542 KINETICS OF BIOLUMINESCENT REACTION. II 



neously in the absence of the enzyme, and without Hght production. 

 It is not possible at present to state whether this spontaneous oxida- 

 tion continues side by side with the catalytic oxidation when the 

 enzyme has been added, or how important a part it plays if it be 

 then present. In any case it proceeds at a much lower rate than the 

 catalytic reaction, and does not affect the monomolecular reaction, 

 and hence only affects the value of k in equation (2), without destroy- 

 ing the logarithmic form itself. 



It is not possible, of course, to follow the kinetics of this secondary 

 reaction in any direct way since it produces no light. I have 

 attempted to ascertain by an indirect method whether the assumption 

 of a monomolecular form for this reaction is correct. To three tubes 

 containing originally identical quantities of luciferin solution (20 cc.) 

 were added identical luciferase solutions (1 cc.) at successive times, 

 the second addition being 10 minutes after the first, and the third 

 addition 20 minutes later. All solutions were stirred from the instant 

 of addition. The light from these tubes was allowed to fall for 90 

 seconds from identical distances upon three small neighboring areas 

 of a strip of film. A calibration series was then impressed upon the 

 record and the film developed. In 90 seconds over 99 per cent of the 

 luciferin in such an aqueous solution has been oxidized with light 

 production. It will later be seen that the total light given out when 

 the reaction has gone to completion is probably a linear function of 

 the initial concentration of luciferin. The decrease in total light 

 therefore, in solutions which have stood until the secondary reaction 

 has reduced the initial luciferin concentration, may be taken as a 

 direct measure of such reduction. In one experiment I found that 

 the total light emitted from the second tube, which had stood for 10 

 minutes, was 52.24 per cent of that emitted by its companion tube 

 run previously. If the spontaneous reaction is monomolecular in 

 character, the third tube run after 20 minutes should have shown a 

 decrease in intensity to 27.29 per cent of the first tube. The actual 

 total intensity observed in this tube was 28.45 per cent which agrees 

 rather well with the expected value. Such data gives fair quantitative 

 support to the hypothesis. 



While this secondary or spontaneous reaction has entered in at 

 various points to complicate the problem, it has also proven of value 

 in one part of the study. It is greatly accelerated by a rise in tem- 



