232 



LIGHT AND LIFE 



L + ATP 



L- AMP + POP 

 CoASH 



Cystein 



COAS - L+AMP 



COXYLUCIFERYL- CoA) 

 GSH 



AOH 



N-OXYLUCIFERYL S-OXYL-GSH L + CoASH 



-CYSTEINE 

 li". 10. The activation of oxvliuiferin and the reaction of coenzyme A. 



of oxyliiciferin by fluorescent increase in a mixture of L-CoA, AMP 

 and pyrophosphate. 



The results presented in Fig. 1 1 represent a spectrophotofluoro- 

 metric study of part of the reaction described in Fig. 10. In this 

 experiment the initial rapid decrease in fluorescence is due to the 

 formation of the CoA derivative of oxyluciferin in the presence of 

 ATP, magnesium, and luciferase. The L-CoA derivative has been 

 isolated and identified by using paper chromatography and enzy- 

 matic procedures. Cysteine is caj^able of reacting with the L-CoA to 

 produce oxyluciferyl-cysteine. The fluorescence increase on the addi- 

 tion of cysteine at 12 minutes (Fig. 11) is due to the formation of 

 the cysteine derivative, which has a fluorescence which is only about 

 50 per cent less than that of free oxyluciferin. The cysteine oxyluci- 

 ferin derivative has also been identified by chromatographic pro- 

 cedures. The en/ymatic activation of oxyluciferin by ATP in the 

 presence of coenzyme A leads to the formation of pyrophosj^hate and 

 free adenylic acid as well as oxyluciferyl-coenzyme A. The results 

 (Fig. 12) indicate that this reaction does occur. Oxyluciferyl-CoA 

 was produced in a reaction mixture consisting of oxyluciferin, luci- 

 ferase, coenzyme A, and ATP. This is shown on the graph by the 

 initial decrease in fluorescence. Upon the addition of adenylic acid 

 after 1 1 minutes there was a small increase in fUiorescence. When 

 high concentrations of pyrophosjihate were added there was a large 

 increase in fluorescence because of the formation of free oxyluciferin. 

 ATP was also formed in this reaction and was measured by the fire- 

 fly system in a separate experiment. The high concentration of pyro- 

 phosphate required to obtain an increase in fluorescence indicates 

 that the equilibrium is in favor of oxyluciferyl-CoA formation. 



