1606 PHOTOCHEMISTRY OF CHLOROPHYLL CHAP. 35 



dant]. Wessels concludes that reoxidation occurs mainly by action of 

 oxygen on the intermediate X, and not on the hydroquinone Q. 



Among the uncertainties of this derivation are, in addition to the neglect 

 of intermediate systems "on the oxidation side," also the use of the concen- 

 trations [X], [Chi], [ox.] and [red.] as if the system were homogeneous 

 (while in fact, chlorophyll, and probably X as well, are structure-bound in 

 the chloroplast fragments). Finally, the independence of R of [ox.] and 

 [Chi] cannot be accepted as a general rule without more varied data. 



For mathematical elaboration of the importance of the last point, we 

 must refer to Horwitz (1954^). We can only mention here the more com- 

 plex kinetic derivations of Gilmour et at., intended primarily to account for 

 observations in flashing light (c/. p. 1479). 



(c) Dark Reactions 



It was mentioned before that cell macerates and chloroplast suspensions 

 undergo various reactions in the dark, which may involve molecular oxy- 

 gen or added oxidants, and complicate quantitative investigation of the Hill 

 reaction. 



Warburg and Liittgens (1946) found that press juices from spinach or 

 beet leaves "respire" {i. e., consume oxygen in the dark); this oxygen con- 

 sumption is not significantly affected by light. 



The suspensions used in this stage of Warburg's work were prepared 

 by grinding the leaves in a meat grinder, and pressing the mash through 

 cloth. Wliole cells and undissolved salts were precipitated from the sus- 

 pension by centrifuging briefly at 1200 g. The supernatant was dark green, 

 had a pH of about 6.5 and contained whole as well as broken chloroplasts. 

 This green extract showed in a respirometer a rapidly declining oxygen up- 

 take. Carbon monoxide (80%) reduced it by 50%. Since this inhibition 

 was not light sensitive, it was considered indicative of oxygen transfer by a 

 copper-containing oxidase (such as the cytochrome oxidase). This hy- 

 pothesis was supported by the observation that the oxygen consumption 

 increased upon the addition of the polyphenol, pyrocatechol. Even in 

 the presence of this substrate, respiration declined with time; it could be 

 further stabilized by the addition of excess hydroquinone. The oxidation- 

 reduction chain, which is operative in the presence of both compounds, 

 probably is 



p-hydroquinone^ f p-quinone 



+ / > \ + 



o-quinone ■, /pyrocatechol 



+ < + 



Cu "*■ oxidase ' ^Cu +2 oxidase 



+ > [ + 



MO2 / I H2O 



(35.36) p-hydroquinone + x2 O2 > p-quinone + H2O 



