170 CARNEGIE INSTITUTION OF WASHINGTON. 



Kinetics of the Bioluminescent Reaction in Cypridina, by W. R. Amherson. 



The rate of decay in the hght produced when aqueous solutions of Cypridina 

 luciferin and hiciferase are mixed has been followed, and the relative hght 

 intensities along the course of the reaction have been evaluated by a method 

 of photographic photometiy. In this method a photographic record is taken 

 of the light issuing from a narrow sht window in a blackened glass container, 

 within which the reacting solutions are placed. A photographic film is re- 

 volved, by an ac curat :; kymograph, at constant speed past the window. 

 Simultaneous studies of two solutions are made by the use of two identical 

 containers, and in this way the effect of changes in luciferase (enzyme and 

 luciferin (substrate) concentrations, the temperature coefficient, and other 

 problems have been studied. 



The densities of the films along the course of the reaction are evaluated, 

 after development, by the use of the optical pyrometer. In addition to the two 

 moving records of the bioluminescence, there is impressed upon each film a 

 series of 15 cahbration exposures, in which Cypridina hght itself is used as the 

 light source. These 15 exposures give a graded series of densities. The relative 

 intensities of the Hght producing these densities is accurately known. A curve 

 of blackening can then be drawn for each separate experiment, and the 

 unknown intensities along the moving records are referred to this curve for 

 evaluation. 



The results of these studies may be summarized as follows : 



(1) The decay curve of the hght produced in the course of the luminescent 

 reaction in Cypridina is, after the first second, in complete agreement with the 

 theoretical expectation for a mono-molecular reaction, if hght intensity at any 

 instant is assumed to be proportional to reaction velocity at that instant. For 

 such a reaction (straight-hne form) : 



Log 1= —kt-\-\og Ak. 

 where Z = light intensity, /c = velocity constant, t = time, ^4= initial concen- 

 tration of the single reactant. 



The experimental values satisfy this equation. 



(2) The first second or two of the reaction is characterized by a briUiant 

 initial flash, whose value is much too high to accord with the succeeding inten- 

 sities and with the above formula. This high initial reaction velocity may 

 be an indication of a heterogeneous system. 



(3) Identical solutions run simultaneously give decay curves with check 

 within the limits of the photographic error. 



(4) Stirring does not affect the reaction velocity or the form of the decay 

 curve. 



(5) Reaction velocity is proportional to enzyme concentration. 



(6) Changes in the concentration of the substrate do not affect the value 

 of k when all other factors are held constant. A diminution of luciferin con- 

 centration results only in a decrease in the value of the y intercept, log Ak, 

 The two straight-hne plottings for two different concentrations are parallel. 



(7) The temperature coefl&cient is high, being about 4.5 for the 15° to 25"* 

 interval and 3.0 for the 25° to 35° interval. 



