422 BIRGIT VENNESLAND 



to be oxidized by OX, with an accompanying reoxidation of R by Og, over 

 an electron transport chain which gives a coupled phosphorylation of 2 

 moles of ATP per atom of O reduced. When the Hill reagent is reduced, 

 it replaces O2 as an oxidant for R, but for each mole of Hill reagent reduced 

 by R, an equal amount is reduced by [CO]. If no net ATP synthesis occurs 

 in the latter reaction, the average F jze ratio must be one. 



Because of the importance of the demonstration that CO, is a required 

 component of the Hill reaction, it seemed desirable to verify Warburg's 

 conclusion, particularly for the indophenol dyes which appear to react 

 more immediately with the oxidation-reduction components of the grana 

 than does a Hill reagent such as ferricyanide. In these experiments (which 

 have been done together with Dr. Babette Stern [35, 55]), we wished to 

 measure both the rate of oxygen evolution and the rate of reduction of Hill 

 reagent. Several /xmoles of Hill reagent are required in order to obtain 

 reasonably accurate rate measurements of O., evolution by the manometric 

 procedures employed in our laboratory. Because of its intense pigmenta- 

 tion, trichlorophenol indophenol could not be employed in these amounts. 

 The reduced dye is rapidly oxidized by ferricyanide, however, in a non- 

 enzymic reaction. We therefore used ferricyanide with a catalytic amount 

 of trichlorophenol indophenol. The procedure involved a determination of 

 the relative rates of photoreduction of the ferricyanide in the presence and 

 absence of CO,,. The CO., was removed by the use of KOH in the centre 

 well of the Warburg vessel. 



If measurements were made with fresh grana after the usual equilibra- 

 tion period in the dark of about 15 min., little or no difference was noted in 

 reaction rates. It is thus easy to understand why the CO., effect on the Hill 

 reaction has often been overlooked. If the dark equilibration was extended 

 over a period of several hours, however, a marked effect of CO., developed. 

 The COo appears to be tenaciously held by the preparation, and its removal 

 by KOH in the centre well required a prolonged preincubation in the dark. 

 The longer this preincubation, the greater the CO., effect, as measured by 

 the ratio of the photoreduction rate in the presence of CO2 to the photo- 

 reduction rate in its absence. 



The above procedures were worked out before the latest papers of 

 Warburg and Krippahl [53, 54] were available to us. It is of interest that 

 the details of procedures we have employed are rather different from those 

 used in the Dahlem laboratory, but that the conclusions are in agreement. 

 We used dye with ferricyanide, saturating light with a small amount of 

 grana, and a long preincubation in the dark. Warburg and Krippahl 

 employed ferricyanide without dye, excess grana, limiting light, and a 

 preincubation period of i hr. in the light. They employed a new mano- 

 metric procedure to show how the rate of photoreduction of ferricyanide 

 varies with CO., tension, and they also demonstrated that the effect of CO2 



