258 



PURIFICATION AND PROPERTIES 



TABLE I 

 Purification of Bacterial Luciferase 



{ Achromobacter fischeri) grown on Farghaly's (1950) liquid medium 

 with 1% peptone added were harvested by centrifugation after 12-18 

 hours and lysed in distilled water (3 g wet weight per 100 ml H2O). 

 After removal of the cell debris by centrifugation the protein was pre- 

 cipitated by acidification and resuspended in a small volume of water 

 at pH 7.0. Further purification was achieved by fractionation with 

 (NH4)2S04, pH 7.0. All procedures were carried out in the cold. Light 

 intensity was measured with a photomultiplier apparatus (Hastings, 

 McElroy, and Coulombre, 1953). The reaction mixture consisted of 

 0.5 ml of buffer (0.135 M KH2PO4, 0.135 M NaoHP04, and 0.01 M 

 NaCl) pH 6.8, 0.1 ml of 2.5 X 10-^ M DPNH, 0.1 ml of 5 X 10"^ M 

 riboflavin-5-phosphate, 1.0 ml of a satin-ated water solution of dodecyl 

 aldehyde, and enzyme, other reagents, and water to a total volume of 

 2.5 ml. The reaction was usually initiated by adding DPNH. 



The enzyme preparation from the 60-70% (NH4)2S04 fraction is 

 colorless, rapidly destroyed at 80° C, and stable for several months in 

 the frozen state. It will emit a weak light with DPNH alone, but for 

 appreciable activity FMN (McElroy, Hastings, Sonnenfeld, and Cou- 

 lombre, 1953) and aldehyde (Cormier and Strehler, 1953) must be 

 added. FMN reduced by bubbling hydrogen in the presence of a 

 catalyst will dispense with the requirement for DPNH ( Strehler, Har- 

 vey, Chang, and Cormier, 1954). Other reduced compounds (reduced 

 riboflavin, reduced dyes) will function in lieu of DPNH, but FMN is 



