264 LIGHT AND LIFE 



Luciferase, isolated and purified in this fashion, is probably not 

 soluble. The addition of Tween 80 to a final concentration of 

 0.03% in the reaction mixttue has been found to stimulate both the 

 initial rate of the reaction and the maximum light intensity attained. 

 This addition now is made routinely during the assay. The homo- 

 genate is not made up in a buffer containing Tween 80, as this causes 

 a more rapid loss in activity. Treatment of the 30-50% (NH4) 2SO4 

 fraction with wheat germ lipase or snake venom has not resulted in 

 increased activity or in reduction of the stimulatory effect of the 

 Tween 80 addition. If the mold luciferase is particulate in nature, 

 then the particle must be of comparatively small dimensions, as 

 activity is still restricted to the supernatant fraction after centri- 

 fugation of the homogenate at 29,000 X g for 30 minutes. In view 

 of these findings, and since facilities for routine high speed centri- 

 fugation are not available, we have proceeded with the objective 

 of learning as much as possible about the system in this state. 

 The results set forth in Fig. 1 could be attributed to an increase 

 in the amount of active enzyme during the course of the reaction. 

 Throughout the initial phases of this work it was noticed that enzyme 

 preparations frequently were completely inactive. Precedent to this 

 possibility is given by the fact that the initial stages of luciferase 

 purification from both the firefly and Photobacteriiim fischeri also 

 have been complicated by the fact that the enzyme is readily inhibited, 

 a fact leading one to believe that the preparation is very unstable (9) . 

 Treatments of the inactive fungal enzyme by dialysis against pH 7.0 

 phosphate buffer, precipitation with cold ethyl alcohol or acetone, 

 and additions of Versene or sulfhydryl reagents, or both, have been 

 unsuccessful in liberating the presumed inhibitor. However, the 

 addition of crystalline bovine plasma albumin (Armour and Com- 

 pany) to the reaction mixture stimulates the reaction. Such an addi- 

 tion has subsequently been found not only to "reactivate" the in- 

 active enzyme but also to stimulate the response from an active 

 preparation. In Fig. 2, albimiin is seen to give about a 5-fold in- 

 crease in the maximum amount of light emitted and a corresponding 

 increase in the rate. This effect is specific for bovine plasma albumin, 

 bovine seriun albumin, or crystalline hinnan albumin; neither crystal- 

 line egg albumin, bovine albumin— fraction V, nor gelatin will elicit 

 the same response. The |)<)ssibility that the crystalline bovine plasma 

 ;ilbimiin protects the huiferase, due to its low concentration, from 

 dcnaturation is inilikely since the en/yme preparation at this stage 

 of |)ui ifi( ntioii (ontiiins from 20 to 30 milligrams of protein per 



