natural proteose, soluble in absolute alcohol and not digested by trypsin or that it belongs to some other group than the proteins. The absence of a biuret reaction would point in that direction and the question must await further study.
Cypridina luciferin is found in the luminous gland of the animal and possibly in parts non-luminous as well as in the luminous organ. This is true of the luciferin from fireflies which is found throughout the body of Luciola, Photuris and Photinus.
Cypridina luciferase.—Luciferase, on the other hand, has all the properties of a complex protein. It will not dialyze through collodion or parchment membranes, is soluble only in aqueous solvents, and hence precipitated by alcohol and acetone, digested by proteolytic enzymes, readily changed by contact with dilute acid and alkali and irreversibly coagulated on boiling. It is completely salted out of solution by saturation with (NH4)2SO4 and nearly completely precipitated by the alkaloidal reagents. Its other properties are given in [Table 8]. Taken together, they point to the group of albumins as the class of proteins with which luciferase most closely agrees.
If luciferase is not a protein it is so closely bound up with protein that it cannot be separated. This is characteristic of many enzymes and luciferase is also an enzyme. We can determine this by finding out whether luciferase will accelerate the oxidation of a large amount of luciferin, for such is the test of a catalytic substance. If we take 1 c.c. of a dilute solution of luciferase (1 Cypridina to 50 c.c. water) and add to it successive 1 c.c. portions of concentrated luciferin (1 Cypridina to 2 c.c. solution) as soon as the light from the preceding addition has disappeared,
after four 1 c.c. additions, no more light is produced. The luciferase is therefore used up and cannot oxidize more than a certain quantity of luciferin. In this experiment, however, we added a concentration of luciferin from one Cypridina 100 times that of the luciferase from one Cypridina, i.e., four additions each 25 times as concentrated. We have, of course, no way of telling what the absolute amount (in milligrams) of luciferin or luciferase is in a single Cypridina, but we do know that the luciferase from one Cypridina cannot oxidize luciferin from more than 100 Cypridinas. If the ratio of luciferin to luciferase in a single animal is 100:1, it would mean that luciferase could oxidize 10,000 times its weight of luciferin. A large excess of luciferin but not an indefinite quantity can be oxidized by luciferase, and I believe this is sufficient justification for considering luciferase an enzyme, although it is not an ideal example of an organic catalyzer. Quite a number of enzymes are known to be diminished during the course of the reaction they accelerate or to be poisoned by their reaction products. Enzyme reactions inhibited by the formation of reaction products again proceed if these are removed or diluted. However, light does not again appear in a mixture of weak luciferase with excess of luciferin upon dilution with water, so that the luciferase cannot have been merely inhibited by some reaction product but must have been actually used up during the reaction. It should be noted in passing that the peroxidases, ordinarily spoken of as oxidizing enzymes, are used up in the reaction and can only oxidize limited amounts of oxidizable substances, a quantity almost in proportion to the concentration of peroxidase present.
Whether luciferase is an oxidizing enzyme made up of
an albumin associated with some heavy metal as iron, copper or manganese is uncertain. From analyses of whole Cypridina, kindly made for me by Prof. A. H. Phillips of Princeton University, all three of these metals, which we know to be associated with biological oxidations, are present, and it is quite possible that one of them is concerned with the oxidation of luciferin.
Although I have tested a great many oxidizers, organic and inorganic, and a large number of oxidizing enzymes from blood and tissue extracts of animals rich in iron, copper and manganese, I have found no material which is capable of taking the place of Cypridina luciferase. Peroxidases or oxidases of plants, hæmoglobin, hæmocyanin, extracts of mussels, manganese containing blood of various marine crustacea and mollusks will give no light on mixing with luciferin. Such active oxidizers as KMnO4, H2O2, BaO2, and many others, will not oxidize Cypridina luciferin with light production, although they can oxidize Pholas luciferin with light production.
The action of Cypridina luciferase is very highly specific. It is found only in the luminous organ of Cypridina hilgendorfii, not in non-luminous parts and not in a non-luminous species of Cypridina closely related to hilgendorfii.
Luciferins and luciferases from closely allied luminous forms will mutually interact to produce light, but no light appears if these substances come from distantly related forms. Thus firefly (Photuris) luciferin will give light with Pyrophorus luciferase and vice versa, but Cypridina luciferin will give no light with firefly (Luciola) luciferase or vice versa, nor with Pholas luciferase or vice versa. The faint luminescences sometimes observed on mixing