MECHANISM OF PHOTOSYNTHESIS I39 



as final: the present measurements are, however, consistent 

 with a reduction of the coenzymes to only a very small 

 extent. 



The discovery that the chloroplast reaction may be 

 coupled with known respiratory systems through the co- 

 enzymes greatly extends the biochemical methods available 

 for the study of photosynthesis. In the original model 

 experiment of Vishniac and Ochoa (mentioned earlier in 

 Chapter 7) the complete malic enzyme system obtained from 

 pigeon liver together with pyruvic acid was added to an 

 illuminated preparation of spinach chloroplasts. The condi- 

 tions were maintained anaerobic by removal of the oxygen 

 by chromous chloride in a separate compartment in the 

 reaction vessel. The oxygen produced was then measured 

 in terms of the chromic chloride and found to correspond 

 with the amount of malate formed from pyruvate. Carbon 

 dioxide was supplied as bicarbonate with 5% CO2 in 

 the gas phase. In this experiment the essential reaction 

 brought about by the illuminated chloroplast preparation 

 must be: 



TPN++H2O— TPNH+H++JO2; AF=50,8oo cals.' 



Ochoa found that other systems dependent on coenzymes 

 could be reversed. For example, pyruvate could be reduced 

 to lactate, by the dehydrogenase depending on DPN, in 

 presence of the illuminated chloroplast preparation. It was 

 also found that the mitochondria from the mung bean would 

 carry out oxidative phosphorylation when in presence of 

 DPN and the illuminated chloroplast preparation. It is thus 

 established that coenzymes I and II can be reduced by 

 chloroplast preparations when a suitable system is also 

 present to accept the *H' originally resulting from the 

 photochemical reactions. This allows measurements to be 

 made of the extent to which a reversal of respiratory process 

 can occur with chloroplasts in vitro. When the experiments 

 with chloroplast preparations are carried out under anaerobic 

 conditions and all the oxygen is removed the results obtained 

 can be explained in terms of the measurement of the 

 reducing properties of the chloroplasts by other methods. 



