DIFFERENT OXIDANTS 1579 



dized by molecular oxygen (perhaps via the cytochrome system) and stor- 

 ing the oxidation energy in phosphate bonds (a coupling demonstrated by 

 Lehninger) . 



In the above-described experiments of Vishniac and Ochoa the proof 

 of the assumed photochemical reduction of pyridine nucleotides by water 

 consisted in the demonstration of reduction and carboxylation of pyruvate 

 (or other metabolic acids) in light ; a complete proof calls also for the dem- 

 onstration of oxygen liberation in stoichiometric proportion. Furthermore, 

 the relevance of these observations to the mechanism of photosynthesis 

 depends on the yield of the reaction. As stated in chapter 4 in the discus- 

 sion of earlier claims of photosynthesis in vitro, "everything is possible in 

 photochemistry," provided one is satisfied with very small yields. Conse- 

 quently, no reaction in vitro can be considered as a significant step in re- 

 constructing photosynthesis outside the living cell unless its yield approaches 

 that of natural photosynthesis. 



Vishniac and Ochoa (1952) were able to demonstrate, using chromous 

 chloride as reagent, that oxygen was in fact formed in the stoichiometri- 

 cally expected amount (specifically, 0.5 mole O2 were liberated per mole of 

 lactic acid formed, in accordance with reactions (35.34Aa,b). The yield 

 was, however, very low — corresponding to about one molecule oxygen per 

 molecule chlorophyll every 3 hours or about 0.1% of the rate of photosyn- 

 thesis in saturating light. Other above-enumerated enzymatic systems 

 gave oxygen yields (calculated from the rate of formation of the reduction 

 products) of one molecule oxygen per molecule chlorophyll between every 

 1.2 and every 60 hours. The light used was relatively weak — about 4 

 klux, but even with allowance for this fact, these rates are of different order 

 of magnitude than those of photosynthesis. 



Tolmach (1951 ^•^) came to a study similar to that of Vishniac and Ochoa 

 from a different side. The evolution of oxygen by illuminated chloroplast 

 suspensions in the absence of specially added oxidants (chapter 4, page 62) 

 points to the presence in plants of a "natural Hill oxidant" (or oxidants). 

 These may, or may not, serve as intermediates in Hill reaction, or photo- 

 synthesis, or both; in any case, it would be important to identify them. 

 Hill had shown that the photochemical oxygen liberation by chloroplasts, 

 without added oxidants, is very brief in washed chloroplasts, but lasts much 

 longer if the chloroplasts are left suspended in the cell sap (or if a chloro- 

 plast-free leaf extract is added to washed chloroplasts). Figure 35.19A 

 shows the course of oxygen liberation which Tolmach was able to demon- 

 strate by suspending a drop of a suspension of spinach chloroplasts in an 

 illumination chamber traversed by a stream of pure nitrogen, and analyzing 

 the gas leaving the chamber for oxygen by the highly sensitive phosphores- 

 cence-quenching method of Pringsheim and Franck. 



