CAN CARBON DIOXIDE SERVE AS OXIDANT IN HILL REACTION? 1535 



on the initial maximum rate, but a marked influence on the final, constant level; subse- 

 quent addition of CO2 in the nitrogen atmosphere caused a marked increase in this rate. 

 "Light saturation" of the steady oxygen production was reached, with isolated chloro- 

 plasts, much earlier than with intact leaves. In flashing light, too, the yield per flash 

 was 50-100 times smaller with isolated chloroplasts than with whole leaves. 



As mentioned above, Franck (1945) first interpreted these results as indicating an 

 "oxygen burst" caused by photochemical utilization of a carbon dioxide derivative 

 (RCOOH, or ACO2, or { CO2! in our earlier designations) accumulated in the chloroplasts 

 before the leaves were macerated, and a much lower, steady oxygen production which 

 may be due to very slow renewed formation of the same compound in isolated chloro- 

 plasts. This hypothesis was revised by Franck when experiments with radiocarbon 

 (Brown and Franck, 1947) produced no evidence of carbon dioxide fixation by chloro- 

 plasts {i. e., of a CO2 uptake not reversed by treatment with boiling acetic acid), either 

 under anaerobic or under aerobic conditions. The sensitivity of the method was high 

 enough to discover an uptake of CO2 equivalent to the amount of oxygen liberated. The 

 enhancing effect of carbon dioxide on oxygen liberation was therefore reinterpreted as a 

 catalytic phenomenon. 



Fager (19520 attempted to obtain chloroplast suspensions retaining a 

 capacity for the reduction of carbon dioxide by using low temperature 

 (<1° C), anaerobic conditions (N2 atmosphere) and only dim light during 

 the preparation of chloroplasts (from spinach leaves). Crude juice, con- 

 taining chloroplasts, grana, etc., but no whole cells, was obtained by press- 

 ing the macerate through a sintered filter plate. The uptake of radiocarbon 

 from C*02 by this juice was increased noticeably (by about 70%) in light; 

 however, this increase corresponded to only 0.01 mole C* taken up in 15 

 min. per mole chlorophyll — -about 0.1% of photosynthetic fixation in intact 

 cells. Centrifugation of the crude juice, and resuspension of the precipitate 

 in buffer, gave a product without CO2 fixing capacity; combining the pre- 

 cipitate with the supernatant restored it. Analysis showed that over 70% 

 of the additional C* fixation in light was in phosphoglyceric acid, PGA, 

 (54%) and pyruvic acid, PA (18%). (Of the C* fixed in the dark, 65% 

 was in phosphoglyceric acid and 20% in pyruvic acid.) After only 1 min. 

 of exposure, the ratio of extra tagged PGA to extra tagged PA was as high as 

 10: 1, indicating that PGA was tagged first, in chloroplast preparation as in 

 live cells (c/. chapter 36). Addition of various low molecular substances 

 increased the extra fixation of C* in light; among these were acetic acid 

 (increase by 40%), acetaldehyde (by 90%), pyruvic acid (by 130%), gly- 

 oxal (by 140%), and 2-phosphoglycol aldehyde (by 40%). The same com- 

 pounds, with the exception of glyoxal and phosphoglycoaldehyde, also in- 

 creased the dark fixation. This seemed to indicate (see, however, below) 

 the presence of an enzyme capable of fixing CO2 in these C2 compounds 

 with the help of light energy — a result that could bear relation to the 

 mechanism of formation of PGA in photosynthesis (c/. chapter 36, section 

 8). However, it now seems most hkely (chapter 36, section 7) that PGA 

 results from carboxylation and splitting of a pentose diphosphate. 



