II . /). Mcelroy and h. h. seliger 249 



acid pH to exhiljit a red fluorescence; (b) the bioluniinescence, and 

 most cheniiluniinescences, occur at neutral or basic pH values, so that 

 acid ()H may have little relation to the actual environment during 

 chcmilumincscencc; and (c) energy transfer should be concentration- 

 ilependent and the residts of the Hash height versus enzyme concen- 

 tration would therefore rule out this type of transfer. 



These data eliminate the possibility that two LH^-AMP molecules 

 would interact with oxygen in a reaction analogous to the one pro- 

 posed by Linschitz, in which one reduced molecide (in our case 

 LH2-AMP) is regenerated. It was conceivable that the exergonic 

 step was creating the excited state of LHo-AMP, whose emission 

 would correspond to the observed fluorescence of this compound. 

 Energy transfer to the oxidized product (L-AMP) would under these 

 conditions give rise to the red emission. 



Thus we must account for a minimum of approximately 60 kcal/ 

 mole in the oxidation of a single LHo-AMP molecule, an amount which 

 is more than is available from the production of a peroxide, obtained 

 from a simple balancing of the equation LHo-AMP -\- O^ -^ LAMP 

 -|- HoOo. We have not yet been able to isolate unambiguously the 

 excited state of the product molecule from which the light quantum 

 is emitted, although we infer that its fluorescence yield must be 

 practically 100 per cent. It is possible that the oxygen activation 

 leading to the formation of hydrogen peroxide could furnish suffi- 

 cient energy. The formation of a double bond in the ring structure 

 of luciferin could contribute as much as 30 kcal of resonance energy. 

 The difference in binding energy between LAMP and LH2-AMP 

 may contribute, as well as energy from the formation of hydrogen 

 peroxide. Further experimental evidence, particularly on the possible 

 formation of H^Os, is necessary before a detailed description of 

 mechanism can be given. It is possible that knowledge of the struc- 

 ture of the luciferin molecule will furnish a better idea as to the 

 chemistry involved, and this is being pursued at the present time. 



Bacterial Luminescence 

 Early attempts to extract the hmiinescent system from bacteria were 

 almost uniformly unsuccessful, although Gerretsen in 1920 succeeded 

 in obtaining a weak light-emitting cell-free extract. This observation 

 was repeated by Shoup and Strehler in 1951. In 1953 Strehler demon- 

 strated that DPN and DPNH would substitute for the hot water ex- 

 tract to restore light emission in a cold water bacterial extract (25, 

 26) . Later experiments by Strehler indicated that DPNH was the 



