Sm LIGHT AND LIFE 



excited by light at 340 ni/x, free a-DPNH yields maximum emission 

 at 465 m/x. This 340 m^a band is the absorption band of the reduced 

 nicotinamide ring, and it disappears when the DPNH is oxidized. 

 When a-DPNH is excited by Hght at 260 mfx, where the absorption 

 is 95 per cent on the part of the adenine moiety of the molecule, there 

 is very little fluorescence (Shifrin and Kaplan) , although under the 

 same conditions yy-DPNH shows stronge fiuorescence excitation 

 (Weber) . These observations can only mean that in the active 

 isomer the absorbed energy is transferred from the adenine to the 

 nicotinamide portions of the molecule, by means of a coupled oscil- 

 lator mechanism, and that in the a-isomer the transfer is impossible. 

 In the case of the energy transfer, since the lifetime of the adenine 

 excited state is very short, the pyridine ring must be in close juxta- 

 position to the adenine ring; and it may therefore be supposed that 

 the a-isomer is inactive because it is incapable of folding so as to 

 bring the adenine and pyridine rings into sufficient proximity. 



Following the demonstration in 1954 (Pullman, San Pietro, and 

 Colowick) that it is position 4 on the pyridinium ring (para to the 

 N atom of the ring) that is reduced by addition of H, it was shown 

 by a deuterium-labeling experiment that the two H atoms on 

 C-4 behave differently in oxidations and reductions. Always the 

 same H atom is removed in oxidations of the nicotinamide ring that 

 has been added when reduction occurs, and this is true of non-enzy- 

 matic oxidations and reductions as much as of enzymatic ones. The 

 explanation for the different reactivity of the two H atoms on C-4 

 probably lies in some difference in their environment depending on 

 the properties of the folded inner complex of the dinucleotide. 



If it is true that free DPNH in solution has a folded conformation, 

 this should further be evident from the effects of temperature, sol- 

 vent, and structural modifications on the existence of the folded 

 complex. Velick shows that increasing temperature does in fact 

 reduce the relative quantum yield of DPNH fluorescence at 340 m^u., 

 and that the efficiency of the excitation at wavelength 260 m^ de- 

 clines even more precipitously. The effects of changing the solvent 

 are even more striking than those of temperature. In organic solvent 

 (methyl carbitol) instead of water the emission maximum is en- 

 hanced about fourfold. The 340 m^x excitation band is correspond- 

 ingly enhanced, but the 260 m^ excitation band completely disap- 

 jiears. Thus a moderate change in the dielectric constant of the 



