162 BIOCHEMISTRY OF FIREFLY LUMINESCENCE 



place in a stepwise manner and that each unique step involves energy 

 changes not greater than 10-15 kcal. The two important and obvious 

 exceptions involve either the absorption of radiant energy (photosyn- 

 thesis) or the emission of light (bioluminescence). In an effort to 

 determine the nature of the oxidative reaction which presumably 

 liberated 40-60 kcal the senior author in 1947 attempted to isolate 

 luciferin from firefly lanterns. The component which was finally 

 isolated as the barium salt was ultimately identified as adenosine 

 triphosphate (McElroy, 1947). Thus by the classical definition ATP 

 was firefly luciferin. Since ATP was known to be the immediate 

 energy source for mechanical activity as well as other processes, this 

 seemed to be a logical extension of the idea of energy transport and 

 utilization in a biological system. Three important questions, however, 

 remained unanswered: (1) Does all the energy for light emission come 

 from phosphate bond energy? (2) What is the importance of the 

 oxidative reaction? (3) What is the nature of the light emitting 

 molecule? Subsequent work on the fireflies indicated that two addi- 

 tional factors were required. One of these turned out to be magnesium 

 ion and the second was a fluorescent substance with an emission 

 spectrum similar to the luminescent spectrum ( see Fig. 1 ) . Since this 

 substance is destroyed during luminescence and because of its fluores- 

 cent properties, it was called luciferin, the phosphor of fireflies. All 

 subsequent work has indicated that the basic reaction in fireflies can 

 be described by the following reaction: 



Luciferin + luciferase + Mg + ATP + oxygen — > light + products 



Both luciferin and luciferase have been highly purified, and the pres- 

 ent but brief review will attempt to describe some of the properties of 

 the individual components as well as the entire system. 



Purification of Luciferase and Luciferin 



Luciferase 



The following procedure is based upon the original report of 

 McElroy and Coulombre (1952). Five grams of the dried lanterns of 

 Photinus pyralis are ground with sand and extracted three times with 

 a total volume of 100 ml HoO. The pH of the extract is adjusted to 8 



