86 J. L. ROSENBERG, S. TAKASHIMA, R. LUMRY 



The chloroplasts were prepared from sugar-beet leaves, washed at 

 least three times, suspended in buffer without added oxidant, and 

 equilibrated with atmospheres of either air, nitrogen, or water vapor 

 only. All measurements were made below 20°C. 



We had expected to find the same short-lived substance which 

 Witt found after illumination at lower intensities (7). In fact, we had 

 thought that this substance might be present in much larger amounts 

 than in his experiments because of the great intensity of our flashes 

 and because of the short time elapsing between excitation and 

 analysis. Accordingly, we had anticipated that we would be able to 

 determine the entire visible absorption spectrum of Witt's inter- 

 mediate. On the contrary, our experiments were all negative. That is, 

 we could observe no change in absorption spectrum as a result of the 

 flash, within the experimental limit of about 5% in the optical den- 

 sity. Negative results were also obtained in a few experiments with 

 Chlorella suspensions. To justify the difference in behavior of our 

 suspensions with that of chlorophyll in organic solutions, the fol- 

 lowing possibilities must be considered. 



1. Chlorophyll molecules in vivo are all equivalent but are so 

 altered that the lifetime of a molecule in the metastable state is shorter 

 than that in dilute organic solutions. A shorter in vivo lifetime would 

 be expected as a result of increased concentration alone if, as suggested 

 by Livingston (5) , the metastable state decays by a bimolecular re- 

 action involving a second excited or unexcited pigment molecule. 

 Such a reaction, however, must be prohibited in the plant since it 

 would not be photochemically productive. Thus the decrease in 

 metastable state lifetime must be due to an accelerated, specifically 

 in vivo reaction leading to useful photoproducts either through the 

 metastable state or through a new state characteristic of chlorophyll 

 plus an additional reactant of photosynthesis. The half-time of such 

 a reaction would have to be less than 10 microseconds. Perhaps this 

 short a reaction time should not be surprising since the primary 

 photochemical step in photosynthesis must compete very favorably 

 with the known rapid and probably wasteful reaction of oxygen with 

 chlorophyll's metastable state. Since the latter reaction can proceed 

 with zero energy of activation, but apparently does not occur to an 

 appreciable extent even at the relatively high oxygen concentration 

 present in air-saturated supensions of healthy cells, the reaction of the 

 metastable state with the substrate of photosynthesis must indeed be 



