506 



Anthony San Pietro, Leo P. Vernon and Dorothy Limbach 



linear relationship at low transhydrogenase concentrations. At higher trans- 

 hydrogenase concentrations, there is some deviation from linearity. The 

 same results are obtained whether or not the purified PPNR is included in the 

 reaction mixture. 



12 24 36 48 



Mg PROTEIN 

 (TRANSHYDROGENASE) 



450 



400 350 



WAVELENGTH m^ 



300 



Fig. 3. (Left) Effect of transhydrogenase upon NADP Reduction 



with Hematoporphyrin and Ascorbate. 



Fig. 4. (Right) Light Minus Dark Difference Spectra. 



It can be seen that the curves presented in Fig. 3 do not go through the 

 origin. This is due to the fact that there is a photobleaching of the hemato- 

 porphyrin when illuminated alone or in the presence of purified PPNR. Light 

 minus dark difference absorption spectra for various reaction mixtures are 

 presented in Fig. 4. When transhydrogenase and hematoporphyrin are both 

 present, an increase in absorbance at 340 nnp is observed (Curve 1). For 

 hematoporphyrin alone (Curve 2) or hematoporphyrin plus purified PPNR 

 (Curve 3), there is a decrease in absorbance at 340 mp upon illumination. 



In contrast to most photos ynthetic reactions which exhibit light saturation 

 at about 1000 foot- candles, this reaction did not exhibit light saturation over 

 the range of light intensities studied. 



DISCUSSION 



The photochemical reduction of pyridine nucleotides by chloroplasts is 

 thought to require both PPNR and a specific flavoprotein ^°' ^^' ^^^ It is 

 generally assumed that the PPNR is reduced by the light-trapping system 

 (chlorophyll a) and that the flavoprotein catalyzes the transfer of electrons 

 from reduced PPNR to NADP. 



In the experiments reported herein, the hematoporphyrin serves as the 



