F. I. TSUJI, A. M. CHASE AND E. N. HARVEY 137 



was prepared by extracting fat-free Cypridirm powder with methyl 

 alcohol, then removing the alcohol by evacuation, extracting the 

 luciferin residue with butyl alcohol, removing this alcohol by evac- 

 uation, and finally redissolving the luciferin in the mobile phase mix- 

 ture for placement on the column. The developed band of luciferin 

 could then be followed on the column by its yellow color and yellow 

 fluorescence and could be collected at the appropriate time with a 

 fraction collector. The luminescence with luciferase served to identify 

 this luciferin. There is some evidence from spectral measurement that 

 the luciferin isolated by the above procedure from the crude extract 

 is different from the luciferin prepared by the Anderson's benzoyla- 

 tion method. 



Electrophoretic Purification 



Filter paper electrophoresis of doubly cycled material was also 

 used to isolate luciferin for spectrophotometric comparison with that 

 obtained by paper chromatography. The electrophoretic method was 

 essentially that of Kunkel and Tiselius (1951). A 0.1 M NaHoP04- 

 H3PO4 buffer of pH 2.55 and ionic strength 0.1 was used to dissolve 

 the luciferin. A potential gradient of approximately 5.9 volts/cm and 

 6.4 ma was appHed to a double layer of Whatman No. 3 paper for 

 14 hours at 10° C. The acid pH was selected because luciferin is more 

 stable at low pH (Anderson, 1936). Dextran and Armour's crystalline 

 bovine albumin were run concurrently and localized in a strip of the 

 paper after electrophoresis with 1% bromophenol blue in absolute 

 ethyl alcohol saturated with mercuric chloride. After dyeing the paper 

 was washed with absolute methyl alcohol, leaving the albumin blue. 

 Luciferin is presumably washed out by the methyl alcohol in which 

 it is extremely soluble. Dextran indicated the degree of electro-osmotic 

 flow of the buffer. 



After electrophoresis, a definite area on another strip of the paper 

 showed a yellow color, a yellow fluorescence in the ultraviolet light 

 of a Mineralite lamp, and luminesced with luciferase. As in the case 

 of chromatography with ethyl acetate, only one intense luminescent 

 area was observed. Luciferin, like albumin, was positively charged at 

 this pH. The ratio of the distance moved by luciferin to that moved 

 by albumin was approximately 0.34 (3.4 cm for luciferin, 10 cm for 



