114 LIGHT AND LIFE 



mum in the organic solvent is enhanced about fourfold and is shifted 

 from 465 m^ to about 440 m^. There is a corresponding enhance- 

 ment of the 340 m/A excitation band but the 260 ni/x excitation band 

 has nearly disappeared. The complex under these conditions, a 

 moderate lowering of the dielectric constant of the solvent, has 

 largely dissociated. These changes are precisely those that occur in 

 the DPNH excitation and emission spectra when it is complexed to 

 many but not all dehydrogenase proteins. The spectra of the com- 

 plexes however involve some complications which will be taken up 

 at a later point in the discussion. 



The behavior of structural analogues. The fluorescence excitation 

 and emission spectra of two structural analogues of DPNH are 

 shown in Fig. 5. Replacement of the carboxamide group of the 

 nicotinamide with acetyl yields the acetyl pyridine analogue, APNH. 

 The emission band is weakened and shifted toward longer wave- 

 lengths, but the 260 m^ excitation band is relatively stronger than 

 before. The amide group is therefore not specifically involved in 

 the formation of the inner complex. In the second analogue, desamino 

 DPNH, the amino group of the adenine is replaced by hydroxyl. 

 Note that the short wavelength excitation band has shifted to 250 

 n\fji, where hypoxanthine absorbs, and is rather weak, possibly because 

 the amino group of the adenine is involved in the formation of the 

 inner complex. Shifrin and Kaplan (21a) report that the 260 m^u, 

 excitation band of the a isomer of DPNH (Fig. 1) is absent. Al- 

 though models of the a isomer may be folded to bring the pyridine 

 and adenine rings in juxtaposition, the allowed conformations are 

 different from those of the (i isomer. Either an inner complex is not 

 formed or one is formed in which the orientations are not favorable 

 for the transfer of excitation energy. 



Conformation and the Stereochemistry of Oxidation 



AND Reduction 

 Isomerism at pyridine C-4. When DPN is reduced enzymatically 

 with a deuterium-labelled substrate, deuterium is transferred to the 

 coenzyme to form DPND. It is the deuterium and not the hydrogen 

 that is removed exclusively when the DPND is reoxidized in the re- 

 verse reaction catalyzed by the same enzyme. The enzyme thus dis- 

 tinguishes between the two hydrogen atoms at pyridine C-4, and this 

 is not surprising in terms of what we know in general about enzyme 

 specificity. However, it was observed by San Pietro and coworkers 

 (19) that the two hydrogen atoms at pyridine C-4 also behave dif- 



