1608 PHOTOCHEMISTRY OF CHLOROPHYLL CHAP. 35 



chlorophyll. Leaf material from vegetables and cereals reacted much 

 slower in the dark than leaf mash from trees, ferns and aquatics. 



Clendenning and Gorham (1950^) later studied the dark reactions of 

 chloroplast suspensions in more detail. Acidification was faster at pH 7.5 

 than at pH 6.0. It required Fe+^ as ferricyanide or oxalate and was non- 

 enzjonatic; it may involve reductants such as ascorbic acid, glutathione 

 or tannins. Two other dark reactions also were observed : boiled sap from 

 bean leaves produced carbon dioxide in the dark when mixed with Hill's 

 solution— apparently by oxidative decarboxylation; crude chloroplast sus- 

 pensions of the same species showed oxygen absorption on the dark (c/. 

 the above-described observations of Warburg and Liittgens). A similar 

 but less rapid oxygen uptake occurred with chloroplasts of other species. 

 The autoxidation was heat sensitive in some species and not in others, indi- 

 cating that both enzymatic and nonenzymatic reactions are involved. 



Gerretsen (1950-) also observed the ''respiration" of crude Avena 

 chloroplast suspension in the dark. It was cyanide-insensitive (up to 

 0.04% HON), not stimulated by glucose, but enhanced by the amino acid 

 asparagine; Gerretsen therefore concluded that the main cyanide-sensitive 

 and glucose-stimulated respiration apparatus of the cells was destroyed in 

 the maceration, while an accessory cyanide-insensitive mechanism, able 

 to utilize amino acids, survived it. Mn++ ions had no influence on the 

 rate of oxygen uptake by the chloroplasts in the dark (as contrasted to 

 light). Gerretsen (1951) observed an increase in acidity of aerobic chloro- 

 plast suspensions in dark, which he ascribed to CO2 formation by respira- 

 tion; under anaerobic conditions a similar trend was observed and ascribed 

 to fermentation. 



{d) Inhibition and Stimulation 



If Hill's reaction is brought about by a part of the photosjmthetic ap- 

 paratus salvaged after the mechanical destruction of the cells, it must con- 

 tain intact the photochemical mechanism and the enzymatic components 

 (designated by Ec and Eo in chapters 7 and 9) involved in the liberation of 

 oxygen from the primary photochemical oxidation product (designated 

 there by Z, A'OH, or {O2}). Whether any enzymes which ordinarily oper- 

 ate between the primary photochemical reduction product (designated as 

 HX in chapter 7) and carbon dioxide are involved in the Hill reaction, we 

 do not know. The substitute oxidants (such as quinone or Fe+^) may re- 

 act with the primary reduction product HX directly, without the help of 

 an enzyme (or they may even take part in the photochemical reaction 

 proper, without the intermediary of the system X/HX which we have as- 

 sumed to be tied to chlorophyll) . Certainly the Hill reaction does not need 

 the carboxylating enzyme, Ea, which catalyzes the dark association of 



