140 CHEMISTRY OF CYPRIDINA LUCIFERIN 



395 mfi. The absorption peak at 265 m/x could be responsible for 

 photochemical changes which have been observed in luciferin after 

 exposure to ultraviolet light (Chase and Giese, 1940). 



The stability as well as the form of the absorption spectrum are 

 very dependent upon the hydrogen ion concentration. The spectrum 

 is relatively stable at pH 1.0 but becomes rapidly less so at higher 

 pH's. For this reason many measurements of the visible and ultra- 

 violet spectra have been made under various experimental conditions 

 in an effort to establish a cui"ve which would with certainty represent 

 luciferin. None of these measurements has previously been published. 

 Recently, application of the techniques of paper chromatography and 

 paper electrophoresis has produced luciferin that is very much purer 

 than any available previously. The details of the methods have al- 

 ready been described. By means of micro attachments for the Beck- 

 man spectrophotometer (Lowry and Bessey, 1946), we have now 

 measured and compared absorption spectra of luciferin isolated by 

 these methods. Since these purification procedures yield products 

 having one and the same absorption spectrum which, furthermore, 

 undergoes identical changes on exposure to air and treatment with 

 base or acid, it would seem justified to assume a high degree of 

 purity for the luciferin. 



In Fig. 3, five sets of absorption spectra are shown. Two different, 

 doubly cycled luciferin preparations are represented, made independ- 

 ently by two of us, using different batches of Cypridinae and slightly 

 different procedures. These preparations will be designated as I and 

 11. The absorption spectra shown in Figs. 3A, 3B, and 3C are from 

 preparation I; those in Figs. 3D and 3E, from preparation II. In all 

 cases the solid line represents the spectrum measured as soon as pos- 

 sible after dissolving the material in 0.1 N hydrochloric acid, with 

 minimal exposure to air. The dash line and dotted line show the spec- 

 tra of the same solutions after exposure to air for the times indicated. 



Figure 3B gives spectra of a luciferin solution which was subjected 

 to no further purification than two cycles of Anderson's (1935) pro- 

 cedure. Considerable general ultraviolet absorption is evident, but 

 inflections in the curves and definite changes during exposure to air 

 are quite apparent. The spectra in Fig. 3C are from a solution of 

 luciferin isolated from preparation I by paper chromatography, while 



