ISOLATED CHLOROPLASTS II7 



(c) The determination of the change in H^ ion concentration. 

 In the reaction: — 



4Fe(CN)6+2H20 - 4Fe(CN)6+4H++02 



all three of the methods, a, b, and c have been applied and 

 have been found to yield concordant results. With such a 

 reaction as that with ferricyanide the consistency of the 

 different methods indicates clearly the part played by water 

 in the in vitro photochemical production of oxygen. 



COMPARISON OF THE CHLOROPLAST REACTION 

 AND PHOTOSYNTHESIS 



If the chloroplast reaction is to be of use in the bio- 

 chemical analysis of reactions taking part in photosynthesis 

 the conditions of oxygen production in vitro must be con- 

 sidered in relation to the production of oxygen in the living 

 cells. When the same hydrogen acceptors, which have been 

 used with chloroplast preparations, are added to living cells, 

 in the absence of carbon dioxide, oxygen is produced upon 

 illumination. This evidence alone does not establish that 

 the hydrogen acceptor is acting as a substitute for carbon 

 dioxide because in the living cell there is the possibility of 

 a dark reaction between the added substance and carbo- 

 hydrate resulting in the production of carbon dioxide. In 

 the light normal photosynthesis will convert such carbon 

 dioxide into oxygen which will be evolved. Such a dark pro- 

 cess apparently occurs when nitrate, benzaldehyde, or ferri- 

 cyanide is added to Chlorella cells, although in the case of 

 nitrate the rate of production of oxygen is greater in the 

 light than that of the carbon dioxide production in the dark. 

 Thus in this case either the oxidation reaction which occurs 

 in the dark is accelerated in the light (owing for example to 

 the increased concentration of some carbon intermediate 

 which reacts with the nitrate) or there is in addition some 

 direct photochemical reduction of nitrate accompanied by 

 oxygen production. When o-benzoquinone is added to 

 Chlorella cells oxygen is liberated in the light and in this 

 case no difficulty in interpretation arises, since both respira- 

 tion and normal photosynthesis are inhibited by the added 



