1538 PHOTOCHEMISTRY OF CHLOROPHYLL CHAP. 35 



following requirements : It must be rich in chlorophyll ; the plants must be 

 easily macerated, with the destruction of cell walls and liberation of cell 

 contents; the cell sap and plasma should not contain compounds which 

 inhibit the Hill reaction, undergo dark reactions with the oxidant, or 

 ferment, with the liberation of carbon dioxide, either in the dark, or in 

 light. 



"Crude" chloroplast preparations, i. e., suspensions obtained from cell 

 macerates simply by removing coarse debris (by filtration through cloth or 

 by low-speed centrifugation) contain all the components of the plasma and 

 cell sap. Such preparations show wide differences in the initial rate of oxy- 

 gen liberation in light, the total amount of oxygen they can produce, and 

 the extent of dark and photochemical side reactions. Their stability, i. e., 

 the rate of deterioration of the photochemical activity in dark storage or in 

 light, also varies widely. Many of these differences probably are due to 

 the presence of cell sap and plasma constituents. If these are removed, 

 e. g., by high-speed centrifugation, the precipitated and resuspended ma- 

 terial, which now consists of whole and broken chloroplasts (with some 

 plasma residues probably still clinging to them, cf. chapter 14, page 369), 

 shows fewer side reactions, but usually has a lower photochemical activity 

 (related to unit mass of chlorophyll) than the crude suspension. The loss of 

 activity is enhanced if the precipitate is washed to better remove the sap 

 and plasma components. However, the activity lost can often be restored, 

 at least partially, by the addition of salts, particularly of potassium chloride 

 (cf. below, section 3(c)). 



The sap and plasma-free chloroplast preparations from different plants 

 still exhibit considerable differences in efficiency. The latter depends not 

 only on the species used but also on the season of the year and the time of 

 the day when the leaves were gathered, their age, the intensity and dura- 

 tion of light exposure prior to collection, and preillumination (or dark 

 storage) before maceration. 



Hill and Scarisbrick (1940) noted that the efficiency of chloroplasts from Stellaria 

 media depended stronglj^ on the time of the day when the leaves were picked. French 

 and Rabideau (1945), in comparing the quantum yields in chloroplast suspensions from 

 Spinacia and Tradescantia (page 1129), found that pretreatment of the material (ex- 

 posure of leaves to darkness or light) affected the yield much more strongly than the 

 genetic difference between the two species. 



Kumm and French (1945) compared the rates of oxygen evolution by 

 chloroplast preparations from 22 species, using as oxidant Hill's mixture 

 (0.5 M potassium oxalate, 0.01 M ferric ammonium sulfate, 0.02 M potas- 

 sium ferricyanide, 0.2 M sucrose, 0.167 M borate buffer, pH 7.0). Many 

 preparations gave no Hill reaction at all; inhibition by substances such as 



