MILTON J. CORMIER 275 



Preparation of Extracts and of Crude Luciferin 



Extracts 



Freshly collected sea pansies were placed in a dark, running sea- 

 water aquarium for 1 to 8 hours. Since these animals have a diurnal 

 rhythm of luminescence (7) , this procedure was necessary to re- 

 juvenate the luminescence system. Approximately 50 animals at 

 a time were destemmed and dropped into a cold mortar containing 

 10 ml of saturated ammonium sulfate, pH 7.5. They were cut into 

 small pieces with scissors and finally ground with sand for 30 minutes. 

 A total of 200 ml of saturated ammonium sulfate was gradually added 

 with stirring, and the suspension was centrifuged at 12,000 X g ^or 

 10 minutes. The precipitate was carefully drained to remove excess 

 ammonium sulfate, and then extracted by grinding in a mortar with 

 a total of 50 ml of 0.05 M potassium phosphate buffer, pH 7.5, con- 

 taining 0.001 M glutathione (phosphate-GSH buffer) . The extract 

 was centrifuged for 10 minutes at 12,000 X g^ and the resulting 

 supernatant centrifuged for 30 minutes at 140,000 X g- The latter 

 centrifugation yields a clear, amber-colored supernatant solution 

 which contains the activity and will be referred to as the crude 

 extract. The activity of the extract was preserved by storing at 

 -20 °C. The giinding, centrifugation, and extraction procedures 

 were done at to 3° C. 



For the experiments reported here, unless otherwise indicated, the 

 crude extract was precipitated once with saturated ammonium sul- 

 fate, pH 7.5 (to 75 per cent saturation) , and the precipitated protein 

 was redissolved in phosphate-GSH buffer. 



Treatment of the animals with ammonium sulfate is an essential 

 feature of the extraction process. Grinding rejuvenated organisms 

 with sand in the presence of phosphate-GSH buffer without am- 

 monium sulfate treatment does not yield an active extract. Am- 

 monium sulfate produces at least two observable effects: first, it 

 allows a greater amount of protein to be extracted; and second, it 

 acts as an anesthetic and thus prevents an excessive luminescent re- 

 sponse during grinding, such as would result in oxidation of Renilla 

 luciferin. 



Luciferin 



The once-extracted Reuilln residue was reextracted with phosphate- 

 GSH buffer in the manner described for preparation of the crude 

 extract, except that centrifugation at 140,000 X g was not required. 



