176 BIOCHEMISTRY OF FIREFLY LUMINESCENCE 



pyrophosphatase (McElroy, Coulombre, and Hays, 1951). The results 

 in Fig. 9 illustrate the effect of adding pyrophosphatase to a purified 

 luciferase. As discussed below, the results indicate that luciferase 

 reacts with the second protein to form an inactive complex which 

 effectively removes most of the enzyme from active participation in 

 the light reaction. Although there is some rapid complexing of the 

 luciferase during the first few seconds, it is apparent that the rapid 

 decay to the low light intensity is not observed unless pyrophosphatase 

 is added. The rate of decay to the steady-state level of luminescence 

 is proportional to the added pyrophosphatase. It is not clear why one 

 obtains an initial rapid decrease of the light intensity to approximately 

 50% of the flash height even in the absence of pyrophosphatase. It may 

 mean that a second luciferase molecule may t unction in the complex- 

 ing reaction. 



Effect of Secondary Addition of Polyphosphates 



In preliminary experiments designed to determine whether pyro- 

 phosphate would influence the utiHzation of ATP in the Hght reaction, 

 it was observed that the addition of the former, after light production 

 by ATP had decreased to a small value, stimulated light production in 

 much the same way as additional ATP (Fig. 10). Since pyrophosphate 

 by itself or pyrophosphate plus adenylic acid failed to initiate light 

 production, it seemed likely that this compound was in some way 

 making the ATP available for light production. A second luminescent 

 response to pyrophosphate occurred in the presence of ADP as well as 

 ATP. With ADP there was a slight lag in the initial light emission 

 in contrast to the instantaneous response to ATP. Furthermore, the 

 maximum light intensity obtained by the addition of pyrophosphate 

 was greater than that obtained with the initial addition of ADP. With 

 ATP the maximum response obtained with a secondary addition of 

 pyrophosphate was approximately 50% of the initial intensity (Fig. 

 10). 



The response of the luminescent system to pyrophosphate was only 

 temporary, as the results in Fig. 10 demonstrate. In many respects it 

 simulates the effect of ATP. The rapid drop in Hght intensity in the 

 experiments described above is due to a rapid hydrolysis of pyrophos- 

 phate by inorganic pyrophosphatase. An analysis for orthophosphate 



