F. I. TSUJI, A. M. CHASE AND E. N. HARVEY 133 



as quantitative determinations of the amino acids present after hy- 

 drolysis by acid. Methods and results are described below. 



Chromatographic Purification 



The starting point for chromatographic procedures was Cijpridina 

 luciferin prepared by two cycles of benzoylation according to the 

 method of Anderson (1935). The final butanol solution of luciferin 

 was stored under an atinosphere of hydrogen and aliquots were with- 

 drawal as required. The butanol could then be removed by evacuation 

 and the residue redissolved in methyl alcohol for work with paper 

 chromatography and in the appropriate buffers for work with paper 

 electrophoresis and ion-exchange resins. All these methods, as well as 

 measurement of the absorption spectrum of doubly cycled luciferin, 

 have revealed the presence of at least several impurities. 



Mason and Davis (1952) were able to chromatograph Cypridina 

 luciferin on No. 1 Whatman paper, using the ascending technique. 

 They worked at room temperature in a hydrogen atmosphere satu- 

 rated with the vapor phase of the developing solvent. They reported 

 that doubly cycled luciferin was separable by several solvents into 

 what they designated as alpha and beta luciferins possessing the fol- 

 lowing Rf values, respectively: chloroform, and 0.25; n-butanol, 

 and 0.55, and n-butanol saturated with water; 0.8 and 0.8. Both 

 luciferins were yellow but did not fluoresce. Their chromatographic 

 studies did not reveal any impurities, either by ordinary or ultraviolet 

 light, or by any of various tests tried. 



The preceding evidence may not necessarily indicate the existence 

 of two kinds of luciferin, for in most of the present work only one 

 luciferin has been observed, as manifested by a single, intense lumi- 

 nous area on the paper when treated with luciferase. Occasionally 

 after paper electrophoresis and more often with paper chromatog- 

 raphy, a less intense luminous spot was detected at the origin, but 

 this might be ascribed either to luciferin strongly adsorbed by the 

 paper or to undissolved particles of luciferin, or both. At other times 

 extremely weak luminescence has been observed at the solvent front 

 after paper chromatography, but this is probably due to luciferin 

 bound to impurities moving at the front. Luminescence at the origin 

 was nearly always observed in paper chromatography where separa- 



