Part 8
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 a.n.a.logy 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 a.n.a.logous, 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 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 princ.i.p.al 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 a.s.sumed 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]
[8] Private communication from R. Dubois.
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, KMnO_{4}, H_{2}O_{2}, haematine and H_{2}O_{2}, BaO_{2}, PbO_{2}, 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 (NH_{4})_{2}SO_{4}. It is not precipitated by NaCl, MgSO_{4} or acetic and carbonic acids, except in presence of neutral salts. It forms an insoluble alkali alb.u.min with NH_{4}OH. Dubois (1887 _a_) stated at one time that it could be crystallized and has spoken of it as belonging to several different cla.s.ses of substances, proteose, nucleoprotein, alb.u.min. Most recently he describes luciferin as a natural alb.u.min 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 anaesthetics, 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 a.s.sociated 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_, _Ostraea_ 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 KMnO_{4} 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. H_{2}SO_{4}. After one day of boiling with 20 per cent.
HCl the luciferin was completely destroyed and with 4 per cent.
H_{2}SO_{4} 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 CaCo_{3}. 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_
========================================================================= Property
Luciferase
Luciferin ------------------------+-----------------------+------------------------ _Salting out_
By saturation NaCl
Not precipitated
Not precipitated.
By half saturation
Do.
Do.
MgSO_{4}
By saturation MgSO_{4}
Nearly completely
Partially
precipitated
precipitated.
By saturation MgSO_{4}
...
Do.
+ acetic acid
By half saturation
(NH_{4})_{2}SO_{4}
Slightly precipitated
Not precipitated.
By saturation
Completely precipitated
Nearly completely (NH_{4})_{2}SO_{4}
precipitated.
By saturation
(NH_{4})_{2}SO_{4} +
...
Nearly completely acetic acid
precipitated.
_Solubility in_
Methyl alcohol
Insoluble
Soluble.
Ethyl alcohol
Do.
Do.
90 per cent.
Do.
Do.
70 per cent.
Do.
Do.
50 per cent.
Slightly soluble
Do.
Propyl alcohol
Insoluble
Do.
Isobutyl alcohol
Do.
Fairly soluble.
Amyl alcohol
Do.
Slightly soluble.
Benzyl alcohol
Do.
Soluble.
Acetone
Do.
Fairly soluble.
90 per cent.
Do.
Soluble.
70 per cent.
Slightly soluble
Do.
50 per cent.
Fairly soluble
Do.
Ethyl acetate
Insoluble
Do.
Ethyl propionate
Do.
Fairly soluble.
Ethyl butyrate
Do.
Do.
Ethyl valerate
Do.
Slightly soluble.
Ethyl nitrate
Do.
Very slightly soluble.
Glycerine
Do.
Soluble.
Glycol
Do.
Do.
Ether
Do.
Insoluble.
Chloroform
Do.
Do.
Carbon disulfide
Do.
Do.
Carbon tetrachloride
Do.
Do.
Benzol
Do.
Do.
Toluol
Do.
Do.
Xylol
Do.
Do.
Petroleum ether
Do.
Do.
Anilin
Do.
Do.
Glacial acetic acid
Do.
Fairly soluble.
