w. D. Mcelroy and j. w. Hastings 165 



TABLE I 

 Purification of Firefly Luciferase 



on ATP is the only immediate labile phosphate group which can be 

 used in the luminescent reaction. These results, as well as others 

 which will be reported later concerning the hexokinase reaction, 

 indicate that an active myokinase had been removed during purifica- 

 tion. 



Luciferin 



Most of the firefly luciferin remains in the supernatant after the 

 calcium phosphate gel treatment. The supernatant is adjusted to pH 

 3.5 and extracted twice with an equal volume of redistilled ethyl 

 acetate. All the active luciferin passes into the ethyl acetate. The 

 ethyl acetate is removed by vacuum distillation and the active 

 luciferin is dissolved in a small volume of water. This crude prepara- 

 tion can be used for enzyme assay. Further purification is achieved 

 by adsorbing the luciferin on an acid (2N HCl) treated Dowex 50 

 column (mesh size less than 80). The column is washed thoroughly 

 with 2N HCl and finally with water. The luciferin is slowly developed 

 on the column by a weak solution of NH4OH (1.5%). The luciferin 

 migrates down the column in a sharp band and is finally eluted. The 

 luciferin can be readily followed on the column by its brilliant yellow- 

 green fluorescent band. The eluates containing the active luciferin 



