84 Papers from the Department of Marine Biology. 
This experiment is of special interest because the blood contains hemo- 
eyanin, which is colorless in the reduced condition and blue in the oxy- 
condition. The color change thus serves as an indicator of the oxygen 
concentration in the blood. A sample of foul-smelling Limulus blood 
full of bacteria will become colorless on standing in a test-tube for 10 to 
15 minutes, but the blue color quickly returns if shaken with air. Such 
a blood has the power of reducing oxyluciferin through the activity of 
the bacteria which it contains. Fresh blood has very little if any 
reducing action. 
As almost all animal tissues contain reductases it is not surprising 
to find that a freshly prepared and filtered extract of Cypridina con- 
taining oxyluciferin and luciferase, which gives no light on shaking, 
will, on standing in a stoppered tube for 24 hours at room tempera- 
ture, give light when air is admitted. While this may be due to the 
development of bacteria with a reducing action, it does not seem 
likely, as under the same conditions methylene blue is not reduced 
in 24 hours and there is no turbidity or smell of decomposition in 
the tube. In 48 hours bacteria appear and methylene blue is also 
reduced. If we add chloroform, toluol, or thymol to the tubes of 
Cypridina extract to prevent the growth of bacteria, and allow them 
to stand 48 hours, upon admitting air the tube with chloroform gives 
no light, but the tubes with toluol and thymol do give light, although 
it is not so bright as if they were absent. I believe that these sub- 
stances have a destructive action on the reductases, most complete 
in the case of chloroform. 
I have not been able to demonstrate that a Cypridina extract will 
reduce methylene blue or nitrates to nitrites, either with or without the 
addition of acetaldehyde. This may be due to the fact that oxylucif- 
erin, which is also present, may be reduced more readily than either 
nitrates or methylene blue, and so is reduced first. 
Dubois? has described in Pholas a precursor of luciferin which he 
ealls proluciferin, which is converted into luciferin by another enzyme, 
coluciferase. The proluciferin is not destroyed by boiling and the 
coluciferase will withstand a higher temperature than luciferase and 
may be freed of luciferase in this manner. He cites an experiment* to 
prove the existence of proluciferin and coluciferase in Pholas, but I 
have been unable to repeat this with Cypridina. One might suppose 
that on allowing an extract of Cypridina (luciferase) to stand in absence 
of oxygen some proluciferin, assuming this to be present, would be con- 
verted into luciferin, which would give light if air was admitted. But 
we can allow a boiled extract of Cypridina (containing no coluciferase) 
to stand with milk or muscle-tissue suspensions in absence of oxygen 
and upon admitting air and adding luciferase obtain light. As lucif- 
1 This experiment may also be performed with Pholas luciferase with a similar result. 
2 Dubois, Compt. rend. Soc. Biol., 1907, 850; 1917, 964. 
3 Dubois, Compt. rend. Soc. Biol., 1917, 964. 
