82 Papers from the Department of Marine Biology. 
when the photogenic substance is oxidized. Thus, the crystals of 
xanthin or some related substance in the reflecting layer of the firefly 
have been regarded as the oxidation products of the luminous material, 
thought to be nucleoprotein. Dubois! regards luciferin as a protein 
and states that it forms the same oxidation products as other pro- 
teins, amino-acids being mentioned as possible substances formed. It 
should be pointed out in this connection that the formation of amino- 
acids from proteins involves no oxidation, but an hydrolysis. 
If we assume that the oxidation of luciferin changes the molecule but 
slightly, we at once think of comparing the change luciferin <—, oxylu- 
ciferin with the change reduced hemoglobin <=; oxyhemoglobin. The 
condition is, however, not so simple as this, for oxyhemoglobin will 
again give up its oxygen, providing the partial pressure of oxygen is 
sufficiently low, whereas oxyluciferin will not do this. We can not re- 
duce oxyluciferin solution by exhausting the oxygen with an air pump. 
There is another oxidation—reduction system which can also be 
easily reversed, but not by merely removing the oxygen—that is, the 
reduction of a dye such as methylene blue to its leuco-base. I believe 
the change which occurs when luciferin is oxidized is similar to that 
which occurs when the leuco-base of methylene blue or sodium indigo- 
sulphonate is oxidized to the blue dye. 
My attempts to reduce the oxidation product of luciferin started from 
the observation that if one places a clear solution of luciferase in a tall 
test-tube, although it may give off no light at first when shaken, after 
standing a day or so a very bright light would appear on shaking. 
This was especially true when the luciferase had become turbid and 
ill-smelling from the growing of bacteria. Thinking that the bacteria 
produced a substance which could be oxidized by the luciferase, I tried 
growing bacteria and also yeast on appropriate culture media and after 
some days of growth mixing the culture media containing the products 
of bacterial or yeast growth with luciferase, expecting to obtain light; 
but no light appeared. However, if a little crude luciferase solution 
was added to the bacterial or yeast cultures and then allowed to stand 
for some hours, light appeared whenever they were shaken. Indeed, 
such cultures behaved much as a suspension of luminous bacteria which 
has used up all the oxygen in the culture fluid and will only luminesce 
when, by shaking, more oxygen dissolves in the culture medium. 
Realizing that in bacterial cultures in test-tubes anaerobic conditions 
soon appear, and also the strong reducing action of bacteria upon many 
substances (for instance, nitrates or methylene blue) under anaerobic 
conditions, it struck me that the bacteria might be utilizing the oxygen 
of the oxidaticn product of luciferin, reducing it to luciferin again. We 
must remember that since crude luciferase solution is a cold-water 
extract of a luminous animal allowed to stand until all the luciferin has 
1 Dubois, Ann. Soc. Linn. de Lyons, 1914, Ixi, 169. 
