116 



LIGHT AND LIFE 



nicotinamide riboside portion of the pyridine nucleotides in the oxi- 

 dized and reduced states are shown in Fig. 6. In DPN the pyridinium 

 ring is aromatic and planar and the ribosidic bond lies in the plane 

 of the ring. The ribose plane therefore lies somewhere between a 

 right angle and a tetrahedral angle to the plane of the pyridine, de- 

 pending on the conformation of the ribose. This is in accord with 

 X-ray crystal structure analyses of simple ribosides (11, 4). Models 

 of the reduced riboside are constructed in two ways: (a) with the 

 trivalent ring nitrogen pyrimidal as in aliphatic amines; and (b) 

 with the lone pair of the ring nitrogen involved in the conjuga- 



50 - 



40 



30 



20 



10 



-1 1 1 



EXCITATION 

 ° — o GPD DPNH 

 • — • GPD 

 DIFFERENCE 



260 300 340 380 



Fig. 7. Fluorescence excitation spcc.iiim, coi reded, ol DPNH (ojkmi circles) in 

 the C.1'1) (Dl'XH):, complex. I he i)iifrcr was O.I M potassium phosphate, pH 7.3, 

 selected to minimize the quenching of DPNH emission by protein. Under these 

 conditions the chief sign of complex formation is polarization and the excitalicm 

 spectrum itself. DI'NH emission was measured at }8.') m^^ to minimize the cor- 

 rection for the tailing protein emission when excitation was in the 280 mn band. 

 Superimposed is the excitation spectrum of the protein fluorescence normalized at 

 liHd niM and measured at :'>'>() uy/x. I he (ur\e on the lower left is the excitation 

 dilference spectrum. 



