248 LIGHT AND LIFE 



OH group, altering in effect the fluorescence or chemiluminescence 

 properties of the luciferyl-adenylate compound. 



One other factor has been shown to modify the emission spectrum 

 of the biohmiinescence reaction in vitro. We have indicated pre- 

 viously that inorganic phosphate is an inhibitor of light emission; 

 the results shown in Fig. 27 indicate that phosphate produces a con- 

 dition even at neutral or alkaline pH which is essentially similar to 

 a reduction of the pH of the solution, that is, the appearance of a 

 red emission band. The shift from the 562 m/i peak to the 616 

 lUfx peak in the presence of phosphate or under acid pH, and the 

 sharp decrease in quantum yield in acid pH, are additional evidence 

 that the enzyme is important in influencing the excited state (23) . 



Discussion of Mechanism 



It would be appropriate at this time to summarize w'hat is known 

 of the mechanism of the bioluminescence and to point out some of 

 the questions still unresolved. We know that during the enzymatic 

 oxidation of luciferin, oxyluciferin is formed. On the basis of quanti- 

 tative analysis on ashing, the chemical formula of oxyluciferin differs 

 from luciferin by only two hydrogen atoms. That oxyluciferin differs 

 only slightly from luciferin is also evidenced by the striking similarity 

 of their absorption, fluorescence, and fluorescence yield curves and 

 their respective variations with pH. This is then different than in 

 the case of the chemiluminescence of luminol, where the release of 

 Na from the phthalhydrazide molecule is sufficiently exergonic to 

 leave the product phthalic acid molecule in an excited state from 

 which blue light can be emitted. We have yet to resolve the steps 

 by which the LHa-AMP active intermediate, bound on the enzyme 

 molecule, reacts with dissolved molecular oxygen to produce L-AMP 

 and a light quantum. From the kinetic data on the fluorescence of 

 E-L-AMP + PP ±^ E + L + ATP and from the flash height data 

 as a function of enzyme concentration, we can safely say that there 

 is only one site for light emission on the enzyme surface. The quantum 

 yield of unity also precludes the utilization of two luciferin radicals 

 on an enzyme molecide. 



The fact that the acid fluorescence of synthetically formed LHg- 

 AMP is close to the bioluminescence emission would imply an energy 

 transfer from an excited jjroduct to the LHo-AMP molecule. This 

 picture is further strengthened by the fact that the acid fluorescence 

 of L-AMP shows no yellow-green peak but does have a red fluores- 

 cence. However, (a) LH^-AMP can also be shown at more strongly 



