STUDIES ON BIOLUMINESCENCE
Authors: Harvey E. Newton 
Published: May 2004

There seems to be very little doubt but that luciferase is a protein or so closely associated with proteins that their removal destroys its characteristic properties. The particular group of proteins to which it belongs may be arrived at by a process of exclusion, and only the group of albumins has properties which agree completely with those of luciferase.
Dubois believes Pholas luciferase to be an oxidizing enzyme similar to the oxydones of Battelli and Stern because it is readily destroyed by fat solvents such as chloroform, strong alcohol, etc. He has detected iron in a luciferase solution which has dialyzed against running water for a long time, and believes it to be made up of protein in combination with iron and to act as an "oxyzymase ferrique." Cypridina luciferase, on the other hand, is not readily destroyed by fat solvents. Toluene and chloroform are good preservatives, and I often make use of them for this purpose, keeping the luciferase solutions for many months. Professor A. H. Phillips of Princeton University has very kindly analyzed some whole dried Cypridinœ for me and finds iron, copper, and manganese but no zinc or vanadium to be present. Whether these metals are connected with the action of Cypridina luciferase is uncertain, but it is significant that all three of the metals thought to be concerned in organic oxidations are present.
Although a large amount of luciferin mixed with a small amount of luciferase will use up all the latter, I agree with Dubois that luciferase has sufficient properties in common with the enzymes as a class to be considered an enzyme. The peroxidases are well known to be used up in the reactions they accelerate. All workers on enzymes agree that the more enzymes are purified the less active they become. The chemical procedures necessary to remove foreign material bring about irreversible changes in the enzyme itself, a characteristic also of many protein groups and of the colloidal state in general. This is true in the case of luciferase, for the crude luciferase solution is the most active preparation that can be obtained.
I believe that Cypridina luciferase should be placed in a class of oxidizing enzymes by itself—a group having the chemical reactions of an albumin, possibly in combination with some heavy metal, and which as far as we know, acts specifically on only one substance, Cypridina luciferin. It resembles the plant peroxidases in resisting the action of chloroform, toluene, etc., but will not oxidize any of the hydroxyphenol or aminophenol compounds so readily oxidized by the peroxidases, nor will the peroxidases oxidize luciferin with light production. Dubois' researches show that Pholas luciferase differs in some properties from Cypridina luciferase, and my own work indicates that firefly luciferase is more like that of Pholas. A comparative study of other species of luminous animals is needed in order to delimit more accurately the class of luciferases as a whole.
Luciferin presents many characteristics in common with the proteins, but two, which, to say the least, throw doubt on its protein nature: (1) its peculiar solubility (in alcohols, esters, and glacial acetic acid), (2) and its resistance to digestion by proteases, even by trypsin which has almost universal digestive action. These two peculiarities have been discussed above. We can only say that if a protein, luciferin must belong to a new group differing from known natural proteins in these respects. In general characteristics this new group would fall somewhere on the border-line between the proteoses and peptones. It would not be surprising to find in nature proteoses or peptones soluble in absolute alcohol. We know also that some NH-CO linkages of proteins are broken down with great difficulty by trypsin as it is difficult to obtain a tryptic digest of protein which does not give the biuret reaction, and the work of Fischer and Abderhalden has shown that certain artificial polypeptides are not digested by pure activated pancreatic juice. We have, then, three possibilities. Luciferin is (1) either a natural proteose not attacked by trypsin, or (2) if attacked by trypsin, its decomposition products (presumably amino-acids) still contain the group oxidizable with light production, or (3) it is not a protein at all. I believe that luciferin has too many protein characteristics to conform to the last possibility. I have been unable to oxidize with light production various mixtures of amino-acids (from beef and casein) by means of luciferase and consequently am led to believe that luciferin is a new natural proteose, soluble in absolute alcohol and not digested by trypsin.
Dubois believes Pholas luciferin to be a natural albumin with acid properties. Cypridina luciferin could not possibly be regarded as an albumin, but it is very likely that the luciferins of different species of luminous animals differ in certain characteristics. As in the case of the luciferases, we know that the luciferins are not identical substances, and only future work can determine in what particulars they differ.
A summary of the properties of Cypridina luciferin and Cypridina luciferase will be found in the tables accompanying this paper.