1540 PHOTOCHEMISTRY OF CHLOROPHYLL CHAP. 35 



cellular and filamentous green algae, liverworts, horsetails, herbaceous 

 monocotyledons and dicotyledons (all these gave positive results) ; and 

 from mosses, ferns, gymnosperms and woody angiosperms (which showed 

 no Hill activity at all). The suspensions from gymnosperms contained 

 much oil, while those from broad leaf trees (Acer, Populus, Quercus) were 

 so acid that the chloroplasts coagulated before the Hill solution was added. 

 (Active chloroplasts from these leaves could probably be obtained by using 

 more strongly buffered media.) With the 70 "active" species, the initial 

 rate of acidification in Hill's mixture in light varied from an equivalent of 50 

 mm.^ O2 to that of 2500 mm.^ O2 per mg. chlorophyll per hr. The highest 

 yields were obtained with material from millet, flax, Swiss chard, spinach, 

 lettuce and lamb's quarters (cf. table 35.X). Chloroplast preparations 

 from Chlorella (obtained by grinding chilled cells in a tissue homogenizer) 

 gave initial rates of 200-500 mm. Vhr., but the reaction stopped after about 

 5 minutes (compare the results of Punnett et al. below). 



About 70 of the investigated crude suspensions produced acid in the 

 dark in the presence of Hill's solution — in some cases (spruce, pine, arbor 

 vitae), as much as an equivalent of 5000 mm.^ Oa/hr. ! Millet, spinach and 

 flax chloroplasts (as well as those of Chlorella) showed practically no such 

 dark reaction, chloroplasts from Swiss chard and lamb's quarters only a 

 very weak one. (These results show that the choice of spinach leaves as 

 the most common experimental material for studying the Hill reaction has 

 been a lucky one.) Crude suspension from Lycopodium and Sedum ex- 

 hibited an alkaline instead of an acid "drift" after mixing with a neutral 

 Hill solution — probably caused by precipitation of calcium oxalate (Clen- 

 denning and Gorham 1950^). 



It was mentioned above that the inactivity of some chloroplast prepa- 

 rations was ascribed by Kumm and French to the presence of tannins 

 forming colored complexes with iron. Clendenning and Gorham, too, ob- 

 served a purplish bro\\ii coloration upon addition of Hill's mixture to some 

 inactive chloroplasts, but the same change was noted also with some of the 

 active preparations; furthermore, chloroplasts which were inactive with 

 Hill's solution, were inactive also with quinone. By adding boiled cell sap 

 from "inactive" species (e. g., bean) to active chloroplast suspensions (e. g., 

 those from millet), it could be sho\vn that this sap contained a heat-stable 

 "inhibitor" of the Hill reaction. 



With New Zealand spinach, chloroplasts from mature leaves were more 

 active (with ferric oxalate as well as with quinone) than those from young 

 leaves. This applied not only to crude suspensions, but also to chloro- 

 plasts separated by high-speed centrifugation from cells fluids and plasma. 

 The activity decreased again in senescent leaves. The deficiency of cer- 

 tain minerals (N, Fe), which produced chlorotic leaves, caused the photo- 



