134 STUDIES ON BIOLUMINESCENCE. X 



ducing light. If the temperature of a luminous bacterium is gradually 

 raised, respiration increases continuously up to a relatively high 

 maximum, whereas the luminescence decreases rapidly above a rela- 

 tively low optimum. The two processes are not equally affected by 

 increasing temperature. 



It is obvious that, to measure carbon dioxide production during 

 luminescence, we must use cell-free solutions of the oxidizable material 

 of luminous animals, luciferin, and oxidize it suddenly in order to 

 have the maximum amount of CO2 produced at one time. This 

 experiment can be carried out with the luciferin of Cypridina Jiilgen- 

 dorfii, an ostracod crustacean. A brilliant luminescence results from 

 adding a small amount of luciferase solution to a solution of Cypridina 

 luciferin. The preparation of these solutions has been described in 

 a previous paper.* 



Carbon dioxide production was tested by determining if any change 

 in acidity, which might come from the CO2 produced, occurs when 

 solutions of luciferin and luciferase are mixed. After several attempts 

 to measure acidity by adding an indicator (thymolsulfonephthalein) 

 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 thymolsulfonephthalein. The elec- 

 trometric determination with the hydrogen and 0.1 n KCl calomel 

 electrode is the most sensitive. A McClendon electrode and 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 hydrogen 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 prepared 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 oxida- 



* Harvey, E. N., /. Gen. Physiol., 1918-19, i, 269. 



