138 CHEMISTRY OF CYPRIDINA LUCIFERIN 



albumin). The luciferin area was eluted with metliyl alcohol, the 

 yellow solution evacuated to dryness, and the residue dissolved in 

 0.1 M HCl for measurements of the absorption spectrum (to be dis- 

 cussed in the next section ) . 



Paper electrophoresis of luciferin was also carried out at room tem- 

 perature and pH 4.0 (acetic acid/sodium acetate buffer), pH 5.8 

 (acetic acid/pyridine buffer), and pH 8.9 ( veronal/ veronal sodium 

 buffer). At these pH values, two intense blue fluorescent spots, prob- 

 ably the same blue fluorescent substance seen in paper chromatog- 

 raphy, were observed moving toward the negative pole. At pH 4.0 

 and pH 5.8, the luciferin was still positively charged, although bo- 

 vine albumin was positively charged at the lower pH but negatively 

 charged at the higher pH. At pH 8.9, the inhibition of lucif erase by 

 sodium barbiturate and the auto-oxidation of luciferin (resulting in 

 low light production) were too great to localize the position of the 

 migrating luciferin. 



Absorption Spectrum 



The first indication that luciferin possessed specific spectral absorp- 

 tion which might prove useful in its isolation and identification was 

 the observation (Chase, 1940) that neutral aqueous solutions had an 

 absorption band at about 435 m/x which was replaced during exposure 

 to air by another at about 465 m/x. The latter subsequently disap- 

 peared, leaving the solution colorless to the eye. A yellow color had, 

 indeed, been associated with Cypridina luciferin since it was first 

 studied by Harvey (1917), and Anderson (1935) had never been 

 able to dissociate color from the ability to give light with luciferase, 

 even after as many as three cycles of purification by his method. 



The visible absorption band of an aqueous luciferin solution* and 

 the changes which it underwent during exposure to air made it evi- 

 dent that information on luciferin structure might come from ultra- 

 violet absorption spectrophotometry, and that the spectrum would 

 certainly be useful as a criterion of purity. Chase and Brigham (1951) 

 obtained the first reliable measurements of the ultraviolet absorption 



" The luciferin used in the work to be described in this section was in 

 all cases obtained by two cycles of Anderson's (1935) purification pro- 

 cedure before being subjected to further purification methods. 



