J. WOODLAND HASTINGS AND VERNON C. BODE 297 



In addition to the cell extract and salt, the luminescent reaction 

 requires molecular oxygen (Fig. 3) . 



The extract may be fractionated with anmionium sulfate to yield 

 an enzyme purified by about 20-fold, as shown in Table I. Dried or 

 fresh cells are extracted three times with a total of 100 ml of dis- 

 tilled water per gram of cells, dry weight. The dark brownish-green 

 extract is then fractionated with ammonium sulfate to give an active 

 fraction which precipitates between 35 and 65% saturation of am- 



>- 

 z 



UJ 





TIME - MINUTES 



Fig. 3. This illustrates the requirement for oxygen in the (iouxfiiilax luminescent 

 reaction. .\ reaction was initiated with air hubbling through it, and about one 

 minute later the gas flow was changed to hydrogen. 1 he light intensity decreased 

 rapidly, but became higher when the gas flow was changed back to air (20% O.). 



monium sulfate. At low ammonium sulfate concentrations a deep 

 green protein precipitates, and at higher concentrations a pink-red 

 protein is precipitated. Upon subsequent purification it is found 

 that the activity of the luciferase does not accompany the colored frac- 

 tions. A second ammonium sulfate fractionation results in further 

 purification. Enzyme stored at — 10°C is stable for at least a year. 



This partially purified enzyme contains no measurable amounts 

 of substrate, i.e., luciferin. Luciferin may be prepared by extracting 

 cells for two minutes with Avater at 90°C. Luciferase activity is 

 thereby destroyed, and when the luciferin is added to a partially 



