184 



BIOCHEMISTRY OF FIREFLY LUMINESCENCE 



first method described above, very good agreement is obtained. This 

 fact is regarded as good evidence that the complexing reactions do in 

 fact proceed independently of the oxygen concentration involved. 

 Had the lowered oxygen affected the binding of the enzyme in one 

 way or another, it would be surprising if the two methods for obtain- 

 ing the relationship between oxygen concentration and Hght intensity 

 would give the same results. 



80 - 



70 



60 -' 



50- 



^ 40 - 



(0 



20 - 



10 



0.5 1 15 2 2 5 



TIME- MINUTES 



35 



Fig. 17. Effect of mixing reactants anaerobically and adding oxygen at different 

 time intervals. Curve A is a control run in air. 



Two additional observations support this conclusion. First, in the 

 above series of experiments air is admitted to the various reaction 

 mixtures after the reading at 2^ minutes. The typical flash is ob- 

 served, after which the intensity returns to the low steady-state level. 

 In all cases, irrespective of the oxygen concentration used, the in- 

 tensity in air returns to a level which corresponds to the intensity of 

 the control reaction in air at the same time after the start of the 

 reaction. Second, if all the components of the reaction are mixed 

 under strictly oxygen-free conditions and air is admitted at various 

 time intervals after mixing, the intensity to which the reaction mix- 



