314 



S. HESTRIN 



VOL. 4 (1950) 



It is apparent that acid shift of pn within the range studied displaces the equihbrium 

 in the direction of synthesis. In the experiments of Figs 3 and 4 the speed of the 

 approach to the equihbrium was found to be dependent upon the esterase concen- 

 tration. To insure a close approach to the equilibrium in a conveniently short time, 

 a much greater enzyme concentration than is conveniently used in a hydrolysis assay 

 was taken. 



30 90 150 210 



Minutes 



Fig. 3. Synthesis of acetylcholine as a function of pn- Solutions were made with 1.15 g each of choline 

 chloride and sodium acetate trihydrate at pn 5-i in a total volume of 6.0 ml, and with 1.2 1 g each of 

 these substrates in the same total volume at pn 5-9 and 7.0. p^ was set with hydrochloric acid and 

 measured with a glass electrode in samples diluted for the purpose with three volumes of water. In 

 one control mixture at each pn, 8 /tM of acetylcholine per ml was added at the outset. Enzyme was 

 added in an amount per ml sufficient to effect hydrolysis of 2 g of acetylcholine chloride per hr in 

 optimum conditions. Temperature 23'' C. Ester was determined on aliquots of 0.5 ml. A standard 

 curve was constructed with known acetylcholine amounts in the same medium. Care is taken in the 

 ester determination to bring the pn of the sample to i. 0-1.2 at the step prior to ferric chloride addi- 

 tion in order to avoid interfering colour by reaction between fatty acid and ferric chloride. In several 

 cases, water was added to a reaction mixture in which the synthesis had come to a rest. A rapid 

 shift of the equilibrium in the reaction direction of hydrolysis could then be observed. In the absence 

 of either acetate, choline, or esterase, no ester formation was observed. 



References p. 321. 



