w. D. Mcelroy and j. w. Hastings 181 



intensity. It should be pointed out that no inorganic phosphate was 

 Hberated when the inorganic triphosphate was added to the reaction 

 mixture. 



Immobilization of Lucif erase— Complexing Reaction 



The results reported above have indicated that the rapid depression 

 of light intensity, after the initial addition of ATP, is not due to the 

 utilization of the various substrates of the reaction mixture. The re- 

 sults presented in Fig. 13 are convincing evidence that the luciferase 

 itself is being inhibited or complexed in the reaction. Successive addi- 

 tions of the enzyme elicit responses similar to the initial reaction. 

 With additional enzyme, however, the baseline intensity increases up 

 to approximately the fifth addition. Although additional luciferase 



LH2 t Mg**t ATP + E (LUCIFERASE) 



1 



ACTIVE INTERMEDIATE 



X/^ PROTEIN \ '^ 



INACTIVE LIGHT 



COMPLEX 



Fig. 14. Scheme for the complexing reaction in firefly luminescence. 



gives a flash, the height of the response gradually decreases. The 

 addition of inorganic triphosphate, after nine additions of enzyme, 

 mobilizes a large fraction of the enzyme in the system, resulting in a 

 brilliant flash which is four times the intensity of any of the individual 

 responses. 



The decline of luminescence after its initiation with ATP to the low 

 steady-state level is believed to be due to the reversible formation of 

 an inactive complex from an active intermediate (see Fig. 14). This 

 intermediate is presumably composed of luciferase, luciferin, Mg, and 

 ATP. Probably through a series of reactions the active intermediate 

 is finally converted, in the presence of oxygen, to an excited state 

 which subsequently emits hght (see below). The low baseline level 

 of luminescence represents, therefore, a steady-state equilibrium 

 between active intermediate and inactive complex. Thus when the 



