Although luciferin is not digested by trypsin, even after five days at 38° C., it does hydrolyze with mineral acids after about 16 hours' boiling. Some proteins, the albuminoids and racemized proteins, resist tryptic digestion but yield to acid hydrolysis. We know also that some NH-CO linkages of proteins are broken down with great difficulty by trypsin as it is difficult to obtain a tryptic digest of protein which does not give the biuret reaction, and the work of Fischer and Abderhalden has shown that certain artificial polypeptides are not digested by pure activated pancreatic juice.

We have, then, three possibilities: Luciferin is (1) either a natural proteose not attacked by trypsin, or (2) if attacked by trypsin its decomposition products (presumably amino-acids) still contain the group oxidizable with light production, or (3) it is not protein at all. I have been unable to oxidize with light production various mixtures of amino-acids (from tryptic digestion of beef and casein, or the acid hydrolysis products of luciferin itself) by means of luciferase, and consequently am led to believe that Cypridina luciferin is either a new natural proteose, soluble in absolute alcohol and not digested by trypsin or that it belongs to some other group than the proteins. The absence of a biuret reaction would point in that direction and the question must await further study.

Cypridina luciferin is found in the luminous gland of the animal and possibly in parts non-luminous as well as in the luminous organ. This is true of the luciferin from fireflies which is found throughout the body of Luciola, Photuris and Photinus.

Cypridina luciferase.—Luciferase, on the other hand, has all the properties of a complex protein. It will not dialyze through collodion or parchment membranes, is soluble only in aqueous solvents, and hence precipitated by alcohol and acetone, digested by proteolytic enzymes, readily changed by contact with dilute acid and alkali and irreversibly coagulated on boiling. It is completely salted out of solution by saturation with (NH₄)₂SO₄ and nearly completely precipitated by the alkaloidal reagents. Its other properties are given in Table 8. Taken together, they point to the group of albumins as the class of proteins with which luciferase most closely agrees.

If luciferase is not a protein it is so closely bound up with protein that it cannot be separated. This is characteristic of many enzymes and luciferase is also an enzyme. We can determine this by finding out whether luciferase will accelerate the oxidation of a large amount of luciferin, for such is the test of a catalytic substance. If we take 1 c.c. of a dilute solution of luciferase (1 Cypridina to 50 c.c. water) and add to it successive 1 c.c. portions of concentrated luciferin (1 Cypridina to 2 c.c. solution) as soon as the light from the preceding addition has disappeared, after four 1 c.c. additions, no more light is produced. The luciferase is therefore used up and cannot oxidize more than a certain quantity of luciferin. In this experiment, however, we added a concentration of luciferin from one Cypridina 100 times that of the luciferase from one Cypridina, i.e., four additions each 25 times as concentrated. We have, of course, no way of telling what the absolute amount (in milligrams) of luciferin or luciferase is in a single Cypridina, but we do know that the luciferase from one Cypridina cannot oxidize luciferin from more than 100 Cypridinas. If the ratio of luciferin to luciferase in a single animal is 100:1, it would mean that luciferase could oxidize 10,000 times its weight of luciferin. A large excess of luciferin but not an indefinite quantity can be oxidized by luciferase, and I believe this is sufficient justification for considering luciferase an enzyme, although it is not an ideal example of an organic catalyzer. Quite a number of enzymes are known to be diminished during the course of the reaction they accelerate or to be poisoned by their reaction products. Enzyme reactions inhibited by the formation of reaction products again proceed if these are removed or diluted. However, light does not again appear in a mixture of weak luciferase with excess of luciferin upon dilution with water, so that the luciferase cannot have been merely inhibited by some reaction product but must have been actually used up during the reaction. It should be noted in passing that the peroxidases, ordinarily spoken of as oxidizing enzymes, are used up in the reaction and can only oxidize limited amounts of oxidizable substances, a quantity almost in proportion to the concentration of peroxidase present.

Whether luciferase is an oxidizing enzyme made up of an albumin associated with some heavy metal as iron, copper or manganese is uncertain. From analyses of whole Cypridina, kindly made for me by Prof. A. H. Phillips of Princeton University, all three of these metals, which we know to be associated with biological oxidations, are present, and it is quite possible that one of them is concerned with the oxidation of luciferin.

Although I have tested a great many oxidizers, organic and inorganic, and a large number of oxidizing enzymes from blood and tissue extracts of animals rich in iron, copper and manganese, I have found no material which is capable of taking the place of Cypridina luciferase. Peroxidases or oxidases of plants, hæmoglobin, hæmocyanin, extracts of mussels, manganese containing blood of various marine crustacea and mollusks will give no light on mixing with luciferin. Such active oxidizers as KMnO₄, H₂O₂, BaO₂, and many others, will not oxidize Cypridina luciferin with light production, although they can oxidize Pholas luciferin with light production.

The action of Cypridina luciferase is very highly specific. It is found only in the luminous organ of Cypridina hilgendorfii, not in non-luminous parts and not in a non-luminous species of Cypridina closely related to hilgendorfii.

Luciferins and luciferases from closely allied luminous forms will mutually interact to produce light, but no light appears if these substances come from distantly related forms. Thus firefly (Photuris) luciferin will give light with Pyrophorus luciferase and vice versa, but Cypridina luciferin will give no light with firefly (Luciola) luciferase or vice versa, nor with Pholas luciferase or vice versa. The faint luminescences sometimes observed on mixing firefly or Cypridina luciferase with boiled extracts of non-luminous forms, or of distantly related luminous forms, are probably caused by photophelein in the boiled extract.

Like the plant peroxidases, Cypridina luciferase is not readily affected by the action of chloroform, toluol, etc. Unlike the plant peroxidases, it will not oxidize (i.e., produce coloration) in either presence or absence of H₂O₂, any of the hydroxyphenol or aminophenol compounds, such as pyrogallol, a-naphthol, para-diamino-benzine, gum guaiac, etc., commonly used as peroxidase reagents. Neither will luciferase produce light with any substances, such as oils, lophin, pyrogallol, gallic acid, esculin, etc., which we know to be capable of oxidation with light production by other means. The luciferases are very highly specific and act only upon the luciferins of the same or closely related species. They must be placed by themselves in a new class of oxidizing enzymes.

According to Dubois, Pholas luciferase is rather readily destroyed by chloroform and my own observations indicate that this is true also of firefly luciferase, so that a certain amount of variation exists in the group of luciferases.

None of the luminescent animals which I have studied are at all affected by cyanides. The luminescence continues in extracts of Cypridina, firefly, and Cavernularia, or in Noctiluca and luminous bacteria after addition of small or high (m/40) concentrations of KCN. In this respect the luciferases are very different from many types of oxidizing enzymes which are inhibited by exceedingly weak concentrations of cyanide. It should be borne in mind, however, that while KCN inhibits catalase and the catalytic decomposition of H₂O₂ by Pt or Ag, it does not affect the catalytic decomposition of H₂O₂ by thallium.

Oxyluciferin.—When luciferin is oxidized it must be converted into some substance or substances and I believe this change involves no fundamental destruction of the luciferin molecule as it is a reversible process. I shall speak of the principal (if not the only) product formed as oxyluciferin.

If we assume that the oxidation of luciferin changes the molecule but slightly, we at once think of comparing the change luciferin ⇆ oxyluciferin with the change reduced hæmoglobin ⇆ oxyhæmoglobin. The condition is, however, not so simple as this, for oxyhæmoglobin will again give up its oxygen providing the partial pressure of oxygen is made sufficiently low, whereas oxyluciferin will not do this, at least in the dark. We can not reduce oxyluciferin solution by exhausting the oxygen with an air-pump.

There is another oxidation-reduction system which can also be easily reversed, but not by merely removing the oxygen from the solution—that is, the reduction of a dye such as methylene blue to its leuco-base. I believe the change which occurs when luciferin is oxidized is similar to that which occurs when the leuco-base of methylene blue or sodium indigo-sulphonate is oxidized to the blue dye. Oxidation of leuco-dye bases occurs spontaneously in presence of oxygen and appears to consist in the removal of hydrogen from the leuco-base with formation of water. Reduction of these dyes may be effected in the same ways that oxyluciferin can be reduced. In the case of methylene blue, reduction consists in the addition of two hydrogen atoms. Whether a similar change occurs when oxyluciferin is reduced or whether oxygen is actually added as in formation of hæmoglobin cannot be definitely stated at present. We may write equations representing these possibilities as follows:

C₁₆H₂₀N₃SCl (leuco-methylene blue) + O ⇆ C₁₆H₁₈N₃SCl (methylene blue) + H₂O

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. Realizing 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.

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 extract 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

It is possible that the action of bacteria (which produces CO₂), muscle tissue (which contains lactic acid), milk (in which lactic acid may be formed by bacteria), or Mg + acid, in forming luciferin, is not the result of their reducing power but of their acidity. Fortunately we can test this matter by the use of reducing fluids which are not acid. If they also form luciferin from oxyluciferin, a reduction must occur. Nascent H can be generated by the action of NaOH on Al, or when finely divided Mg or Zn or Al is placed in water. With Mg the water becomes only slightly alkaline from formation of almost insoluble Mg(OH)₂. If we add some Al powder and dilute NaOH to an oxyluciferin solution, H is given off and luciferin is formed. As oxyluciferin cannot be formed by the addition of alkali alone we must have in this experiment a reduction of oxyluciferin in alkaline medium by the nascent H produced. Luciferin can also be formed by merely adding Al or Zn or Mg dust to an oxyluciferin solution. Methylene blue can also be readily reduced to its leuco-base by Zn dust or Al + NaOH.

Indeed, if one adds some Al or Zn or Mg powder to a solution of luciferase, light will appear whenever the solution is shaken. Luciferase solution must always contain the oxidation product of luciferin, oxyluciferin. In presence of nascent H this is reduced to luciferin, and since the reaction of the medium is alkaline and luciferase is present this is oxidized with light production, when, by shaking, air is dissolved. The light can never become very bright except at the surface because of the deficiency of oxygen in the solution. It would seem, then, that the action of bacteria, yeast, muscle cells, etc., on oxyluciferin must be due not entirely to their acid reaction but to their reducing power as well.

The above experiment is a very striking and instructive one. Given a test tube of luciferase solution containing, as it does, oxyluciferin, add some Zn dust or Mg powder, and the evolution of hydrogen begins. Conditions are now favorable for the reduction of oxyluciferin and this occurs. Shake the contents of the tube to dissolve oxygen and light appears. Allow the tube to stand and the light soon disappears. Shake again and the light reappears. The luminescence reduction and oxidation process can be demonstrated many times.

A similar experiment can be performed with luciferase and oxyluciferin solution by addition of NH₄SH. This will serve also as another example of the reduction of oxyluciferin in an alkaline medium. Whenever we shake a tube of luciferase, oxyluciferin and NH₄SH, light will appear. When the tube is at rest it becomes dark. Even the merest touch is sufficient to agitate the tube contents, cause solution of oxygen and appearance of light. It is just as if we stimulate the tube to produce light and I believe the phenomenon has a deeper significance and a more fundamental similarity to the phenomena of stimulation than may at first appear. What more simple means of controlling a process can we think of than by admission or withdrawal of oxygen? The firefly turns on its light by stimulation through nerves of the luminous organ. Noctiluca flashes on stimulation of any kind, even the slightest agitation causing a brilliant emission of light. If the stimulation process means merely the admission of oxygen to the photogenic cells we have a mechanism in the cell itself for automatically producing the light. The admission of oxygen results in aërobic conditions and luciferin in presence of luciferase can then oxidize to oxyluciferin with luminescence. When the oxygen is used up, the light ceases, anaërobic conditions prevail, and the oxyluciferin is reduced to luciferin again. Thus, luciferin is reformed during the rest period of Noctiluca or between the flashes of the firefly. What more efficient type of light than this is to be desired?

Again, methylene blue offers an interesting parallel to oxyluciferin. A little NH₄SH added to methylene blue solution will reduce (decolorize) it to the leuco-base. If the tube is now shaken the blue color returns. On standing reduction again occurs. The process can be repeated a number of times, the reaction going in one or the other direction, depending on the oxygen content of the mixture.

As methylene blue contains no oxygen, its reduction consists in the addition of two atoms of hydrogen. When leuco-methylene blue oxidizes, water is formed by the union of these two atoms of hydrogen with oxygen, thus:

C₁₆H₂₀N₃SCl + O ⇆ C₁₆H₁₈N₃SCl + H₂O
(leuco-methylene blue)    (methylene blue)

Briefly—MH₂ + O ⇆ M + H₂O

To reduce methylene blue we can add the two hydrogen atoms directly from nascent hydrogen formed in the solution or we can split up water by a catalyzer in the presence of some substance, which will take up the oxygen of water, thus:

NaH₂PO₂ + H₂O + Pd = NaH₂PO₃ + H₂ + Pd
(Sodium hypophosphite)    (Sodium phosphite)

This reaction occurs in presence of finely divided palladium. Methylene blue can be reduced by the H₂ and the hypophosphite oxidized.

Since oxyluciferin can be reduced by palladium and sodium hypophosphite (Harvey, 1918), it is probable that we can write the equation for reduction of oxyluciferin and oxidation of luciferin in a similar manner to that of methylene blue:

Luciferin + O ⇆ Oxyluciferin + H₂O

Briefly—LH₂ + O ⇆ L + H₂O.

Just as in the case of methylene blue the reaction proceeds in the right hand direction spontaneously if the pressure of O is sufficiently high. If luciferase is also present we have luminescence.

LH₂ + O + luciferase ⇆ L + H₂O + luciferase (luminescence)

The reaction proceeds in the left hand direction under low oxygen pressure, in the presence of nascent hydrogen or with some catalyzer which is able to split water, transferring the H₂ to oxyluciferin and the O to an acceptor (A). NaH₂PO₂ plays the part of the acceptor.

L + H₂O + A + Pd = LH₂ + AO + Pd.

This appears to be the way in which the reducing enzymes or perhydridases (comparable to the Pd) of living tissues act (Bach, 1911-13) and the action of yeast cells, bacteria, muscle suspensions, etc., in reducing oxyluciferin must occur in the same manner.

If we assume that the LH₂ (luciferin) compound is dissociated to even the slightest extent into L and hydrogen, the hydrogen ion will shift the equilibrium toward the formation of that substance which involves the taking up of hydrogen. Consequently we may obtain a partial formation of luciferin by adding an acid to oxyluciferin. Reduction of the H-ion concentration tends to shift the equilibrium in the opposite direction. Consequently, addition of alkali favors the oxidation of luciferin, and it is quite generally true that biological oxidations are favored by an alkaline reaction. In addition oxygen in alkaline medium has a higher oxidation potential than in neutral or acid media. I believe this is the explanation of the action of acid in formation of luciferin from oxyluciferin.

Addition of acid is not the only means of favoring the formation of luciferin from oxyluciferin. Any reaction which proceeds in one direction with evolution of light should, theoretically, proceed in the opposite direction under the influence of light. So far as I know the case of a reaction, photogenic in one direction and photochemical in the other direction, has never been described, unless we are to accept the cases of phosphorescence, for instance, the absorption of light by CaS and its emission in the dark. However, the reaction which occurs during phosphorescence cannot be stated.

It is a fact that light will cause the reduction of oxyluciferin. A tube of oxyluciferin exposed to sunlight for six hours, or the mercury arc for two hours, will be partially converted into luciferin. It will luminesce when luciferase is added, while a control tube kept in darkness shows no trace of luciferin. The action is more marked with the ultra-violet as a solution of oxyluciferin in a quartz tube showed more reduction than one in a glass tube when exposed for the same length of time to the quartz mercury arc. The reduction is not dependent on the formation of acid under the influence of light since two tubes of oxyluciferin, one kept in darkness and the other exposed to sunlight for six hours, had the same reaction, Ph = 9.3. Of course some reducing substance might be formed under the influence of light but this is not very probable.

We may therefore write the reaction for luminescence in the following way:

Acid and light favor reduction while alkali and darkness favor oxidation in the luciferin ⇆ oxyluciferin reaction. Whether the luciferin be really oxidized by removal of H₂ or whether by addition of oxygen is, of course, uncertain, but the analogy with methylene blue is striking and may serve as a working hypothesis until the composition of luciferin and its oxidation product are known.

While I have not studied the properties of oxyluciferin as fully as those of luciferin, so far as I can judge, both substances give the same general reactions and possess identical properties. Both crude luciferin and crude oxyluciferin solution are yellow in color, but I do not believe that either pure luciferin or oxyluciferin are yellow in color, because an ether or benzine extract of Cypridina is also yellow, although luciferase, luciferin, and oxyluciferin are insoluble in ether and benzine. The yellow pigment which can be observed to make up part of the luminous gland of Cypridina is not luciferin or luciferase. It may be a pigment related to urochrome.

When tests are applied and precipitating reagents are added to crude luciferin and crude oxyluciferin solution, they give identical results in each case. A more complete account of the chemistry of luciferin has been given in this chapter, and there is no need of duplicating these statements regarding oxyluciferin. Like luciferin, the oxyluciferin will pass porcelain filters, dialyze through parchment or collodion membranes, and is undigested by salivary diastase, pepsin HCl, Merck's pancreatin in neutral solution, and erepsin. The salivary diastase and the pancreatin (containing amylopsin, trypsin, and lipase) were allowed to digest for four days at 38° C. without showing any evidence of digestive action.

As luciferin is so easily oxidizable a substance, we should expect to find that it will reduce just as glucose will reduce. However, a concentrated solution of luciferin has no reducing action on Fehling's (alkaline Cu), Barfoed's (acid Cu), Nylander's (alkaline Bi) or Knapp's (alkaline Hg) reagent. Glucose will reduce methylene blue in alkaline (not in neutral solution), but luciferin will not reduce methylene blue in alkaline or neutral solution. It would seem, then, that luciferin must contain no aldehyde group. If so, we should expect to obtain reduction of some of the above reagents. Just what group is concerned in the oxidation is unknown at the present time, and in the absence of more experimental data, speculation regarding it can be of little value.

SUMMARY

In summing up we may say that the luminescence of at least three groups of luminous animals, the beetles, Pholas, and Cypridina, has been definitely shown to be due to the interaction of two substances, luciferin and luciferase, in presence of water and oxygen. Luciferin and luciferase have quite different properties and may be easily separated from each other by various chemical procedures. As the luciferins and luciferases from different luminous animals have somewhat different properties, they may be designated by prefixing the generic name of the animal from which they are obtained.

Cypridina luciferin differs from Pholas luciferin in that it can not be oxidized with light production by KMnO₄, H₂O₂, with or without hæmoglobin, or similar oxidizing agents. Cypridina luciferase differs from Pholas and firefly luciferase in that it is not readily destroyed by the fat-solvent anæsthetics, such as chloroform, ether, etc.

When Cypridina luciferin is oxidized, no fundamental splitting of the molecule occurs, because the product, oxyluciferin, can be readily reduced to luciferin again. This reduction is brought about under conditions similar to those necessary for the reduction of dyes, such as methylene blue. Oxyluciferin can be reduced to luciferin, which will again give light with luciferase, by the reductases of muscle tissue, liver, etc., or by bacteria; by Schardinger's enzyme of milk; by H₂S; by the nascent hydrogen from the action of acetic acid on magnesium or of water or NaOH on aluminium, zinc or magnesium; and by palladium black and sodium hypophosphite, all well-known reducing methods. Reduction of oxyluciferin no doubt occurs even in presence of luciferase if oxygen is absent, and reduction of oxyluciferin no doubt occurs in animals which burn luciferin within the cell, thus tending for conservation of material. Dilute alkali favors oxidation and dilute acid favors the reduction. Light favors the reduction of oxyluciferin.

Apparently luciferin and oxyluciferin have identical chemical properties. Neither is digested by the enzymes: malt diastase, ptyalin, yeast invertase, pepsin, trypsin, steapsin, amylopsin, rennin, erepsin, urease or enzymes occurring in the water extracts of dried spleen, kidney, or liver. Luciferase is destroyed only by pepsin (probably), trypsin, erepsin, and something in spleen and liver extract.

Luciferase is unquestionably a protein and all its properties agree with those of the albumins. Although used up in oxidizing large quantities of luciferin, it behaves in many ways like an enzyme and may be so regarded.

Luciferin, on the other hand, is not digested by proteolytic enzymes, is dialyzable, almost but not completely precipitated by saturation with (NH₄)₂SO₄, and is soluble in absolute alcohol, acetone, and some other organic solvents, but not in the strictly fat-solvents like ether, chloroform, and benzol. There are, however, certain CO-NH linkages which are not attacked by proteolytic enzymes and some peptones soluble in absolute alcohol, so that these two characteristics do not bar it from the group of proteins. Luciferin, in fact, has many properties in common with the proteoses and peptones but its chemical nature cannot be definitely stated at present.

CHAPTER VII
DYNAMICS OF LUMINESCENCE

Luciferin and luciferase from Cypridina will also luminesce in exceedingly small concentration. If one grinds a single Cypridina in a mortar with water and dilutes the extract to 25,600 c.c., light can be observed if luciferin is added to this dilute luciferase solution. By determining the volume of the luminous gland of Cypridina and even assuming that this volume is all luciferase, one can calculate that one part of luciferase in 1,700,000,000 parts of water will give light when luciferin is added. Likewise, a similar dilution of luciferin will give visible light when luciferase is added.

The sensitivity of our eye is largely responsible for the detection of so small an energy change. As we have seen, recent determinations have proved that the dark adapted eye can detect 18 × 10^-10 ergs per second. From the heat of complete oxidation of pyrogallol it is possible to calculate the amount of pyrogallol necessary to give 18 × 10^-10 ergs if completely oxidized. This quantity is infinitesimally small. When pyrogallol is oxidized by K₄Fe(CN)₆ and H₂O₂, it is not completely oxidized and probably only a small amount of the energy is converted into light; otherwise we should be able to see the luminescence of a very much weaker concentration of pyrogallol. As the reaction luciferin ⇆ oxyluciferin is so easily reversible, very little energy must be liberated, and, as experiments indicate, very little heat, if any, accompanies light production. Even though this be true, it is still possible for a very small amount of luciferin to produce a very large amount of light.

A very small amount of luciferase only is necessary because it behaves as an enzyme and follows the general rule that catalysts act in minute concentrations.

On the assumption that luciferase is an enzyme, an organic catalyst oxidizing luciferin with light production, we may appropriately inquire into the relation between the concentration of luciferin and luciferase and intensity and duration of luminescence. Oxygen tension, hydrogen ion concentration and temperature must be maintained constant as these all affect both intensity and duration of luminescence. Before considering luciferin and luciferase, however, let us study a few well-known chemiluminescent oxidations with special reference to concentration of reacting substances and temperature.

The effect of temperature on luminescence is of special interest because it gives us a means of analysis for determining if the luminescence depends on reaction velocity. We know that photochemical reactions are very little affected by temperature because the reaction is dependent on the absorption of light, a physical process, and this increases only a small per cent. for a rise of temperature of 10° C. To put it in the usual way, its temperature coefficient (Q₁₀) for a 10° interval is usually less than 1.1. On the other hand, we should expect photogenic reactions, in which some of the chemical energy is converted into radiant energy, to give off much more light the greater the reaction velocity. As reaction velocity increases so rapidly with temperature (Q₁₀ = 2 to 3), luminescence intensity should rapidly increase with increase in temperature.

Trautz (1905), from his extensive study of the chemiluminescence of phenol and aldehyde compounds came to the conclusion that luminescence intensity was proportional to reaction velocity. He based his conclusions largely on the effects of temperature and concentration of reacting substances and went so far as to declare that any reaction would produce luminescence if the reaction velocity were sufficiently increased. It is quite true that increasing the temperature does increase the intensity of chemiluminescence, but this is only within certain limits. As we raise the temperature, chemiluminescence becomes more intense but we soon reach a temperature for maximum luminescence and above this the intensity diminishes. This is especially well seen in the action of various oxidizers on pyrogallol and H₂O₂ recorded in Table 10. At 100° C. practically no light is produced by many oxidizers which are themselves unaffected at 100°. If we are to connect reaction velocity with intensity of luminescence we must conclude that the evolution of light is dependent rather on an optimum than a maximum reaction velocity.

TABLE 10
Temperature and Light Production. The Oxidizer is Mixed with an Equal Amount of M/100 Pyrogallol + 3 per cent. H₂O₂

Quite a number of instances are known in which increasing the mass of reacting substances leads not to an increase but to an actual cessation of luminescence. This fact does not confirm the theory that reaction velocity is a determining factor in luminescence. The conditions for the luminescence of white phosphorus are most interesting and unusual. (See van't Hoff, 1895; Ewan, 1895; Centnerszwer, 1895; Russell,1903; Scharff, 1908.) Phosphorus will only begin to luminesce at a certain small pressure of oxygen. This "minimum luminescence pressure" of oxygen is very low, so low that earlier observers, failing to remove traces of oxygen, thought that luminescence might occur in absence of oxygen. Curiously enough there is also a "maximum luminescence pressure" of oxygen above which no luminescence occurs. Phosphorus will not luminesce in pure oxygen. Between the minimum and maximum is an "optimum luminescence pressure" where luminescence of the phosphorus is brightest. The exact values of these pressures vary with degree of water vapor present and with temperature. According to Abegg's Handbuch der anorganischen Chemie, the maximum luminescence pressure with water vapor present, is 320 mm. Hg at 0° and increases 13.19 mm. Hg for each degree rise in temperature. This means that for a definite temperature, say, 20°, phosphorus will not luminesce with an oxygen pressure of 583 mm. Hg, but will luminesce with pressures under this. If, however, we raise the temperature, luminescence will occur with an oxygen pressure of 583 mm. Hg.

A somewhat analogous case is presented by the oxidation of pyrogallol solution in contact with ozone, except that in this reaction too high a concentration of pyrogallol will hinder the oxidation. I have not studied the effect of varying concentrations of ozone. If oxygen, passed through an ozonizer (the silent electric discharge tube), is bubbled through m/100 pyrogallol, no luminescence occurs at 0°, a fair luminescence at 20°, a good luminescence at 50°, and a bright luminescence at the boiling point. If the pyrogallol is of m concentration, no luminescence occurs at 0° or 20°, a fair luminescence at 50°, and a bright luminescence at the boiling point. For a definite temperature, say 20°, no light appears if the pyrogallol is of m concentration, but if we raise the temperature, luminescence can occur. The similarity to phosphorus is obvious. Thus the "maximum luminescence pressure" of pyrogallol increases with increase of temperature.

We have already seen that pyrogallol can also be oxidized, if H₂O₂ is present, by a great variety of substances, such as peroxidases of potato or turnip juice, hæmoglobin, KMnO₄, K₄Fe(CN)₆, CrO₃, MnO₂, hypochlorites and hypobromites, or colloidal Pt and Ag. For convenience we may collectively speak of these as oxidizers. They are recorded in Table 13. No light occurs if H₂O₂is absent. In the case of some of these oxidizers pyrogallol will luminesce in dilute concentrations but not in strong. Also, dilute pyrogallol will luminesce with a dilute solution of oxidizer but not with a concentrated solution of oxidizer. The effect of rise in temperature in these cases also is to increase the "maximum luminescence concentration" of pyrogallol and the "maximum luminescence concentration" of oxidizer. Table 11 shows this effect of temperature with K₄Fe(CN)₆ and varying concentrations of pyrogallol, and Table 12 shows the effect of temperature with pyrogallol and varying concentrations of K₄Fe(CN)₆. Table 10 shows the relation between temperature and intensity of luminescence with pyrogallol and various oxidizers. The terms faint, fair, good, and bright are purely relative designations of brightness as estimated by the eye, for accurate measurements of weak intensities are very difficult to make.

From Table 10 it should be noted that the intensity of luminescence of pyrogallol oxidized with most oxidizers is actually less at the boiling point, a fact which I have repeatedly verified. Let us now see how these facts are to be explained. If we assume that luminescence is dependent on reaction velocity, the intensity of luminescence should increase with increasing temperature. Up to a certain limit this is what we find, but at temperatures above this limit the intensity of luminescence actually decreases. The duration of luminescence also decreases. There is an optimum temperature for luminescence in many cases and we can only conclude that luminescence depends not on a very rapid reaction velocity but on a certain definite reaction velocity. Assuming that this is true, how can we account for the anomalous fact that in high concentrations of oxygen, phosphorus will not luminesce or that in high concentrations of pyrogallol, there is no luminescence in presence of ozone or of oxidizer and H₂O₂. Of course with high active mass of oxygen (in case of phosphorous luminescence) or of pyrogallol (in case of pyrogallol luminescence) the reaction velocity must be greater than the optimum. If that is the case, then lowering the temperature should reduce the reaction velocity to the optimum and light should appear. However, as we have seen, not lowering but raising the temperature causes luminescence with high oxygen concentration or high pyrogallol concentration.