It was found that even after the luciferase and luciferin solutions came to the same temperature within the thermos bottle, this was not necessarily the same as that of the room and a slow rise or fall occurred as indicated by a slow drift of the galvanometer coil. Readings of each thermocouple on the galvanometer scale were therefore taken at one-minute intervals for some time before and after mixing the luciferin and luciferase solutions and plotted as curves. Control experiments were also carried out in exactly the same manner as the luciferin-luciferase experiments, but water was placed in the two tubes instead of luciferin and luciferase. Figs. 34 and 35 give typical experiments with water and with luminescent solutions, respectively.

With both control (water) and luciferin experiments there was a slight rise in temperature on mixing the liquids in the two tubes. The average rise of five control (water) experiments was .0054° C. and the average rise of five luciferin experiments was .0048° C.

The average rise in temperature is no doubt due to heat from friction in mixing of the liquids and breaking of the glass tube. The difference in the average rise of control and of luciferin experiments is so small (.0006° C.) as to have little significance. We may therefore conclude that if any temperature change occurs during the luminescent reaction it is certainly less than 0.001° C. and probably less than 0.0005° C., too small to be measured by this method.

To prepare the luciferin solution, two grams of dried Cypridina were dissolved in 20 c.c. hot water and 10 c.c. of this 10 per cent. solution was used in the thermos bottle in the above experiments. If we assume that 1 per cent. of the dried Cypridina is luciferin, 0.01 gram of luciferin on oxidation was not able to raise the temperature of the 10 c.c. (in reality 11 c.c., since 1 c.c. luciferase solution was mixed with the 10 c.c. luciferin solution) .001° C. This means that 1 gram luciferin liberates at least less than 10 calories during the luminescence accompanying oxidation.

Since 1 gram glucose liberates 4000 calories on complete oxidation to CO₂ and H₂O, it will be seen that the oxidation of luciferin is a very different type of reaction from the oxidation of glucose. As we shall see, it is probably similar to the oxidation of reduced hæmoglobin or the oxidation of leuco methylene-blue to methylene blue. According to Barcroft and Hill (1910), 1.85 calories are produced per gram of hæmoglobin oxidized. I have been unable to find figures for the heat exchange during oxidation of leuco-dyes, but it is no doubt also small. Since luciferin evolves no measurable amount of heat on oxidation, we have very good evidence in support of that obtained by electrometric measurements of H-ion concentration, that no carbon dioxide is produced during luminescence of luminous animals.

In most animal cells it is perfectly clear that luminescence does not accompany respiration, since respiration is a continuous process, whereas light is only produced on stimulation. It is true that on stimulation respiration is accelerated, and we might suppose that luminescence is an accompaniment of accelerated respiratory oxidations; but this is not the case, for in luminous animals a rise in temperature of ten degrees centigrade will accelerate the respiratory oxidations 250 per cent. without necessarily causing the production of light.

In fungi and bacteria, on the other hand, which continually emit light, it is quite natural to suppose that the light is an accompaniment of respiration, just as we know the heat of these forms to be. This view was accepted by such of the earlier workers as Fabre in 1855, who found that luminous portions of a mushroom, Agaricus olearius, gave off more CO₂ (4.41 c.c. CO₂ per gram in 36 hours at 12° C.) than non-luminous portions (2.88 c.c. CO₂ per gram in 36 hours at 12° C.). This experiment has never been repeated and there are many reasons besides luminescence why one piece of fungus might have a more rapid respiratory rate than another piece. It is not true that rapidly respiring plant tissues, such as germinating seeds or the spadix of Araceæ, are luminous, although they produce considerable heat.

On the other hand, it is very easy to prove that luminescence, even in bacteria, is not connected with respiration. Thus, Beijerinck (1889 c) found that of several species of luminous bacteria studied by him, one, Bacterium phosphorescens, was a facultative anaërobe and would grow, i.e., multiply, but not luminesce in the absence of oxygen. Some forms, ordinarily producing light, will grow, but fail to luminesce at high temperatures. Beijerinck (1915) has recently found that these individuals may, by continued cultivation at high temperatures, form non-luminous strains which fail to luminesce when again brought into lower temperatures, favorable for luminescence. These non-luminous mutants occasionally give rise to atavistic brilliantly luminous forms. Beijerinck also finds that after exposure of Photobacter splendidum to ultra-violet or strong sunlight, radium or mesothorium rays, luminescence continues but no growth occurs. There is thus ample evidence that growth and respiration are properties quite distinct and separable from luminescence. Indeed, respiration increases continuously up to a relatively high maximum whereas luminescence falls off rapidly above a relatively low optimum. McKenney (1902) found also that Bacillus phosphorescens could grow rapidly in 0.5 per cent. ether without producing light.

Luminescence has been compared in bacteria to pigment formation, as rather definite cultural conditions are necessary for realization of both chromogenic and photogenic function. Some pigment-formers, as Bacillus pyocyaneus, which produces a water-soluble green pigment, remain colorless under anaërobic conditions. A colorless chromogen is formed, which oxidizes to the green pigment in the air. If this colorless chromogen produced light during its oxidation as well as green pigment, we would have a case of both chromogenic and photogenic function combined in one species of bacterium. Luminescence involves something more than respiration, an oxidation of a very definite and particular kind.

Since Spallanzani's observation that the luminous material of medusæ could be dried, and upon moistening would again give light, many confirmatory observations have been made on other forms. Pyrosoma, Pholas, Phyllirrhoë, fireflies, Pyrophorus, copepods, ostracods, pennatulids, fungi, and bacteria can all be dessicated and the photogenic material preserved for a greater or less time. In a dessicator filled with CaCl₂, dried luminous bacteria lose, after a few months, their power to give light on being moistened. On the other hand, ostracods and copepods will still luminesce after years of dessication. The luminous material in the latter case appears capable of indefinite preservation, but it is possible that the quick loss of photogenic power with dried luminous bacteria is merely an indication that they contain very little photogenic substance and that the dried ostracods would also in time lose their power to luminesce. It is certainly a fact that the amount of luminous material in a single gland cell of an ostracod is vastly greater than that in the same mass of bacterial colony.

When the dried powdered luminous material of an ostracod is sprinkled over the surface of water, it goes into solution and leaves luminous diffusion and convection trails plainly visible in the water. Many luminous marine forms give off a phosphorescent slime when they are handled, which adheres to the fingers. It is not surprising that this luminous matter should have early received a name. In 1872, Phipson called it noctilucin and described some of its properties. He regarded the luminous matter which can be scraped from dead fish (luminous bacteria) and the mucous secretion of Scolopendra electrica or the luminous matter of the glowworm to be this material, noctilucin, which, "in moist condition, takes up oxygen and gives off CO₂ and when dry appears like mucin." Phipson says that it forms an oily layer over the seas in summer (he probably refers to masses of dinoflagellates), is liquid at ordinary temperatures and less dense than water, smells a little like caprylic acid, is insoluble in water but miscible with it, insoluble in alcohol and ether, dissolves with decomposition in mineral acids and alkalies and contains no phosphorus. We can see from this description that the word "noctilucin" does not indicate a chemical individual, but it is the earliest attempt to definitely designate the luminous substance.

The idea of a definite substance oxidizing and causing the light has been upheld by a number of investigators, and many years later Molisch called this substance the photogen. He contrasts the "photogen theory" with certain other views of light production, which may be spoken of as "vital theories," notably those of Pflüger (1875), who looked upon luminescence as a sign of intense respiration, and of Beijerinck (1915), who regarded the light as an accompaniment of the formation of living matter from peptone.

Fortunately biological science has advanced beyond the stage where a living process can be explained by calling it a vital process, and we must fall back upon the idea of a photogen oxidizing with light production. Indeed, it is now possible to go much further than this and describe the properties of the photogen, but we must not lose sight of the fact that it was recognized very early in the history of Bioluminescence, that water, oxygen, and a photogenic substance were necessary for light production.

A very great advance in our knowledge of the chemistry of the problem was made by Dubois in 1885. He showed that if one dips the luminous organ of Pyrophorus in hot water, the light disappears and will not return again. Also if one grinds up a luminous organ the mass will glow for some time but the light soon disappears. If one brings the previously heated organ in contact with the unheated triturated organ it will again give off light. Later, Dubois showed that the same experiment could be performed with the luminous tissues of Pholas dactylus. A hot-water extract of the luminous tissue, and a cold-water extract of the luminous tissue, allowed to stand until the light disappears, will again produce light if mixed together. Dubois (1887 b) advanced the theory that in the hot-water extract there is a substance, luciferin, not destroyed by heating, which oxidizes with light production in the presence of an enzyme, luciferase, which is destroyed on heating. The luciferase is present together with luciferin in the cold-water extract, but the luciferin is soon oxidized and luciferase alone remains. Mixing a solution of luciferin and luciferase always results in light production until the luciferin is again oxidized. Similar substances have been found by me in the American fireflies, Photinus and Photuris, the Japanese firefly, Luciola, and in the ostracod crustacean, Cypridina hilgendorfii. Crozier[6] reports that they exist also in Ptychodera, a balanoglossid. I have been unable to demonstrate their existence in luminous bacteria; in the annelid, Chætopterus; the pennatulids, Cavernularia and Pennatula; the squid, Watasenia; and the fish, Monocentris japonica. E. B. Harvey (1917) could not demonstrate them in Noctiluca. There are several reasons why the existence of such bodies might be difficult to demonstrate, but these reasons cannot be considered here. We thus see that the photogen is in reality of dual nature, that two substances are necessary for light production and that they may be very readily separated because of difference in resistance to heating. In this respect Bioluminescence is similar to some other biological processes, notably to certain immune reactions and to certain enzyme actions.

Thus, for the hæmolysis of foreign red blood corpuscles, a specific immune body (amboceptor or substance sensibilatrice) not destroyed by moderate heating, and a thermolabile complement (alexin) are necessary.

For the alcoholic fermentation of glucose by the zymase of yeast juice two substances are also necessary. The zymase is made up of a heat resistant, dialyzing component, the co-enzyme, and a non-dialyzing substance, destroyed on boiling, the enzyme proper. Both must be present for alcoholic fermentation of glucose to proceed and the two may be separated by dialysis or by their difference in resistance to heating. Several other characteristics of living cells are known to depend on the joint action of two substances, one thermolabile, the other thermostable. The reducing action of tissues, according to Bach, requires a reducing enzyme proper or perhydridase and some easily oxidizable substance, such as an aldehyde. The aldehyde has been spoken of as the co-enzyme.

Because of the necessity of thermostable and thermolabile substances for light production in luminous animals and because I was unable to oxidize the thermostable material of Cypridina with such oxidizing agents as KMnO₄, H₂O₂, blood and H₂O₂, BaO₂, etc., I called the heat resistant substance of Cypridina, "photophelein" (from phos, light and opheleo, to assist), comparable to co-zymase, and the heat sensitive substance of Cypridina, "photogenin" (from phos, light and gennao, to produce), comparable to the zymase proper of yeast. In mode of preparation and properties, the photophelein of Cypridina was also comparable to the luciferin of Pholas and the photogenin of Cypridina to the luciferase of Pholas. I also regarded photogenin as the source of the light (hence the name), because a solution of Cypridina photogenin (=Pholas luciferase) will give light on mixing with crystals of salt and other substances which could not possibly be oxidized. I later found, however, that this result was due to the fact that the photogenin solution contained some of the thermostable substance (luciferin) bound (combined or adsorbed), and that this was freed by the salt crystals and oxidized with light production. I have consequently abandoned the view that the system of substances concerned in light production is similar to the zymase—co-zymase system of yeast—and have adopted Dubois' term, luciferase (=photogenin) for the thermolabile material, and luciferin (=photophelein) for the thermostable material.

The luciferin of Cypridina differs from that of Pholas in that it will not oxidize with light production with any oxidizing agents that I have tried, and will give no light with luciferase from Pholas. It does, however, oxidize spontaneously in solution, although no light accompanies this oxidation.

I believe that for accuracy and definiteness we must designate the luciferins and luciferases from different animals by prefixing the generic name of the animal and speak of Pholas luciferin, Cypridina luciferase, Pyrophorus luciferase, etc. In extracts of many non-luminous animals Dubois has found oxidizing agents which can oxidize Pholas luciferin with light production and I have confirmed this for Pholas, but I have not found any such substances in non-luminous animals which will oxidize Cypridina luciferin with light production. I have found in extracts of non-luminous animals substances which will liberate the bound luciferin in a concentrated Cypridina luciferase solution. The luciferin can then be oxidized by the luciferase and light appears. Their effect is similar to that of salt crystals and I suggest that they be called photopheleins, substances that assist in the luciferin-luciferase reaction by liberating bound luciferin. One of the best ways of freeing a solution of luciferase from bound luciferin is to shake with chloroform. We can then do away with the disturbing effects of bound luciferin.

It is obvious that luciferin must be formed from some precursor in the cell and following the usual biochemical terminology, Dubois has called it proluciferin or preluciferin, and believes that it is converted into luciferin by an enzyme co-luciferase. The experiments to prove the existence of proluciferin were first made by Dubois on Pholas in 1907 and have since been amplified (1917 a; 1918 a and b).

In order to understand these experiments it must be borne in mind that Dubois prepares luciferin from Pholas in three ways: (1) By precipitating the viscid luminous fluid from the siphons with 95° alcohol and dissolving the precipitate in water (1901a, 1907). (2) By extracting the luminous organs with 90° alcohol in a closed vessel for twelve hours and filtering (1896). (3) By heating the viscid luminous fluid to 70° C. Apparently Pholas luciferin is sparingly soluble in alcohol as it can be obtained either in an alcoholic extract (method 2) or by precipitation with alcohol (method 1). Proluciferin (called preluciferine in a later paper, 1917 a, 1918 a), is prepared by methods 1 or 2 except that fatigued siphons, from which luciferin has been removed by washing, are used (1907, 1917 a, 1918 a). Preluciferin can also be obtained on boiling an extract of the luminous organ of Pholas because luciferin (at 70°), luciferase (at 60°) and a co-luciferase are all destroyed below the boiling point (1917 a).

Co-luciferase is prepared (1) by heating a luciferase solution to 65° (1917 a) or (2) by extracting with water portions of the siphon of Pholas which have previously been macerated and well extracted with alcohol (1918 a). Long-continued treatment with alcohol apparently destroys the luciferase without affecting the co-luciferase. On mixing a solution of preluciferin with one of co-luciferase and allowing them to stand for 8-10 hours, luciferase is formed and can be recognized by the fact that it will give light with a crystal of KMnO₄. Preluciferine does not do this.

Recently Dubois (1918 a) states that preluciferine is nothing but taurine and that taurine occurs in large quantities in Pholas and is transformed into luciferine by the action of co-luciferase. Not only taurine, but also Byla's peptone, egg lecithin, and esculin can be converted into luciferine by co-luciferase, and since esculin, a glucoside, is so transformed, Dubois believes this proves that co-luciferase belongs to the hydrolases. Indeed, it proves too much. Luciferin must have an extraordinary chemical structure if it can be formed by hydrolysis of such diverse compounds as peptone, lecithin, esculin and taurine. A glance at the structural formula of esculin and taurine is sufficient to emphasize the diverse nature of these two substances.

I believe that in these experiments Dubois has been working with an oxidation product of luciferin, what I have called oxyluciferin, rather than a pro-substance. The mode of preparation of Pholas preluciferin and Pholas co-luciferase is such as could be used in the preparation of Cypridina oxyluciferin, and it seems more logical to look for the presence of Pholas oxyluciferin in one or both of Dubois' extracts rather than believe that luciferin can be formed from both taurine and esculin. When the co-luciferase solution stands with the preluciferin solution we would in reality have not the formation of luciferin from preluciferin, but the formation of luciferin from oxyluciferin, by some reducing agent in the mixture. Indeed, in a very recent paper Dubois (1919c) takes the view that his co-luciferase is a reducing enzyme which forms luciferin by reduction (presumably from oxidized luciferin) and no mention is made of preluciferin.

It is, of course, obvious that when luciferin oxidizes, some oxidation products must be formed. Most observers have assumed the oxidation products of luciferin to be relatively simple and to represent a rather complete breaking down of the luciferin molecule. Carbon dioxide was mentioned by Phipson (1872) as being formed. We have just seen that no carbon dioxide is formed during the oxidation of Cypridina luciferin and there is evidence that no fundamental change at all occurs. It is for this reason that I have called the oxidation product of luciferin oxyluciferin.[7] As we shall later see, the change luciferin oxyluciferin is to be compared to the oxidation of colorless dyes (leuco-compounds) to the colored dye. The chemical properties of oxyluciferin are similar to those of luciferin and the oxyluciferin can be readily reduced to luciferin again.

Finally, we have the fluorescent substance of Pyrophorus and fireflies, which Dubois first called pyrophorin, but later, adopting McDermott's terminology, speaks of as luciferesceine. This Dubois regards as a substance intensifying the light and modifying its color by changing invisible into visible rays. As we have seen, this theory, while attractive, will not stand the test of critical examination.

Phipson's noctilucin, while the first name for the photogen of luminous animals, is too vague a substance, chemically, to warrant a retention of the term. Of the names, luciferin, luciferase, preluciferin or proluciferin, co-luciferase, photogenin, photophelein, oxyluciferin, luciferesceine, I believe that only proluciferin, luciferin, oxyluciferin, luciferase and photophelein stand for substances which are really significant for the theory of light production. Luciferin is the heat resistant, dialyzable substance which takes up oxygen and oxidizes with light production in the presence of the heat sensitive, non-dialyzing, enzyme-like luciferase. The luciferin must come from some precursor, proluciferin, but I have been unable to demonstrate the existence of this body in Cypridina and know nothing definite of its properties. The luciferin oxidizes to oxyluciferin which has the same chemical properties as the luciferin itself and may be reduced to luciferin again by reducing substances in luminous and other animals or by inorganic reducing agents. Photophelein is a name for substances in various animal or plant extracts which are capable of liberating luciferin from some bound condition in solutions containing luciferase. Under this term are included a number of unknown, probably quite different substances, some of which are thermostable and others thermolabile.

We have seen that Bioluminescence is an oxyluminescence, that the light is probably due to the oxidation of a compound, luciferin, in presence of air and water and that the oxidation is accelerated by an enzyme-like substance, luciferase. We also saw in Chapter 2 that light production is of fairly common occurrence during the oxidation of many organic compounds, provided the oxidation is carried out in the proper way. Many of these organic compounds must be oxidized by relatively strong alkali or such strong oxidizing agents as would have a very deleterious action on living cells. In 1913, Ville and Derrien, in a short note to the French Academy, "Catalyse Biochemique d'une Oxydation Luminescente," show that lophin could be oxidized by vertebrate blood in the presence of H₂O₂. In the same year Dubois (1913) found that esculin, the glucoside from horse chestnut bark, would also oxidize and luminesce in presence of blood and H₂O₂. In these cases the hæmoglobin of the blood acts as a catalyst, transferring oxygen from the H₂O₂ to esculin or lophin and is to be compared to luciferase, except that luciferase does not require the presence of H₂O₂.

As the hæmoglobin does not lose this power on boiling, whereas luciferase does, the analogy is far from perfect. Many oxygen carriers are known, however, which may be destroyed on boiling their solutions, namely, the peroxidases of plant juices. Esculin will not luminesce with peroxidase and H₂O₂, but pyrogallol or gallic acid will. If one mixes a test tube containing pyrogallol solution + H₂O₂ with potato or turnip juice or almost any plant extract, a yellowish luminescence appears. The plant extract loses the power to cause such luminescence on boiling and the peroxidase will not dialyze. It is, of course, comparable to luciferase and acts on the thermostable, dialyzable pyrogallol-H₂O₂ mixture, which is comparable to luciferin. Curiously enough, although many hydroxyphenol and amino-phenol compounds can be oxidized by peroxidase and H₂O₂, only pyrogallol and gallic acid will oxidize with light production. Many other oxidizers can take the place of the peroxidase. A list of these is given on page 151. No other peroxide can take the place of H₂O₂ with peroxidases as oxidizers, but a few can replace H₂O₂ with other oxidizers. This is brought out in Table 7.

Table 7
Peroxides Giving Light with Pyrogallol and Oxidizers

Our knowledge of the existence of such analogous, purely organic chemical oxidations, which proceed with light production, greatly strengthens Dubois' theory that the luciferin-luciferase reaction really represents a catalytic oxidation of similar nature. As Dubois (1914 a) expresses it, we are dealing in luminous organisms with "1° une luminescence; 2° une chemiluminescence; 3° une oxyluminescence; 4° une zymoluminescence.

"Ou si l'on bien admettre que les zymases sont encore quelque chose de vivant, une Biozymoöxyluminescence." Perhaps it is not really necessary to admit that the enzymes are living in order that we may adequately visualize the nature of the photogenic process.

In the next chapter the properties of the three principal substances, luciferin, oxyluciferin and luciferase, will be studied more carefully.

CHAPTER VI
THE CHEMISTRY OF LIGHT PRODUCTION, PART II

Since Radziszewski's experiments on the oxidation of oils in alcoholic solutions of alkali, most of the early workers on Bioluminescence tacitly assumed that the oxidizable material was fat or a fat-like substance. Support was given to this view by the occurrence in cells of granules or globules from which the light was seen to come. We now know that these bodies are not fat droplets and that neither luciferin nor luciferase are soluble in such fat solvents as ether, chloroform, benzol or benzine. Phipson's description of the properties of noctilucin are too crude and inaccurate to be considered. Dubois did not study the chemical properties of luciferin and luciferase from Pyrophorus, the first form with which he worked, except to point out that Pyrophorus luciferase was destroyed on heating and was precipitated by alcohol while the Pyrophorus luciferin was not so affected. Luciferin was found only in the luminous organ of Pyrophorus, not in the blood; luciferase probably exists throughout the animal.[8]

Pholas luciferin.—In a series of papers since 1887 Dubois has studied the chemical properties of Pholas luciferin and Pholas luciferase. He finds the luciferin to be destroyed above 70° C., to dialyze slowly, to oxidize with light production in the presence of Pholas luciferase, KMnO₄, H₂O₂, hæmatine and H₂O₂, BaO₂, PbO₂, hypochlorites, and the blood of various marine mollusks and crustacea. It is insoluble in fat solvents but forms a colloidal solution in water from which it is precipitated unchanged by picric acid, alcohol at 82°, and (NH₄)₂SO₄. It is not precipitated by NaCl, MgSO₄ or acetic and carbonic acids, except in presence of neutral salts. It forms an insoluble alkali albumin with NH₄OH. Dubois (1887 a) stated at one time that it could be crystallized and has spoken of it as belonging to several different classes of substances, proteose, nucleoprotein, albumin. Most recently he describes luciferin as a natural albumin having acid properties. It occurs only in luminous, not in non-luminous animals, and is found in all parts of the mantle, especially the siphons. It does not occur in non-luminous parts of the mollusk. No photographs of luciferin crystals have ever been published.

Pholas luciferase.—Pholas luciferase has all the properties of an enzyme, is destroyed at 60° C., is non-dialyzable, insoluble in fat solvents, but forms a colloidal solution in water. It is not affected by 1 per cent. NaF but its activity is suspended in saturated salt solutions, sugar or glycerine, and it may be preserved in this way, its activity returning on dilution. It is digested by trypsin and slowly destroyed by the fat solvent anæsthetics, such as chloroform. For this reason Dubois regards it as an oxidizing enzyme similar to the oxydones of Batelli and Stern. Because he found iron in an extract of Pholas dialyzed for a long time against running water, Dubois considers that it is associated with iron, and reports that it will oxidize the ordinary oxidase reagents, such as pyrogallol, gum guaiac, a-naphthol and para-phenylene-diamine, etc. It remains to be proved, however, that luciferase and not the oxidizing systems such as occur in all cells are responsible for the coloration of these reagents. Dubois has found luciferases or substances capable of giving light with Pholas luciferin in the blood of many non-luminous crustacea and mollusks (in Barnea candida, Solen, Cardium edulis, Ostræa and Mytilus).

Cypridina luciferin.—Despite the large amount that has been written on luminous animals, Dubois' work on Pholas and my own on Cypridina and the firefly are the only truly chemical studies that give us any idea of the nature of the photogenic substances in any luminous animal. In many ways Cypridina luciferin is similar to Pholas luciferin, but the two differ in a sufficient number of points to make certain that they are not identical substances. As I have emphasized above, we should speak not of luciferin and luciferase but of the luciferins and the luciferases. The luciferins, as the oxidizable substances, must claim first attention. They are more simple substances than the luciferases. If we are to produce light artificially in the same way that animals do, the luciferins must be synthesized. The luciferin of Pholas will luminesce with KMnO₄ and other oxidizing agents, and, although I have not yet succeeded in oxidizing Cypridina luciferin with oxidizing agents, I have no doubt but that some inorganic catalyzer will be found to take the place of luciferase and accelerate oxidation of Cypridina luciferin with light production.

The most remarkable peculiarity of Cypridina luciferin is its stability. In my first paper on Cypridina I stated that luciferin was not destroyed by momentary boiling but would be destroyed if boiled four or five minutes; also, that it was unstable at room temperatures and would disappear from solution in the course of a day or so. The reason for this is that luciferin oxidizes even in absence of luciferase and will then no longer give light with luciferase. This spontaneous oxidation, which occurs without light production, can be prevented by keeping the luciferin in a hydrogen atmosphere or by the addition of acid. Under these conditions the luciferin can be boiled without destruction or preserved for months without deterioration. The rapid disappearance of luciferin from neutral or alkaline solution on boiling in the air is entirely due to the more rapid oxidation at the boiling point. As the oxidation product, oxyluciferin, can be readily reconverted into luciferin again, we can not consider luciferin unstable in the sense that its molecule is actually destroyed as is the case when luciferase is boiled.

Not only is luciferin stable on boiling but it will actually withstand boiling for 10 hours with 20 per cent. HCl (by weight, sp. gr. = 1.1) or with 4 per cent. H₂SO₄. After one day of boiling with 20 per cent. HCl the luciferin was completely destroyed and with 4 per cent. H₂SO₄ destruction was almost complete. In these cases there was no question of a mere oxidation to oxyluciferin, as no oxyluciferin could be demonstrated after boiling with such strong acids. An actual destruction, probably an hydrolysis of the luciferin molecule, occurred. We shall have occasion to refer to this again in considering the protein nature of luciferin. It must be borne in mind that many proteins require four or five days' boiling with 20 per cent. HCl for complete hydrolysis to amino-acids. Luciferin forms a solution in water, probably colloidal, although the luciferin will dialyze through parchment or collodion membranes. It is rather readily adsorbed by various finely divided materials such as bone black, Fe(OH)₃, kaolin, talc and CaCo₃. It is not destroyed by any of the enzyme solutions which I have tried. These include such as are widely divergent in action: pepsin HCl, trypsin, erepsin, salivary and malt diastase, yeast invertase, urease, rennin and the enzymes of dried spleen, kidney and liver substances.

By extracting the dried Cypridinas ground to a powder, the solubility of luciferin in non-aqueous solvents could be easily studied, and by adding such reagents as dilute acids, alkalies, neutral salts and the alkaloidal reagents to an aqueous solution of luciferin the general biochemical behavior of luciferin can be quite accurately stated. For convenience the results of this study are given in Table 8.

Table 8
Properties of Photogenic Substances from Cypridina

On the other hand, luciferin has two properties which to say the least are unusual for proteins. I refer to its solubility in alcohols, acetone, esters, etc., and non-digestibility by trypsin or erepsin, which have almost universal proteolytic power.

The best known class of proteins soluble in alcohol is the prolamines of plants, but the prolamines are insoluble in water and in absolute alcohol. Zein, the prolamine of corn, is soluble in 90 per cent. ethyl, methyl, and propyl alcohols, in glycerol heated to 150° C., and in glacial acetic acid. Recently Osborne and Wakeman (1918) have described a protein from milk having solubilities similar to those of gliadin, the prolamine of wheat. Welker (1912) has described a substance, obtained from Witte's peptone, giving the biuret, Millon, and Hopkins-Cole tests, which is soluble in water and absolute alcohol but not in ether, and it is possible that others of the peptones are soluble in absolute alcohol. On the other hand, some proteins in the absence of salts form colloidal solutions in strong alcohol from which they may be precipitated by an appropriate salt. As the absolute alcohol extract of Cypridinæ was made from dry material containing the salts of sea water, some salt was present, but there is always the possibility of sol formation.

If we extract dried Cypridinæ, which have previously been thoroughly extracted with benzine or ether, with 800 c.c. of boiling absolute alcohol for an hour, filter the alcohol extract through blotting paper and hardened filter paper, quickly evaporate the filtrate to dryness on the water bath, and dissolve the residue in a small quantity of water saturated with CO₂,[9] we obtain a yellow opalescent solution which gives a bright light with luciferase. This solution contains some protein or protein derivatives as it gives a very faint Millon reaction, a good positive ninhydrin test, reddish blue in color, but no biuret reaction. It precipitates with tannic and phosphotungstic acids but not with picric, acetic, trichloracetic, or chromic acids. The extract gives a faint Molisch reaction for carbohydrates. As the evidence points to the presence of some protein products in the absolute alcohol extract of Cypridinæ, it is possible that this protein is luciferin. It should be emphasized, however, that the Millon reaction was very faint, although the ninhydrin was quite marked and the biuret negative.