164 BIOCHEMISTRY OF FIREFLY LUMINESCENCE 



protein the gel is washed twice with cold alkaline water and then with 

 a 2% solution of (NH4)2S04 at pH 8. Elution of the enzyme is ob- 

 tained by washing the gel twice with a 7% solution of (NH4)2S04 at 

 pH 8 (prep. III). The final volume of combined eluates of preparation 

 III is 95 ml. Preparation III is then fractionated with (NH4)2S04 in 

 successive steps of 10% saturation up to 50% saturation and then in units 

 of 2 to 3% saturation up to 65%. The pH during this procedure is main- 

 tained at 8.0, The major part of the active enzyme is recovered 

 between 57-65% (NH4)2S04 saturation. The latter precipitate is dis- 

 solved in 25 ml of water (prep. IV) and the enzyme is readsorbed 

 onto calcium phosphate gel as described above. The supernatant is 

 discarded. The enzyme is eluted with 7% (NH4)2S04 at pH 8 (prep. 

 V) and precipitated by adding sohd (NH4)2S04 to 70% saturation (pH 

 8). The precipitate is dissolved in 5 ml of H2O and the pH is adjusted 

 to 8 (prep. VI). A further treatment of preparation VI with the low 

 concentration of calcium phosphate gel removes some inert protein 

 (prep. VIII). The activity of the various fractions is summarized in 

 Table I. In this procedure the enzyme was purified approximately 70 

 times on a protein basis with a total recovery of 15%. In addition, the 

 preparation is completely free of luciferin, and under these conditions 

 no light is emitted upon the addition of ATP. 



Preparation VI (Table I) remained stable for several weeks at 

 temperatures below 0°, but was rapidly inactivated at room tempera- 

 ture, especially in dilute solutions. At 40°, approximately 50% loss of 

 activity was encountered in 10 minutes. The preparation was colorless 

 with a sharp absorption peak at 278 millimicrons. The total amount of 

 light which could be obtained from preparation VI was greatly re- 

 duced by purification. Within a few minutes after the addition of 

 ATP, the light disappeared completely and reappeared only when 

 additional purified luciferin was added. The results demonstrated that 

 under these conditions a definite, but limited amount of light was 

 obtained for a given amount of luciferin, indicating an irreversible 

 inactivation such as observed in the Cypridina system. Although 

 preparation VI responded normally to ATP, no light was emitted with 

 ADP, in contrast to the crude extract. Similarly, ATP which had been 

 treated with hexokinase and glucose failed to initiate light emission 

 in preparation VI. The results demonstrate that the terminal phosphate 



