F. I. TSUJI, A. M. CHASE AND E. N. HARVEY 129 



or at low temperature, involves extraction of dried Cypridinae with 

 benzene to remove lipids, extraction with methanol to dissolve the 

 luciferin, replacement of methanol with n-butanol, treatment with 

 benzoyl chloride, partition between ether and water, hydrolysis of 

 the benzoylated luciferin with aqueous HCl, partition of the freed 

 luciferin with 71-butanol, and storage of the butanol solution under 

 hydrogen. At least two cycles of benzoylation must be carried out for 

 greatest purity. In this paper, material so treated will be referred to 

 as doubly cycled luciferin. 



Removal of the butanol in vacuo leaves a resin-like amorphous 

 brownish-yellow film with high luminescent activity. Each milliliter 

 of the butanol luciferin solution contains from 0.05 to 0.115 mg. of 

 solid and the purification of the material as measured in hght units 

 per unit of weight is about 2000 times that of the dried powdered 

 Cypridinae. The purified luciferin is much more stable against spon- 

 taneous oxidation than are crude aqueous solutions, especially in 

 dilute acid. 



Anderson (1936) also demonstrated that only the product of spon- 

 taneous oxidation of luciferin, or the oxidation carried out with oxi- 

 dants like ferricyanide, was reversible with reducing agents, and that 

 the yield of luciferin on reduction of oxidized luciferin is only high 

 (70%) if the reduction is carried out immediately after oxidation. If 

 allowed to stand, little reduction occurs. 



When luciferin is oxidized with luciferase, and reduction is imme- 

 diately attempted, a slight amount of luciferin can be obtained, at- 

 tributed by Anderson to reduction of a small amount of spontaneously 

 oxidized luciferin. This type of oxidation proceeds simultaneously 

 with the light-emitting oxidation in presence of luciferase and com- 

 phcates the interpretation of reaction kinetics (Chase and Lorenz, 

 1945). Anderson (1937) also studied the relation between salt con- 

 tent and pH of the medium and total hght emitted. Salts are not 

 necessary for luminescence, although NaCl in 0.01 M concentration 

 increases the total light 2.3 times, while KI decreases total light to 

 0.08 of that in distilled water. Kinetics of the Ctjpridina luminescent 

 reaction, involving quantitative studies of the effects of temperature, 

 light, pH, and various inhibitors have been carried out by Chase and 

 collaborators during the years 1940-1952. A resume of this work and 



