Bioluminescence

Bioluminescence is a word applied to the light emitted by various living organisms. At least 40 different orders of animals contain luminous species and two groups of plants, the bacteria, responsible for the luminescence of flesh and dead fish, and the fungi, which live in phosphorescent wood. Luminous bacteria are so small that individuals cannot be seen by their own light but colonies are visible. They are easily cultured and are nonpathogenic to man but may infect living animals, giving rise to a luminescent disease of sand-fleas, shrimps and midges, which is eventually fatal. Luminous bacteria may also live symbiotically in special organs of certain fish, notably Photoblepharon and Anomalops, of the Banda islands.

Bacteria and fungi emit light continuously day and night, while all other forms luminescence only when stimulated. The phosphorescence of the sea appears when many different kinds of small organisms are disturbed by the breaking of waves or motion of a boat. Among the groups of animals containing luminous species are flagellates, radiolaria, sponges, jelly-fish hydroids, sea pens, ctenophores, nemerteans, earthworms and many marine worms, shrimp, ostracods and copepods, myriapods, several groups of insects, monuses, squid, brittle stars, balanoglossids, tunicates and fish.

Bioluminescence is never dependent on a previous illumination of the cells or a previous radiation of any kind, nor is it connected with crystallization, friction, or rubbing, but is the result of oxidation by molecular oxygen of a definite substance produced in the luminous cell. It is a chemiluminescence. The luminous material or photogen is almost universally manufactured by living cells as granules, which may normally undergo oxidation within the cell, as in the fire-fly, (intracellular luminescence) or be extruded as a luminous slime or secretion (extracellular luminescence) as in a small ostracod crustacean, Cypridina. Most is known concerning the chemistry of extracellular luminescence, especially that of Cypridina.

In Cypridina the granules in the secretion dissolve on contact with sea water and the homogeneous luminescence is emitted by the resultant colloidal solution. Two kinds of granules are distinguishable in the cells of Cypridina, one large and yellow, the other small and colorless. In fact, in five of twenty-five different groups of luminous animals tested, it can be demonstrated that luminescence is due to two chemical substances, luciferin (yellow) and luciferase (colorless), which can be easily separated because of a difference in resistance to heating and other properties.

Crude luciferin solution is prepared by making a hot water extract of a luminous organ. Heating destroys the luciferase but does not harm the luciferin. Crude luciferase solution is prepared by making a cold water extract of a luminous organ, when both luciferin and luciferase dissolve and luminescence occurs. The extract is then allowed to stand in the air until the light disappears, evidence that the luciferin has been completely oxidized, leaving the luciferase, an enzyme, in solution. A luciferase solution, by virtue of this mode of preparation, must contain the oxidation product of luciferin as well as luciferase.

Luciferin and luciferase are quite specific. Luciferin from one animal will not luminesce if mixed with the luciferase of another luminous form unless the animals are closely related, such as two species of the same genera or two genera within the same order. Even if the color of the luminescence is different in the two different species light will appear, provided the species are closely related. In this case it is interesting to note that the color of luminescence of the resultant "cross" is determined by the animal supplying the luciferase. Luciferase must be the source of the light. It is convenient to designate the luciferins and luciferases by prefixing the name of the animal from which the substances are obtained.

Most luminous animals, if dried rapidly will again luminesce on moistening. Dried Cypridinae have been kept for 26 years without deterioration and can be used for preparing luciferin and luciferase.

Cypridina luciferin is purified by extraction of the dry Cypridinae with methyl alcohol. Ten per cent of butyl alcohol is then added and the methyl alcohol removed in vacuo. The supernatant butyl alcohol extract is chilled and benzoylated with benzoyl chloride. After fifteen minutes this solution is diluted with ten volumes of water and the new inactive benzoyl luciferin derivatives extracted with pure ether. After removing the ether in vacuo the residual liquor is hydrolyzed with hydrochloric acid in absence of oxygen. The free active luciferin is left in the acid solution and can be extracted with butyl alcohol. By repeating the benzoylation and hydrolysis, a product purified 2,000-fold, as compared with dry Cypridinae, can be obtained.

To purify luciferase it is usually sufficient to dialyze a cold, wellstirred water extract of rapidly dried, powdered Cypridinae against cold running water for twenty hours. Dialysis removes pigment, and a precipitate forms which can be filtered off. A few drops of toluene added to this solution will preserve it for months with little loss in activity if kept in a refrigerator.
Cypridina luciferin is slowly dialyzable; not destroyed by trypsin; soluble in water, absolute methyl, ethyl, and propyl alcohol but insoluble in acetone, benzene and ether; readily adsorbed on fine particles. It does not act as an antigen.

Cypridina luciferase is non-dialyzable; destroyed by trypsin; soluble in water, but insoluble in alcohols and all fat solvents; readily adsorbed on surfaces. It is capable of forming an antiluciferase when injected into the blood of a rabbit. Other luciferins and luciferases have different properties.

The oxidation of luciferin with production of light in the presence of luciferase gives an oxidation product which cannot be reduced, whereas the oxidation without luminescence by oxidants like potassium ferricyanide is reversible.

This Product is called oxidized lucifenn. The change with ferricyanide occurs in two steps, one of which is the reversible oxidation previously referred to; the second is irreversible and probably also an oxidation, although this has not been definitely demonstrated. The spontaneous oxidation of luciferin without emission of light in crude solutions (without luciferase) is probably catalyzed by traces of heavy metals in the solution and proceeds much more slowly when the luciferin has been purified. Both the non-luminescent oxidation and the luminescent oxidation undoubtedly take place simultaneously when luciferin is mixed with luciferase.

Cyanide does not affect luciferase but forms an irreversible combination with purified Cypridina luciferin. Azide combines reversibly with luciferin whereas urethane, sulfanilamide, sulphathiazol, sulphapyridine and p-aminobenzoic acid probably act reversibly to inhibit luciferase activity.