CHASE, BALL, CORNELIUS, AND LEDERMAN 25<J 



PH 



II 



13 



Fig 1. The relative first-onler reaction-rate constant lor the non-linnincscent 

 oxidation of Cypiidina luciferin during exposure, at 26°C. to the l>H's indicated. 



Ascorbic acid was found to be a most effective inhibitor of this 

 non-luminescent oxidation of hiciferin. Selecting pH 8.6, where the 

 luciferin is very unstable, experiments were performed in the presence 

 of (a) 0.01 M ascorbic acid, (b) 0.001 M ascorbic acid, and (c) no 

 ascorbic acid. The lower half of Fig. 2 shows typical results. It is 

 evident that 0.01 M ascorbic acid almost completely prevents the 

 loss of luciferin by non-luminescent oxidation, and 0.001 M ascorbic 

 acid is nearly as effective— this, at 26 °C and pH 8.6, where, in the 

 absence of the ascorbic acid, half the luciferin is lost through oxida- 

 tion in less than ten minutes. 



The addition of a trace of ascorbic acid to a reaction mixture of 

 Cypridina luciferin and luciferase completely abolishes the dim light 

 emission which has been attributed to reversibly oxidized luciferin 

 (1) , and a reaction mixture so treated shows only the regular bright 

 limiinescence characteristic of luciferin which has not undergone any 

 oxidation. Evidently any reversibly oxidized luciferin in such a 

 mixture is reduced by the ascorbic acid. 



This effect of ascorbic acid on the course of light emission when 

 luciferase is added to partially oxidized luciferin is illustrated very 

 strikingly in the upper part of Fig. 2, which is traced from the 

 chart of the recording photoelectric light integrator. The compound 

 nature of the reaction in the absence of ascorbic acid, and the de- 

 layed dim light emission due to reversibly oxidized luciferin (1) 

 are very evident. This luminescence curve cannot be described by 



