310 TITRIMETRIC METHODS 



for each titration since a gradual bluing of the color occurs when the 

 soln. stands at a pH near neutrality. 



Glick Acidinietric Method for Cholinesterase 



SPECIAL REAGENTS 



Enzyme Extraction Medium. 30% glycerol. 



Buffered Substrate Solution. 0.4% acetylcholine chloride in veronal 



buffer, pH 8.0 (7.15 ml. 0.1 M sodium diethylbarbiturate + 2.85 



ml. 0.1 M hydrochloric acid.) Dissolve the substrate in the buffer 



just before use. 

 Eserine-Indicator Solution. Add 10 ml. 0.1% eserine sulfate to 1.5 



ml. 0.04% bromothymol blue, or, according to Sawyer (1943), to 



1.5 ml. 0.04% bromocresol purple. 

 0.05 N Hydrochloric Acid. 

 End Point Color Standard (jdH 6.2 or 5.9). 10 ml. i¥/15 phosphate 



buffer -\- 1 ml. 0.04% bromothymol blue or bromocresol purple. 



PROCEDURE 



Identical with that in the preceding method with the substitution 

 of the special reagents required for this enzyme. 



CATALASE 



Holter and Linderstr0m-Lang (1936) and Holter and Doyle 

 ( 1938) described an adaptation to the micro level of the iodometric 

 catalase method of Stern (1932). The estimation of the decomposi- 

 tion of hydrogen peroxide by the enzyme is made by thiosulfate 

 titration of the iodine formed by oxidation of iodide placed in the re- 

 action mixture. The precision of the titration is 0.06 /a1. 0.02 N thio- 

 sulfate, which is equivalent to the decomposition of 0.02 /xg. hydrogen 

 peroxide. 



Holter and Doyle Method for Catalase 



SPECIAL APPARATUS 



A 15 ix\. constriction pipette for the substrate is surrounded by a 

 water jacket, as shown in Figure 54 (page 173). Circulate ice water 

 through the jacket to keep the soln. cold during the pipetting. 



