BENT LEY GLASS 841 



() tryptophan residues, or S0% of the energy Ironi the 7 resickies 

 within its domain. From Forster's equation, the distance Ro at 

 which there is equal jjrobabihty ol a transfer between donor and 

 acceptor and ol emission ol a quantum of light is calculated to be 

 25 A for tryptophan-DPNH. If the transferred energy comes from 

 seven different sources, it is to be expected that polarization of the 

 excited fluorescence woidd be low, as was in tact noted above. Velick 

 however utters a caution against regarding protein quenching as a 

 quantitative indicator of coenzyme binding in all cases. 



Turning to flavin mononucleotide (FAIN) and flavin adenine 

 dinucleotide (FAD) , it is once again apparent that changes in the 

 absorption spectra afford useful clues to the presence of excited states 

 and enzyme-coenzyme interactions. To start with, the quantum yield 

 of the fluorescence emission in the dinucleotide is only one-tenth as 

 great as that of the mononucleotide. This fact coupled with a spec- 

 tral shift to a longer wavelength in the 460 m^ l^and, a small de- 

 crease in absorption, and a pronounced shoulder in the 460-490 m^u. 

 region, lead to the suggestion that FAD, like DPNH, occurs as a 

 folded inner complex, with perhaps a face-to-face orientation of the 

 adenine and isoalloxazine structures and an open-chain ribotol in- 

 stead of a ring ribose on the flavin. Beinert points out that the 460- 

 490 m/x shoulder, which appears in a number of flavo-proteins, either 

 when jDiue or when substrate is added, may indicate the presence of 

 a jDrotein-coenzyme complex. A good example: D-amino acid oxi- 

 dase-FAD-benzoate (Yagi) . The absorption peak in the near ultra- 

 violet (300-400 ni/x.) is shifted toward 370 m^ by hydrogen-bonding 

 solvents, toward 330 m^u, by those that do not form hydrogen bonds. 

 An absorption band at 315 m^ in the complex between yellow butyrul 

 dehydrogenase and crotonyl-CoA can be positively assigned to the 

 interaction of the isoalloxazine ring system with a nucleotide (Bein- 

 ert) , and presumably the similar band of microsomal cytochrome 

 reductase (see below) is produced by the same type of interaction. 



The folded FAD complex behaves like DPNH in respect to the 

 independence of fluorescence from temperature and the enhancement 

 of fluorescence in methyl carbitol as compared with water. The 

 fluorescence excitation spectrum of FAD agrees closely with the ab- 

 sorption spectrum, except that at 260 m^ the quantum yield is re- 

 duced. In this coenzyme most of the absorption at 260 m/x, is due to 

 the isoalloxazine moiety rather than to the adenine portion of the 



