Pur/'fication and Properties 

 of Bacterial Luciferase 



J. W. Hastings and W. D. McElroy 



Department of Biological Sciences, Northwestern University, Evanston, 



Illinois, and McCollum-Pratt Institute and Department of Biology, 



The Johns Hopkins University, Baltimore, Maryland 



The demonstration of luminescence in cell-free bacterial extracts, 

 which had long been sought by workers in the field, was finally 

 achieved by Strehler early in 1953. After the demonstration that di- 

 phosphopyridine nucleotide (DPN) markedly stimulated lumines- 

 cence, work progressed rapidly. Within the year this bioluminescent 

 reaction was the first in which all known stimulatory factors had been 

 chemically identified. Although many unanswered questions concern- 

 ing the mechanism of the reaction remain, it seems worth while to 

 review some of our studies on the system, particularly where our 

 results differ from or amplify those reported by Strehler. 



Preliminary experiments with relatively crude extracts had indi- 

 cated that ( 1 ) DPN was functional in the luminescent reaction only 

 when reduced and might be replaced by other reducing compounds 

 (TPNH), and (2) that a heat stabile fraction of the crude extract 

 contained a stimulatory factor not identical with DPNH. The puri- 

 fication of the enzvme and a studv of other factors was thus un- 

 dertaken. Conventional enzymological methods resulted in a forty- 

 to sixty-fold purification of the enzyme (see Table I).* Bacteria 



" Repeated (NH4)oS04 fractionations of the active fractions have resulted 

 in even greater purity. Green and McElroy (unpublished) find that their 

 best fractions are homogeneous by ultracentrifugation (mol. wt. ca. 85,000) 

 and contain three components with slightly different electrophoretic mo- 

 bilities at pH 7.6. The active fraction constitutes over 70% of the total 

 protein. 



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