134 CHEMISTRY OF CYPRIDINA LUCIFERIN 



tions were poor as evidenced by either high or low luciferin Rf values 

 accompanied by considerable tailing. At no time, however, have two 

 luminous regions of the same area and intensity been observed; when 

 two regions have been observed, one has always been at the origin. 



The work of Mason and Davis was repeated in every detail. For 

 chloroform, the principal luminescent area was at Rf — 0, with a 

 broad, unbroken band extending forward to about Rf = 0.14. When 

 n-butanol was used, the principal luminescent region was at Rf = 0.77 

 and a very small, less intense spot occurred at Rf = 0. The diameter 

 of the spot at R/ = was practically the same as that of the luciferin 

 spot at the origin before chromatography. With n-butanol saturated 

 with water, the Rf of luciferin was about 0.88; there was no other 

 luminous region. In addition to these solvents two other solvents, 

 phenol and collidine ( 2,4,6-trimethylpyridine ) , each saturated with 

 water, were also tried with little success. With the former, the residual 

 phenol remaining in the paper after drying strongly inhibited the 

 luminescent reaction with luciferase, whereas with the latter, rapid 

 destruction of luciferin occurred. 



With Whatman No. 3 filter paper, the ascending technique was 

 again employed and the chromatography run in the cold room at 

 2.0-2.5° C for a period of four or more hours. Doubly cycled luciferin 

 in methanol was used to spot the paper. Solvents tried in various com- 

 binations were benzene, ?j-propanol, water, acetone, n-butanol, eth- 

 ylene glycol, n-amyl alcohol, n-hexyl alcohol, and ethyl ether. They 

 gave from very poor to fair separations and considerable streaking 

 occurred in most of them. The best chromatography was obtained 

 with a developing solvent consisting of ethyl acetate, ethyl alcohol, 

 and water (5,2,3 by volume, upper layer used). The paper, spotted 

 with doubly cycled luciferin, stood in the developing chamber, in 

 contact with the vapor, for one-half hour before lowering into the 

 developing solvent. After the solvent front had moved a distance of 

 about 26 cm (4 hours), the paper was removed from the chamber 

 and dried in air, this process taking only a few minutes. On visual 

 examination, the paper showed two main areas: a yellow-brown one 

 at the solvent front and a yellow one between the front and the 

 origin (see Fig. 1). When examined under a Mineralite lamp, two 

 brightly fluorescent areas were observed: one of yellow fluorescence, 



