LUCIFERIN AND LUCIFERASE.
The light from the natural secretion of Cypridina lasts for some time in sea-water and then disappears, and no amount of shaking will cause it to appear again. If we add to this natural secretion an extract of Cypridina heated to boiling, a brilliant light again results; or if we mix a water extract of Cypridina whose light has disappeared on standing with a similar extract whose light has been destroyed by boiling, light again results. As already mentioned, Dubois first demonstrated this for Pyrophorus and a year later for Pholas dactylus. In Dubois's phraseology we have mixed two substances—luciferin (in the boiled tube) and luciferase (in the tube allowed to stand)—necessary for light production. According to Dubois (io), one of these, luciferase, is an oxidizing enzyme and is destroyed by heat; the other, luciferin, a sub stance not destroyed by heat, is capable of oxidation with light-produc tion by means of luciferase. When the natural secretion of Cypridina is allowed to stand all the luciferin is oxidized and the luciferase is left; when a luminous extract of Cypridina is boiled the luciferase is de stroyed before all the luciferin is oxidized. The two substances alone in solution are non-luminous.
At one time I believed Dubois's interpretation of this experiment to be correct, but results on Cypridina have led me to wholly different conclusions regarding the existence of luciferin and luciferase. Dubois's interpretation is indeed attractive. We know that the light-produc tion is an oxidation; that two substances are concerned; that these substances give light in very small concentration comparable with enzyme activity (see p. 188); that one of them can use up a large amount of the other (see p. 189) and possesses certain properties (destruction by heat, phosphotungstic and tannic acid) characteristic of enzymes. Further, we actually know of oxidative reactions taking place with the production of light under the action of oxidizing enzymes from plants and animals (see p. 225).
occurs only in the luminous organs of Pholas dactylus (io). I find that Cypridina luciferin (a substance not readily destroyed by heat and giving light with luciferase) will not give light with the above-men tioned oxidizing agents and is found in many other non-luminous animals and in the non-luminous parts of Cypridina hilgendorfii.
Dubois finds that Pholas luciferase (a substance destroyed easily by heat and using up Pholas luciferin with light-production) occurs in many other non-luminous animals (io), whereas I find a body with the same properties only in the luminous organs of Cypridina. This sub tance, which we may temporarily call Cypridina luciferase, in concen trated solutions, will give light (as before mentioned) with extracts of many non-luminous animals. It will also give light if mixed with many pure substances, as chloroform, ether, benzol, thymol, saponin, oleic acid, atropin, NaC1, and others. Since most of the above sub stances could not possibly be oxidized by the luciferase, I conclude that they cause in some way the giving out of light in what Dubois terms luciferase. On this view luciferase is the source of the light and the luciferin (in the boiled extract of Cypridina) is something which causes the luciferase to emit light.
The substances causing the emission of light from a concentrated solution of luciferase are similar to those producing cytolysis of cells, and I have considered the possibility that the concentrated extract might contain fragments of the luminous gland-cells which cytolyze with light-production or possibly granules which dissolve with light production, as described in many other forms, and as especially prom inent in the juice of Cavernularia. I am, however, convinced that there are in the extract of cypridinas which will give light with unoxidizable substances no cell fragments present and no granules above those of submicroscopic colloidal dimensions, for the following reasons: First, the light is always perfectly homogeneous and in marked con trast to the points of light of Cavernulartia juice, where visible granules and cell fragments do occur.
Second, Cypridina extract (luciferase) will give light with thymol, butyl alcohol, or NaC1 crystals after filtration through a Chamberland or a Berkefeld filter, which removes all cell fragments.