Part 7 (2/2)

[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 a.s.sist), 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 a.s.sist 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 (1901_a_, 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 was.h.i.+ng, 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 KMnO_{4}. Preluciferine does not do this.

Recently Dubois (1918 _a_) states that preluciferine is nothing but taurine and that taurine occurs in large quant.i.ties 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.

[Ill.u.s.tration: Taurine]

[Ill.u.s.tration: Esculin]

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 a.s.sumed 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.

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