188 



BIOCHEMISTRY OF FIREFLY LUMINESCENCE 



pyrophosphate and the time to reach the secondary peak are shown 

 in Fig. 21. 



If inhibitory concentrations of pyrophosphate are incubated with 

 the luciferase preparations containing pyrophosphatase for various 

 time intervals prior to the addition of ATP, a similar recovery is 

 observed, i.e., ATP is not required for this effect. The results of such 

 an experiment are shown in Fig. 22. In curve A the reaction was 

 started in the usual manner with ATP at zero time. After the steady- 



I 23456789 

 PYROPHOSPHATE CONC. M x 10^ 



Fig. 21. The relationship between pyrophosphate concentration and light emission 

 (McEIroy et ah, 1953). The black circles represent initial light intensity 

 while the white circles represent the time required to reach the secondary 

 peak of luminescence as recorded in Fig. 20. 



state luminescence had been reached, an inhibitory concentration of 

 pyrophosphate was added. The response is at first a depression fol- 

 lowed by an increase in the light intensity, which reaches a maximum 

 2 minutes later. In the other experiments, ATP was not added ini- 

 tially; however, the pyrophosphate was added at the same relative 

 time after mixing enzyme, buffer, luciferin, and Mg+ + . At various 

 intervals after the addition of pyrophosphate, the ATP was introduced 

 to initiate light emission. The results clearly indicate that the longer 

 the incubation time the greater is the initial effect of ATP, in so far as 

 light intensity is concerned. One obtains the secondary peak of lu- 

 minescence only at certain critical times of incubation. This diphasic 



