548 KINETICS OF BIOLUMINESCENT REACTION. H 



theoretical expectation. Even the values calculated for the initial 

 brightness from the intersections of the straight line plottings upon 

 the zero time axis give poor approximation to the theoretical values. 

 I believe that the presence of the initial flash is responsible in some 

 way for this lack of coincidence, masking, as it does, the fundamental 

 nature of the decay during the first second or two. I consider the 

 very good approximation of the experimental values for reaction 

 velocity with the expectation for a direct proportionality between it 

 and enzyme concentration as being more significant and important 

 than these initial light observations and calculations. 



5. Influence of Luciferin Concentration. 



Returning again to equation (2) we observe that if the value of A, 

 or luciferin concentration, be reduced with all other factors held 

 constant, the value of k is not affected by this reduction, and the 

 slope of the straight line plotting, its graphical counterpart, is also 

 unaffected. In other words, the theoretical expectation is that with 

 two different luciferin concentrations the straight line forms should 

 run parallel to each other. 



In my first attempt to investigate this factor I adopted a dilution 

 method similar to that used with success in reducing enzyme concen- 

 trations to known lower values. In diluting I first used distilled 

 water, and then later, because this procedure diluted the yellowish 

 pigments always present as well, I diluted with oxyluciferin solutions, 

 boiled until complete oxidation of the original luciferin had been 

 effected. For I found that the yellowish pigments gave a rather 

 high extinction coefficient (about 0.3) for blue light which to the eye 

 was a fair match for Cypridina light, and it at once became e\ddent 

 that while dilution with water would presumably not affect the shape 

 of the decay curve, it would affect the magnitude of the light inten- 

 sities observed all along it. 



It appeared therefore that in order to obtain comparable values 

 for two different luciferin concentrations it was necessary to control 

 the pigment concentration. I did this by diluting with oxyluciferin 

 solutions, whose pigment concentration was matched with that of 

 the newly prepared luciferin solution which was to be studied, the 

 two solutions being observed through a colorimeter until the concen- 



