LIGHT- INDUCED RAPID ABSORPTION CHANGES DURING PHOTOSYNTHESIS. 

 II. 430 mp ABSORPTION CHANGES IN AGED CHLOROPLASTS IN THE 

 PRESENCE OF PMS AND ASCORBATE 



Bacon Ke 



Chemical separation of two light reactions in photosynthesis and the re- 

 constitution of the reaction system with artificial reagents have been amply 

 demonstrated. Vernon and Zaugg ^^^ have shown that aged chloroplasts which 

 had lost the capacity for oxygen evolution were able to photoreduce TPN when 

 reduced DPIP was added. Other evidence for the chemical separation of two 

 light reactions has been obtained from experiments on the relief of CMU 

 inhibition of TPN reduction by ascorbate and redox dyes ^'^' and from experi- 

 ments with mutants of Chlamydomonas reinhardi ^^i and Scenedesmus ^^K 



Some preliminary observations on the 430 m^ absorption changes in aged 

 chloroplasts and the response of these absorption changes to ascorbate and 

 PMS will be reported in this note. In aged chloroplasts the 515 nn^ absorption 

 increase was either negligible or completely absent, indicating that the reac- 

 tion associated with oxygen evolution was inactivated. 



Rapid absorption changes were studied with an instrument basically simi- 

 lar to that reported by Witt (^). Construction details of the instrument used 

 in the present work will be reported elsewhere (6). Polychromatic red (620 - 

 720 n^j) flashes with a duration of 2 x 10-5 gee were used for excitation. 

 Aging of spinach chloroplast was done at 4° C for one week. The chloroplast 

 suspension usually contained about 30 ;ig chlorophyll in a total volume of 3. 

 ml 0. 1 M pH 7 phosphate buffer. 



Whereas fresh chloroplasts do not show any absorption change at 430 m;j, 

 at least not under the experimental conditions and sensitivity used here, aged 

 chloroplasts show a light- induced absorption decrease (Fig. 1, curve a). At 

 the time resolution available, the rise time of the signal was estimated to be 

 less than 10''* sec, and the half life of decay was 4 - 5 x 10' sec. 



The 430 m^ absorption change in aged chloroplasts can be completely 

 abolished by 2 x 10"^ M ferricyanide, but is unaffected by ferrocyanide at a 

 concentration as high as 10" '^ M. In a reducing medium of 2 x 10" ^ M ascor- 

 bate, the absorption change was enhanced and the half life was shortened to 

 2 X 10" '^ sec (Fig. 1, curve b). The loss of 430 m>i absorption change caused 

 by ferricyanide can be fully restored by adding ascorbate. 



Upon addition of PMS to a concentration of 3 x 10' ^ M to the aged 



153 



