w. D. Mcelroy and j. w. Hastings 



189 



response appears to be directly related to the amount of free and 

 pyrophosphate-bound luciferase, the latter being released by the action 

 of pyrophosphatase. The maximum and normal response to ATP is 

 obtained when the incubation time is extended to a point where all 

 the added pyrophosphate has been hydrolyzed. The phosphate anal- 



-300 



-250 



-200 



TIME-MINUTES 



Fig. 22. The effect of delayed addition of ATP on the pyrophosphate response. 

 In curve A, the reaction was started with ATP; 2 minutes later 0.5 ml of 

 0.01 M sodium pyrophosphate was added. In curves B, C, and D, the ATP 

 addition was delayed until 2.5, 3.5, and 4 minutes respectively. Pyrophos- 

 phate (0.5 ml of 0.01 M), however, was added at 2 minutes to all the 

 reaction mixtures. Curve E represents the average inorganic phosphate con- 

 centration in the reaction mixtures. 



ysis of the reaction mixture indicates that over 95% of the pyrophos- 

 phate is decomposed at the time the secondary peak in luminescence 

 has been reached. The phosphate analysis is also recorded in Fig. 22 

 and was approximately the same for all the reaction mixtures. It is 

 apparent from this curve that no detectable phosphate is released 



