REVERSIBLE BLEACHING OP CHLOROPHYLL in VIVO 69 



toluene, has absorption bands at 518 m/x and 585 ni/z. Both bands were 

 attributed to a semiquinone: the 518-m/i band to its ion and the 585- 

 m/i band to the nondissociated form.* Krasnovskii (6) has suggested 

 that chlorophyll participates in photosynthesis by reversible reduc- 

 tion to the semiquinone state. 



Studies of reversible photobleaching of chlorophyll in 02-free 

 methanol (9,10,14) and of its reversible photooxidation by Fe+++ in 

 methanol (15) and by quinone in rigid solvents (8) revealed an en- 

 hanced absorption in the region 450 to 530 m/i, but no sharp bands 

 were detected. The brown intermediate in the phase test (probably, 

 an ionized enol form) of chlorophyll a shows a strong band at 524 

 m/i, with a shoulder at 486 m/x, and weaker bands at 645 and 683 

 mn (16). 



It thus seems that, in vitro, reduced chlorophyll a is characterized 

 by bands at 525 m/x and 585 m/c, metastable chlorophyll a by a band 

 at 475 m/i, and ionized chlorophyll by bands at 486 m/x and 524 m/i. 

 Reversible oxidation increases absorption in the same general region, 

 apparently without producing a sharp new band. The absorption in 

 the red decreases in every case. 



Duysens (2) (and also Witt (17)) noted that illuminated Chlorella 

 cells show, in addition to spectral changes attributable to the oxida- 

 tion of a cytochrome (and perhaps also to the reduction of a pyridine 

 nucleotide) (1), a sharp rew absorption band at 515 m/x and a some- 

 what smaller "negative" band (i.e., selective decrease of absorption) 

 at 478 m/i. Duysens attributed the two changes to the transformation 

 of an unidentified pigment, whose "dark" form absorbs at 478 m/x 

 and whose "phototropic" form absorbs at 515 m/x. Witt noted the 

 515-m/t band also in plants exposed to an intense light flash. Duysens 

 observed no change in the absorption in the red region, thus apparently 

 precluding the attribution of the effect at 515 m/i to chlorophyll. 



Using an apparatus similar in principle to that of Duysens (3) 

 but somewhat different in construction, we have been able to observe 

 a decrease in absorption of illuminated Chlorella cells in the red. 

 In our apparatus, the modulated photomultiplier output was ampli- 

 fied through three sharply tuned and six narrow-band staggered 

 stages; by means of a phase-inverting parallel twin-T tuned network, 

 a considerable portion of the signal was negatively fed back from the 

 fourth stage to the input. The ultrasharp tuning and increased feed- 



* Linscliitz and co-workers (8A) found no evidence of the presence of free radi- 

 cals in this system by the method of paramagnetic resonance. 



