E. NEWTON HARVEY 141 



perhydridase the oxygen of water oxidizes the aldehyde and the hy- 

 drogen set free reduces any easily reducible substance which may be 

 present. There is a perhydridase in fresh milk, spoken of as Schard- 

 inger'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 

 coenzyme. Milk will reduce methylene blue without aldehyde if 

 bacteria are present in large numbers. There is also no reduction if 

 the milk, methylene blue, and aldehyde are agitated with air. The 

 temperature optimum is rather high, 60-70°C. 



I find that milk is a favorable and convenient medium for the re- 

 duction 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 admitted. 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 horseshoe crab (Limulus) allowed to stand until bacteria develop. ^^ 

 This experiment is of special interest because the blood contains hemo- 

 cyanin 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 smelHng Limulus 

 blood full of bacteria will become colorless on standing in a test-tube 

 for 10 or 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. 



^^ Schardinger, F., Chem. Ztg., 1904, xxviii, 704. 

 12 Alsberg, C. L., /. Biol. Chem., 1915, xxiii, 495. 



