VOL. 4 (1950) 



ACYLATIONS BY ACETYLCHOLINE ESTERASE II 



311 



A. HYDROLYSIS OF ACETYLCHOLINE AS A FUNCTION OF Pn 



An enzyme concentration assay curve is reproduced in Fig. i. The hydrolysis-time 

 curves in phosphate solution at Ph 74 depart from a stra'ght line to a measurable extent 

 only alter about 30% of the substrate at an initial concentration of 4 ^M per ml has 

 been split. The plot of the initial reaction velocity against enzyme concentration in the 

 range studied yields a straight line. 



Fig. I. Acetylcholine hj'drolysis as a function 

 of enzyme concentration. Mixtures contain i .0 

 M potassium dihydrogen phosphate adjusted 

 with sodium hydroxide to pn 7-4. gelatin 

 0.07%, acetylcholine 4 //M/ml. Temperature 

 23° C. The Ph remained constant within 0.2 pn 

 units during the course of the hydrolysis. The 

 non-enzymatic hydrolysis in these conditions 

 was barely detectable. Curves 1-5 show fin- 

 dings with enzyme dilutions i : 4 000, i : 8 000, 

 1:12000, 1:20000 and 1 : 30000 respectively. 

 In the inset, relative enzyme concentration 

 is plotted on the abscisca and the corres- 

 ponding relative initial reaction velocity on 

 the ordinate. 



,t?80 



36 i,2 



Minctes 



TABLE I 



ACETYLCHOLINE HYDROLYSIS IN PHOSPHATE SOLUTION AS A FUNCTION OF pH IN THE ACID RANGE 



The solutions contained a constant amount of enzyme, 0.07% gelatin, o.i M potassium phosphate, 

 sodium hydroxide in varying amounts and acetylcholine chloride in a concentration of 4 //M/ml. 

 The ph remained constant during the course of the hydrolysis within 0.2 pn units. Temperature 

 21° C. Non-enzj^matic hydrolysis proved negligible in the conditions used. Control mi.xtures to which 

 no acetylcholine was added failed to produce colour when examined with the reagent. The solutions 

 remained clear and removal of the protein present in the reaction mixture was unnecessary. 



Variation of esterase activity accompanied shift of pn on the acid side of the scale 

 in a range which is still of physiolog'cal interest. The course of the reaction in phosphate 

 buffer is illustrated by the experiment recorded in Table I. It is evident that increase 

 of Ph from 5.5 to 7.4 results in a progressive and marked rise of reaction rate in phosphate 

 buffer. Between pn 7.4 and 7.8 in phosphate and between p^ 7.6 and 9.4 in borate the 

 enzyme-mediated hydrolysis exhibited a constant initial reaction rate. At pn higher 

 than 9.4 inactivation of enzyme occurred at 21° C, the inactivation was retarded con- 

 siderably at 17° C. Non-enzymatic hydrolysis of the substrate was found to become 

 relatively appreciable at pn 9.2 and rose rapidly with further increase of the pn (Table II). 

 A summary of findings is presented in Fig. 2. The p^ range in which the acetylcholine 

 Jie/erences p. 321. 



