Discussion 



Bacterial Luciferin 



Dr. Harvey: The striking luminescence which appears when reduced 

 flavin mononucleotide (reduced riboflavin phosphate, FMN-H2) is 

 added to cell-free luminous bacterial extracts containing a long-chain 

 aliphatic aldehyde is easily visible to the dark adapted eye and re- 

 minds one of the luciferin-luciferase reaction of other organisms. The 

 experiment raises a number of questions: (1) whether the FMN-Ho 

 is reducing some other compound in the bacterial extract which is 

 responsible for the light; (2) whether other reduced flavin compounds 

 will luminesce when mixed with bacterial extracts; (3) whether 

 FMN-Ho may be regarded as bacterial luciferin. The first question 

 has been answered in the negative by showing that a reduced com- 

 pound with a lower redox potential than FMN-Ho (£'o at pH 7 = 



— 0.185), when added to the bacterial extract, does not cause light 

 emission. Strehler et al. (1954) found that reduced anthraquinone-2-6- 

 disodium sulfonate (E'o at pH 7 — —0.192), a substance harmless for 

 luminous bacteria (Harvey, 1929), evokes no luminescence when 

 mixed with the bacterial extract. However, if oxidized riboflavin phos- 

 phate (FMN) has been previously added, then the reduced anthra- 

 quinone reduces the FMN and a bright light appears. The experiments 

 suggest that reduced FMN may be designated bacterial luciferin. 



In the same paper it was reported that riboflavin (Merck) emitted 

 light when mixed with cell-free bacterial extracts of Achromobacter 

 fischeri. Recently I have tested a number of other flavin compounds 

 with a crude cell-free extract of a luminous bacterium having a low 

 (12° C) temperature optimum. The cell-free extract was prepared by 

 Dr. B. L. Strehler from a strain of luminous bacteria obtained from 

 Dr. C. B. Van Niel and known as "Gest." The active acetone bacterial 

 powder had been extracted with water and centrifuged at 70,000 X g 

 for one hour. Although the temperature rose somewhat during centri- 

 fuging, the water extract always gave a bright light when mixed with 

 reduced riboflavin phosphate, indicating that the enzyme essential for 

 light production had not been denatured. The extract was stored at 



— 10" C before use. The dilute flavin solutions in distilled water were 

 reduced in small centrifuge tubes in a stream of hydrogen after adding 

 platinized asbestos. The tubes were then stoppered, centrifuged at 



241 



