142 CHEMISTRY OF CYPRIDINA LUCIFERIN 



In Fig. 3D are drawn the spectra of this same preparation after fur- 

 ther purification by paper electrophoresis. 



It is at once apparent on inspection of the five sets of absorption 

 spectra in Fig. 3 that they all show, not only the same absorption 

 maxima, but also the same changes when the solutions stand in con- 

 tact with air at room temperature. Even in the presence of consider- 

 able impurity, as in Fig. 3B, the same absorption maxima ( represented 

 by inflections) and the same changes in the spectrum are easily dis- 

 cernible. The definite difference between the initial spectrum in Fig. 

 3A and those of Figs. 3C, 3D, and 3E is undoubtedly due to the fact 

 that, in the first case, a longer time elapsed between eluting the 

 material from the paper and measuring the initial spectrum. It is 

 practically certain that the changes observed in the absorption spectra 

 of luciferin solutions during exposure to air are the result of oxidation 

 of some sort. 



Disregarding Fig. 3B, the initial spectra in Figs. 3A, 3C, 3D, and 

 3E are practically identical. They have an absorption maximum at 

 265 m/jL, a smaller peak (or shoulder) at about 310 m/i, and a broad 

 absorption band in the visible region, centering at about 465 m^n. On 

 standing, exposed to air at room temperature, the 265-m/x peak be- 

 comes less, the peak at 310 m^ becomes much more pronounced (is 

 possibly unmasked ) , a new band appears at 380 m/A and much of the 

 visible absorption disappears. Finally, as the dotted line curves show, 

 the 380-m/x band and practically all visible absorption disappear and, 

 at the same time, there is a further decrease in the absorbance of the 

 bands at 265 and 310 m/x. By this time the spectrum has become 

 practically stable. 



It seems reasonably certain that Cypridirm luciferin has now been 

 isolated in a rather high state of purity and that, consequently, the 

 absorption spectra of Fig. 3 may be characteristic of luciferin at pH 

 1.0 and of the changes which it undergoes at this pH on exposure to 

 air. 



A comparison of Figs. 2 and 3 shows that the spectrum of a lucif- 

 erin solution is not only less stable at pH 6.8 than at pH 1.0, but also 

 that the changes on exposure to air are different. Clearly, the hydro- 

 gen ion concentration affects one or more components of this system. 

 Furthermore, dilute hydrochloric acid solutions of doubly cycled lucif- 



