110 



LIGHT AND LIFE 



Spectral evidence for the folded conformation. The absorption spec- 

 trum of DPNH is shown in Fig. 2. At 260 m^ there is a strong ab- 

 sorption band that arises chiefly from the adenine. At 340 m^i, is 

 the absorption band of the reduced nicotinamide ring. This band 



100 



80 



60 - 



40 



20 - 



1 1 1 • 1 1 1 ^ 



DPNH 

 -^- Excitation 

 -•- Absorption 



360 m/< 



Fig. 2. 1 he al)s()rpti()n and fluorescene excitation spectra of DPNH in water, 

 emission was measured at 46'i m^ as a function of wavelength of excitation. Cor- 

 rections were apphed at each excitation point to account for the spectral energy 

 distril)ution of the caliljrated light source, a high pressure xenon arc ojierated on 

 1).C. The arbitrary units of the excitation spectrum were normalized to coincide 

 with the absorption spectrum at SU) ni/^. Lack of perfect coincidence of the 

 spectra in the .'540 lUfj. band arc prol)ably not real but the result of limitations 

 in technique. 



disappears when the coenzyme is reoxidized. It is restored by reduc- 

 tion and also by the non-enzymatic addition of certain luicleophilic 

 reagents, such as cyanide (5) , which attack the reactive position at 

 pyridine C-4. In the latter case the absorption band is shifted to 

 the 325 mix region. The reduced pyridine ring is fluorescent with an 

 emission maxinuun in water at 465 m^x. The open circles in Fig. 2 



