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.