KINETICS 1615 



eluding also hydroxylamine and narcotics) which affect photosynthesis 

 more or less uniformly at all light intensities. 



(e) Survival of Photochemical Reductant in the Dark 



In the next chapter we will deal with the controversial question of 

 whether illumination of live cells produces in the latter a strong reductant 

 which survives long enough to permit the demonstration of its "reducing 

 power" after the cessation of illumination. This controversy has extended 

 also to the Hill reaction. 



Mehler (1951 ^) attempted to find a surviving reductant in illuminated 

 (crude) spinach leaf juice. From the data of Calvin, and Fager, on live 

 cells (c/. chapter 36) , coupled with estimates of the recovery of chlorophyll 

 and other cell constituents in the preparation of this juice, he expected to 

 find an amount of reductant equivalent to about 4% of that of chlorophyll. 

 The suspension was illuminated in phosphate buffer in N2 atmosphere, 

 pipetted into a solution of dichlorophenol-indophenol (DCPI), and the 

 absorption at 610 m/x measured about 15 seconds later. No difference 

 could be noticed between the effect on the optical density of chloroplasts 

 illuminated for 3^, 1 or 2 minutes, or not illuminated at all. The tempera- 

 ture of the experiment was not stated. 



Krasnovsky and Kosobutskaya (1952), who made practically identical 

 experiments with chloroplast suspensions from Phaseolus, arrived at the 

 opposite conclusion. They illuminated the suspension for 3 minutes at 

 0° C. in a vacuum Thunberg tube, added DCPI from a side tube 1-5 sec- 

 onds after the end of illumination, and found the optical density at 600 m/x, 

 measured 20-40 sec. after the addition of DCPI, distinctly different from 

 that measured in a similar experiment with not preilluminated suspension. 

 This difference (AD) varied strongly, depending on the ''physiological 

 state" of the material; it was much smaller in a suspension of washed 

 chloroplasts (<0.05) than in the crude leaf juice (up to 0.15). The AD 

 remained unchanged after 3^, 1 or 5 minutes, indicating that the "reducing 

 power" survived for over 5 min. in the dark (at 0° C. and in the absence of 

 O2). Similar results were obtained with thionine, but AD was only one- 

 half as large as with DCPI. A slow reduction of DCPI in the dark was 

 found to be superimposed on the light reaction (or reaction of preillumi- 

 nated chloroplasts) ; this dark reaction was attributed to the presence, in 

 the juice, of dehydrogenases and hydrogen donors, such as ascorbic acid. 

 Preillumination appeared to increase the quantity of the reductants in the 

 chloroplast juice by 10-30%, equivalent to 5-8% of the amount of chloro- 

 phyll present. The light-activated reductant did not seem to be a reduced 

 form of chlorophyll, since the absorption spectrum of the latter was not 



