136 CHEMISTRY OF CYPRIDINA LUCIFERIN 



0.2 M disodium hydrogen phosphate (pH 8), and O.l N NaOH. Elu- 

 tion occurred only with alkahne solutions of pH 8 or greater, but 

 here luciferin lost its activity so rapidly that alkaline eluants appeared 

 undesirable. 



A carboxyhc acid resin, XE-97 (Rohm and Haas), an analog of 

 IRC-50, was also tried in fine mesh form in a column 2.26 sq cm X 

 17.5 cm at 2.0-2.5° C. Luciferin was readily taken up from pH 6.4 

 phosphate buflFer, forming a thin yellow fluorescent band at the top 

 of the column. One-cubic centimeter fractions were collected with a 

 fraction collector. The following eluants were tried without success: 

 pH 6.4 phosphate buffer, pH 5.0 citrate buffer, and pH 3.4 citrate 

 buffer. None of the fractions, adjusted to pH 6.8, gave light when 

 tested with luciferase, and the yellow fluorescent band remained at 

 the top of the column. When pH 3.4 citrate buffer containing 25% by 

 volume of ethyl alcohol was used, the yellow fluorescent band moved 

 slightly, but the effluent fractions gave no light when tested with 

 luciferase (after making the pH 6.8 and diluting to minimize lucif- 

 erase inhibition by the alcohol ) . 



The yellow band of the resin column was carefully removed and 

 transferred to a 15-ml centrifuge tube where, after centrifuging off 

 the buffer, the resin was washed with methyl alcohol and then cen- 

 trifuged. A clear yellow supernatant was obtained which gave bril- 

 liant luminescence with luciferase. Some of this was evacuated to dry- 

 ness and the yellow residue redissolved in 0.1 N HCl. The absorption 

 spectrum of this solution resembled that of those obtained by paper 

 chromatography and also those from paper electrophoresis, although 

 it showed slightly greater density at the short wavelengths. Using 

 spectral absorption as a criterion, it is obvious that chromatographic 

 procedures have resulted in removal of many extraneous substances 

 and the preparation of luciferin in a high degree of purity. 



Further attempts have been made to isolate luciferin in larger 

 amounts by employing column-partition chromatography techniques. 

 Preliminary experiments with powdered cellulose columns operated in 

 the cold room at 2.5° C have yielded rather good separation of luciferin 

 from crude extracts of the material. Water represented the stationary 

 phase, and a mixture of ethyl acetate and ethyl alcohol, saturated 

 with water, made up the mobile phase. The cmde luciferin solution 



