190 



BIOCHEMISTRY OF FIREFLY LUMINESCENCE 



from ATP during the rapid complexing reaction, a point that has been 

 estabhshed and discussed previously. 



If pyrophosphate is added to a highly purified enzyme mixture 

 containing relatively little or no pyrophosphatase activity, the light 

 intensity does not rapidly decrease from the initial flash height to the 



en 



2 

 UJ 



t- 



X 



o 



TIME - MINUTES 

 Fig. 23. The effect of delayed addition of pyrophosphatase. The luciferase used 

 was free of pyrophosphatase activity. Pyrophosphate was added initially to 

 give a final concentration of 5 X 10* M. The luminescent reaction was 

 allowed to proceed for 5 minutes before the experiment recorded in the 

 graph was started. The arrow indicates when two different concentrations of 

 pyrophosphatase were added to the reaction mixture. To reaction mixtures 

 B and A were added 0.1 and 0.2 ml of partially purified pyrophosphatase 

 ( 1.0 mg protein/ml) respectively. The initial depression, as well as the steady- 

 state baseline level, after hydrolysis of pyrophosphate, are proportional to the 

 protein added. There was no phosphate liberated during the 6.5 minute"^ 

 prior to the addition of pyrophosphatase. 



low baseline level. The results in Fig. 23 show that the luminescence 

 is maintained at a high steady-state level. Under these conditions no 

 phosphate is liberated, and the secondary rise in light emission is not 

 observed. If, however, a partially purified pyrophosphatase is added, 

 there is at first a depression which varies directly with the amount of 

 protein added, followed by an increase in the light intensity. The rate 



