On the Chemistry of Light Production in Luminous Organisms. 83 
been oxidized, it must contain oxyluciferin as well as luciferase and will 
give light if the oxyluciferin is again reduced and oxygen admitted. 
This appears to be the correct explanation of the above experiments. 
Not only bacteria but also tissue extracts have a strong reducing 
action in absence of oxygen. Thus, muscle tissue stained in methylene 
blue will very quickly decolorize (reduce) the methylene blue if oxygen 
(air) is kept away, but the blue color immediately returns if air is 
admitted. Oxyluciferin (i. e., a solution of luciferin which has been 
completely oxidized by boiling or standing in air until it no longer gives 
light with luciferase), if mixed with a suspension of ground frog’s muscle 
and kept in a well-filled and stoppered test-tube for some hours, is re- 
duced to luciferin and gives a bright light if now poured into luciferase 
solution. Frog-muscle suspension alone or oxyluciferin alone give no 
light with luciferase, nor will a mixture of frog-muscle suspension and 
oxyluciferin, if shaken with air for several hours. Only if this last 
mixture be kept under anaerobic conditions is the oxyluciferin reduced. 
The reducing action of tissues is said to be due to a reducing enzyme 
(reducase or reductase), itself composed of a perhydridase and some 
easily oxidized body such as an aldehyde.’ In the presence of the 
perhydridase the oxygen of water oxidizes the aldehyde and the hydro- 
gen set free reduces any easily reducible substance which may be 
present. There is a perhydridase in fresh milk, spoken of as Schar- 
dinger’s enzyme,” which is destroyed by boiling. If some aldehyde is 
added fresh milk will reduce methylene blue to its leuco-base or 
nitrates to nitrites, upon standing a short time. If shaken with air the 
blue color returns. There is no reduction unless an aldehyde is added 
or unless some boiled extract of a tissue such as liver is added. The 
boiled-liver extract has no reducing action of its own, but supplies a 
substance similar to the aldehyde which has been spoken of as a co- 
enzyme. Milk will reduce methylene blue without aldehyde if bacteria 
are present in large numbers. Also, there is no reduction if the milk, 
methylene blue, and aldehyde are agitated with air. The temperature 
optimum is rather high, 60° to 70° C. 
I find that milk is a favorable and convenient medium for the reduc- 
tion of oxyluciferin and that it acts without the addition of an aldehyde 
or the presence of bacteria. There is probably a substance acting as 
the aldehyde in the luciferase-oxyluciferin solution. No light appears 
if milk is added to a luciferase-oxyluciferin solution, but if the mixture 
is allowed to stand in absence of oxygen light will appear when air is ad- 
mitted. The air can be conveniently kept out by filling small test-tubes 
completely with the solution and closing them with rubber stoppers. 
Oxyluciferin may also be readily reduced by the use of the blood of 
the horse-shoe crab (Limulus) allowed to stand until bacteria develop.* 
1 Bach, A., Biochem. Z., 1911, xxxi, 443; xxxiii, 282; 1912, xxxviii, 154; 1913, li, 412. 
2 Schardinger, F., Chem. Zeit., 1904, xxviii, 704. 
3 Alsberg, C. L., Journ. Biol. Chem., 1915, xxiii, 495. 
