adding 150 /xl. acetone-alcohol-indicator 

 mixture. 



3. Titrate with the tetramethylammonium hydroxide to the shade 

 of the greenish end point color standard. Use one of the devices to ex- 

 clude carbon dioxide during the titration (Figs. 85, 86, pp. 257, 258). 



ESTERASES AND LIPASES 



An acidimetric micro method for esterase was described by Glick 

 (1934) ; this method was later applied to lipase by changing the sub- 

 strate (Glick and Biskind, 1935). The method is based on the con- 



