134 



LIGHT AND LIFE 



nucleotide. These results suggest that the dinucleotide does not emit 

 at all and that the emission from FAD comes from about 10 per 

 cent of the open form which is in thermal equilibrium with the 

 inner complex. The small or zero temperature coefficient of FAD 

 fluorescence is thus probably a coincidence. Thermal quenching of 

 the emission of the open conformation is compensated by the in- 

 creased concentration of this form as a function of temperature. 



Spectral changes during oxidation and reduction. Unlike the pyri- 



Absorption 



Fluorescence 

 excitation 



i-ig. 1 



ciitles), 

 370 ma 



300 340 380 420 460 500 

 Wavelength, nyz 



7. 1 he conettcd fluorescence excitation spectrum of FAD in water (open 

 normalized to coincide with the absorption spectrum (solid line), at about 



dine nucleotides, it is the oxidized forms of FMN and FAD which 

 have strong absorption bands in the visible and near ultraviolet, 

 and it is tiie oxidi/.ed and not the reduced forms that fluoresce. The 

 spectral changes in FMN at various stages of reduction are shown in 

 Fig. 18. Using a raj)id scanning spectrophotometer, Beinert (2) ob- 

 served two additional, very weak bands at longer wavelengths during 

 the reduction of FMN by hydrc:)sulfite (Fig. 19) . The band at 565 

 niyu, rises to a maxinuim at 50 per cent reduction and then begins to 

 disapjjcar and is absent when reduction is complete. The band at 

 880 lUjx cxhil)its a similar behavior but is strongly dependent upon 

 concentration and temjjerature. Beinert therefore assigned the 560 

 myu, band to a scmiquinone or free radical intermediate and the 880 

 ni/x band to the semiquinone dimer. Tlie occurrence of a semiquinone 



