270 STUDIES ON BIOLUMINESCENCE. IX 



determine, the properties of oxyluciferin and luciferin are identical^ 

 so that what is here recorded regarding luciferin will apply to oxy- 

 luciferin also. Unless otherwise specified, luciferin, oxyluciferin, and 

 luciferase refer to these substances obtained from Cypridina 

 hilgendorfii. 



Method. 



The living animals are dried quickly in desiccators over CaCl2 

 and may then be kept indefinitely in well stoppered bottles contain- 

 ing a few lumps of CaCl2 to remove any dampness in the air con- 

 tained in the bottle. The Cypridince are well ground to a powder 

 which lights brilliantly if moistened with water, both of the photo- 

 genic substances going into solution. It serves as the raw material 

 for the isolation of luciferin and luciferase. From this material an 

 extract may be prepared with distilled water which contains luciferin, 

 luciferase, all the proteins of the animal soluble in water, salts, and 

 other water-soluble material. The extract, filtered through filter 

 paper, is yellow colored and slightly opalescent and glows for some 

 time. The light finally disappears due to the oxidation of the luci- 

 ferin. There is no change of color on standing. If the extract so- 

 lution is not too concentrated and well shaken with air, all the lucif- 

 erin will be oxidized and luciferase alone of the photogenic sub- 

 stances together with oxyluciferin will remain. Salts and other 

 crystalloidal substances may be removed by dialysis since luciferase 

 does not dialyze. The solution is remarkably stable. I have allowed 

 it to dialyze against running tap water in a Schleicher and Schiill 

 parchment diffusion thimble for 2 weeks and then against distilled 

 water for 1 more week without any marked loss of luciferase. In- 

 deed, solutions of luciferase may stand until they become foul and 

 ill smelling from bacterial decomposition without destruction of the 

 luciferase. Solution may be preserved free of bacterial development 

 with toluene or chloroform for many months, but a slow destruction 

 of the luciferase occurs and at the same time a precipitate forms. 

 The luciferase is present in the colloidal state as it does not pass col- 

 lodion or parchment paper membranes. A solution prepared in the 

 above manner will be known as crude luciferase solution. Although 

 the luciferase can be purified by repeated precipitation with a va- 



