II . I). McELRO) AM) H. H. SELIGER 



239 



One mighl cxjjcct that some product would occur in the li^lit- 

 eniitting step which would have lluoiescence properties similar to the 

 bioluniinescence emission and which coidd possibly be identified as 

 the li^ht-cmilting species. 



For this reason caretid measiuements were made of the absorjjtion 

 and emission properties of a niunber of luciferin and oxyluciierin 

 derivatives. The results, presented in Fig. 18, show normalized 

 idtraviolet absorption spectra of luciferin and oxyluciferin in acid 

 and base. At neutral pH or below, luciferin shows an afjsorption 

 peak at 327 millimicrons which corresponds to the activation peak 

 for its fluorescence. When the pH is made higher than 9, the absorp- 

 tion peak shifts from 327 to 381 millimicrons. The small absorption 

 in the short idtraviolet also shifts to a longer wavelength. The molar 

 absorbance at 327 millimicrons is proportional to concentration. The 

 327/263 ratio is used as a criterion of purity, and for crystalline luci- 

 ferin is approximately 4.5. 



The absorption spectrum of oxyluciferin under the corresponding 

 conditions is also shown in Fig. 18. The absorption spectrum of 

 oxyluciferin shows the same general shape and shift with pH as that 

 of luciferin. The shift of approximately 20 millimicrons to longer 

 wavelength as we go from the dihydro form to the oxidized form 

 might be just what woidd be expected from the addition of a double 

 bond to the aromatic structure. The retention of both absorption 



250 



300 



350 



400 



450 



m p. 



Fig. 18. \oiniali/ed absorbance spectra ot luciterin and oxyluciferin. The points 

 of crossing of the acid and base ciir\es are also isobestic points. 



