The Nature of Animal Light

by E. Newton Harvey

Hæmoglobin + O ⇆ oxyhæmoglobin.

Let us now turn to the methods which may be used in reduction of oxyluciferin. We may then endeavor to write an equation which will represent the fundamental changes in the luminescence reaction.

My attempts to reduce the oxidation product of luciferin started from the observation that if one places a clear solution of luciferase in a tall test tube, although it may give off no light at first when shaken, after standing a day or so a very bright light would appear on shaking. This was especially true when the luciferase had become turbid and ill-smelling from the growth of bacteria. Thinking that the bacteria produced a substance which could be oxidized by the luciferase, I tried growing bacteria and also yeast on appropriate culture media, and after some days of growth mixing the culture media containing the products of bacterial or yeast growth with luciferase, 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. Real

izing that in bacterial cultures in test tubes, anaërobic conditions soon appear, and also the strong reducing action of bacteria upon many substances (for instance, nitrates or methylene blue) under anaërobic conditions, it struck me that the bacteria might be reducing the oxidation product of luciferin to luciferin again. We must remember that since crude luciferase solution is a cold-water extract of a luminous animal allowed to stand until all the luciferin has 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.

Oxyluciferin may also be readily reduced by the use of the blood of the horse-shoe crab (Limulus) allowed to stand until bacteria develop. This experiment is of special interest because the blood contains hæmocyanin, 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.

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 reduced 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 anaërobic 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 hydrogen set free reduces any easily reducible substance which may be present. There is a perhydridase in fresh milk, spoken of as Schardinger'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 co-enzyme. The aldehyde is oxidized to its corresponding acid. Milk will reduce methylene blue without aldehyde if bacteria are present in large numbers. 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 reduction 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.