88 Papers from the Department of Marine Biology. 
color on standing. If the extract solution be not too concentrated and 
is well shaken with air, all the luciferin will be oxidized and luciferase 
alone of the photogenic substances, 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 two weeks and 
then against distilled water for one more week without any marked loss 
of luciferase. Indeed, solutions of luciferase may stand until they 
become foul and ill-smelling from bacterial decomposition without 
destruction of the luciferase. Solutions may be preserved free of bac- 
terial development with toluol or chloroform for many months, but a 
slow destruction of the luciferase occurs; at the same time a precipitate 
forms. The luciferase is present in the colloidal state,as it does not 
pass collodion or parchment-paper membranes. A solution prepared 
in the above manner will be known as crude luciferase solution. For 
many purposes it is not necessary to previously remove the fat. 
Although the luciferase can be purified by repeated precipitation with 
a variety of substances,it loses strength during the process and the 
crude luciferase solution is the most powerful that can be obtained. 
From the dried, powdered, fat-free Cypridinz an extract may be pre- 
pared with hot water which contains the second of the photogenic sub- 
stances, luciferin, all of the proteins of the animal not coagulated by 
heating, the remaining salts, and other material soluble in hot water. 
My procedure is to add boiling water directly to the dry powder, boil 
about 20 seconds, and filter quickly while hot. The filtrate is slightly 
opalescent, yellow-colored, and does not darken on standing. On cool- 
ing, this solution often glows faintly, but if heated to the boiling-point a 
second time the glow ceases and does not return on cooling, although its 
content of luciferin is diminished. This hot-water extract of Cyprid- 
inze will be spoken of as crude luciferin solution. If the hot-water 
extract stands in a shallow dish at room temperature, the luciferin also 
disappears in the course of some hours, the time depending on its 
concentration. As already mentioned, this is due to oxidation of the 
luciferin apart from luciferase, as may be very easily determined by 
keeping the luciferin extract in absence of oxygen. I have kept such a 
solution in a test-tube covered with a layer of vaseline 1 inch in depth 
for 90 days, and at the end of that time it was capable of giving a 
brilliant light when mixed with luciferase. Luciferin and luciferase 
together in solution are likewise both stable in the absence of oxygen. 
I have kept such solutions in an evacuated tube or in a hydrogen atmos- 
phere for many months and at the end of that time on admitting air a 
briluant light appears. 
The oxidative disappearance of luciferin, like other chemical reac- 
tions, is greatly accelerated at the higher temperatures. A solution of 
