F. I. TSUJI, A. M. CHASE AND E. N. HARVEY 145 



On adjusting the pH of such an alkahne luciferin solution back to 

 pH 1.0 from 13, the absorption band at 355 m/A largely disappears 

 and some of the specific absorption of tlie acid solution in the visible 

 region and at 265 and 310 irifi is restored. A spectrum measured under 

 such conditions is shown in Fig. 4A (dotted line curve), and it is 

 apparent that the final acid absorption spectrum resembles qualita- 

 tively that of the acid luciferin solution as originally prepared, al- 

 though the densities are considerably decreased. An obvious interpre- 

 tation of these results (although not necessarily the only one) might 

 be that during the interval when the luciferin was exposed to dis- 

 solved oxygen at pH 13 an appreciable amount was oxidized, so that 

 upon making the solution acid once more, and thereby restoring the 

 original form of the absorption spectrum, much of the absorbance of 

 the peaks had been lost. The slight qualitative differences between 

 the initial and the final spectra might be attributed to contribution 

 by the spectrum of the oxidized form of the material. 



It is evident from the following experiment that reaction with dis- 

 solved oxygen certainly plays a role in the above changes. If all oxy- 

 gen is displaced from an acid luciferin solution immediately after its 

 preparation, by passing pure hydrogen through the solution in the 

 absorption cell, and if such an oxygen-free solution is then adjusted 

 to a pH of 13, an absorption peak centering at 380 m/x is found and 

 this peak is perfectly stable as long as the solution remains oxygen 

 free. 



As has already been demonstrated in some detail, the absorption 

 spectrum of aqueous luciferin solutions undergoes specific changes on 

 standing exposed to air, and these changes are probably due to 

 oxidation of the luciferin. If a luciferin solution of pH 1.0 is allowed 

 to stand, exposed to air, until the spectrum has reached a practically 

 stable condition, and if the pH is then adjusted to 13 with sodium 

 hydroxide, a different absorption spectrum is obtained than when 

 freshly prepared luciferin solutions of pH 1.0 are adjusted to pH 13. It 

 will be recalled that fresh solutions show a new band at 380 m^n 

 which rapidly shifts to a stable position at about 355 m/i,. On the other 

 hand, the solution which has stood at pH 1.0 until its spectrum is 

 stable, and is then adjusted to pH 13, shows a band which is relatively 

 stable at 330 m/A as shown in Fig. 4B. This result indicates that a 



