126 



LIGHT AND LIFE 



band appears (Figs. 7, 8) , and concomitantly the protein emission is 

 partially quenched. Three examples of these effects are illustrated in 

 Figs. 11 to 13. The amount of quenching that occurs when all bind- 

 ing sites are occupied on LDH is 67 per cent with DPNH and ap- 

 proximately 80 per cent with APNH. This difference is correlated 



320 360 400 440 

 Emission (m/x) 



Fig. 12. Ihe emission of LDH (curve A) and of the I,DH-(DPNH):, complex 

 (curve B) excited at 290 iii/x. Although the quenching of LDH emission by bound 

 coenzyme is appreciable, the high tryjnophan content and high fluorescence 

 quantum yield of LDH obscures the coenzyme emission Ijand, which is clearly re- 

 vealed only in the diflevcnce spectrum, curve C. 



with the oscillator strengths of the coenzyme and its analogue, ^\•hich 

 exhibit molar extinction coefficients of 6200 and 8100 as their respec- 

 tive long wavelength absorption maxima. The polarization of APNH 

 emission excited by transfer from LDH is also about 4 per cent higher, 

 indicating a geometrical factor that might be in its favor. 



It is useful to view the energy transfer in terms of normalized ex- 

 citation spectra. In Fig. 14 the coenzyme excitation spectrum of the 

 LDH-DPNH complex has been normalized to coincide with the 

 absorption si)ectrum of the complex at 355 n\fi. This is analogous 

 to the i>ro(cdure employed with free DPNH (Fig. 2) . The 280 m^ 

 excitation band is superimposed on two absorption curves, the ab- 

 sorption cinvc of the protein after subtraction of the coenzyme con- 

 tribution, and the absorption curve of the constituent tryptophan 



