424 BIRGIT VENNESLAND 



CO2 eflFect. The results show that the rate of photoreduction of ferricyanide 

 alone, though stimulated by COo, is still relatively slow even in the presence 

 of COg. When dye is present together with CO2, however, the rate of 

 photoreduction of ferricyanide is considerably faster than the rate observed 

 when either dye or CO2 is absent. Since it seems unlikely that CO2 should 

 be required for the reduction of ferricyanide by reduced dye, the data 

 suggest that it is the photoreduction of the dye itself which is COg- 

 dependent. It also seems unlikely that the CO2 effect is a pH effect, since 

 the addition of a small amount of dye has no appreciable effect on the pH. 

 Table II shows an experiment to verify this conclusion. The small decrease 

 in pH brought about by added COg was duplicated by addition of HCl, 

 with no stimulatory effect on the Hill reaction. 



Finally, and most importantly, the CO2 effect could be shown to be 

 freely reversible [55]. A preparation of grana which has lost activity by 

 prolonged incubation in the presence of KOH is rapidly reactivated if COg 

 is added back a few minutes prior to the photoreduction assay. A repre- 

 sentative experiment illustrating the reactivation is shown in Table III. 



We regard the above experiments as a partial confirmation of Warburg's 

 results. The requirement of COo for the Hill reaction is another discovery 



TABLE III 

 Reversibility of the CO., Effect on the Hill Reaction 



Reaction rate in /xmoles per 

 mg. chlorophyll per hour 



rrocedure Ferricyanide Pressure increase 



reduced calculated as O2 



(4 X ytimoles O2) 



I '5% CO., in No present in dark and light 108 (144) 



N2, and no CO2 in dark and light 66 62 

 No CO2 in dark, 1-5% COo in N2 added 15 



min. before assay in light loi (142) 



The reaction mixtures contained 100 /imoles of sodium pyrophosphate buffer 

 of pH 6-8, 40 /tmoles of KCl, 0-07 ;umole of trichlorophenol indophenol, and 

 spinach grana containing 0-2 mg. chlorophyll. Samples were preincubated in the 

 dark for 2 • 5 hr. Assay in light was for 40 min. Other conditions are those given for 

 Table I. Figures in brackets include CO2 given off from the bicarbonate of the 

 medium as the result of acid formation attending ferricyanide reduction. 



of major significance, which should be listed with the many notable 

 achievements of the Dahlem laboratory. Although our experimental 

 techniques are not as elegant as those employed by Warburg and Krippahl, 

 we feel our results have additional reinforcing value because they were 



