140 PHOTOGENIC REACTION IN CYPRIDINA 



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 occurred to 

 me that the bacteria might be utilizing the oxygen of the oxidation 

 product of luciferin, reducing it to luciferin again. We must remem- 

 ber that since crude luciferase solution is a cold water extract of a 

 luminous animal allowed to stand until all the luciferin has been oxi- 

 dized, 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 oxy- 

 gen (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 boihng or standing in air until it no longer 

 gives light with luciferase, if mixed with a suspension of ground frog 

 muscle and kept in a well filled and stoppered test-tube for some 

 hours, is reduced to luciferin and gives a bright light if now poured 

 into luciferase solution. Frog muscle suspension alone or oxyluci- 

 ferin alone gives 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 is 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. i° In the presence of the 



10 Bach, A., Biochem. Z., 1911, xxxi, 443; xxxiii, 282; 1912, xxxviii, 154; 1913, 

 m, 412. 



