w. D. Mcelroy and j. w. Hastings 191 



of light-intensity increase, as well as the time required to reach the 

 maximum intensity, depends upon the amount of pyrophosphatase 

 added. The curves A and B of Fig. 23 illustrate this point. Phosphate 

 liberation parallels the light-intensity curve in a manner similar to 

 the results presented in Fig. 22. The initial depression of the light 

 intensity, as well as the depression of the steady-state baseline level, 

 by the addition of pyrophosphatase is additional evidence for the im- 

 portance of a second protein in the complexing reaction. That pyro- 

 phosphatase is the important protein for the complexing reaction is 

 indicated by other evidence than was presented above. The ability of 

 a preparation to complex the luciferase parallels the pyrophosphatase 

 activity during purification of the latter. Factors such as temperature 

 and a variety of inhibitors depress the complexing and pyrophospha- 

 tase activity in a similar manner. 



Inorganic triphosphate is not broken down even by the partially 

 purified luciferase preparations, and it might be expected that a differ- 

 ent type of response would be observed with this compound. Several 

 different experiments have supported this viewpoint. In none of these 

 experiments has there been observed the secondary rise after the 

 initial response to added inorganic triphosphate. There is, however, a 

 flash which rapidly decays to a steady-state level. The level of this 

 baseline luminescence is, however, higher with each successive addi- 

 tion of triphosphate, in contrast to pyrophosphate response in the 

 presence of pyrophosphatase. The results of one such experiment are 

 recorded in Fig. 24. Curves A, B, and C represent three successive 

 additions of triphosphate at 2-minute intervals. Although the initial 

 flash response is progressively decreased, the steady-state basehne 

 increases in proportion to the triphosphate concentration. Similar 

 results with different concentrations of triphosphate have been re- 

 ported previously. Curves 2, 3, and 4 show the effect of successive 

 additions of pyrophosphate to a similar reaction mixture, while curve 

 1 represents a similar experiment, but with one-haff the amount of 

 pyrophosphatase added. 



Although the preparation containing pyrophosphatase does not 

 catalyze the hydrolysis of inorganic triphosphate, it influences the 

 luminescent response to the compound. During purification of the lu- 

 ciferase the maximum flash elicited by the secondary addition of tri- 



