242 



LIGHT AND LI IE 



Q 



>- 



LJ 



o 



LiJ 

 CJ> 

 C/) 

 UJ 



cr 

 o 



Z) 



10 



PH 



12 



Fig. 21. Fluorescence yields of luciferin and oxyluciferin as functions of jiH. 

 The measurements were complicated by the absorption shifts, shown in Fig. 18. 

 "Fhe (|nality of the emission, however, was imchanged by /;H. Measurements 

 were made relative to fluorescein in alkaline .solution. The rather sharp pK at 

 j>H 8.4 corresponds to the ionization of tiie hydroxyl group of the molecule, and 

 it is apparently the if)nized species wliidi emits light in bioluminescence. 



and a spectrophotometer, taking account of the spectral absorption 

 shifts shown in Fig. 18. The rehitive data were then compared 

 cHrectly with fhiorescein in basic sohition, since the spectral emission 

 of this compound is very near to that of luciferin and its absolute 

 fluorescence yield is well known. The curve indicates that both luci- 

 ferin and oxyluciferin have the same ionizable groups, a carboxyl 

 group showing a pK between pH 3 and 4 and a phenolic OH group 

 with a pK at pH 8.4. The carboxyl group has been confirmed on the 

 basis of infra-red spectra and other chemical tests. The fluorescence 

 yield exhibits the most dramatic change depending upon the ioniza- 

 tion of the hydroxyl group and it is most likely this group which is 

 j)revented effectively from ioni/ing when oxyluciferin is bound on 

 the en/.yme surface, since the fluorescence of L-AMP relative to L 

 shoAvs the same relative decrease in intensity. 



The absorption and fluore.scence levels of oxyluciferin at three dif- 

 ferent pH's and at different temperatures are shown in Fig. 22. At 

 liquid nitrogen tcmperatines regardless of pH there is only one major 

 emi.ssion, and this is at 480 millimicrons. At minus 70 degrees and 

 pH 10 there is a 480 millimicron jjcak Avhich changes gradually to 

 540 millimicrons as the temperature is raised. A similar change in 



