CYPRIDINA LUCIFERIN OXIDATION AS A FUNCTION 



OF pU, AND REDUCTION OF LUCIFERIN 



BY ASCORBIC ACIDi 



A. M. Chase^ J. H. Ball, C. E. Cornelius, and R. J. Lederman 



Physiological Laboratory, Princeton University, Princeton, New Jersey 



Studies, in vitro, of the kinetics of the luminescent reaction of 

 luciferin and luciferase from Cypridina have long been plagued by 

 a complication due to the presence of reversibly oxidized luciferin 

 (1) . The curve relating total light emitted to time (the integral 

 curve) becomes distorted when more than a small amoiuit of reversibly 

 oxidized luciferin is present, and this often makes it difficult or im- 

 possible to analyze data in terms of effects of experimental variables 

 upon the activity of the enzyme. Also, procedures for purifying 

 Cypridina luciferin are complicated by the fact that this luciferin 

 is so unstable in the presence of dissolved oxygen. 



We have extended earlier studies on the effects of temperature 

 upon the non-luminescent oxidation of Cypridina luciferin (2) to 

 include measurements of the rate of this process as a function of 

 pH, and have found that this luciferin is least stable at about /?H 9 

 and becomes considerably more resistant to oxidation at pH's more 

 acid or more alkaline than this. 



The experimental procedure and materials used Avere essentially 

 the same as in the earlier work involving temperature (2) . Luciferin 

 was allowed to stand in 0.1 M phosphate buffers of the desired 

 pH at 26 °C. Samples from the reaction mixtures were periodically 

 mixed with luciferase, and the amount of light still remaining was 

 measured with a recording photoelectric light integrator. The first- 

 order reaction-rate constant for the disappearance of the luciferin 

 by non-huTiinescent oxidation was then calculated for each pW. 



Fig. 1 shows the results of these experiments. It is evident that 

 non-luminescent oxidation is most pronoiniced at about pW 9, being 

 nearly fifty times as fast there as at /?H 5 and about three times as 

 fast as at pH 13 or 14. Anderson stated some years ago (1) that this 

 luciferin was relatively stable in dilute acid or alkali but gave no 

 details. 



'Supported by a National Sdfiuc Koimdation giant. 



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