DEPARTMENT OF MARINE BIOLOGY. 199 



light production, is probably an albumin or very closely associated with an 

 albumin. Evidence was also presented to show that the oxidation product of 

 luciferin, which I called oxyluciferin, has properties similar to luciferin itself 

 and represents only a shght oxidative change, not a fundamental splitting of 

 the luciferin molecule. The oxyluciferin can be readily reduced to luciferin 

 again by various reducing agencies, among them the nascent hydrogen pro- 

 duced by action of acids on metals. Acid favors the reduction of oxyluciferin, 

 and alkali favors the oxidation of luciferin. 



Thanks to the kindness of Professor C. Ishikawa, of the Agricultural Col- 

 lege, Imperial University of Tokyo, Japan, I have received an additional sup- 

 ply of Cypridina material, with which the researches have been continued. 

 My present research deals with the action of acids in facilitating reduction of 

 oxyluciferin, the possible production of CO2, and of heat during oxidation of 

 luciferin, and the general nature of the luciferin <zz:> oxyluciferin reaction. 



Carbon dioxid production was tested by determining if any change in 

 acidity, which might come from CO2 produced, occurs when solutions of luci- 

 ferin and lucif erase are mixed. After several attempts to measure acidity by 

 adding an indicator (thymol-sulphone-phthalein) to the solution, this method 

 was given up because the luciferin and luciferase solutions are yellowish in 

 color, which interferes with the yellow-blue color change of the thymol- 

 sulphone-phthalein. The electrometric determination with the hydrogen 

 and N/10 KCl calomel electrode is the most sensitive. A McClendon elec- 

 trode and a Leeds and Northrup potentiometer were used. The acidity of 

 the luciferin solution, luciferase solution, and the two after mixing was found 

 to be the same, Ph = 9.04. Therefore, not enough CO2 is produced to affect 

 the H-ion concentration. 



As both luciferin and luciferase solutions contain proteins, and as luciferase 

 is certainly and luciferin probably a protein, it will be seen that their buffer 

 value is relatively high. The luciferin and luciferase solutions, although pre- 

 pared with distilled water, no doubt contain also a small amount of buffer 

 salts. Our experiments show this much, however, that not enough CO2 is 

 produced during luminescence to saturate the proteins in solution, including 

 luciferin and luciferase themselves. The reaction responsible for luminescence, 

 the oxidation of luciferin, is therefore not to be compared to the reactions in 

 cells giving rise to the carbon dioxid of respiration. 



The production of heat during luminescence was determined by bringing 

 solutions of luciferin and luciferase to the same temperature and then mixing 

 them. One can thus measure any increase or decrease of temperature which 

 occurs during the luminescence which results from mixing, and gain some idea 

 of the heat of oxidation of luciferin. Although the experiment sounds very 

 simple, it is actually somewhat difficult to carry out. After many attempts 

 it was found necessary to bring the luciferin and luciferase solution to tem- 

 perature equihbrium in two separate tubes within one thermos bottle, and to 

 mix the solution by breaking the tubes. Two thermo-couples of copper 

 advance wire were used to measure the temperature — one in the luciferase, 

 the other in the luciferin solution. These were connected through a copper 

 double-throw switch with a galvanometer of a sensitivity such that 1 mm. 

 deflection represented a temperature change of 0.003° C. As mixing the 

 solutions heats them sUghtly, control experiments with water in each tube 

 were carried out. 



With both control (water) and luciferin experiments there was a shght rise 

 in temperature on mixing the liquids in the two tubes. The average rise of 

 5 control (water) experiments was 0.0054° C. and the average rise of 5 luciferin 

 experiments was 0.0048° C. The difference in the average rise of control and 

 of luciferin experiments is so small (0.0006° C.) as to have Uttle significance. 



