ESTERASES AND LIPASES 309 



tinuous neutralization of the acid liberated by an alkaline buffer 

 medium. The loss in alkalinity of the buffer is then measured by 

 titration with acid to pH 6.5, the magnitude of the titration being 

 inversely proportional to the amount of substrate hydrolyzed. The 

 greatest deviation in the determination is equivalent to about 0.14 

 ,al. 0.05 N acid. 



An adaptation was also made for the determination of cholin- 

 esterase (Glick, 1938). The sensitivity of this method is equivalent 

 to the hydrolysis of 1 X 10"^ mole of ester. The titration is carried to 

 an end point of pH 6.2. Sawyer (1943) reported a sharper end point 

 and an increase in sensitivity when bromocresol purple is substi- 

 tuted for bromothymol blue and the end point is taken at pH 5.9. 

 For a gasometric method see page 393. 



Glick Acidimetric Method for Esterase and Lipase 



SPECIAL REAGENTS 



Enzyme Extraction Medium. 30% glycerol. 



Buffered Substrates. Esterase: 1% methyl butyrate in a buffer 

 soln. 0.1 A^ to sodium hydroxide and 0.4 A'' to glycine, pH 8.7 at 

 40°; prepare fresh before use. Lipase: Shake thoroughly a drop 

 of tributyrin in a few ml. of the buffer soln. above; let stand for 

 about 1 hr. in a refrigerator to allow the larger droplets to settle 

 and use the supernatant homogeneous emulsion, or filter through 

 paper and use the filtrate; prepare fresh before use. 



Phenol-Indicator Solution. 10 ml. 2% phenol -[-1.5 ml. 0.04% 

 bromothymol blue. 



0.05 N Hydrochloric Acid. 



End Point Color Standard (pH 6.5). 3.1 ml. M/15 secondary 

 sodium phosphate -j- 6.9 ml. M/15 primary potassium phosphate 

 -}- 1 ml. 0.04% bromothymol blue. 



PROCEDURE 



1-4. These steps are carried out in the same fashion as the cor- 

 responding ones in the procedure for proteolytic enzymes (page 304). 



5. Stop the reaction by adding 50 fA. phenol-indicator soln. with 

 a constriction pipette. 



6. Titrate with 0.05 A'' hydrochloric acid, matching the greenish 

 color to that of the end point color standard. Take a uniform time 



