CHAPTER V
THE CHEMISTRY OF LIGHT PRODUCTION, PART I
Two experiments, both performed very early in the history of Bioluminescence, are of great importance in understanding the nature of animal light. Boyle (1667), as already mentioned, proved the necessity of air for the luminescence of wood and fish and Spallanzani (1794) showed that parts of luminous medusae gave no light when dried but if moistened again would emit light as before. We see then, that air (oxygen), water, and some photogenic substance are necessary for the light production. Spallanzani's experiment, which has been confirmed for a great many luminous forms, shows also that animal luminescence is not a _vital_ process, in the same sense that the conduction of a nerve impulse is a vital process. A nerve loses its characteristic property of conduction on drying or maceration while luminous cells still possess the power to luminesce after drying or maceration. Using the terminology of the older physiology we may say that "living protoplasm" is not necessary for light production.
The experiments of Boyle (1626-91) are of great interest, especially those in which he studied the behavior of shining wood under the receiver of his air pump. On October 29, 1667, he wrote:
"Exp. I.: Having procured a Piece of _shining Wood_, about the bigness of a groat or less, that gave a vivid Light, (for _rotten Wood_) we put it into a middle sized _Receiver_, so as it was kept from touching the Cement; and the _Pump_ being set a-work, we observed not, during the 5 or 6 first Exsuctions of the Air, that the splendor of the included Wood was manifestly lessened (though it was never at all increased;) but about the 7th Suck, it seemed to glow a little more dim, and afterwards answered our Expectation, by losing of its Light more and more, as the Air was still farther pumped out; till at length about the 10th Exsuction, (though by the removal of the Candles out of the Room, and by black Cloaths and Hats we made the place as dark as we could, yet) we could not perceive any light at all to proceed from the _Wood_.
"Exp. II.: Wherefore we let in the outward Air by Degrees and had the pleasure to see the seemingly extinguished Light revive so fast and perfectly, that it looked to us almost like a little Flash of Lightning, and the Splendor of the Wood seemed rather greater than at all less, than before it was put into the Receiver."
Boyle proved that light from the wood was able to pass a vacuum and later showed that "shining fish" behaved as the "shining wood," but that a piece of white hot iron would not regain its light on readmitting air to the exhausted receiver and that the iron lost its glow under the air-pump merely because it cooled off. A piece of glowing coal, however, did lose its light in the absence of air and regained it on again admitting air, provided the air had not been removed for too long. Boyle was apparently impressed with the similarity of the light giving process in glowing coal and shining wood as he draws a comparison between the two which brings out the fundamental similarity of combustion processes.
"Resemblances:
VII. The Things wherein I observed a Piece of _shining Wood_ and a _burning Coal_ to agree or _resemble_ each other are principally these _five_:
1. Both of them are _Luminaries_, that is, give _Light_, as having it (if I may so speak) _residing in them_; and not like _Looking-glasses_, or _white Bodies_, which are conspicuous only by the _incident Beams_ of the _Sun_, or some other _luminous Body_, which they _reflect_....
2. Both _shining Wood_ and a _burning Coal_ need the Presence of the Air (and that too of such a _Density_ to make them continue _shining_)....
3. Both _shining Wood_ and a _burning Coal_, having been deprived, for a Time, of their _Light_, by the withdrawing of the contiguous _Air_, may presently recover it by letting in fresh _Air_ upon them....
4. Both a _quick Coal_ and _shining Wood_ will be easily quenched by _Water_ and _many other Liquors_....
5. As a _quick Coal_ is not to be _extinguished_ by the Coldness of the _Air_, when it is greater than ordinary; so neither is a Piece of _shining Wood_ to be deprived of its _Light_ by the same Quality of the _Air_....
Differences:
1. The first Difference I observed betwixt a _live Coal_ and a _shining Wood_ is, that whereas the _Light_ of the _former_ is readily _extinguishable_ by _Compression_ (as is obvious in the Practice of suddenly _extinguishing_ a piece of _Coal_ by treading upon it), I could not find that such a _Compression_ as I could conveniently give without losing sight of its operation, would _put out_, or much injure the _Light_, even of small Fragments of _shining_ Wood....
2. The next _Unlikeness_ to be taken notice of betwixt _rotten Wood_ and a _kindled Coal_ is, that the latter will, in a very few _Minutes_, be totally _extinguished_ by the withdrawing of the _Air_; whereas a Piece of _shining Wood_, being eclipsed by the Absence of the _Air_, and kept so for a Time, will immediately _recover_ its _Light_ if the _Air_ be let in upon it again within half an hour after it was first withdrawn....
3. The next _Difference_ to be mentioned is, that a _live Coal_, being put into a small close Glass, will not continue to _burn_ for very many _Minutes_; but a Piece of _shining Wood_ will continue to shine for _some_ whole _Days_....
4. A _fourth Difference_ may be this: that whereas a _Coal_, as it _burns_, sends forth Store of _Smoke_ or _Exhalations_, _luminous Wood_ does not so.
5. A _fifth_, flowing from the former, is, that whereas a _Coal_ in _shining_ wastes itself at a great Rate, _shining Wood_ does not....
6. The last Difference I shall take notice of betwixt the bodies hitherto compared is, that a _quick Coal_ is actually and vehemently hot; whereas I have not observed _shining Wood_ to be so much as sensibly _lukewarm_."
It should be clearly borne in mind that if we place luminous organisms, say bacteria or fungi, in an atmosphere devoid of oxygen and find that no light is produced, this may merely mean that certain functions of the cell are interfered with, including light production, but does not necessarily indicate that oxygen is actually used up in the photogenic process. If we find, however, that extracts of luminous cells or luminous secretions devoid of cells cease to light when the oxygen is removed and again luminesce when it is returned, we may be quite certain that the photogenic process itself requires free oxygen. As luminous extracts of fireflies, pennatulids, ostracods, _Pholas_ and others give off no light when the oxygen is removed, we may safely conclude that for these luminescences, oxygen is necessary. Bacteria, fungi, and _Noctiluca_, whose light also disappears in absence of oxygen, although they are whole cells, we may by analogy also assume to require oxygen in the photogenic process.
Some of the earlier workers on fireflies and _Noctiluca_ obtained light even after placing these organisms in absence of oxygen, but they did not realize how low is the amount of oxygen necessary to produce light. It is difficult to remove traces of oxygen from the water, traces which are nevertheless sufficient to cause luminescence. If the organisms are numerous, as in an emulsion of luminous bacteria, they will themselves use up all the oxygen and the liquid soon ceases to glow except at the surface in contact with air. We may gain an idea of the amount of oxygen necessary for luminescence from an experiment of Beijerinck (1902). He mixed luminous bacteria with an emulsion of clover leaves containing chloroplasts and kept the two in the dark until all the oxygen was used up and the bacteria ceased to glow. If now a match was struck for a fraction of a second, sufficient oxygen was formed by photosynthesis to cause the bacteria to luminesce for a short time.
Exact figures on the minimal concentration of oxygen for luminescence cannot be given. The luminescent secretion of _Cypridina hilgendorfii_ will still give off much light if hydrogen containing only 0.4 per cent. of oxygen is bubbled through it, _i.e._, a partial oxygen pressure of 1/250 atmosphere (3.04 mm.Hg). However, addition of a fresh emulsion of yeast cells to a glowing _Cypridina_ secretion is sufficient to rapidly extinguish the light, because the yeast is capable of utilizing the last trace of oxygen in the mixture. Light only appears when, by agitation, we cause more air to dissolve. The minimal concentration of oxygen for luminescence of _Cypridina_ lies somewhere between 3.04 mm. and the amount which living yeast fails to extract from solution, a concentration approaching zero. It is probably nearer the latter figure.
As the oxygen pressure is increased from 0 to about 7 mm., the intensity of the _Cypridina_ luminescence increases and at the latter figure the light is just as bright as if the solution were saturated with air (152 mm.O_{2}). Thus, the luminescence requires only a low pressure of oxygen and the similarity to the saturation of haemoglobin with oxygen is obvious. Just as haemoglobin is nearly saturated with oxygen at low pressures and becomes bright red in color, so the luminous material becomes saturated with oxygen at low pressures and glows intensely.
Boyle also made many experiments to show that air was necessary for the life of animals and the germination of seeds and showed that repeatedly respired air was unfit for further breathing. About the same time R. Hooke discovered the true meaning of respiratory movements and by forcing a blast of air continuously through the lungs with bellows, was able to keep animals alive. He concludes "that as the bare Motion of the _Lungs_, without fresh air, contributes nothing to the life of the Animal, he being found to survive as well as when they were not moved as when they were; so it was not the Subsiding or Movelessness of the _Lungs_ that was the immediate cause of death, or the stopping of the circulation of the Blood through the _Lungs_, but the Want of a sufficient Supply of fresh Air." The cause of death on collapse of the lungs could not be better stated to-day. Thus combustion, respiration and luminescence of flesh or wood were early recognized as related phenomena.
Although the "gas sylvestre" (CO_{2}) of burning charcoal and fermentation of wine was known to van Helmont (1577-1644) and Mayow (1646-1679) in 1674 showed that "spiritus nitroaerens" (oxygen) was responsible for the life of animals and for combustion, a century elapsed before the true significance of these gases became known. In the meantime the phlogiston theory of combustion had been developed, Black (1728-1799) in 1755 had rediscovered carbon dioxide ("fixed air") in the expired air and Priestley (1733-1804) and Scheele (1742-1786) had both rediscovered oxygen ("dephlogisticated air") in 1774. About the same time Lavoisier overthrew the phlogiston doctrine and showed that in the combustion of organic substances water and CO_{2} are formed.
Later it was realized that this slow combustion did not take place in the lungs, or in the blood, but in the tissues cells themselves and respiration in the chemical sense has come to mean this universal slow combustion in the cells of the body rather than the breathing movements of the lungs themselves. In anaerobic respiration, CO_{2} is given off, but no oxygen absorbed. In aerobic respiration, oxygen is absorbed and CO_{2} given off. In addition we know of many substances which oxidize by taking up oxygen without giving off CO_{2}. We have seen that oxygen must be absorbed for luminescence of animals and we may now inquire whether CO_{2} is given off and the relation between respiration and light production.
To determine if CO_{2} is given off during luminescence it is necessary to work with fairly pure luminous materials, obtained from luminous organisms. It is impossible to use the living organisms themselves as the CO_{2} continually respired becomes a very disturbing factor. From _Cypridina_, a small crustacean, two materials soluble in water may be prepared (_luciferin_ and _luciferase_), which will give a brilliant luminescence on mixing. It is possible to determine the H-ion concentration of the two solutions separately and of the mixture of the two after the luminescence has occurred.
If CO_{2} is produced during luminescence the H-ion concentration of the luminous solution should increase. Measurements made electrometrically with the hydrogen electrode have failed to demonstrate any increase in acidity. The PH of both solutions and of a mixture of the two is 9.04. This would indicate that CO_{2} is not produced. As both luminous solutions contain proteins and the luminous substances themselves are probably proteins, which have a high buffer value, a method of this kind is none too sensitive. However, we can definitely state that not enough CO_{2} is produced to be detected and that this may be due to the buffer action of the luminous substances themselves. After all, unless luminescence is connected with respiration, we should hardly expect CO_{2} to be produced.
Another method of testing CO_{2} production is to measure the amount of heat produced during luminescence. Substances burned during respiration give off considerable heat, one gram of glucose to CO_{2} and H_{2}O, as much as 4000 calories. We have seen in Chapter III that no infra-red radiation is produced in the light of the firefly. This does not mean, however, that no heat is produced by the reaction which produces the luminescence. A temperature change of a few thousandths or hundredths of a degree would evolve no measurable radiation. Coblentz (1912) first studied the problem of heat production in the firefly, using a thermocouple as the measuring instrument. He came to the conclusion that the temperature of the insect was slightly lower than the temperature of the air and that the luminous segments were slightly hotter than the non-luminous segments, whereas a dead firefly is of the same temperature as its surroundings. No definite increase or decrease in temperature could be established during the flash of the firefly. However, further work on the firefly is much to be desired.
The use of a living animal for such measurements introduces a possible source of error in that any contraction of the muscles of the animal will produce heat which may add to an increase or mask a decrease of temperature during luminescence. Utilization of extracts of luminous animals containing the luciferin and luciferase mentioned above avoids the complications due to muscular contraction. By bringing the solutions of luciferin and luciferase to the same temperature and then mixing them one can measure any increase or decrease of temperature which occurs during the luminescence which results from mixing. We can thus gain some idea of the heat of oxidation of luciferin.
As a determination of heat production is of considerable interest the method will be given in some detail. Although the experiment sounds very simple, it is actually somewhat difficult to carry out. The attainment of temperature equilibrium between two solutions is very slow when one wishes to obtain them to within 0.001 deg. C. of the same temperature. After many attempts, the following arrangement of apparatus (Fig. 33) was found most satisfactory. About 10 c.c. luciferin solution was placed in the inner tube (_D_) of a special non-silvered thermos bottle (_A_). About 1 c.c. of luciferase solution was placed in a very thin-walled glass tube (_E_) which was immersed in the luciferin solution and connected with a small motor so that it could be slowly but constantly rotated, thus stirring the solutions. Thermocouples (_L_ and _M_) of advance (.008 in)--copper (No. 30, _B_ and _S_, enamel insulated) wire were paraffined and placed in each tube and the copper wires connected through a copper double throw switch (_C_) with a Leeds and Northrup d'Arsonval wall galvanometer (No. 34637, silver strip suspension) of 35 ohms resistance and 310 megohms sensitivity. The constant temperature junctions (_N_) were placed in a large Dewar flask (_B_) filled with water at approximately the same temperature as the luciferin solution. One mm. galvanometer scale division represented 0.003 deg. C. and the division readings could be estimated to tenths. By means of a glass rod (_F_) placed in the tube containing luciferase solution, this tube could be broken and the luciferase and luciferin solution mixed.
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 deg. C. and the average rise of five luciferin experiments was .0048 deg. 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 deg. 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 deg. C. and probably less than 0.0005 deg. 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 deg. 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 CO_{2} and H_{2}O, 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 haemoglobin or the oxidation of leuco methylene-blue to methylene blue. According to Barcroft and Hill (1910), 1.85 calories are produced per gram of haemoglobin 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 CO_{2} (4.41 c.c. CO_{2} per gram in 36 hours at 12 deg. C.) than non-luminous portions (2.88 c.c. CO_{2} per gram in 36 hours at 12 deg. 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 _Araceae_, 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 anaerobe 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 anaerobic 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 medusae could be dried, and upon moistening would again give light, many confirmatory observations have been made on other forms. _Pyrosoma_, _Pholas_, _Phyllirrhoe_, 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 CaCl_{2}, 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 CO_{2} 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 Pflueger (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, _Chaetopterus_; 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.
[6] Private communication.
Thus, for the haemolysis 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 KMnO_{4}, H_{2}O_{2}, blood and H_{2}O_{2}, BaO_{2}, 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 deg. alcohol and dissolving the precipitate in water (1901_a_, 1907). (2) By extracting the luminous organs with 90 deg. alcohol in a closed vessel for twelve hours and filtering (1896). (3) By heating the viscid luminous fluid to 70 deg. 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 deg.), luciferase (at 60 deg.) 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 deg. (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 KMnO_{4}. 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 (1919 _c_) 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.
[7] It is unfortunate that Dubois (1918 b) has used the term oxyluciferine in a quite different sense from the present use. He regards oxyluciferine as a substance still capable of giving light by autooxidation, and represents the steps in luminescence as follows:
"Co-luciferase + preluciferine = luciferine.
Luciferase + luciferine = oxyluciferine.
Oxyluciferine + oxygene = lumiere."
I should represent them as follows:
Luciferin + oxygen <=> oxyluciferin.
The reaction proceeds to right with light production only in presence of luciferase.
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 H_{2}O_{2}. 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 H_{2}O_{2}. In these cases the haemoglobin of the blood acts as a catalyst, transferring oxygen from the H_{2}O_{2} to esculin or lophin and is to be compared to luciferase, except that luciferase does not require the presence of H_{2}O_{2}.
As the haemoglobin 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 H_{2}O_{2}, but pyrogallol or gallic acid will. If one mixes a test tube containing pyrogallol solution + H_{2}O_{2} 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-H_{2}O_{2} mixture, which is comparable to luciferin. Curiously enough, although many hydroxyphenol and amino-phenol compounds can be oxidized by peroxidase and H_{2}O_{2}, 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 H_{2}O_{2} with peroxidases as oxidizers, but a few can replace H_{2}O_{2} with other oxidizers. This is brought out in Table 7.
TABLE 7
_Peroxides Giving Light with Pyrogallol and Oxidizers_
Key to column headings: [A]: Oxidizer. (Equal parts added to a mixture of M/100 pyrogallol and the peroxide) [B]: H_{2}O_{2} 3 per cent. [C]: Benzoyl hydrogen peroxide (insoluble powder) [D]: Ozonized turpentine (one drop) [E]: Na_{2}O_{2} (powder) [F]: BaO_{2} (powder) [G]: MnO_{2} (insoluble powder) [H]: PbO_{2} (insoluble powder) [I]: K persulfate M/10 [J]: Na perborate M/20 [K]: K perchlorate M/10 [L]: Quinone (insoluble crystals)
----------------------------------------------------------------------------- [A] |[B]|[C]|[D]| [E] | [F] |[G]|[H]| [I] | [J] |[K]|[L] -------------------------+-- +---+---+-----+-----+---+---+-----+-----+---+--- Turnip juice | + | - | - | - | - | - | - | - | | | - 1 percent blood extract | + | - | - |Faint| - | - | - | - | - | - | - | | | |flash| | | | | | | M 20 K_{4}Fe(CN)_{6} | + | - | - | - | - | - | - | - | - | - | - M 100 KMnO_{4} | + | - | - | - | - | - | - |Faint|Fair | - | - | | | | | | | |flash|flash| | M 10 FeCl_{3} | + | | | | | | | | - | - | M 100 CrO_{3} | + | | | | | | | | - | - | Na hypobromite | + | - | - |Faint|Faint| - | - |Fair |Fair | - | - | | | |flash|flash| | |flash|flash| | Ca hypochlorite | + | - | - | - | - | - | - |Faint|Fair | - | - | | | | | | | | |flash| | MnO_{2} | + | | | | | | | | | | Mn(OH)_{3} sol in peptone| + | | | | | | | | - | - | Colloidal Ag | + | | | | | | | | | | -------------------------+---+---+---+-----+-----+---+---+-----+-----+---+---
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 deg. une luminescence; 2 deg. une chemiluminescence; 3 deg. une oxyluminescence; 4 deg. une zymoluminescence.
"Ou si l'on bien admettre que les zymases sont encore quelque chose de vivant, une Biozymooxyluminescence." 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.