F. I. TSUJI, A. M. CHASE AND E. N. HARVEY 147 



It has been pointed out in previous sections that paper chroma- 

 tography and paper electrophoresis techniques demonstrate that in 

 the doubly cycled luciferin there are at least two fluorescent sub- 

 stances, one of which is luciferin, yellow fluorescent, and the other an 

 impurity, blue fluorescent. For further study of luciferin fluorescence, 

 the yellow fluorescent region of the filter paper was eluted with 

 methanol, the solvent removed in vacuo, and the residue dissolved 

 in 0.1 N HCl. In a beam of white light, observed from the side, no 

 fluorescence can be detected, indicating a relatively weak intensity, 

 but in the Wood light (maximum energy 365 mju) and also in a 

 Mineralite (ultraviolet without the visible; maximum energy 253.6 

 m^) the luciferin solution is yellow fluorescent. In the light from a 

 "Purple ultra" incandescent bulb the fluorescent color was green to 

 bluish green, probably due to a combination of the yellow fluorescence 

 and the blue light transmitted by this bulb. Dilution of the 0.1 N HCl 

 luciferin with 9 parts of water nearly abolishes the fluorescence, in- 

 dicating its low intensity. Paper chromatographed luciferin is also 

 yellow fluorescent when dissolved in methanol and butanol. It is clear 

 that Cypridina luciferin does exhibit fluorescence,* although it is far 

 less bright than the fluorescence of many other chemiluminescent sub- 

 stances. 



The effect of acid-base change and oxidation of chromatographed 

 luciferin in aqueous solution was studied by preparing three tubes 

 with luciferin in 0.1 N HCl: leaving one tube acid, neutrahzing 

 another with NaOH, and making a third alkaline with NaOH to 

 about pH 13. Table I shows the relative fluorescence of the fresh, 

 active luciferin and of the material allowed to stand for 14 days until 

 the luciferin had completely oxidized. 



It is apparent from the observations noted in the table that the 

 fluorescence of a 0.1 N HCl solution of luciferin is very much less 

 after it has been exposed to air until completely oxidized than when 

 freshly made up. This can quite probably be interpreted in terms of 

 the decrease of the ultraviolet absorption of such a solution which 



° In certain solvents (e.g., ethanol) at room temperature, Cypridina 

 luciferin exhibits an apparent "phosphorescence" after irradiation with 

 ultraviolet light. This effect iS believed to be a sustained chemiluminescence 

 induced by the radiation (see Tsuji and Harvey, 1954). 



