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 anæsthetics, 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 associated 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, Ostræa 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 KMnO4 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. H2SO4. After one day of boiling with 20 per cent. HCl the luciferin was completely destroyed and with 4 per cent. H2SO4 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 CaCo3. 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].