Il6 PHOTOSYNTHESIS 



METHODS OF MEASURING THE CHLOROPLAST REACTION 



(a) Determination of the oxygen production. The most 

 reliable data are obtained when the production of oxygen is 

 measured in relation to a known added amount of hydro- 

 gen acceptor. With hydrogen acceptors of more negative 

 reducing potential the measurement of oxygen may be made 

 spectroscopically using the blood pigment haemoglobin. The 

 whole reaction is observed in the liquid phase and both the 

 total amount of Og and the concentration of O2 in equili- 

 brium with the haemoglobin can be determined at any 

 instant during the progress of the reaction. With reagents of 

 more positive potential the direct manometric determination 

 of the O2 produced has been found satisfactory. The polaro- 

 graphic and electrode potential methods which have been 

 successfully employed for the determination of Og in photo- 

 synthesis are less easy to apply to cell-free preparations. 

 The very sensitive method for detecting O2, depending on 

 the quenching of the luminescence of the dye trypoflavine 

 (an acridine derivative), has been used to determine O2 

 at pressures of the order of io~^ mm. Hg. This last 

 method, developed by Franck, Pringsheim, and Lad (1945) 

 replaced the earlier qualitative methods of great sensi- 

 tivity, depending on bacterial luminescence or on bacterial 

 motility, by a quantitative one only perhaps one order less 

 sensitive. 



(h) Determination of the amount of H acceptor reduced. 

 When a dye is used as a hydrogen acceptor the progress of 

 the reaction can be followed by a photometric method. The 

 method using dichlorphenolindophenol (a blue dye suffici- 

 ently soluble in water) had been developed by French and 

 co-workers and shown to give reliable results. The danger 

 of this method is that the dye is liable to reduction by pro- 

 cesses involving the direct oxidation of the cellular consti- 

 tuents. It is not always certain that these effects can be 

 accounted for by a dark control. Chemical analysis can be 

 also used with any hydrogen acceptor for the chloroplast re- 

 action. In some cases the degree of reduction can be deter- 

 mined by an electrode measurement. 



