LEAF POWDERS AND ISOLATED CHLOROPLASTS 63 



for not more than an hour. All this seems to point to a decomposition 

 of a limited quantity of a peroxide, either left in the leaves as a residue 

 from normal metabolism, or accumulated by post mortem processes. 

 Yamafuji and coworkers have observed that dried vegetable and animal 

 tissues form small quantities of peroxide {cf. page 78). This peroxide, 

 slowly accumulated in darkness or in diffuse light, could decompose 

 suddenly upon exposure to strong light (particularly if chlorophyll is 

 present as a sensitizer), thus producing the "burst" of oxygen observed 

 by Molisch and Inman. 



If this explanation is correct, the oxygen evolution by dried leaves is 

 not directly related to photosynthesis (except for the fact that it, too, 

 may be sensitized by chlorophyll). However, the experiments of Hill 

 (1937, 1939, 1940) favor another hypothesis — that the oxygen evolution 

 by leaf powders bears a significant relation to the production of oxygen 

 in photosynthesis. 



In Hill's first experiments (1937, 1939), a chloroplast suspension was 

 obtained from leaves of Stellaria media, Lamium album and other plants, 

 by grinding in a phosphate-buffered 10% sucrose solution (pH 7.9), and 

 filtering through glass wool. The suspension was mixed with a solution 

 of hemoglobin, under exclusion of air. The evolution of oxygen (in 

 light of 40,000 lux) was measured spectrophotometrically by observing 

 the conversion of hemoglobin into oxyhemoglobin. However, oxygen was 

 found only when an aqueous leaf extract was added to the suspension. 

 (This extract was prepared by grinding leaves under acetone, filtering, 

 drying the filtrate and extracting the residue with water.) In further 

 developing these experiments. Hill observed that the leaf extract can be 

 replaced by a yeast extract, and that the efficiency of the latter was 

 dependent on its content of organic iron compounds. Finally, he 

 found that the oxygen evolution can also be brought about by the 

 addition of ferric potassium oxalate, thus allowing him to dispense with 

 cell extracts of unknown composition. The illumination of an air-free 

 mixture of chloroplasts with ferric potassium oxalate and hemoglobin, 

 causes a rapid appearance of oxyhemoglobin, and a reduction of Fe+++ 

 to Fe++ (detectable, for example, by means of dipyridyl). In the dark, 

 the original state is slowly restored again. The total quantity of oxyhemo- 

 globin produced in this experiment, depends on the quantity of ferric 

 salt taken, while the initial velocity of oxidation is determined by the 

 quantity of the chloroplasts. The most active wave lengths are those 

 around 600 mju (thus pointing to chlorophyll as the sensitizing agent). 



The maximum rate of oxygen evolution by a chloroplast suspension in 

 the presence of ferric oxalate, was about equal to the rate of oxygen 

 liberation by a suspension of whole cells (from the same leaves) in the 

 presence of carbon dioxide, but was only one-tenth of the rate of photo- 



