The Nature of Animal Light

by E. Newton Harvey

As almost all animal tissues contain reductases it is not surprising to find that a freshly prepared and filtered extract of Cypridina containing oxyluciferin and luciferase, which gives no light on shaking, will, on standing in a stoppered tube for 24 hours at room temperature in the dark 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 substances have a destructive action on the reductases, most complete in the case of chloroform. Dubois (1919c) also has recorded the occurrence of a reducing enzyme in Pholas, a "hydrogenase," which is able to form hydrogen from cane sugar, and luciferin from a boiled ex

tract of Pholas. He now regards it as identical with his co-luciferase.

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 oxyluciferin, which is also present, may be reduced more readily than either nitrates or methylene blue, and so is reduced first.

We can also reduce oxyluciferin by means which do not involve the use of animal extracts. Perhaps the best of these is reduction by palladium black and sodium hypophosphite. The latter is oxidized in presence of palladium and nascent hydrogen is set free. The nascent hydrogen reduces any easily reducible substance which may be present, such as methylene blue or oxyluciferin. Oxyluciferin is not reduced by palladium alone or hypophosphite alone, but methylene blue is reduced by palladium black alone.

If hydrogen sulphide is passed through a solution of methylene blue the dye is very quickly reduced and becomes colorless. If the H₂S is driven off by boiling the colorless methylene-blue solution, the blue color again returns on cooling. Oxyluciferin can also be reduced by H₂S.

If one adds some Mg powder to oxyluciferin and then dilute acetic acid in successive additions as the acetic acid is used up in formation of Mg acetate, the oxyluciferin will be reduced relatively quickly. Nascent hydrogen is produced in the reaction and is no doubt the active reducing agent.

Dilute acid favors the reduction of oxyluciferin. If

one saturates an oxyluciferin solution with CO₂ or adds a little dilute acetic acid, HCl, HNO₃ or H₂SO₄, to it, a certain amount of reduction will occur. No reduction occurs if the solution is saturated with pure hydrogen, even if allowed to stand 24 hours. The action of the acid begins when the solution of oxyluciferin, ordinarily slightly alkaline (Ph = 9), is made neutral (Ph = 7.1) as indicated in [Table 9]. The action of the acid must be on the oxyluciferin, as no luciferin or other enzymes destroyed on boiling are present.

Table 9
Effect of Acid on Reduction of Oxyluciferin

Solution	Ph	Luminescence with luciferase	Remarks
20 c.c. Oxyluciferin alone	9.01	Negative
20 c.c. Oxyluciferin + .05 c.c. 5 per cent. acetic acid	8.8	Negative
20 c.c. Oxyluciferin + .15 c.c. 5 per cent. acetic acid	7.1	Fair
20 c.c. Oxyluciferin + .30 c.c. 5 per cent. acetic acid	5.9	Good	Acid forms precipitate in this oxyluciferin sol.
20 c.c. Oxyluciferin + .50 c.c. 5 per cent. acetic acid		Good	Acid forms precipitate in this oxyluciferin sol.
20 c.c. Oxyluciferin + .75 c.c. 5 per cent. acetic acid		Good[10]	Acid forms precipitate in this oxyluciferin sol.