118 



LIGHT AND LIFE 



200 



Emission rryx 

 -o- 350 

 —480 

 ^\ -X- polarization 



Pu 

 0.3 



02 



240 280 320 360 

 Excitation, m^ 



Fig. 8. The fluorcscenrc excitation spectrum of DPNH in the LDH-(DP\H):, 

 complex in tris acetate, 0.1 M, j>H 7..S. This buffer augments the enhancement of 

 Dl'NH emission by the protein. On the scale of the spectrum the contributicm 

 of protein emission excited at 280 m^ and measured at 480 m^a is negligible. Ex- 

 citation of protein emission, measured at ;i')0 m^u. is shown by the closed circles. 

 Ihe crosses show the changes in polarization of the emission of bound DPNH as 

 a function of the wavelength of excitation with unjiolarized incident light. If the 

 260 m^ excitation band of the DPNH had been retained in the complex its con- 

 tribution would ha\e been given by the dotted curve. Note that the polarization 

 is not constant throughout the 340 m^ excitation band. This is the result of 

 perturbation by the virbrational structure. The low polarization of DPNH 

 fluorescence excited in the 280 m/x absorption band of the protein is discussed 

 later. 



ratio of the heights of the 260 and 340 ni/t excitation peaks remains 

 constant as a function of temjicrature. This is to be expected, since 

 the complex is (atalytically active and the geometry of the bound 

 (ocn/yme should therefore be rigorously fixed. Since the standard 

 free energy change in complex formation is approximately 11,000 

 calories per mole and since the dissociation constant shows only a 

 very small temperature dependence, a change in nucleotide conforma- 



