150 CHEMISTRY OF CYPRIDINA LUCIFERIN 



thus indicating the presence of either an amino acid, protein, cycHc 

 peptide, diketopiperazine, acylated amino acid, or acylated peptide; 

 and (5) beta luciferin gave a positive dye retention test (Robinson 

 and Fehr, 1952) for proteins and peptides. The amino acids obtained 

 from 24-hour hydrolysis of beta luciferin with 4 N HCl at 135° C 

 were identified by two-dimensional paper chromatography and micro- 

 biological assay as follows: glycine, threonine, proline, lysine, aspartic 

 acid, glutamic acid, and either leucine, isoleucine, or phenylalanine. 

 In view of Mason's findings, the following experiments were under- 

 taken, using 28 mg of doubly cycled luciferin. The absorption spec- 

 trum of this material indicated the presence of some impurity, but 

 the following tests were negative: biuret, ninhydrin, anthrone (for 

 carbohydrates), and molybdenum blue (for phosphorus after com- 

 plete digestion). Quantitative protein determination by the method 

 of Lowry et al. ( 1951 ) indicated that this luciferin preparation was 

 28% protein or polypeptide, calculated in terms of equivalence of 

 crystalline bovine albumin. This doubly cycled luciferin was hydro- 

 lyzed by refluxing with 6 N HCl (ninhydrin negative) for 16 hours 

 according to the method of Stein and Moore (1948).* The hydrolyzate 

 after filtration had a clear golden-orange color, was moderately 

 fluorescent (ultraviolet excitation) and gave weak luminescence with 

 luciferase. It also showed a strong positive ninhydrin test according 

 to the method of Moore and Stein ( 1948 ) . The ninhydrin color value 

 obtained indicated an equivalence of 35% leucine. The hydrolyzate 

 was analyzed for amino acids, using both the long and short Dowex 

 50 columns, by the method of Moore and Stein (1951). Very good 

 separations of the amino acids were obtained. Recovery of ninhydrin 

 color value, expressed as equivalence of leucine introduced into both 

 columns, was 107%. By this technique, the following substances were 

 found in the luciferin hydrolyzate: taurine, aspartic acid, threonine, 

 serine, sarcosine, glutamic acid, proline, glycine, alanine, valine, 

 methionine, isoleucine, leucine, tyrosine, phenylalanine, lysine, ammo- 

 nia (from degradation of amino acids and amides), histidine, and 

 arginine. The presence of beta alanine, tryptophane, ethanolamine, 

 hydroxylysine, and ornithine was uncertain. On the other hand, there 



' It is a pleasure to acknowledge the collaboration of Dr. Harold H. 

 Williams of Cornell University in the amino acid analysis. 



