ISOLATED CHLOROPLASTS II5 



Alternatively benzoquinone may be used as a hydrogen 

 acceptor, when it is quantitatively reduced to hydroquinone 

 with the production of a corresponding amount of oxygen. 

 This reaction was first demonstrated by Warburg and 

 Liittgens (1946) and with this substance they demonstrated 

 that 100 moles of quinone per mole of chlorophyll present, or 

 a weight of quinone equal to the total w^eight of the granules, 

 could be reduced, thus finally removing the possibility that 

 the 'chloroplast reaction' might be due to the liberation of 

 oxygen from a limited quantity of a peroxide originally 

 present in the chloroplast preparation. 



2C6H4O2 + 2H2O - 2C6H4(OH)2 + 02 



Ai^= + i2,300 cals. per hydrogen atom transferred at pH 7 



Dyestuffs such as phenol indophenol and 2:6-dichlorophenol 

 indophenol have also been used as hydrogen acceptors. 

 Oxygen itself can also serve as a hydrogen acceptor in the 

 chloroplast reaction as shown by Mehler (1952). 



In all these reactions a molecule of water is involved, 

 suggesting that the oxygen might originate from the water 

 rather than from the hydrogen-accepting reagent. This latter 

 point was established by Holt and French (1948) who, using 

 water enriched with i^O, showed the isotopic composition 

 of the oxygen produced to be the same as that of the water 

 present. 



Attempts have been made to determine the smallest unit 

 of the chloroplast which is capable of reaction. Preparation 

 of small particles of fairly uniform size may be made by 

 grinding the chloroplasts with powdered glass; suspensions 

 of very small chloroplast fragments in which the grana are 

 largely disintegrated have been obtained by the u^e of either 

 ultrasonic vibrations or by forcing the chloroplast suspen- 

 sion under high pressure through a small opening. Even 

 with whole chloroplast suspensions good activity is only 

 obtained if the preparation is made at low temperature, the 

 activity decreasing rapidly at room temperature. With each 

 successive fragmentation the activity of the preparation 

 decreases but some partial restoration is obtained with the 

 smallest particles after coagulation by the addition of salts. 



