Enzymes 209 



choline and demonstrating the acid moiety as in the Tween 

 method and ( 2 ) using esters of thiochoHne and precipitating 

 free thiochohne as its cupric salt. 



A. The long-chained fatty acid ester technique.— 



Esters of choline with higher fatty acids are hydrolyzed by 

 some cholinesterases at rates very much slower than acetyl- 

 chohne^^^ but still high enough to be useful for histochem- 

 ical purposes. 



The chlorides of lauroyl-, myristoyl-, palmitoyl- and stear- 

 oylcholine can be synthesized with ease. Myristoylcholine is 

 available commercially. ^^^ All these esters are white crystal- 

 line solids, fairly soluble in distilled water. Pamitoyl and 

 stearoylcholine will precipitate in the presence of salts unless 

 some propylene glycol is added to the mixture. Stearoyl- 

 choline is hydrolyzed so slowly that its use is not recom- 

 mended. 



The principle of the technique^^^ is identical with that of 

 the Tween method for esterase-lipase. However, the tech- 

 nical details had to be modified because of the necessity of 

 using relatively short-chained substrates (C12-C16). The Ca 

 soaps of the shorter fatty acids are very coarsely crystalline, 

 and for this reason Ca is replaced by Co, the corresponding 

 salts of which are relatively fine granular precipitates. (Co 

 is not recommended for use in the Tween technique because 

 it markedly inhibits esterase-hpase. ) The best buffer is tris- 

 maleate, which prevents the impregnation of the tissue by 

 Co, which otherwise may produce an unpleasant darkish 

 background. 



Method 



Fixation as in the case of esterases. Collodion protection of 

 the sHdes is advisable. 



1. Prepare a 0.02 M ( ± 0.7 per cent) stock solution of the 



118. Click, D.: J. Biol. Chem., 137:357, 1941. 



119. From Dajac Laboratories, 511 Lancaster Ave., Leominster, Mass. 



120. Gomori, G.: Proc. Soc. Exper. Biol. & Med., 68:354, 1948. 



