854 LIGHT AND LIFE 



flash is similar in shape to the normal firefly's flash but lasts much 

 longer, and it has not been possible, to date, to duplicate the natural 

 flash on the laboratory by altering any of the known variables. This 

 failure is one reason for supposing that in the firefly itself the flash 

 is controlled by pyrophosphate, acting by reversing the product inhibi- 

 tion of the enzyme. 



The emission spectrum shows a smooth peak at 5(32 m^i, the width 

 of the band extending from 500 to 630 m/x. The energy requirement 

 must therefore be at least 57 kcal per mole. The absorption spectra 

 of luciferin and oxyluciferin are very similar, the major peak of the 

 latter being shifted some 20 m^u, toward the red (in acid) in com- 

 parison with the former. This evidence indicates that the difference 

 between the two forms is simply the addition of a double bond in 

 conjunction with the loss of two protons from the dihydro form. 

 The fluorescence spectra are also very similar, with maxima at 535 ni/n 

 and 544 m/j. respectively. The fluorescence spectrum of oxyluciferin 

 exhibits a slight shoulder at 460 m/x at acid pH. The shoulder remains 

 rather constant as the pH changes until it disappears in the increas- 

 ingly intensified 544 ni/x band. The fact that the absorption maximum 

 of oxyluciferin, like that of luciferin, shifts about 50 lUfx toward longer 

 wavelength at pH 8 and above, while the fluorescence maximum re- 

 mains constant, implies that different parts of the molecule are in- 

 volved in the absorption and the emission of light. The efficiency of 

 this intramolecular transfer of energy is shown by the sharp increase 

 in the fluorescence yield of both luciferin and oxyluciferin at pH 

 8.4, the point at which the hydroxyl group of the molecule under- 

 goes ionization. The similarity of these fluorescence yield curves for 

 the reduced and oxidized forms of luciferin indicates that both have 

 the same ionizable groups, and the corresponding decreases in fluores- 

 cence when L-AMP and L are bound on the enzyme indicate that it 

 is the hydroxyl group which is prevented from ionizing when oxyluci- 

 ferin is bound on the enzyme surface. At no pH or temperature, 

 over the range from — 196°(^ to normal temperatures, is there any 

 fluorescence emission agreeing with the liioluminescent emission spec- 

 trum. However, the fluorescence spectra of LH^-AMP and LAMP 

 are radically different from those of LH2 and L, respectively, and 

 LHo-AMP does have a 570 mfx peak in acid that corresponds to the 

 bioluminescence spectrum. The L-AMP fluorescence does not so 

 correspond. 



The quantum yield of the bioluminescent reaction is unity; that 

 is, one light quantum is emitted for every luciferin molecule oxidized. 



