CHLOROPHYLL-SENSITIZED OXIDATION-REDUCTIONS 1517 



illumination with the one obtained after similar treatment without DPN, 

 showed a difference in the region 300-360 mix, which indicated the forma- 

 tion of a compound with an absorption band at 345 mju (fig. 35.6)— the 

 well-known position of the absorption band of DPNH2. 



The band at 525 mn, which appeared in illuminated chlorophyll-ascorbic 

 acid mixtures in the absence of DPN and which was ascribed by Kras- 

 novsky to reduced chlorophyll (HChl, or HoChl?), "sometimes" did not 

 appear when DPN was present; this result was attributed to the high rate 

 of the second reaction in the sequence (35. 27a, b) : 



(35.27a) Chi + AH, "—^ HjChl + A (or HChl + AH) 



(35.27b) HsChl + DPN (or 2 HChl + DPN) > DPNH2 + Chi 



(AH2 = ascorbic acid). 



To prove the occurrence of reaction (35.27b) directly, a mixture of 

 chlorophyll and ascorbic acid was illuminated alone in an evacuated Thun- 

 berg tube, and DPN (or other oxidants) was then added from a side tube. 

 The disappearance of the absorption band at 675 m^ in light, and its re- 

 appearance in dark after the addition of oxidants, is illustrated by figure 

 35.7. This figure shows that recoloration was far from complete — the 

 optical density, which had dropped in light from 0.7 to 0.1, was restored 

 by the oxidants only to a value of 0.45. The figure also shows that saf- 

 ranin and riboflavin caused a much faster return of the color than did DPN, 

 or air. 



These results are suggestive, and Krasnovsky's conclusion that reduc- 

 tion of DPN by reduced chlorophyll is the link by which the photochemical 

 process is tied, in photosynthesis, to the sequence of enzymatic reactions 

 leading to fixation and reduction of CO2, is plausible; it fits well into the 

 picture of the reaction mechanism of photosynthesis derived from C(14) 

 experiments (chapter 36). However, just because of this suggestiveness 

 and the crucial importance of the conclusions, much more rigorous experi- 

 ments will be needed than those described by Krasnovsky and Brin, before 

 the capacity of chlorophyll to sensitize the reduction of the oxidized form 

 of coenzyme II, by hydrogen donors such as ascorbic acid (thus overcoming 

 an opposing normal potential difference of about 0.24 volt, at pH 7), can be 

 considered as proved. Spectroscopic proof of the formation of DPNH2 

 (based on a small difference between two high optical densities), as well 

 as the proof of the reversible reduction of chlorophyll, may prove to be 

 correct, but are as yet not quite convincing. The fact that the absorption 

 at 675 m/i is restored to only one half its original value indicates a con- 

 siderable irreversible change. Further development of these experiments, 

 with better spectroscopic technique, supplemented by chemical separation 

 and enzymatic tests, appears desirable. 



